Nutritional supplement comprising lactoferrin protein complexes in subjects in need thereof

Abstract

The present invention discloses a nutritional supplement for oral administration comprising a protein component, including heat-treated lactoferrin, whey protein hydrolysate, alpha-lactalbumin, and a naturally occurring milk. The combination of these components provides a synergistic effect for improved oral delivery of proteins, enhancing bioavailability and efficacy. The nutritional supplement offers a novel approach to delivering essential proteins for various applications, such as nutritional supplements, pharmaceuticals, and functional foods, with potential benefits for human health and well-being.

Description

NUTRITIONAL SUPPLEMENT COMPRISING LACTOFERRIN PROTEINCOMPLEXES IN SUBJECTS IN NEED THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims priority to U.S. Provisional Application No. 63 / 575,296 filed on April 5, 2024. which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Nutritional supplements, such as infant formulas, comprise combinations of proteins, fats, carbohydrates and often vitamins and minerals. The present disclosure is directed to a surprising and unexpected nutritional supplement compnsing protein complexes comprising lactoferrin wherein the nutritional supplement has a bioactivity approximately the same as a nutritional supplement including lactoferrin not included in a protein complex.SUMMARY

[0003] Embodiments described herein are directed to protein complexes for nutritional supplements including lactoferrin and alpha-lactalbumin.

[0004] Embodiments described herein are directed to a protein complex for nutritional supplements, the protein complex including lactoferrin and whey protein hydrolysate proteins.

[0005] Embodiments described herein are directed to a protein complex for nutritional supplements, the protein complex including lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.

[0006] Embodiments described herein are directed to a nutritional supplement, the nutritional supplement including a protein complex including lactoferrin and alphalactalbumin.

[0007] Embodiments described herein are directed to a nutritional supplement, the nutritional supplement including a protein complex including lactoferrin and whey protein hydrolysate proteins.

[0008] Embodiments described herein are directed to a nutritional supplement, the nutritional supplement including a protein complex including lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.BRIEF DESCRIPTION OF DRAWINGS

[0001] FIG. 1 A shows native PAGE of undigested proteins.

[0002] FIG. IB shows western blotting of undigested lactoferrin.

[0003] FIG. 1C shows western blotting of undigested alpha-lactalbumin.

[0004] FIG. ID shows native PAGE of simulated gastrointestinal digestion.

[0005] FIG. IE shows western blotting of simulated gastrointestinal digestion of lactoferrin.

[0006] FIG. IF shows western blotting of simulated gastrointestinal digestion of alpha-lactalbumin.

[0007] FIG. 2A shows SDS PAGE of undigested proteins.

[0008] FIG. 2B shows western blotting of undigested lactoferrin.

[0009] FIG. 2C shows western blotting of undigested alpha-lactalbumin.

[0010] FIG. 2D shows SDS PAGE of simulated gastrointestinal digestion.

[0011] FIG. 2E shows western blotting of simulated gastrointestinal digestion of lactoferrin.

[0012] FIG. 2F shows western blotting of simulated gastrointestinal digestion of alpha-lactalbumin.

[0013] FIG. 3 shows internalization into human intestinal epithelial cells (HIECs).

[0014] FIG. 4A shows effect of proteins on proliferation of HIECs.

[0015] FIG. 4B shows effect of proteins on differentiation of Caco-2 cells.

[0016] FIG. 5A shows effect of proteins on transcription.

[0017] FIG. 5B shows effect of proteins on transcription.

[0018] FIG. 6 shows effect of proteins on transcription of TGF-(31.

[0019] FIG. 7 shows effect of proteins on IL-18 secretion by Caco-2 cells.

[0020] FIG. 8 shows effect of proteins on growth of EPEC.

[0021] FIG. 9A shows effects of proteins on the inflammatory response induced byEPEC infection.

[0022] FIG. 9B shows effects of proteins on the inflammatory response induced by EPEC infection.DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of variousaspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0024] DEFINITIONS

[0025] As used in this document, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term “comprising” means “including, but not limited to."

[0026] While various compositions, methods, and devices are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions, methods, and devices can also "consist essentially of' or "consist of the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0027] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0028] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation isintended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (for example, “a” and / or ‘'an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0029] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0030] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can beeasily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently- broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0031] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly- disclosed.

[0032] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

[0033] As used herein, the term ’‘about” when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27.500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation.

[0034] The terms “administer,” “administering” or “administration” as used herein refer to directly administering a compound or a composition to a subject.

[0035] The term “bioactivity ” as used herein refers to the intended effect on a living organism. As used herein, lactoferrin promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promotes differentiation of intestinal cells, induces differentiation of intestinal cells, inhibits growth of enteropathogenic Escherichia coli in the digestive system, inhibits bacterial infection in the intestinal lumen, increases interleukin- 18 secretion by intestinal cells, modulates interleukin- 18 secretion by intestinal cells, increases transforming growth factor [31 (TGF-01) secretion by intestinal cells, modulates transforming growth factor |31 (TGF-p l ) secretion by intestinal cells, increases intestinal immunity, lowerscell permeability, increases expression of tight junction proteins, increases gut health, and combinations thereof. A protein complex or nutritional supplement comprising a protein complex with a bioactivity substantially similar to the individual proteins not in a protein complex or a nutritional supplement that does not comprise protein complexes will be within a standard deviation of the individual proteins not in a protein complex or a nutritional supplement that does not comprise protein complexes.

[0036] As used herein, the term ‘"effective amount7’ refers to an amount that results in measurable result. As used herein, an effective amount of a nutritional supplements maintains or restores nutrition in the subject.

[0037] The term “naturally occurring milk” as used herein refers to milk from any natural source. Naturally occurring milk may contain the same or similar ingredients as the nutritional supplements described herein, but these ingredients are not used in calculating the amount of each ingredient in the nutritional supplements described herein. A naturally occurring milk may be from a non-human source, such as a cow (bovine), sheep, goat, yak, water buffalo, horse, reindeer, or camel. A naturally occurring milk may be non-animal milk, such as soy, rice, hemp, pea, oat. almond or other nuts. A naturally occurring milk may be whole milk, reduced fat milk, low fat milk, and fat-free milk.

[0038] The term “nutritional support” as used herein refers to delivery of nutrition to maintain or restore nutritional status.

[0039] The term “preventing” may be taken to mean to prevent a specific disorder, disease or condition and / or prevent the reoccurrence of a specific disorder, disease or condition.

[0040] The term “protein complex” as used herein is a group of two or more associated polypeptide chains. A protein complex can comprise two or more proteins, peptides, or combinations thereof. The association within a protein complex may be achieved by encapsulation, covalent or non-covalent bonds, hydrophobic interactions, electrostatic interactions, van der Waals forces, interstitial interactions, and combinations thereof.

[0041] The term “subject” as use herein, refer to any recipient of the nutritional supplement described herein. The subject can be human or non-human. The subject may be in need of a nutritional supplement. As used herein the terms “subject” and “subject in need thereof’ are interchangeable.

[0042] The term “substantially free” as used herein refers to 1% or less of a substance.

[0043] The term “substantially similar” as used herein refers to having a shared property within 10% of its counterpart.

[0044] As used herein the terms “treat’", “treated”, or “treating"’ refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to protect against (partially or wholly) or slow down (for example, lessen or postpone the onset of) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results such as partial or total restoration or inhibition in decline of a parameter, value, function or result that had or would become abnormal. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent or vigor or rate of development of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether or not it translates to immediate lessening of actual clinical symptoms, or enhancement or improvement of the condition, disorder or disease. Treatment seeks to elicit a clinically significant response without excessive levels of side effects.

[0045] As used herein, "whey protein hydrolysate" refers to whey proteins that have been partially broken down by enzymatic processes. This partial hydrolysis may result in smaller peptide fragments that may be more easily absorbed by the body compared to intact whey proteins.

[0046] The term "lipid-based delivery system" as used herein refers to a carrier system composed of lipids that may encapsulate, protect, and / or enhance the delivery of bioactive compounds. Such systems may include but are not limited to liposomes, micelles, emulsions, or solid lipid nanoparticles.

[0047] As used herein, "protective matrix" refers to a surrounding structure or material that may shield or preserve the integrity of encapsulated compounds. This matrix may provide protection against environmental factors, enhance stability, or control the release of the encapsulated substances.

[0048] The term "cognitive development" as used herein refers to the process by which a child learns to think, reason, understand, and remember. This may include the development of skills such as problem-solving, language acquisition, and decision-making.

[0049] As used herein, "early childhood" typically refers to the period of human development from birth to around eight years of age. This stage may be characterized by rapid physical, cognitive, and social-emotional grow th and development.

[0050] The term "micellar structure" as used herein refers to an aggregation of amphiphilic molecules in a solution, typically forming a spherical or cylindrical structure witha hydrophobic core and hydrophilic exterior. In the context of this disclosure, micellar structures may be used for encapsulation or delivery’ of bioactive compounds.

[0051] PROTEIN COMPLEXES

[0052] Embodiments described herein are directed to protein complexes for nutritional supplements. In some embodiments, the protein complex comprises lactoferrin and alpha-lactalbumin. In some embodiments, the protein complex comprises lactoferrin and whey protein hydrolysate proteins. In some embodiments, the protein complex comprises lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.

[0053] In some embodiments, the protein complexes further comprise proteins from naturally occurring milk. In some embodiments, the naturally occurring milk is nonfat milk. In some embodiments, the naturally occurring milk is whole milk.

[0054] In some embodiments, the protein complex is encapsulated within a lipid- based delivery system. In some embodiments, the lipid-based delivery system enhances stability or bioavailability of the protein complex. In some embodiments, the protein complex is encapsulated in a micelle.

[0055] In some embodiments, the protein complex is encapsulated in a protective matrix. In some embodiments, the protective matrix enhances stability' or bioavailabili ty of the protein complex.

[0056] In some embodiments, the protein complex comprises individual proteins held together in a tertiary structure. In some embodiments, the protein complex comprises two or more proteins. In some embodiments, the individual proteins are bound together by covalent bonds, non-covalent bonds, hydrophobic interactions, electrostatic interactions, van der Waals forces, interstitial interactions, encapsulated, and combinations thereof.

[0057] In any embodiment described herein, the protein complex is bioactive, resistant to gastrointestinal digestion and is capable of binding to a lactoferrin receptor on a cell membrane of intestinal epithelial cells. In some embodiments, the bioactive protein complex promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promoting differentiation of intestinal cells, inducing differentiation of intestinal cells, inhibiting growth of enteropathogenic Escherichia coli in the digestive system, inhibiting bacterial infection in the intestinal lumen, increasing interleukin- 18 secretion by intestinal cells, modulating interleukin- 18 secretion by intestinal cells, increasing transforming growth factor [31 (TGF- i) secretion by intestinal cells, modulating transforming growth factor pi (TGF- i) secretion by intestinal cells, increasing intestinal immunity, lowering cell permeability,increasing expression of tight junction proteins, increasing gut health, and combinations thereof.

[0058] NUTRITIONAL SUPPLEMENT

[0059] The nutritional supplement described herein comprise a protein component, a fat component, a naturally occurring milk component, a carbohydrate component, and combinations thereof. In some embodiments, the nutritional supplement comprises any protein complex described by any embodiment of disclosed herein. In some embodiments, the nutritional supplement is pasteurized. In some embodiments, the nutritional supplement comprises lactoferrin, whey protein hydrolysate, alpha-lactalbumin. In some embodiments, the nutritional supplement comprises, whey protein hydrolysate, alpha-lactalbumin, and a naturally occurring milk.

[0060] In some embodiments, a protein complex or nutritional supplement comprising a protein complex with a bioactivity substantially similar to individual proteins not in a protein complex or a nutritional supplement that does not comprise protein complexes. In some embodiments, a protein complex or nutritional supplement comprising a protein complex with a bioactivity the same as individual proteins not in a protein complex or a nutritional supplement that does not comprise protein complexes.

[0061] In some embodiments, the bioactive nutritional supplement promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promoting differentiation of intestinal cells, inducing differentiation of intestinal cells, inhibiting growth of enteropathogenic Escherichia coli in the digestive system, inhibiting bacterial infection in the intestinal lumen, increasing interleukin- 18 secretion by intestinal cells, modulating interleukin- 18 secretion by intestinal cells, increasing transforming growth factor pi (TGF-[31) secretion by intestinal cells, modulating transforming growth factor [31 (TGF-J31) secretion by intestinal cells, increasing intestinal immunity, lowering cell permeability, increasing expression of tight junction proteins, increasing gut health, and combinations thereof.

[0062] In some embodiments, the bioactive lactoferrin promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promoting differentiation of intestinal cells, inducing differentiation of intestinal cells, inhibiting growth of enteropathogenic Escherichia coli in the digestive system, inhibiting bacterial infection in the intestinal lumen, increasing interleukin- 18 secretion by intestinal cells, modulating interleukin- 18 secretion by intestinal cells, increasing transforming growth factor [31 (TGF-J31) secretion by intestinal cells, modulating transforming growth factor (31 (TGF-[31) secretion by intestinal cells, increasingintestinal immunity, lowering cell permeability, increasing expression of tight junction proteins, increasing gut health, and combinations thereof.

[0063] In some embodiments, the nutritional supplement comprises about 5% to about 20% protein by weight of the nutritional supplement, about 15% to about 40% fat by weight of the nutritional supplement, about 1% to about 40% milk by weight of the protein component, about 40% to about 70% carbohydrate by weight of the nutritional supplement, and combinations thereof.

[0064] Protein Component of the Nutritional Supplement

[0065] In some embodiments, the protein component of the nutritional supplement comprises lactoferrin, whey protein hydrolysate, alpha-lactalbumin, osteopontin, (3-casein, a- casein, K- casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, amylase, ai-antitrypsin, folate-binding protein, insulin-like growth factor-1, epidermal growth factor, cytokines, lactadherin, lactoperoxidase, milk glycans, milk fat globule membrane (MF GM), and combinations thereof. In some embodiments, the protein component comprises nutrient carrier proteins comprising lactoferrin with iron, alpha-lactalbumin with calcium and zinc, [>- casein with calcium and phosphorous, folate binding protein with folate, haptocorrin with vitamin B12, and combinations thereof. In some embodiments, the protein component comprises proteins naturally occurring in milk from any source, such as human milk, bovine milk, or plant-based milks. In some embodiments, the protein component comprises a protein disclosed in the commercial product: '‘Whole Nutritional Infant Formula’ available from ByHeart, of w hich the product label is incorporated herein by reference in its entirety. Further additional proteins are disclosed in Bardanzellu et al. ("' Omics1in human colostrum and mature milk: looking to old data with new eyes," Nutrients, 2017. vol. 9. no. 8, E843), which is incorporated herein by reference in its entirety’.

[0066] In some embodiments, the protein component of the nutritional supplement comprises partially hydrolyzed proteins. In some embodiments, the hydrolyzed proteins are treated with enzymes to break down some, most, or all of the proteins in the protein component. Hydrolyzed proteins can reduce allergic reactions, reduce intolerance and sensitization. In some embodiments, the partially hydrolyzed proteins may be whey protein hydrolysate (WPH) protein. WPH protein generally has a size of about 3 kDa to 10 kDa, which is small and more easily digested. The degree of hydrolysis is the extent to which peptide bonds are broken by any method of protein hydrolysis known to a person skilled in the art. In some embodiments, the degree of protein hydrolysis is about 3% to about 25%. In some embodiments, the degreeof protein hydrolysis is about 5% to about 15%. In some embodiments, the degree of protein hydrolysis is about 3%, about 4%, about 5%, about 6%. about 7%, about 8%. about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25% of the protein, and ranges between any two of these values.

[0067] In some embodiments, the protein component of the nutritional supplement comprises proteins that are intact. In some embodiments, the protein component comprises proteins that are not hydrolyzed. For example, the protein component may comprise alphalactalbumin enriched whey protein concentrate or isolate ("alpha-lactalbumin" is used to denote the alpha-lactalbumin protein in the nutritional supplement provided by the concentrate or isolate). Alpha-lactalbumin is a small protein (14 kDa) that is nearly the size of a hydrolyzed protein. Alpha-lactalbumin is a protein present in human breast milk. It has been demonstrated to reduce gastrointestinal events and has a high content of essential amino acids, which enables having a lower protein nutritional supplement. Further, peptides generated from proteolysis of alpha-lactalbumin have shown in vivo to have bactericidal, opioid agonist, and immunostimulating activity.

[0068] In some embodiments, the protein component of the nutritional supplement is about 5% to about 20% by weight of the nutritional supplement. In some embodiments, the protein component is about 8% to about 16% or about 10% to about 15% by weight of the nutritional supplement. In other embodiments, the protein component is about 5% to about 10%, about 10% to about 15%, about 15% to about 20% by weight of the nutritional supplement. Specific examples of the protein component include about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 14.1%, about 14.3%, about 14.5%, about 14.7%, about 14.9%. about 15%, about 16%, about 17%, about 18%, about 19%, about 20% by weight of the nutritional supplement, and ranges between any two of these values. In some embodiments, the protein component comprises about 5 g / L to about 30 g / L of the nutritional supplement. In some embodiments, the protein component comprises about 10 g / L to about 25 g / L of the nutritional supplement. In some embodiments, the protein component is about 5 g / L. about 6 g / L. about 7 g / L. about 8 g / L. about 9 g / L, about 10 g / L, about 11 g / L, about 12 g / L, about 13 g / L, about 13.2 g / L, about 13.4 g / L, about 13.6 g / L, about 13.8 g / L, about 14 g / L, about 15 g / L, about 16 g / L, about 17 g / L, about 18 g / L, about 19 g / L, about 20 g / L, about 21 g / L, about 22 g / L, about 23 g / L, about 24 g / L, about 25 g / L, about 26 g / L, about 27 g / L. about 28 g / L, about 29 g / L, about 30 g / L of the nutritional supplement and ranges between any two of these values.

[0069] In some embodiments, the lactoferrin includes apolactoferrin, hololactoferrin, or a combination of both. Lactoferrin is an iron-binding glycoprotein that has been proposed to play a role in iron uptake by the intestinal mucosa and to act as a bacteriostatic agent by withholding iron from iron-requiring bacteria. It is also present in neutrophils and is released during inflammation, which suggests that lactoferrin is involved in the immune response. Lactoferrin may function also as a growth factor and / or a bactericidal agent, and may promote maturation of the infant gut.

[0070] In some embodiment, lactoferrin is isolated from the milk of a non-human animal or produced by a genetically modified organism. The lactoferrin may be isolated by any method known to one skilled in the art. The process generally involves an absorbing step to obtain lactoferrin from raw milk material using a weakly acidic cationic exchanger, a washing step to remove non-absorbed substances, and a desorbing step to obtain purified lactoferrin. Varying methods of producing lactoferrin are disclosed in U.S. Pat. Nos. 4,791,193, 5,849, 885, 5,861,491, 5,919,913, and 7,368,141, which are all incorporated herein by reference.

[0071] In some embodiments, the lactoferrin is about 0. 1% to about 30% by weight of the protein component of the nutritional supplement. In some embodiments, the lactoferrin is about 0.5% to about 6% by weight of the protein component. In some embodiments, the lactoferrin is about 4% to about 30% by weight of the protein component. In some embodiments, the lactoferrin is about 0.5% to about 6%, about 1% to about 4%. about 6% to about 7%, about 5% to about 25%. about 6% to about 20%, or about 8% to about 22% by weight of the protein component. Specific examples of the lactoferrin include about 0.1%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 6.2%, about 6.4%, about 6.6%. about 6.8%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 15%, about 16%, about 18%, about 20%, about 22%, about 24%, about 25%, about 26%, about 28%, about 30% by weight of the protein component, and ranges between any two of these values. In some embodiments, the lactoferrin is about 0.05 g / L to about 3 g / L. In some embodiments, the lactoferrin is about 0.3 g / L to about 3 g / L of the nutritional supplement. In some embodiments, the lactoferrin is about 0.5 g / L to about 2 g / L of the nutritional supplement. In some embodiments, the lactoferrin is about 0.1 g / L to about 1 g / L. In some embodiments, the lactoferrin is about 0.05 g / L, about 0.1 g / L, about 0.12 g / L, about 0.125 g / L, about 0.126 g / L. about 0.127 g / L, about 0.128 g / L, about 0.129 g / L, about 0.13 g / L. about 0.14 g / L. about 0.15 g / L, about 0.175 g / L. about 0.2 g / L, about 0.25 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.85g / L, about 0.9 g / L, about 1 g / L, about 1.2 g / L, about 1.4 g / L, about 1.5 g / L, about 1.6 g / L, about 1.8 g / L, about 2 g / L, about 2.2 g / L, about 2.4 g / L, about 2.6 g / L. about 2.8 g / L, about 3 g / L of the nutritional supplement and ranges between any two of these values.

[0072] In some embodiments, the nutritional supplement comprises whey protein hydrolysate. In some embodiments, the whey protein hydrolysate is about 10% to about 75% by weight of the protein component of the nutritional supplement. In some embodiments, the whey protein hydrolysate is about 20% to about 50%. about 45% to about 70%, about 45% to about 60%, or about 55% to about 70% by w eight of the protein component. Specific examples of the whey protein hydrolysate include about 10% about 12%, about 15%, about 18%, about 20%, about 22%, about 25%, about 25.1%, about 25.2%, about 25.3%. about 25.4%, about 25.5%. about 25.6%, about 25.7%, about 25.8%, about 25.9%. about 26%, about 27%, about 27.1%, about 27.2%, about 27.3%, about 27.4%, about 27.5%, about 27.6%, about 27.7%, about 27.8%, about 27.9%, about 28%, about 30%, about 32%, about 35%, about 38%, about 40%, about 42%, about 45%, about 48%, about 49%, about 50%, about 52%, about 55%, about 58%, about 59%. about 60%. about 62%. about 65%, about 68%, about 69%, about 70% weight of the protein component and ranges between any two of these values. In some embodiments, the whey protein hydrolysate comprises about 1 g / L to about 13 g / L of the nutritional supplement. In some embodiments, the whey protein hydrolysate comprises about 3 g / L to about 11 g / L of the nutritional supplement. In some embodiments, the whey protein hydrolysate comprises about 1 g / L, about 2 g / L. about 3 g / L, about 3.1 g / L, about 3.3 g / L. about 3.5 g / L, about 3.7 g / L, about 3.9 g / L, about 4 g / L, about 4. 1 g / L, about 4.3 g / L, about 4.5 g / L, about4.7 g / L, about 4.9 g / L, about 5 g / L, about 5.1 g / L, about 5.3 g / L, about 5.5 g / L, about 5.7 g / L, about 5.9 g / L, about 6 g / L, about 7 g / L, about 7.1 g / L, about 7.3 g / L, about 7.5 g / L, about 7.7 g / L, about 7.9 g / L, about 8 g / L, about 9 g / L. about 9.1 g / L, about 9.3 g / L. about 9.5 g / L, about9.7 g / L, about 9.9 g / L, about 10 g / L, about 1 1 g / L, about 12 g / L, about 13 g / L of the nutritional supplement, and ranges between any two of these values.

[0073] In some embodiments, the nutritional supplement comprises alphalactalbumin. In some embodiments, the alpha-lactalbumin comprises about 5% to about 25% by weight of the protein component of the nutritional supplement. In some embodiments, the alpha-lactalbumin comprises about 10% to about 20%, about 12% to about 20%, about 13% to about 16%, or about 15% to about 20% by weight of the protein component. Specific example of the alpha-lactalbumin include about 5%, about 6%, about 7%, about 8%, about 9%, about 10%. about 11%, about 12%. about 13%, about 13.1%, about 13.2%, about 13.3%, about 13.4%, about 13.5%, about 13.6%, about 13.7%, about 13.8%, about 13.9%, about 14%, about15%, about 16%, about 17%, about 17.1%, about 17.2%, about 17.3%, about 17.4%, about 17.5%, about 17.6%, about 17.7%, about 17.8%, about 17.9%. about 18%, about 19%, about 20%, about 22%, about 24%, about 25% by weight of the protein component and ranges between any two of these values. In some embodiments, the alpha-lactalbumin comprises about 0.5 g / L to about 6 g / L of the nutritional supplement. In some embodiments, the alphalactalbumin comprises about 1 g / L to about 4 g / L of the nutritional supplement. In some embodiments, the alpha-lactalbumin comprises about 0.5 g / L. about 0.8 g / L, about 1 g / L, about 2 g / L, about 2.2 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.8 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L of the nutritional supplement, and ranges between any two of these values.

[0074] In some embodiments, the nutritional supplement comprises osteopontin. Osteopontin is a multifunctional protein present in most tissues and body fluids, with the highest concentrations found in breast milk. Osteopontin is thought to be involved in cell- mediated immune response, chemotaxis of inflammatory cells, anti-inflammatory responses, induction of T-helper type 1 (Thl)-type immunity, and enhanced host defense against pathogens.

[0075] In some embodiment, the osteopontin comprises about 0.1 % to about 4% by weight of the protein component of the nutritional supplement. In some embodiments, the osteopontin comprises about 0.5% to about 1.5%, about 1% to about 3%. about 1.5% to about 2%, about 2% to about 2.5% by weight of the protein component. Specific example of the osteopontin include about 0.1 %, about 0.5%, about 0.6%, about 0.7%, about 0.71%, about 0.73%, about 0.75%, about 0.77%, about 0.79%, about 0.8%, about 0.9%, about 0.92%, about 0.94%, about 0.96%, about 0.98%, about 1%. about 1.5%, about 2%, about 2.5%. about 3%, about 3.5%, about 4% by weight of the protein component and ranges between any two of these values. In some embodiments, the osteopontin comprises about 0.05 g / L to about 0.5 g / L of the nutritional supplement. In some embodiments, the osteopontin comprises about 0.08 g / L to about 0.3 g / L of the nutritional supplement. In some embodiments, the osteopontin comprises about 0.05 g / L, about 0.06 g / L, about 0.07 g / L, about 0.08 g / L, about 0.09 g / L, about 0. 1 g / L, about 0.1 1 g / L. about 0.12 g / L, about 0.13 g / L, about 0.14 g / L, about 0.15 g / L, about 0.16 g / L. about 0.17 g / L, about 0.18 g / L, about 0.2 g / L, about 0.22 g / L, about 0.24 g / L, about 0.26 g / L, about 0.28 g / L, about 0.3 g / L of the nutritional supplement and ranges between any two of these values.

[0076] In some embodiments, the nutritional supplement comprises one or more digestion aiding proteins. In some embodiments, the protein component of the nutritionalsupplement comprises one or more digestion aiding proteins. In some embodiments, the nutritional supplement comprises two or more digestion aiding proteins. In some embodiments, the nutritional supplement comprises three or more digestion aiding proteins. In some embodiments, the digestion aiding proteins are selected from whey protein hydrolysate, alphalactalbumin, K-casein, a-casein, p-casein, bile-salt stimulated lipase, amylase, ai-antitrypsin, and combinations thereof. In some embodiments, the digestion aiding protein is a nutrient carrier protein selected from lactoferrin with iron, alpha-lactalbumin with calcium and zinc, (3- casein with calcium and phosphorous, folate binding protein with folate, and haptocorrin with vitamin B12. A digestion aiding protein is a protein that is easy to digest and is tolerated by the body. Nutritional supplements that are easy to digest are especially important for infants who may be relying on formulas as their primary or sole source of nutrition.

[0077] In some embodiments, the nutritional supplement comprises one or more than one immunoprotective proteins. In some embodiments, the protein component of the nutritional supplement comprises one or more immunoprotective proteins. In some embodiments, the one or more immunoprotective proteins are selected from the group consisting of lactoferrin, osteopontin, K-casein, a-casein. (3-casein. haptocorrin. lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin, lactoperoxidase, milk fat globule membrane (MF GM), and combinations thereof. In embodiments in which the nutritional supplement is applied to infant formulas, the quantities of immunoprotective proteins such as lactoferrin and osteopontin described herein are not found in typical infant formulas. It is believed that the levels of lactoferrin and osteopontin provide the present nutritional supplement with greater immunoprotective properties as compared to infant formulas that are currently on the market.

[0078] In some embodiments, the nutritional supplement comprises a protein component comprising one or more digestion aiding proteins and one or more immunoprotective proteins. Such a nutritional supplement is designed to provide both comfort and immunity', which is in contrast to many knoyvn infant formulas that are designed to address either comfort or immunity not both. For example, infant formulas that focus on providing digestive comfort may comprise 100% hydrolyzed proteins, and therefore lack many of the other types of proteins that infants would otherwise receive through breast milk. On the other hand, infant formulas that focus on providing immunity' may comprise only intact proteins and as a result may not be well-tolerated for digestion.

[0079] In some embodiments, the nutritional supplement comprises a protein component comprising one or more digestion aiding proteins and one or moreimmunoprotective proteins. In some embodiments the one or more digestion aiding proteins and one or more immunoprotective proteins comprise about 5% to about 95% by weight of the protein component. In some embodiments, the one or more digestion aiding proteins and one or more immunoprotective proteins comprise about 5% to about 20%, about 20% to about 50%, about 50%-75%, about 75%-95% by weight of the protein component. Specific examples of the one or more digestion aiding proteins and one or more immunoprotective proteins are about 5%, about 10%, about 15%, about 20%. about 25%. about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% by weight of the protein component and ranges between any two of these values.

[0080] Fat Component of the Nutritional Supplement

[0081] In some embodiments, the fat component of the nutritional supplement comprises innate milk fat globule membrane (MFGM), added MFGM, phospholipids, cholesterol, oil, non-hexane extracted docosahexaenoic acid (DHA), hexane extracted arachidonic acid (AA). non-hexane extracted AA. ascorbyl palmitate or combinations thereof. In some embodiments, the fat component comprises monoglycerides, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate, monoglyceryl linoleate, and combinations thereof. In some embodiments, the fat component comprises tocopherols, such as RRR-tocopherols concentrate (70% concentrate in edible vegetable oil), and combinations thereof. In certain embodiments, the oil comprises vegetable oil, soy oil, palm oil, avocado oil, and combinations thereof. Suitable fat or lipid sources for the fat component of any nutritional supplement described herein include but are not limited to and fat know to the art including, animal sources, such as milk fat, butter, butter fat, egg yolk lipid, and SN2 palmitate oil : marine sources, such as fish oils, marine oils, single cell oils; vegetable and plant oils, such as com oil, canola oil (low erucic acid rapeseed oil), sunflower oil, soybean oil, palm oil, palm olein oil, coconut oil, avocado oil, high oleic sunflower oil, safflower oil, high-oleic safflower oil, evening primrose oil, rapeseed oil. olive oil, flaxseed (linseed) oil, cottonseed oil, palm stearin oil. palm kernel oil, wheat germ oil; medium chain triglyceride oils and emulsions and esters of fatty acids; and any combinations thereof. In certain embodiments, MFGM comprises milk fat globules (MFGs) in a bioactive membrane system and makes up the fat in bovine milk. Fat is the second largest constituent of bovine milk dry matter having nutritional significance, and MFGM as a whole or some of its associated individual components have physiological andnutritional functions, including supporting antiviral and antibacterial mechanisms that combat gut-derived infections.

[0082] In some embodiments, the fat component of the nutritional supplement comprises a combination of one or more of: low erucic acid rapeseed oil (canola oil), sunflower oil, high oleic sunflower seed oil, coconut oil, an oil source for arachidonic acid (AA), and oil source for docosahexaenoic acid (DHA), and monoglycerides.

[0083] In some embodiments, the fat component of the nutritional supplement is about 15% to about 40% by weight of the nutritional supplement. In some embodiments, the fat component is about 20% to about 35% by weight of the nutritional supplement. In some embodiments, the fat component is about 15%, about 16%, about 18%, about 20%, about 22%, about 24%. about 25%, about 26%, about 28%, about 30%, about 32%, about 34%, about 35%, about 36%, about 38%, about 40% by weight of the nutritional supplement, and ranges between any two of these values.

[0084] In some embodiments, the fat component of the nutritional supplement comprises about 25 g / L to about 50 g / L of the nutritional supplement. In some embodiments, the fat component comprises about 30 g / L to about 45 g / L or about 35 g / L to about 40 g / L of the nutritional supplement. In some embodiments, the fat component comprises about 25 g / L, about 26 g / L, about 27 g / L, about 28 g / L, about 29 g / L, about 30 g / L, about 31 g / L, about 32 g / L, about 33 g / L, about 34 g / L, about 35 g / L. about 36 g / L, about 37 g / L, about 37.1 g / L, about 37.2 g / L. about 37.3 g / L, about 37.4 g / L, about 37.5 g / L, about 37.6 g / L, about 37.8 g / L, about 37.9 g / L, about 38 g / L, about 39 g / L, about 40 g / L, about 41 g / L, about 42 g / L, about 43 g / L, about 44 g / L, about 45 g / L, about 46 g / L, about 47 g / L, about 48, g / L, about 49 g / L, about 50 g / L of the nutritional supplement and ranges between any two of these values.

[0085] In some embodiments, the fat component of the nutritional supplement comprises arachidonic acid (AA) in an amount of about 0.1% to about 1.2% by weight of the fat component. In some embodiments, the fat component comprises AA in an amount of about 0.5% to about 0.8% by weight of the fat component. In some embodiments, the fat component comprises AA in an amount of about 0. 1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.51%, about 0.52%. about 0.53%, about 0.54%, about 0.55%. about 0.56%, about 0.57%, about 0.58%, about 0.59%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2% by weight of the fat component and ranges between any two of these values. In some embodiments, the fat component comprises AA in an amount of about 0. 1 g / L to about 0.2 g / L of the nutritional supplement. In some embodiments, the fat component comprises AA in an amount of about 0. 10 g / L, about 0. 11 g / L, about 0. 12 g / L, about 0. 13 g / L,about 0.14 g / L, about 0.15 g / L, about 0.16 g / L, about 0.17 g / L, about 0.18 g / L, about 0.19 g / L, about 2.0 g / L of the nutritional supplement and ranges between any two of these values. In some embodiments, the fat component comprises an oil source for AA, such as ARASCO® oil, in an amount of about 0.1% to about 8% by weight of the fat component. In some embodiments the oil source for AA, such as ARASCO® oil is in an amount of about 0.5% to about 6% or about 1% to about 4% by weight of the fat component. In some embodiments, the oil source for AA. such as ARASCO® oil is in an amount of about 0.1%. about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.51%, about 1.52%, about 1.53%, about 1.54%, about 1.55%, about 1.56%, about 1.57%, about 1.58%, about 1.59%. about 1.6%, about 1.7%, about 1.8%. about 1.9%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% by weight of the fat component and ranges between any two of these values. In some embodiments, the oil source for AA, such as ARASCO® oil is in an amount of about 0. 1 g / L to about 3 g / L of the nutritional supplement. In some embodiments, the oil source for AA, such as ARASCO® oil is in an amount 0.2 g / L to about 2 g / L of the nutntional supplement. In some embodiments, the oil source for AA, such as ARASCO® oil is in an amount 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.51 g / L, about 0.52 g / L, about 0.53 g / L, about 0.54 g / L, about 0.55 g / L, about 0.56 g / L, about 0.57 g / L, about 0.58 g / L, about 0.59 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.2 g / L, about 1.4 g / L, about 1.5 g / L, about 1.6 g / L. about 1.8 g / L, about 2 g / L, about 2.2 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.8 g / L, about 3 g / L of the nutritional supplement and ranges between any two of these values.

[0086] In some embodiments, the fat component of the nutritional supplement comprises docosahexaenoic acid (DHA). In some embodiments, the fat component comprises non-hexane extracted DHA. In some embodiments, the fat component comprises DHA in an amount of about 0.05% to about 0.5% by weight of the fat component. In some embodiments, the fat component comprises DHA in an amount of about 0.2% to about 0.4% by w eight of the fat component. In some embodiments, the fat component comprises DHA in an amount of about 0.05%, about 0.06%. about 0.07%, about 0.08%, about 0.09%. about 0.10%, about 0.12%, about 0.15%, about 0.18%, about 0.20%, about 0.22%, about 0.25%, about 0.28%, about 0.30%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, about 0.40%, about 0.42%, about 0.45%, about 0.48%, about 0.50% of the fat component and ranges between any two of these values. In some embodiments, the fat component comprises DHA in an amount of about 0.05 g / L toabout 0.3 g / L of the nutritional supplement. In some embodiments, the fat component comprises DHA in an amount of about 0.08 g / L to about 0. 15 g / L of the nutritional supplement. In some embodiments, the fat component comprises about 0.05 g / L, about 0.06 g / L, about 0.07 g / L, about 0.08 g / L, about 0.09 g / L, about 0.10 g / L, about 0. 11 g / L, about 0.12 g / L, about 0.13 g / L, about 0. 14 g / L, about 0.141 g / L, about 0. 142 g / L, about 0.143 g / L, about 0. 145 g / L, about 0. 146 g / L, about 0. 147 g / L, about 0. 148 g / L, about 0. 149 g / L, about 0. 15 g / L of the nutritional supplement and ranges between any two of these values. In some embodiments, the fat component comprises an oil source for DHA, such as DHASCO® or DHASCO-B® oil, in an amount of about 0.1% to about 4% by weight of the fat component. In some embodiments, the oil source for DHA, such as DHASCO® or DHASCO-B® oil is in an amount of about 0.3% to about 2% or about 0.5% to about 1.5% of the fat component. In some embodiments, the oil source for DHA, such as DHASCO® or DHASCO-B® oil is in an amount of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 0.91%, about 0.92%, about 0.93%, about 0.94%, about 0.95%, about 0.96%, about 0.97%, about 0.98%, about 0.99%. about 1%, about 1.5%, about 2% by weight of the fat component and ranges between any two of these values. In some embodiments, the oil source for DHA, such as DHASCO® or DHASCO-B® oil is in an amount of about 0.1 g / L to about 3 g / L of the nutritional supplement. In some embodiments, the oil source for DHA, such as DHASCO® or DHASCO-B® oil is in an amount 0.2 g / L to about 1 g / L. In some embodiments, the oil source for DHA. such as DHASCO® or DHASCO-B® oil is in an amount 0.1 g / L, about 0.2 g / L, about 0.3 g / L, 0.31 g / L, about 0.32 g / L, about 0.33 g / L, about 0.34 g / L, about 0.35 g / L, about 0.36 g / L, about 0.37 g / L, about 0.38 g / L, about 0.39 g / L, about 0.4 g / L, about 0.5 g / L, about 0.51 g / L, about 0.52 g / L, about 0.53 g / L, about 0.54 g / L, about 0.55 g / L, about 0.56 g / L. about 0.57 g / L, about 0.58 g / L, about 0.59 g / L. about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.2 g / L, about 1.4 g / L, about 1.5 g / L, about 1.6 g / L, about 1.8 g / L, about 2 g / L, about 2.2 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.8 g / L, about 3 g / L of the nutritional supplement and ranges between any two of these values.

[0087] In some embodiments, the fat component of the nutritional supplement comprises rapeseed oil. In some embodiments, the fat component comprises low erucic acid rapeseed oil (canola oil). In some embodiments, the low erucic acid rapeseed oil (canola oil) is in an amount of about 10% to about 40% by weight of the fat component. In some embodiments, the canola oil is in an amount of about 15% to about 35% or about 20% to about 30% by weight of the fat component. In some embodiments, the canola oil is about 10%, about 12%, about 14%, about 15%, about 16%, about 18%, about 20%, about 22%, about 24%, about25%, about 25.1%, about 25.2%, about 25.3%, about 25.4%, about 25.5%, about 26%, about 28%. about 30%, about 32%, about 34%. about 35%, about 36%, about 38%. about 40% by weight of the fat component, and ranges between any two of these values. In some embodiments, the canola oil is in an amount of about 1 g / L to about 20 g / L of the nutritional supplement. In some embodiments, the canola oil is in an amount of about 5 g / L to about 15 g / L or about 8 g / L to about 12 g / L of the nutritional supplement. In some embodiments, the canola oil is in an amount of about 1 g / L. about 2 g / L, about 3 g / L, about 4 g / L. about 5 g / L. about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 9.1 g / L, about 9.2 g / L, about 9.3 g / L, about 9.4 g / L, about 9.5 g / L, about 9.6 g / L, about 9.7 g / L, about 9.75 g / L, about 9.76 g / L, about 9.77 g / L, about 9.78 g / L, about 9.79 g / L, about 9.8 g / L, about 9.9 g / L, about 10 g / L, about 11 g / L, about 12 g / L, about 13 g / L. about 14 g / L, about 15 g / L, about 16g / L, about 17 g / L, about 18 g / L, about 19 g / L, about 20 g / L of the nutritional supplement and ranges between any two of these values.

[0088] In some embodiments, the fat component of the nutritional supplement comprises sunflower oil. In some embodiments the sunflower oil is in an amount of about 1% to about 15% by weight of the fat component. In some embodiments the sunflower oil is in an amount of about 4% to about 12% or about 6% to about 10% by weight of the fat component. In some embodiments, the sunflower oil is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%. about 8.7%, about 8.8%, about 8.9%, about 9%. about 10%, about 11%, about 12%, about 13%, about 14%, about 15% by weight of the fat component and ranges between any two of these values. In some embodiments, the sunflower oil is in an amount of about 1 g / L to about 6 g / L of the nutritional supplement. In some embodiments, the sunflower oil is in an amount of about 2 g / L to about 4 g / L of the nutritional supplement. In some embodiments, the sunflower oil is in an amount of about 1 g / L, about 2 g / L, about 3 g / L, about 3.1 g / L, about 3.2 g / L, about 3.22 g / L, about 3.24 g / L, 3.26 g / L, about 3.28 g / L, about 3.3 g / L, about 3.4 g / L, about 3.5 g / L, about 4 g / L, about 5 g / L, about 6 g / L of the nutritional supplement and ranges between any two of these values. In some embodiments, the fat component comprises high oleic sunflower seed oil. In some embodiments, the high oleic sunflower seed oil is in an amount of about 20% to about 50% by weight of the fat component. In some embodiments, the high oleic sunflower seed oil is in an amount of about 25% to about 45% or about 30% to about 40% by weight of the fat component. In some embodiments, the high oleic sunflower seed oil is in an amount of about 20%, about 22%, about 25%, about 28%, about 30%, about 32%, about 33%, about 33.1%, about 33.2%, about 33.3%, about 33.4%, about33.5%, about 33.6%, about 33.7%, about 33.8%, about 33.9%, about 34%, about 35%, about 38%. about 40%, about 42%, about 44%, about 45%, about 48%. about 50% by weight of the fat component and ranges between any two of these values. In some embodiments, the high oleic sunflower oil is in an amount of about 5 g / L to about 20 g / L of the nutritional supplement. In some embodiments, the high oleic sunflower oil is in an amount of about 8 g / L to about 18 g / L or about 10 g / L to about 15 g / L of the nutritional supplement. In some embodiments, the high oleic sunflower oil is in an amount of about 5 g / L. about 6 g / L, about 7 g / L. about 8 g / L. about 9 g / L, about 10 g / L, about 11 g / L, about 12 g / L, about 13 g / L, about 13.01 g / L, about 13.02 g / L, about 13.03 g / L, about 13.04 g / L, about 13.05 g / L, about 13.08 g / L, about 13.1 g / L, about 13.2 g / L, about 13.3 g / L, about 13.4 g / L, about 13.5 g / L, about 13.6 g / L, about 13.8 g / L, about 14 g / L, about 15 g / L, about 16 g / L. about 17 g / L, about 18 g / L, about 19 g / L. about 20 g / L of the nutritional supplement and ranges between any two of these values.

[0089] In some embodiments, the fat component of the nutritional supplement comprises coconut oil. In some embodiments, the coconut oil is in an amount of about 5% to about 30% by weight of the fat component. In some embodiments, the coconut oil is in an amount of about 10% to about 25% or about 12% to about 20% by weight of the fat component. In some embodiments, the coconut oil is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 15%, about 16%, about 16.1%. about 16.2%, about 16.3%, about 16.4%, about 16.5%, about 16.6%, about 16.7%, about 16.8%, about 16.9%, about 17%. about 18%, about 20%, about 22%, about 24%, about 25%, about 26%, about 28%, about 30% by weight of the fat component and ranges between any two of these values. In some embodiments, the coconut oil is in an amount of about 1 g / L to about 15 g / L of the nutritional supplement. In some embodiments, the coconut oil is in an amount of about 3 g / L to about 10 g / L or about 5 g / L to about 8 g / L of the nutritional supplement. In some embodiments, the canola oil is in an amount of about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 6. 1 g / L, about 6.2 g / L, about 6.3 g / L, about 6.4 g / L, about6.5 g / L, about 6.51 g / L, about 6.52 g / L, about 6.53 g / L, about 6.54 g / L, about 6.55 g / L, about6.6 g / L, about 6.7 g / L, about 6.8 g / L, about 6.9 g / L. about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 11 g / L. about 12 g / L, about 13 g / L, about 14 g / L. about 15 g / L of the nutritional supplement and ranges between any two of these values.

[0090] In some embodiments, the fat component of the nutritional supplement comprises monoglycerides, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate, monoglyceryl linoleate, and combinations thereof. In some embodiments, the monoglycerides are in an amount of about 0.1% to about 5% by weight ofthe fat component. In some embodiments, the monoglycerides are in an amount of about 0.5% to about 2% by weight of the fat component. In some embodiments, the monoglycerides are in an amount of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2% by weight of the fat component and ranges between any two of these values. In some embodiments, the monoglycerides are in an amount of about 0.2 g / L to about 1 g / L of the nutritional supplement. In some embodiments, the monoglycerides are in an amount of about 0.25 g / L to about 0.4 g / L of the nutritional supplement. In some embodiments, the monoglycerides are in an amount of about 0.2 g / L, about 0.3 g / L, 0.31 g / L, about 0.32 g / L, about 0.33 g / L, about 0.34 g / L, about 0.35 g / L, about 0.36 g / L, about 0.37 g / L, about 0.38 g / L, about 0.39 g / L. about 0.4 g / L, about 0.5 g / L. about 0.6 g / L, about 0.7 g / L. about 0.8 g / L, about 0.9 g / L, about 1 g / L of the nutritional supplement and ranges between any two of these values.

[0091] In some embodiments, the fat component of the nutritional supplement comprises tocopherols, such as RRR-tocopherols concentrate (70% concentrate in edible vegetable oil), in an amount of about 0.015% to about 0.04% by weight of the fat component. In some embodiments, the tocopherols, are in an amount of about 0.02% to about 0.035% by weight of the fat component. In some embodiments, the tocopherols are in an amount of about 0.015%, about 0.02%, about 0.021%, about 0.022%, about 0.023%, about 0.024%, about 0.025%, 0.026%, about 0.027%. about 0.028%, about 0.029%, about 0.03%, by weight of the fat component and ranges between any two of these values.

[0092] In some embodiments, the fat component of the nutritional supplement comprises ascorbyl palmitate in an amount of about 0.001% to about 0.025% by weight of the fat component. In some embodiments, the fat component comprises ascorbyl palmitate in an amount of about 0.005% to about 0.02% or about 0.008% to about 0.018% by weight of the fat component. In some embodiments, the ascorbyl palmitate is in an amount of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.012%, about 0.014%, about 0.015%, or 0.016%, about 0.018%, about 0.02%, about 0.022%, about 0.024%, about 0.025% by weight of the fat component and ranges between any two of these values.

[0093] In certain embodiments, the fat component of the nutritional supplement comprises a combination of low erucic acid rapeseed oil (canola oil) in an amount of about 10% to about 40%, or about 15% to about 35%, or about 20% to about 30%, or about 25%, or 25.3%. by weight of the fat component; sunflower oil in an amount of about 1% to about 15%, or about 4% to about 12%, or about 6% to about 10%, or about 8%, or 8.4%, by weight of thefat component; high oleic sunflower seed oil in an amount of about 20% to about 50%, or about 25% to about 45%, or about 30% to about 40%, or about 35%, or about 34%, or 33.8%. by weight of the fat component; coconut oil in an amount of about 5% to about 30%, or about 10% to about 25%, or about 12% to about 20%, or about 15%, or about 17%, or 16.9%, by weight of the fat component; an oil source for AA, such as ARASCO® oil, in an amount of about 0.1% to about 8%, or about 0.5% to about 6%, or about 1% to about 4%, or about 2%, or about 1%. or about 1.5%, or 1.54%. by weight of the fat component; an oil source for DHA. such as DHASCO® or DHASCO-B® oil, in an amount of about 0.1% to about 4%, or about 0.3% to about 2%, or about 0.5% to about 1.5%, or about 1%, or 0.99%, by weight of the fat component; and monoglycerides, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate, monoglyceryl linoleate, and combinations thereof, in an amount that provides about 0.1% to about 5%, or about 0.5% to about 2%, or about 1%, or 0.9%, by weight of the fat component.

[0094] In certain embodiments, the fat component of the nutritional supplement comprises a combination of canola oil in an amount of about 1 g / L to about 20 g / L, about 5 g / L to about 15 g / L, about 8 g / L to about 12 g / L, about 10 g / L, or about 9.77 g / L; sunflower oil in an amount of about 1 g / L to about 6 g / L, or about 2 g / L to about 4 g / L, or about 3 g / L, or 3.26 g / L; high oleic sunflower seed oil in an amount of about 5 g / L to about 20 g / L, or about 8 g / L to about 18 g / L, or about 10 g / L to about 15 g / L, or about 13 g / L, or 13.02 g / L; coconut oil in an amount of about 1 g / L to about 15 g / L, or about 3 g / L to about 10 g / L, or about 5 g / L to about 8 g / L, or about 6 g / L, or about 7 g / L, or 6.51 g / L; an oil source for AA, such as ARASCO® oil, in an amount of about 0. 1 g / L to about 3 g / L, or about 0.2 g / L to about 2 g / L, or about 1 g / L, or about 0.6 g / L, or about 0.5 g / L, or 0.59 g / L; an oil source for DHA, such as DHASCO® or DHASCO-B® oil. in an amount of about 0. 1 g / L to about 2 g / L. or about 0.2 g / L to about 1 g / L, about 0.3 g / L, or about 0.4 g / L, or 0.38 g / L; and monoglycerides, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate, monoglyceryl linoleate, and combinations thereof, in an amount of about 0.2 g / L to about 1 g / L, or about 0.25 g / L to about 0.4 g / L, about 0.3 g / L. or about 0.35 g / L, or 0.33 g / L.

[0095] In certain embodiments, the fat component of the nutritional supplement comprises a combination of canola oil in an amount of about 20% to about 40%, or about 25% to about 35%, or about 28% to about 32%, or about 30%, or 29.7%, by weight of the fat component; sunflower oil in an amount of about 1% to about 20%, or about 5% to about 15%, or about 8% to about 12%. or about 10%, or 9.9%, by weight of the fat component; high oleic sunflower seed oil in an amount of about 30% to about 50%, or about 35% to about 45%, orabout 38% to about 42%, or about 39%, or about 40%, or 39.6%, by weight of the fat component; coconut oil in an amount of about 10% to about 30%. or about 15% to about 25%, or about 18% to about 22%, or about 19%, or about 20%, or 19.8%, by weight of the fat component; monoglycerides, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate, monoglyceryl linoleate, and combinations thereof, in an amount that provides about 0.1% to about 2%, or about 0.5% to about 1.5%, or about 0.8% to about 1.2%, or about 1%, by weight of the fat component; tocopherols, such as RRR-tocopherols concentrate (70% concentrate in edible vegetable oil), in an amount that provides about 0.015% to about 0.04%, or about 0.02% to about 0.035%, or about 0.025%, or about 0.03%, or 0.028%, by weight of the fat component; and ascorbyl palmitate in an amount that provides about 0.001% to about 0.025%, or about 0.005% to about 0.02%, or about 0.008% to about 0.018%, or about 0.01%, or about 0.015%, or 0.014%, by weight of the fat component.

[0096] Milk Component of the Nutritional Supplement

[0097] In some embodiments, the naturally occurring milk of the nutritional supplement comprises milk from a non-human source, such as a cow (bovine), sheep, goat, yak, water buffalo, horse, reindeer, or camel. In some embodiments, the naturally occurring milk is non-animal milk, such as soy, rice, hemp, pea, oat, almond or other nuts. In some embodiments, the naturally occurring milk comprises whole milk, reduced fat milk, low fat milk, and fat-free milk. In some embodiments, the naturally occurring milk comprises whole milk from a bovine source. Whole milk can help the nutritional supplement to attain innate levels of MFGM, phospholipids, and cholesterol that are important, especially for infant development. Whole milk also allows less reliance on other sources of fat, such as oil, to achieve optimal nutrition. In some embodiments, the naturally occurring milk is organic and / or from grass-fed animal sources. Such milk has been linked with higher levels of conjugated linoleic acid and other important vitamins and nutrients, due to the animal spending more time on pasture and consuming higher amounts of grass. Further, organic farming practices reduce exposure to environmental toxins, which are fat soluble and therefore more likely to be found in fat.

[0098] A skilled artisan would understand that the terms skimmed milk or skim milk are the same as fat-free milk. A skilled artisan would also understand that whole milk comprises approximately about 3.25% milk fat, reduced fat milk comprises approximately about 2% milk fat. low fat milk comprises approximately about 1% milk fat, and fat free milk comprises approximately about 0% to about 0.5% milk fat.

[0099] In some embodiments, the naturally occurring milk protein is about 1% to about 40% by weight of the protein component of the nutritional supplement. In some embodiments, the naturally occurring milk protein is about 8% to about 40%, about 8% to about 35%, or about 12% to about 30% by weight of the protein component. In other embodiments, the naturally occurring milk protein is about 1% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40% by weight of the protein component. Specific examples of the naturally occurring milk protein include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 15%. about 15.1%, about 15.2%, about 15.3%, about 15.4%, about 15.5%, about 15.6%, about 15.7%, about 15.8%. about 15.9%, about 16%, about 17%, about 17.1%, about 17.2%, about 17.3%, about 17.4%, about 17.5%, about 17.6%, about 17.7%, about 17.8%, about 17.9%, about 18%, about 20%, about 22%, about 24%, about 24.1%, about 24.2%, about 24.3%, about 24.4%, about 24.5%, about 24.6%, about 24.7%, about 24.8%. about 24.9%, about 25%, about 26%, about 27%. about 28%, about 29%, about 30%. about 32%, about 34%, about 35%, about 36%. about 38%. about 40% by weight of the protein component, and ranges between any two of these values.

[0100] In some embodiments, the naturally occurring milk fat is about 0% to about 20% of the fat component of the nutritional supplement. In some embodiments, the naturally occurring milk fat is about 5% to about 20% by weight of the fat component. In some embodiments, the naturally occurring milk fat is about 8% to about 15% or about 10% to about 13% by weight of the fat component. In some embodiments, the naturally occurring milk is about 0%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%. about 9%, about 10%, about 11%, about 11.1%. about 11.2%, about 11.3%, about 11.4%, about 11.5%, about 11.6%, about 11.7%, about 11.8%, about 11.9%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% by weight of the fat component, and ranges between any two of these values.[0101 J Carbohydrate Component of the Nutritional Supplemem

[0102] In some embodiments, the carbohydrate component of the nutritional supplement comprises lactose, glucose, fructose, sucrose, human milk oligosaccharides (HMO), bovine milk oligosaccharides (MOS), galactooligosaccharide (GOS), fructooligosaccharide (FOS), fermented carbohydrates, inulin, com syrup solids, dextrin, maltodextrin, dextrose, polydextrose, tapioca, starch, tapioca starch, rice syrup solids, waxycom, waxy rice starch, or combinations thereof. In some embodiments, the nutritional supplement comprises lactose. Lactose has important natural prebiotic benefits, and also provides a natural sweetness to the nutritional supplement, which avoids the need for artificial sweeteners. In some embodiments, the nutritional supplement comprises HMO. In some embodiments, the nutritional supplement comprises MOS. In some embodiments, the prebiotic fibers are from HMO or MOS. In some embodiments, the nutritional supplement comprises GOS. GOS is a prebiotic derived from lactose that drives stool softening and other potential digestive benefits. In some embodiments, the nutritional supplement may comprise FOS, which is a prebiotic.

[0103] In some embodiment, the carbohydrate component of the nutritional supplement is about 40% to about 70% by weight of the nutritional supplement. In some embodiments, the carbohydrate component is about 50% to about 60% by weight of the nutritional supplement. In some embodiments, the carbohydrate component comprises about 40%, about 42%, about 44%, about 45%, about 46%, about 48%, about 50%, about 52%, about 54%, about 55%. about 56%. about 58%, about 60%, about 62%, about 64%, about 65%, about 66%. about 68%, about 70% by weight of the nutritional supplement and ranges between any two of these values. In some embodiments, the carbohydrate component is about 60 g / L to about 90 g / L of the nutritional supplement. In some embodiments, the carbohydrate component is about 70 g / L to about 80 g / L of the nutritional supplement. In some embodiments, the carbohydrate component is about 60 g / L, about 62 g / L. about 64 g / L, about 65 g / L. about 66 g / L, about 68 g / L, about 70 g / L, about 71 g / L, about 72 g / L, about 73 g / L, about 74 g / L, about 75 g / L, about 76 g / L, about 77 g / L, about 78 g / L, about 79 g / L, about 80 g / L, about 82 g / L, about 84 g / L, about 85 g / L, about 86 g / L, about 88 g / L, about 90 g / L of the nutritional supplement and ranges between any two of these values.

[0104] In some embodiments, the carbohydrate is GOS. In some embodiments, the GOS comprises about 2% to about 10% by weight of the carbohydrate component of the nutritional supplement. In some embodiments, the GOS comprises about 4% to about 6% by weight of the carbohydrate component. In some embodiments, GOS comprises about 2% about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% of the carbohydrate component and ranges between any two of these values. In some embodiment, the GOS comprises about 1 g / L to about 8 g / L of the nutritional supplement. In some embodiments, the GOS comprises about 3 g / L to about 6 g / L of the nutritional supplement. In some embodiments, the GOS comprises about 1 g / L, about 2 g / L. about 3 g / L. about 4 g / L,about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L of the nutritional supplement and ranges between any two of these values.

[0105] In some embodiments, the nutritional supplement is free of lactoperoxidase, com syrup, gluten, genetically modified organisms (GMO), maltodextrin, soy, hexane extracted DHA, artificial growth hormones, palm oil, or combinations thereof. In some embodiments, the nutritional supplement is substantially free of lactoperoxidase, com syrup, gluten, genetically modified organisms (GMO), maltodextrin, soy. hexane extracted DHA. artificial growth hormones, palm oil, or combinations thereof. In some embodiments, the nutritional supplement comprises no com syrup, gluten, GMO, maltodextrin, soy, hexane extracted DHA, artificial growth hormones, palm oil, or combinations thereof. In some embodiments, the nutritional supplement comprises no intentionally added lactoperoxidase, com syrup, gluten, genetically modified organisms (GMO), maltodextrin, soy, hexane extracted DHA, artificial growth hormones, palm oil, or combinations thereof. In some embodiments, the nutritional supplement comprises trace amounts of lactoperoxidase, com syrup, gluten, genetically modified organisms (GMO), maltodextrin, soy, hexane extracted DHA, artificial grow th hormones, palm oil, or combinations thereof. In some embodiments, the nutritional supplement comprises less than 20 parts per million (ppm) of com syrup, gluten, GMO, maltodextrin, soy, hexane extracted DHA, artificial growth hormones, palm oil, or combinations thereof.

[0106] In some embodiments, lactoferrin in the nutritional supplement is protected from degradation by the protein matrix and antioxidants created by any nutritional supplement disclosed herein. In some embodiments, the lactoferrin is incorporated into a micellar structure prior to spray drying. In some embodiments, any nutritional supplement described herein comprises no lactoperoxidase. In some embodiments, any nutritional supplement described herein comprises substantially no lactoperoxidase. In some embodiments, any nutritional supplement described herein comprises lactoferrin with less than about 1% of lactoperoxidase. In some embodiments, the presence of lactoperoxidase is measured by electrophoresis and no lactoperoxidase is detected from nutritional supplements of the present invention compared to the same concentration of lactoferrin that has not been heat-treated. In some embodiments, the nutritional supplement comprises less than about 0.01% lactoperoxidase.

[0107] In some embodiments, the nutritional supplement may comprise one or more than one vitamins and minerals. The selection of vitamins and minerals and their quantities will vary based on the application of the nutritional supplement, as one of skill in the art would recognize. For example, an infant may have different vitamin and mineralrequirements than a child of age one to thirteen years old, or an adult. In some embodiments, the vitamins or minerals in the nutritional supplement may be provided, in part of or in full, from the naturally occurring milk. In certain embodiments, the vitamins or minerals in the nutritional supplement may be provided from non-milk sources know n in the art in addition to, or instead of, from the naturally occurring milk.

[0108] In some embodiments, a nutritional supplement according to any of the embodiments described herein comprises one or more than one vitamin. In some embodiments, the vitamins comprise vitamin B 1 (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B2 (riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B3-precursor tryptophan, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate, panthenol), folate (folic acid, folacin, pteroylglutamic acid), vitamin B12 (cobalamin, methylcobalamin, deoxy adenosylcobalamin, cyanocobalamin, hydroxy cobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esters with other long-chain fatty acids, retinal, retinoic acid, retinol esters), vitamin D (calciferol, cholecalciferol, vitamin D3, 1,25, -dihydroxy vitamin D), vitamin E (a-tocopherol, a-tocopherol acetate, a-tocopherol succinate, a-tocopherol nicotinate, a-tocopherol), vitamin K (vitamin KI, phylloquinone, naphthoquinone, vitamin K2, menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8, menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10, menaquin one-11, menaquinone-12, mena-quinone-13), choline, inositol, 6-carotene, and any derivative or combination thereof.

[0109] In some embodiments, a nutritional supplement according to any of the embodiments described herein comprises one or more than one mineral. In some embodiments, the minerals comprise boron, calcium, calcium acetate, calcium gluconate, calcium chloride, calcium lactate, calcium phosphate, calcium sulfate, chloride, chromium, chromium chloride, chromium picolonate, copper, copper sulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyl iron, ferric iron, ferrous fumarate, ferric orthophosphate, iron trituration, polysaccharide iron, iodide, iodine, magnesium, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese, molybdenum, phosphorus, potassium, potassium phosphate, potassium iodide, potassium chloride, potassium acetate, selenium, sulfur, sodium, docusate sodium, sodium chloride, sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate, and any derivative or combination thereof. Non-limiting exemplary derivatives of mineral compounds include salts, alkaline salts, esters, and chelates of any mineral compound.

[0110] In any embodiment described herein, the nutritional supplement comprises ingredients wherein each ingredient has at least about 40% purity. In any embodiment described herein, the nutritional supplement comprises ingredients wherein each ingredient has at least about 70% purity. In some embodiments, the ingredients are at least about 40% pure to at least about 100% pure. In some embodiments, the ingredients are at least about 70% pure to at least about 100% pure. In some embodiments, the ingredients have a purity of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 82%, about 84%, about 85%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%. about 97%, about 98%. about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.99%, about 100% and ranges between any two of these values.[OHl] The nutritional supplement according to any of the embodiments described herein may be in a dry form or a liquid form. In some embodiments, the nutritional supplement may be in a dry powder form. In some embodiments, the nutritional supplement may be in a liquid emulsion. In some embodiments, the nutritional supplement may be in a ready-to-feed nutritional supplement. Any nutritional supplement described herein may be in any form suitable for administration including those known in the art, such as a powder, a gel, a suspension, a paste, a solid, a liquid, a liquid concentrate, a reconstitute-able powdered milk substitute, or a ready-to-use product. In some embodiments, the nutritional supplements is a food, beverage, tablet, capsule, or powder.

[0112] In some embodiments, administration of any nutritional supplement disclosed herein may be selected from oral administration, tube (oral, nasal, percutaneous, gastric, intestinal, and any combination thereof), gavage administration to the enteral tract, or parenteral. In some embodiments, the tube administration may be oral gastric, oral intestinal, nasal gastric, nasal intestinal, percutaneous gastric, and percutaneous intestinal.

[0113] In some embodiments, the nutritional supplement is in a form selected from the group consisting of pellets, beads, beadlets, granules, powder, or a combination thereof. In some embodiments, the nutritional supplement is in powder form, having a particle size in the range of about 2 micrometers (pm) to about 3 millimeters (mm). In some embodiments, the particle size is in the range of about 10 pm to about 500 pm.

[0114] In some embodiments, the nutritional supplement is prepared as a tablet or capsule. In some embodiments, the tablets or capsules contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. In an embodiment, the tablets or capsules are coated according to methods well known in the art.

[0115] In embodiments in which the nutritional supplement is prepared as a tablet, the nutritional supplement is compacted into the dosage form. The disintegrant used in such a tablet is not particularly limited, as far as it is a disintegrant used for pharmaceutical preparations. Examples include, but are not limited to, one or more of crospovidone, crystalline cellulose, hydroxypropylcellulose with a low degree of substitution, croscarmellose sodium, carmellose calcium, carboxy starch sodium, carboxymethyl starch sodium, potato starch, wheat starch, com starch, rice starch, partly pregelatinized starch, and hydroxypropyl starch.

[0116] In some embodiments, the nutritional supplement comprises pharmaceutically acceptable additives comprising excipients, lubricants, pH adjusters, tastemasking agents, sweeteners, acidifiers, refrigerants, foaming agents, preservatives, fluidizers, antioxidants, colorants, stabilizers, surfactants, buffering agents, flavors, binders and drug solubilizers. A person skilled in the art may immediately list specific examples of these additives.

[0117] In some embodiments, the lubricant is light anhydrous silicic acid, magnesium stearate, stearic acid, calcium stearate, aluminum stearate, aluminum monostearate, sucrose fatty acid esters, polyethylene glycol, sodium stearyl fumarate, stearyl alcohol, talc, titanium oxide, hydrous silicon dioxide, magnesium silicate, synthetic aluminum silicate, calcium hydrogen phosphate, hardened castor oil, hardened rapeseed oil, Carnauba Wax, bees wax, microcrystalline wax and sodium lauryl sulfate. One or two or more lubricants can be used.

[0118] In some embodiments, one or more hydrophilic polymers are used in a dosage form of the invention. In some embodiments, the hydrophilic polymers are natural or partially or totally synthetic hydrophilic gums such as acacia, gum tragacanth, locust bean gum, guar gum, and karaya gum; cellulose derivatives such as methyl cellulose, hydroxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, and carboxymethyl cellulose; proteinaceous substances such as agar, pectin, carrageen, and alginates; hydrophilic polymers such as carboxypolymethylene; gelatin; casein; zein; bentonite; magnesium aluminum silicate; polysaccharides; modified starch derivatives; and other hydrophilic polymers known in the art. An addition example is a carbomer, such as Carbopol 97 IP.

[0119] In some embodiments, the nutritional supplement comprises diluents. Diluents increase the bulk of a dosage form and may make the dosage form easier to handle. In some embodiments, the diluent is lactose, dextrose, saccharose, cellulose, starch, and calcium phosphate for solid dosage forms (tablets and capsules); olive oil and ethyl oleate for soft capsules; water and vegetable oil for liquid dosage forms (suspensions and emulsions). In some embodiments, the diluent is sucrose, dextrates, dextrin, maltodextrin, microcrystalline cellulose (for example, PH 102 or PH200. Avicel®), microfine cellulose, powdered cellulose, pregelatinized starch (e.g., Starch 1500®), calcium phosphate dihydrate, soy polysaccharide (e.g., Emcosoy®), gelatin, silicon dioxide, calcium sulfate, calcium carbonate, magnesium carbonate, magnesium oxide, sorbitol, mannitol, kaolin, polymethacrylates (e.g., Eudragit®), potassium chloride, sodium chloride, and talc. One or more diluents may be used in the dosage form.

[0120] In some embodiments the nutritional supplement is in a solid dosage form, such as a tablet. In some embodiments, the solid dosage form comprises one or more binders can help the ingredients hold together. In some embodiments, the binders comprise sugars such as sucrose, lactose, and glucose; com syrup; soy polysaccharide, gelatin; povidone (e.g., Kolli- don®, Plasdone®); Pullulan; cellulose derivatives such as microcrystalline cellulose, hydroxypropylmethyl cellulose (e.g., Methocel®), hydroxypropyl cellulose (e.g., Klucel®), ethylcellulose, hydroxyethyl cellulose, carboxymethylcellulose sodium, and methylcellulose; acrylic and methacrylic acid copolymers; carbomer (e.g., Carbopol®); polyvinylpolypyrrolidine, polyethylene glycol (Carbowax®); pharmaceutical glaze; alginates such as alginic acid and sodium alginate; gums such as acacia, guar gum, and arabic gums; tragacanth; dextrin and maltodextrin; milk derivatives such as whey; starches such as pregelatinized starch and starch paste; hydrogenated vegetable oil; and magnesium aluminum silicate.

[0121] When the nutritional supplements are in the form of a tablet, the shape of the tablet is not particularly limited, as far as it can be produced without difficulty using an ordinary manufacturing apparatus or a manufacturing apparatus with some modifications. A disc shape that is a general concept for tablets can be mentioned as a typical example. The whole size is not particularly limited. For example, the shorter diameter (diameter for a disc tablet) is appropriately in the range of about 6 mm to about 20 mm, or about 8 mm to about 12 mm. The thickness is neither particularly limited, but appropriately about 1 mm to about 10 mm, or about 2 mm to about 8 mm.

[0122] In some embodiments, the nutritional supplements of the present invention may be in a ready-to-use composition. The ready-to-use composition may comprise one or more stabilizing agents, which include, but are not limited to, buffering agents, tonicity agents, polymers, preservatives, antioxidants, sugars and salts, and combinations thereof.

[0123] METHODS OF PROVIDING NUTRITIONAL SUPPORT

[0124] Various embodiments of the present disclosure relate to methods of using any nutritional supplement as described herein for nutritional support of a subject. Some embodiments relate to a method of using a nutritional supplement comprising a protein component, a fat component, a naturally occurring milk, a carbohydrate component, and combinations thereof. In some embodiments, the nutritional supplement comprises lactoferrin, whey protein hydrolysate, and alpha-lactalbumin. Some embodiments relate to a method of using a nutritional supplement comprising lactoferrin, whey protein hydrolysate, alphalactalbumin, and a naturally occurring milk including milk protein naturally occurring in cow's milk.

[0125] In any embodiment described herein, the method comprises administering any nutritional supplement described in any embodiment disclosed herein.

[0126] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein results in easy digestion, immune support, brain heath, gut health, or combinations thereof.

[0127] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in less spit up, softer stools, more efficient weight gain, longer time between nighttime feeds, enhanced nutrient absorption, brain development, eye development, immune health, gut health, or combinations thereof.

[0128] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in promoting proliferation of intestinal cells in a subject.

[0129] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in inducing proliferation of intestinal cells in a subject.

[0130] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in promoting differentiation of intestinal cells in a subject.

[0131] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in inducing differentiation of intestinal cells in a subject.

[0132] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in inhibiting growth of enteropathogenic Escherichia coli in the digestive system of a subject.

[0133] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in inhibiting growth of enteropathogenic Escherichia coli in the digestive system of a subject.

[0134] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in increasing interleukin- 18 secretion by intestinal cells in a subject.

[0135] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in increasing intestinal immunity’ in a subject.

[0136] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in increasing gut health in a subject, the method comprising administering to the subject any nutritional supplement described herein.

[0137] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, wherein increasing gut health includes promoting or inducing proliferation of intestinal cells, promoting or inducing differentiation of intestinal cells, preventing of inhibiting growth of enteropathogenic Escherichia coli in the digestive system, preventing or inhibiting bacterial growth in the intestinal lumen, modifying an immune response to an infection by enteropathogenic Escherichia coli, lowering cell permeability, increasing expression of tight junction proteins, modulating interleukin- 18 secretion (IL- 18) by intestinal cells, modulating transforming growth factor (31 (TGF-(31) secretion by intestinal cells, or combinations thereof.

[0138] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, results in increasing intestinal immunity in a subject, the method comprising administering to the subject any nutritional supplement described herein.

[0139] In some embodiments, the method of providing nutritional support according to any nutritional supplement described herein, wherein increasing intestinalimmunity includes modulating interleukin- 18 transforming growth factor (31 (TGF-(31) secretion by intestinal cells.

[0140] In some embodiments, efficacy of the methods described herein can be determined by measuring an at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%. at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% improvement or change relative to a measurement in a control. In some embodiments, the efficacy of the nutritional supplement of the present invention may be determined by establishing a significant difference from a control, in which p<0.10, or p<0.05, or p<0.01. The control may be a normal healthy control, a pre-treatment control, or an earlier timepoint control.

[0141] In some embodiments, efficacy of the methods described herein can be determined by measuring an at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%. at least about 45%. at least about 50%. at least about 55%. at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, and ranges between any two of these values, improvement or change relative to a measurement in a competitor’s nutritional supplement. In some embodiments, the efficacy of the nutritional supplement of the present invention may be determined by establishing a significant difference from a competitor’s nutritional supplement, in which p<0.10, or p<0.05, or p<0.01.

[0142] In some embodiments, the nutritional supplement is expelled directly into a subject's intestinal tract. In some embodiments, the nutritional supplement is expelled directly into the gut. In some embodiments, the composition is administered enterally. In some embodiments, the composition is administered enterally under the supervision of a physician. In some embodiments, the composition is administered as dietary management of a disease or condition. In some embodiments the disease or condition is celiac disease, food allergies, and combinations thereof.

[0143] In some embodiments, the method comprises administering any nutritional supplement as described herein is an infant formula. In some embodiments, the nutritional supplement is delivered to an infant starting at birth. In some embodiments, delivery of the nutritional supplement continues from birth until about two years of age. In some embodiments, deliver of the nutritional supplement continues through about one month of age, about threemonths of age, about six months of age, about nine months of age, about one year of age, about 15 months of age, about 18 months of age, about 21 months of age, about 2 years of age, and ranges between any two of these values. In some embodiments, the infant formula is administered until the infant has transitioned fully to solid foods. In some embodiments, the infant formula is administered as a supplemental source of nutrition. In some embodiments, the infant formula is administered as a supplemental source of nutrition after solid foods are introduced.

[0144] In some embodiments, any nutritional supplement as described herein is administered to infants bom prematurely. In some embodiments, the nutritional supplement is administered until a time that matches full-term gestation. In some embodiments, the nutritional supplement is delivered to an infant until at least about three months corrected age, about six months corrected age, about nine months corrected age, about one year corrected age, about 15 months corrected age, about 18 months corrected age, about 21 months corrected age, about two years corrected age, and ranges between any two of these values. In some embodiments, the nutritional supplement is delivered to a subject as long as is necessary’ to correct nutritional deficiencies.

[0145] In some embodiments, any nutritional supplement as described herein is administered in a growing-up milk. Growing-up milks are fortified milk-based beverages intended for children over about 1 year of age (generally from about one to about three years of age, or from about four to about six years of age. or from about one year to about six years of age). Growing-up milks can compensate for nutritional deficiencies or can complement a diet to provide additional insurance that a child is receiving all necessary nutrition.

[0146] In some embodiments, any nutritional supplement as described herein is administered as a daily or multiple-times-a-day supplement.ADDITIONAL EMBODIMENTS

[0147] Clause 1. A protein complex for nutritional supplements, the protein complex comprising lactoferrin and alpha-lactalbumin.

[0148] Clause 2. A protein complex for nutritional supplements, the protein complex comprising lactoferrin and whey protein hydrolysate proteins.

[0149] Clause 3. A protein complex for nutritional supplements, the protein complex comprising lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.

[0150] Clause 4. The protein complex of any one of clauses 1-3, wherein the protein complex further comprises proteins from naturally occurring milk.

[0151] Clause 5. The protein complex of clause 4, wherein the naturally occurring milk is nonfat milk.

[0152] Clause 6. The protein complex of clause 4, wherein the naturally occurring milk is whole milk.

[0153] Clause 7. The protein complex of any one of clauses 1-6, wherein the protein complex is encapsulated within a lipid-based delivery system.

[0154] Clause 8. The protein complex of clause 7. wherein the lipid-based delivery system enhances stability or bioavailability of the protein complex.

[0155] Clause 9. The protein complex of any one of clauses 1-6, wherein the protein complex is encapsulated in a protective matrix.

[0156] Clause 10. The protein complex of clause 9. wherein the protective matrix enhances stability or bioavailability of the protein complex.

[0157] Clause 11. The protein complex of any one of clauses 1-10, wherein the protein complex is resistant to gastrointestinal digestion and is capable of binding to a lactoferrin receptor on a cell membrane of intestinal epithelial cells.

[0158] Clause 12. A nutritional supplement, the nutritional supplement comprising a protein complex comprising lactoferrin and alpha-lactalbumin.

[0159] Clause 13. A nutritional supplement, the nutritional supplement comprising a protein complex comprising lactoferrin and whey protein hydrolysate proteins.

[0160] Clause 14. A nutritional supplement, the nutritional supplement comprising a protein complex comprising lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.

[0161] Clause 15. The nutritional supplement of any one of clauses 12-14, wherein the protein complex further comprises proteins from naturally occurring milk.

[0162] Clause 16. The nutntional supplement of clause 15, wherein the naturally occurring milk is nonfat milk.

[0163] Clause 17. The nutritional supplement of clause 15, wherein the naturally occurring milk is whole milk.

[0164] Clause 18. The nutritional supplement of any one of clauses 12-17, wherein the protein complex is encapsulated within a lipid-based delivery system to enhance absorption.

[0165] Clause 19. The nutritional supplement of clause 18, wherein the lipid-based delivery' system enhances stability or bioavailability of the protein complex.

[0166] Clause 20. The nutritional supplement of any one of clauses 12-17, wherein the protein complex is encapsulated in a protective matrix.

[0167] Clause 21. The nutritional supplement of clause 20, wherein the protective matrix enhances stability or bioavailability’ of the protein complex.

[0168] Clause 22. The nutritional supplement of any one of clauses 12-21, wherein the protein complex is resistant to gastrointestinal digestion and is capable of binding to a lactoferrin receptor on a cell membrane of intestinal epithelial cells.

[0169] Clause 23. The nutritional supplement of any one of clauses 12 to 22, wherein the nutritional supplement comprises one or more digestion aiding proteins.

[0170] Clause 24. The nutritional supplement of clause 23, wherein the one or more digestion aiding proteins are selected from whey protein hydrolysate, alpha-lactalbumin, K- casein, a-casein, |3-casem. bile-salt stimulated lipase, amylase, al -antitry psin, and combinations thereof.

[0171] Clause 25. The nutritional supplement of any one of clauses 12-22, yvherein the nutritional supplement comprises one or more immunoprotective proteins.

[0172] Clause 26. The nutritional supplement of clause 25. wherein the one or more immunoprotective proteins are selected from lactoferrin, osteopontin. K-casein. a-casein, (3- casein. haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin. lactoperoxidase, milk fat globule membrane (MFGM), and combinations thereof.

[0173] Clause 27. The nutritional supplement of clause 26, wherein the lactoferrin comprises apolactoferrin, hololactoferrin, or a combination of both.

[0174] Clause 28. The nutritional supplement of any one of clauses 12-27, yvherein the nutritional supplement comprises about 5% to about 20% protein, about 15% to about 40% fat, and about 40% to about 70% carbohydrate.

[0175] Clause 29. The nutritional supplement of clause 28, wherein the protein is lactoferrin, whey protein hydrolysate, alpha-lactalbumin, osteopontin. |3-casein. a-casein, K- casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, amylase, al- antitrypsin, folate-binding protein, insulin-like growth factor- 1, epidermal growth factor, cytokines, lactadherin, lactoperoxidase, milk glycans, milk fat globule membrane (MFGM), or combinations thereof.

[0176] Clause 30. The nutritional supplement of clause 28, yvherein the fat is innate milk fat globule membrane (MFGM), added MFGM, phospholipids, cholesterol, oil, nonhexane extracted docosahexaenoic acid (DHA), hexane extracted arachidonic acid (AA), nonhexane extracted AA, ascorbyl palmitate, or combinations thereof.

[0177] Clause 31. The nutritional supplement of clause 28. wherein the carbohydrate is lactose, glucose, fructose, sucrose, galactooligosaccharide (GOS),fructooligosaccharide (FOS), inulin, com syrup solids, dextrin, maltodextrin, dextrose, polydextrose, tapioca, starch, tapioca starch, rice syrup solids, waxy com, waxy rice starch, or combinations thereof.

[0178] Clause 32. The nutritional supplement of any one of clauses 12-31, wherein the nutritional supplement does not comprise lactoperoxidase.

[0179] Clause 33. The nutritional supplement of any one of clauses 12-32, wherein the lactoferrin is incorporated into a micellar structure.

[0180] Clause 34. The nutritional supplement of any one of clauses 12-33, wherein the protein complex promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promotes differentiation of intestinal cells, induces differentiation of intestinal cells, inhibits growth of enteropathogenic Escherichia coli in a digestive system, inhibits bacterial infection in an intestinal lumen, increases interleukin- 18 secretion by intestinal cells, modulates interleukin- 18 secretion by intestinal cells, increases transforming growth factor (31 (TGF-(31) secretion by intestinal cells, modulates transforming grow th factor (31 (TGF-J31) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability, increases expression of tight junction proteins, increases gut health, and combinations thereof.

[0181] Clause 35. The nutritional supplement of any one of clauses 12-33, wherein the nutritional supplement promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promotes differentiation of intestinal cells, induces differentiation of intestinal cells, inhibits growth of enteropathogenic Escherichia coli in a digestive system, inhibits bacterial infection in an intestinal lumen, increases interleukin- 18 secretion by intestinal cells, modulates interleukin- 18 secretion by intestinal cells, increases transforming growth factor (31 (TGF-J31) secretion by intestinal cells, modulates transforming growth factor pi (TGF-(31) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability, increases expression of tight junction proteins, increases gut health, and combinations thereof.

[0182] Clause 36. The nutritional supplement of any one of clauses 12-33, wherein the nutritional supplement provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

[0183] Clause 37. The nutritional supplement of any one of clauses 12-33, wherein the nutritional supplement provides less spit up, softer stools, more efficient weight gain, longer time between nighttime feeds, enhanced nutrient absorption, brain development, eye development, immune health, gut health, or combinations thereof

[0184] Clause 38. The nutritional supplement of any one of clauses 12-37, wherein the nutritional supplement is for infants.

[0185] Clause 39. The nutritional supplement of clause 38, wherein the nutritional supplement is formulated to enhance cognitive development.

[0186] Clause 40. The nutritional supplement of clause 39, wherein the nutritional supplement is formulated to facilitate healthy intestinal and immune development in early childhood.

[0187] Clause 41. A method of providing nutritional support to a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0188] Clause 42. The method of clause 41, wherein the nutritional supplement provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

[0189] Clause 43. The method of clause 41, wherein the nutritional supplement results in less spit up, softer stools, more efficient weight gain, longer time between nighttime feeds, enhanced nutrient absorption, brain development, eye development, immune health, gut health, or combinations thereof.

[0190] Clause 44. A method of promoting proliferation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0191] Clause 45. A method of inducing proliferation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to clause 12-33.

[0192] Clause 46. A method of promoting differentiation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0193] Clause 47. A method of inducing differentiation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0194] Clause 48. A method of inhibiting growth of enteropathogenic Escherichia coli in a digestive system of a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0195] Clause 49. A method of inhibiting bacterial infection in an intestinal lumen of a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0196] Clause 50. A method of increasing interleukin- 18 secretion by intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0197] Clause 51. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0198] Clause 52. A method of increasing gut health in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0199] Clause 53. The method of clause 52, wherein increasing gut health comprises promoting or inducing proliferation of intestinal cells, promoting or inducing differentiation of intestinal cells, preventing of inhibiting growth of enteropathogenic Escherichia coli in a digestive system, preventing or inhibiting bacterial growth in an intestinal lumen, modifying an immune response to an infection by enteropathogenic Escherichia coli, lowering cell permeability, increasing expression of tight junction proteins, modulating interleukin- 18 secretion (IL-18) by intestinal cells, modulating transforming growth factor pi (TGF-p I ) secretion by intestinal cells, or combinations thereof.

[0200] Clause 54. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a nutritional supplement according to any one of clauses 12-33.

[0201] Clause 55. The method of clause 54, wherein increasing intestinal immunity comprises modulating interleukin- 18 transforming growth factor pi (TGF-pi) secretion by intestinal cells.EXAMPLES

[0202] The below examples are merely illustrative and are not inclusive of any additional data or experimental results that can be derived from any embodiments provided in the instant disclosure.

[0203] Example 1 : Effects of forming milk lactoferrin-protein complexes on lactoferrin functionality and intestinal development in infancy

[0204] Materials and Methods

[0205] Preparation of protein blends: Lactoferrin (Lf)was blended with each of the following individual proteins as described in Table 1 : bovine milk a-lactalbumin (a- Lac. Aria Foods, Slagelse, Denmark), bovine milk whey protein hydrolysate (WPH) (Hilmar 8350,Hilmar Ingredients, Hilmar, CA), or bovine nonfat milk powder (MP) (Organic Valley, LaFarge, WI). MP was intentionally chosen for this investigation to ensure the impact of MP proteins was studied without the impact of naturally occurring fats in MP.

[0206] Table 1. Protein Blends

[0207] Blue native PAGE and immunoblotting: Complexes in protein blends were evaluated by native PAGE using the NativePAGE Novex Bis-Tris Gel System (Thermo Fisher Scientific, Rockford, IL), and 4-16% native Bis-Tris gels (Thermo Fisher Scientific) were used following the manufacturer’s instructions for Coomassie blue R-250 (Sigma- Aldrich, St. Louis, MO) staining. For immunoblotting, proteins were transferred from the native gel to polyvinylidene difluoride (PVDF) membranes by using the Turbo Blotting System (Bio-Rad, Hercules, CA). Membranes were blocked using a blocking solution (3% bovine serum albumin in TBST: Tris buffered saline with 1% Tween-20) for 1 h at room temperature and then incubated for 1 h at room temperature with primary antibodies (anti-Lf, A10-126A, Bethyl Laboratories. Montgomery', TX; anti-a-Lac, Al 0-125 A, Bethyl Laboratories) in blocking buffer. After three washes with TBST. membranes were incubated for 1 h at room temperature with a horseradish peroxi das e-conjugated anti -goat IgG (Invitrogen, Carlsbad, CA) or antirabbit (Cell Signaling Technology7, Beverly, MA) secondary antibody. Pro Signal TM Femto (Genesse Scientific, San Diego, CA) was used to visualize the proteins. All the images were generated with the ChemiDocTM MP Imaging System (Bio-Rad Laboratories, Mississauga, ON) using the optimal exposure setting. All raw images were quantitated using both ImageLab (Bio-Rad) and ImageJ (NIH).

[0208] In vivo digestion: Lf, a-Lac, WPH, NFMP and each Lf-protein blend w ere digested by enzymes in vitro to simulate gastrointestinal digestion

[0024] , Briefly, each protein or protein blend was dissolved in water. To mimic infant gastric digestion, porcine pepsin (2% in 1 mmol / L HC1, 1 : 12.5 ratio of pepsin to protein; Sigma- Aldrich) was added at pH 4.0, and samples were incubated in an incubator shaker (120-140 rpm) for 30 min at 37°C. After the simulated gastric incubation, the pH value of the samples was adjusted to 7.0 with NaHCO3 (1 mol / L), and then pancreatin (0.4% in 0. 1 mol / L NaHCO3. 1 :62.5 ratio of pancreatin to protein; Sigma-Aldrich) was added. The samples were further incubated in an incubator shaker (120-140 rpm) for 30 min at 37°C. After the incubation, pancreatin was inactivated in an 85°C water bath for 3 min. To evaluate the effects of pH changes and 37°C incubation alone on the proteins, samples without enzyme treatments were also analyzed. Proteins with and without in vitro digestion were mixed with native loading buffer or boiled for 5 min in Laemmli sample buffer (Bio-Rad) with 0-mercaptoethanol (5%) and then subjected to Blue Native PAGE or SDS- PAGE followed by Coomassie blue R-250 staining or western blotting.

[0209] Cell Culture: Non-transformed human crypt-like intestinal epithelial cells (HIECs) (a gift from Dr. Jean-Franpois Beaulieu, Universite de Sherbrooke, Quebec, Canada) were maintained in a humidified incubator at 37°C under an atmosphere of 5% CO2 in Opti MEM (Life Technologies Inc., Gaithersburg, MD) supplemented with fetal bovine serum (FBS, 5%, Gemini Bio-products, West Sacramento. CA). NaHCO3 (2.4 g / L; Sigma-Aldrich), penicillin-streptomycin solution (0.5%; Gemini Bio-products), HEPES buffer (1%; Thermo Fisher Scientific), GlutaMax (1%, Thermo Fisher Scientific), and epidermal Growth Factor (EGF, 5 ng / mL, Gemini Bio-products). Medium was changed even,' other day, and cells between passages 18-26 were harvested at 90% confluence.

[0210] Human colon adenocarcinoma Caco-2 cells (American Type culture collection, Rockville, MD) were maintained in a humidified incubator at 37°C under an atmosphere of 5% CO2 in minimal essential medium (MEM, Gibco Invitrogen) containing FBS (10%, Gemini Bio-products), NaHCO3 (2.2 g / L, Sigma-Aldrich), and penicillinstreptomycin solution (1%, Gemini Bio-products) in a humidified cell culture incubator with CO2 (5%) at 37°C. Experiments were conducted using cells between passages 20 to 30.

[0211] Internalization of Lf by HIECs: Whether Lf alone or in protein blends bind to the LfR on the membrane of intestinal epithelial cells was evaluated by confocal microscopy. HIECs were seeded and grown on gelatin coated glass coverslips (Neuvitro Corporation, Camas, WA) using 24-well plates overnight. After HIECs were treated with Lf or each of the protein blends in serum free medium (SFM) at 37 °C for 1 h, cells were rinsed with PBS, fixed with phosphate-buffered paraformaldehyde (4%, 0.4 mL / well) for 10 min at room temperature. Cells were blocked with blocking buffer (5% heat-inactivated rabbit serum in PBS, 0.5 mL / well) for 20 min. After the blocking buffer was removed, cells were rinsed with PBS. Protein (Lf) and receptor (LfR) were probed with primary antibodies [anti-Lf, Bethyl Laboratories, or anti-LfR

[0025] and fluorescence conjugated secondary antibodies (Alexa 488- conjugated-antibodies or Fluor-633-conjugated antibodies, Invitrogen, Carlsbad, CA). After several rinses, coverslips were mounted with a ProLong Gold anti-fade reagent (Invitrogen), seeded on glass slides, and sealed with nail polish. Immunofluorescence imaging was obtainedby a confocal laser scanning microscope (FV1000, Olympus America Inc., Melville, NY) with 60 x magnification under an oil-immersion lens and analyzed with complete integrated image analysis software systems (Olympus America Inc.).

[0212] Proliferation and differentiation assays: Effects on intestinal proliferation were assessed using HIECs. For all experiments, Lf, a-Lac, WPH, NFMP or each Lf-protein blend were diluted proportionally, which means that the concentrations, as shown in the Table I, are lx; when the samples are diluted lOx and 5x, the concentrations are O. lx and 0.2x. respectively. After HIECs were treated with Lf, a-Lac, WPH, NFMP or each Lf-protein blend (lx, n=5) for 24 h, effects of these proteins on intestinal proliferation were evaluated by using a BrdU cell proliferation assay kit (Millipore, Bedford, MA).

[0213] To determine effects of these proteins and protein blends on cell differentiation, Caco-2 cells (90% confluence) were fasted for 2 h with SFM and then incubated with the proteins and protein blends (0.05x, n=5) in medium containing 1% fetal bovine serum (FBS) at 37 °C for 72 h. Cells were washed 3 times with ice-cold PBS, and cell lysates were prepared in a cell homogenization buffer (PBS buffer containing 0.1% Triton X-100, and complete protease inhibitor cocktail; Roche, Indianapolis, IN). Alkaline phosphatase activity, an indicator of intestinal differentiation, was measured using p-nitrophenylphosphate as a substrate (Thermo Fisher Scientific).

[0214] Transepithelial electrical resistance (TEER) measurement: To evaluate whether Lf, a-Lac, WPH, NFMP. and Lf-protein complexes influence permeability and expression of tight junction proteins in human intestinal epithelial cells, polarized Caco-2 cells were used as an enterocyte model

[0023] , After Caco-2 cells were cultured on Trans wells for 14 days, they were treated with individual proteins and Lf-protein blends (0.5x, in SFM; n=5) up to 24 h, and TEER was then measured. To investigate how individual proteins and protein blends influence permeability of Caco-2 cells, effects on transcription of tight junction proteins including claudinl, occludin, and ZO1 were assessed by qRT-PCR as described below .

[0215] RNA extraction and quantitative real-time poly merase chain reaction (PCR) (qRT-PCR): Total RNA was isolated from differentiated Caco-2 cells treated with individual proteins and Lf-protein blends (0.5x, in SFM: n=5) for 24 h with Trizol reagent (Invitrogen). treated with DNase I (New7England Biolabs, Ipswich, MA), then purified by an RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA) and then reverse-transcribed to cDNA using a High-capacity cDNA Reverse Transcription Kit (Applied Biosystems. Foster City, CA) following the manufacturer's instructions. Gene-specific primers to specific genes were selected using the Primer design tool from NCBI and ordered from Operon Technologies(Alameda, CA). qRT-PCR was conducted on the cDNA reaction mixture (2 pL) and SYBR Green (Bio-Rad. Hercules, CA) using an iCycler Real-time PCR System (Bio-Rad). Cycling parameters were 95°C for 15 min and 40 cycles including 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec. Linearity of the dissociation curve was analyzed using the iCycler software, and mean cycle time of the linear part of the curve was designated as Ct. Each sample was analyzed in triplicate and normalized to GAPDH using the following equation: fold change = 2(Ct Gene- Ct GAPDH). Values are shown as mean fold change ± standard deviation, relative to control (set to 1).

[0216] IL-18 secretion: Caco-2 cells (day 14) were fasted for 2 h with SFM and then treated with Lf, , a-Lac, WPH, NFMP, and Lf-protein complexes for 48 h in cell culture medium containing FBS (1%) at 37 °C for 72 h. Cell culture media were collected and concentrated by YM-3 (3K MWCO; Millipore) to measure concentrations of secreted interleukin 18 (IL-18) using an ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

[0217] Effects on transcription of the TGF-01 gene: Caco-2 cells (day 8) on 6-well plates were treated with Lf, a-Lac, WPH, NFMP. and Lf-protein complexes (O. lx) for 72 h at 37°C. RNA extraction and qRT-PCR were conducted as described above.

[0218] Effects on grow th of Enteropathogenic Escherichia coli (EPEC): Effects of the of proteins and protein blends on growth of enteropathogenic E. coli (EPEC) (BAA-2440, ATCC) were determined by counting colony forming units (CFUs) on agar plates. Samples of EPEC (5 x 105 CFUs / mL) were cultured with Lf, a-Lac, WPH, NFMP, and Lf-protein complexes (0.5x of each in Difco Nutrient Broth medium) and incubated for 16 h at 37 °C with shaking at 225 rpm. Aliquots of the medium (10 pL) were plated onto Luria Bertani (LB) agar, and CFUs were counted after overnight incubation at 37 °C.

[0219] Effects of protein and protein blends on the inflammatory response induced by EPEC infection: IL-1 / ?, IL-6, and TNF-a: Caco-2 cells (day 16) were fasted in SFM without antibiotics at 37 °C for 1 h, and then incubated overnight with EPEC (250 pl, optical density approximately 0.4 at 600 nm) and with Lf, a-Lac+WPH+NFMP, WPH, NFMP. and Lf-protein complexes (lx in SFM without antibiotics) at 37 °C for 4 h. After infection, cells were washed twice with PBS, and the medium was replaced by complete medium containing gentamicin (1 pg / ml) in order to prevent the ox ergrow th of extracellular bacteria and host cell death. After 4h, Caco-2 cells were collected for RNA extraction and qRT-PCR analysis of transcripts of pro-inflammatory cytokines (TNF- a, IL- 1(3, and IL-6) as described above.

[0220] Statistical analysis: Data are presented as means ± standard deviation from 2-3 independent experiments. Comparisons between control and treatment groups were conducted using Student’s t-test or one-way ANOVA (GraphPad Prism, GraphPad Software Inc., San Diego, CA). P<0.05 was considered statistically significant.

[0221] Results

[0222] Preparation of Lf-protein blends: Protein blends were prepared as shown in Table 1.

[0223] Lf-protein complexes and resistance to in vitro digestion: To evaluate whether Lf forms complexes with a-Lac and other proteins in WPH and NFMP, blue native PAGE and native Western blotting were conducted (FIG. 1 A, FIG. IB, FIG. 1C, FIG. ID. FIG. IE. FIG. IF). The protein bands of Lf alone and a-Lac alone differ from bands of Lf and a-Lac when blended together or with other proteins, indicating that Lf and a-Lac form a complex with each other and with milk proteins from WPH and NFMP. To evaluate whether Lf and Lf- protein complexes are resistant to in vitro simulated gastrointestinal digestion, these proteins were digested with pepsin at pH 4.0 and pancreatin at pH 7.0 for 30 min at 37°C, respectively. The digested protein samples were then analyzed by blue native PAGE and native Western blotting. Both Lf and a-Lac are more resistant to in vitro digestion when complexed with each other or other proteins. Lf formed similar bands in undigested samples as well as in digested samples (FIG. IB, FIG. IE).

[0224] FIG. 1 shows a native PAGE and western blotting demonstrating that Lf and protein blends are relatively resistant to in vitro simulated gastrointestinal digestion. Protein and protein blends (Img / mL) were digested with pepsin and pancreatin for 30 min, respectively. Undigested and digested proteins and protein blends (10 pL for each) were subjected to native PAGE and stained with Coomassie Blue R-250 or probed with antibodies (anti-Lf or anti- a-Lac). A-C non-digested Lf and blends; D-F digested Lf and blends. A&D: Native PAGE; B&E: Western blotting (anti-Lf); C&F: Western blotting (anti- a-Lac). M: protein molecular marker; 1: Lf; 2: a-Lac; 3: WPH; 4: NFMP; 5: blend 1 (Lf+ a-Lac); 6: blend 2 (Lf+WPH); 7: blend 3 (Lf+ a-Lac+WPH+NFMP). Abbreviations: Lf=lactoferrin, a-Lac= a- lactalbumin, WPH = whey protein hydrolysate, MP= bovine milk powder.

[0225] SDS PAGE and Western blotting were also performed before and after in vitro simulated gastrointestinal digestion to show the size (molecular weight) of Lf alone, a- Lac alone, and each in complexes with other proteins (FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F). SDS PAGE of the undigested proteins show similar molecular weights of Lf alone or in blends and a-Lac alone or in blends. Both Lf alone and a-Lac alone were partlyresistant to in vitro gastrointestinal digestion, but were relatively protected from digestion when in protein blends. Forming complexes with a-Lac and other proteins from WPH and NFMP protects Lf and a-Lac from digestion by gastric and intestinal enzymes (FIG. 2E, FIG. 2F).

[0226] FIG. 2 shows SDS PAGE and western blotting demonstrating that Lf and protein blends are relatively resistant to in vitro simulated gastrointestinal digestion. Protein and protein blends (Img / mL) were digested with pepsin and pancreatin for 30 min, respectively. Undigested and digested proteins and protein blends (10 pL for each) were subjected to SDS PAGE and stained with Coomassie Blue R-250 or probed with antibodies (anti-Lf or anti- a-Lac). A-C non-digested Lf and blends; D-F digested Lf and blends. A&D: Native PAGE; B&E: Western blotting (anti-Lf); C&F: Western blotting (anti- a-Lac). M: protein molecular marker; 1: Lf; 2: a-Lac; 3: WPH; 4: NFMP: 5: blend 1 (Lf+ a-Lac); 6: blend 2 (Lf+WPH); 7: blend 3 (Lf+ a-Lac+WPH+NFMP). Abbreviations: Lf=lactoferrin, a-Lac= a- lactalbumin, WPH = whey protein hydrolysate, MP= bovine milk powder, NFMP=non-fat bovine milk powder.

[0227] Binding of Lf to the LfR and internalization by intestinal epithelial cells: We have previously demonstrated that the LfR mediates Lf internalization by intestinal epithelial cells via clathrin-dependent endocytosis

[0025] , We therefore used confocal microscopy to evaluate Lf alone and in complex forms are able to bind to the LfR on the cell membrane and are internalized by intestinal epithelial cells. After HIECs were treated with Lf or Lf-protein blends for Ih, HIECs were fixed for confocal microscopy analysis. As shown in FIG. 3, the heat treatment did not influence binding of Lf to the LfR and uptake by HIECs, and Lf in complex forms (in all the protein blends) was able to bind to the LfR and was subsequently internalized by HIECs.

[0228] FIG. 3 shows that Lf binds to the LfR and is internalized by human intestinal epithelial cells (HIECs). HIECs grown on glass cover slips were treated with Lf and protein blends for Ih at 37 °C for 1 h, then fixed and then probed with primary antibodies (anti-Lf and anti-LfR antibodies) and fluorescent secondary antibodies. Lf (green), LfR (red).

[0229] Effects of Lf. a-Lac, WPH, NFMP, and Lf-protein complexes on proliferation and differentiation of intestinal epithelial cells: It is known that Lf promotes intestinal proliferation and differentiation

[0026] , HIECs were treated with Lf, a-Lac, WPH, NFMP, and Lf-protein complexes (lx for each) for 24 h at 37°C, and then the BrdU assay was carried out. As shown in FIG. 4A, Lf, a-Lac, WPH, NFMP, and Lf-a-Lac, markedly increased proliferation of HIECs. However, this increase was not found when NFMP or WPH were present in the protein blend. After 72h treatment of Caco-2 cells with proteins and proteinblends, the effects on differentiation of Caco-2 cells were evaluated by measuring activity of alkaline phosphatase, an intestinal differentiation marker

[0027] , As shown in FIG. 4B, all proteins and blends promoted differentiation of Caco-2 cells. Four blends, Lf-a-Lac, Lf-WPH, and Lf-a-Lac-WPH-NFMP, exhibited greater effects than Lf alone.

[0230] FIG. 4A and FIG. 4B demonstrate the effects of proteins and protein blends on proliferation of HIECs (A) and differentiation of Caco-2 cells (B). (A) HIECs were grown on 96-well plates (approximately 40% confluence) and treated Lf or protein blends (lx of each) for 24 h at 37 °C. Cell proliferation was evaluated by a BrdU ELISA kit. (n = 6). (B) Caco-2 cells grown on 24-w ell plates until 90% confluence were treated with Lf or protein blends (0.05x of each) for 72 h at 37 °C. Alkaline phosphatase activity w as measured by using pNPP as a substrate. Results are shown as mean ± SD (n = 5). Bars without a common letter are significantly different (P<0.05).

[0231] Effects of proteins and protein blends on intestinal barrier functions: Caco- 2 cells were cultured on Transwells for 14 days and then treated with Lf, a-Lac, WPH, NFMP, and Lf-protein complexes (0. lx for each) for 48 h. Trans -epitheli al electrical resistance (TEER) was measured at 24h to determine protein effects on intestinal barrier functions (FIG. 5A). All the proteins and protein blends significantly increased TEER. a-Lac, and LF+ a-Lac showed a greater increase in TEER than Lf alone. Next, we examined if the proteins and blends enhanced TEER by increasing expression of tight junction proteins. As shown in FIG. 5B, all proteins and blends (except Lf+WPH) increased transcription of occludin, some more than others. Additionally, WPH, and Lf-a-Lac also increased transcription of ZO1 .

[0232] FIG. 5A and FIG. 5B demonstrate the effects of proteins and protein blends on TEER and transcription of tight conjunction proteins. After Caco-2 cells were cultured 14 days on Transwells, cells were treated with Lf and protein blends (0.5x for each) for 48 h. TEER was measured by using the Milhcell Electrical Resistance System (Millipore). (n=6) Bars without a common letter are significantly different (P<0.05).

[0233] Effects of proteins and protein blends on transcription of the TGF-(31 gene: Caco-2 cells were cultured for 8 days and then treated with Lf. a-Lac, WPH, NFMP, and Lf- protein complexes (0.5x for each) for 24 h. RNA was then extracted for real-time PCR. Only Lf and Lf-WPH increased transcription of the TGF-pi gene (FIG. 6). The effect of Lf was greatest. Although NFMP, Lf-a-Lac, Lf-WPH, and Lf- a-Lac- WPH-NFMP, also increased transcription of the TGF-[31 gene, the results were not significantly different from the control.

[0234] FIG. 6 demonstrates the effects of protein blends on transcription of theTGF-betal gene. After differentiated Caco-2 cells were treated with Lf and protein blends for72 h, total RNA was extracted for quantitative real-time PCR. Results are shown as mean fold changes ± SD (n = 6). Bars labeled with different letters are significantly different (P < 0.05).

[0235] Effects of proteins and protein blends on IL- 18 secretion by Caco-2 cells: Differentiated Caco-2 cells (DI 4) were treated with Lf, a-Lac, WPH, NFMP, and Lf-protein complexes (0. lx for each) for 48 h, and cell culture media were collected for IL-18 ELISA. As shown in FIG. 7, all proteins and blends markedly increased secreted IL- 18 in cell culture media.

[0236] FIG. 7 demonstrates the effects of proteins and protein blends on IL- 18 secretion by Caco-2 cells. After Caco-2 cells (DI 4) were treated with Lf and protein blends for 72 h at 37 °C, the cell culture medium was concentrated by YM-3 and then analyzed with an IL- 18 ELISA kit (R&D Systems). Results are shown as mean ± SD (n = 5).

[0237] Effects of proteins and protein blends on growth of EPEC: EPEC was cultured with Lf, a-Lac, WPH, NFMP and Lf-protein complexes (0.2x for each) at 37 °C for 16 h, the culture medium was grown on agar plates, and EPEC colonies were counted at 24 h. As shown in FIG. 8, all proteins and protein blends significantly inhibited growth of EPEC. a- Lac and WPH showed the least inhibition.

[0238] FIG. 8 demonstrates the effects of protein blends on growth of EPEC. EPEC (5 x 105 CFUs / mL) w as cultured with Lf or protein blends (0.2x of each in Difco Nutrient Broth medium) at 37 °C for 16 h with shaking at 250 rpm. Aliquots of the medium (10 LIL) were plated onto LB agar, and CFUs were counted after overnight incubation at 37 °C. Results are shown as mean ± SD (n = 5). Bars labeled with different letters are significantly different (P < 0.05).

[0239] Effects of proteins and protein blends on immune responses to EPEC infection: After differentiated Caco-2 (D16. cultured in 6 well plates) were infected by EPEC and incubated with Lf, a-Lac, WPH, NFMP, and Lf-protein complexes, effects on immune responses of the cells to EPEC infection were evaluated. As shown in FIG. 9A, infection significantly increased transcription of IL-1 [3, IL-6, and TNFa. All proteins and protein blends inhibited transcription of IL-10 and TNF-a to different extent. All proteins and protein blends, except NFMP. and LF+ a-Lac. decreased transcription of IL-6. FIG. 9B demonstrates the effects of Lf-protein blends on the inflammatory response induced by EPEC infection.

[0240] Discussion

[0241] Lf forms complexes with a-Lac as well as with proteins / peptides fromNFMP and WPH, and these complexes protect Lf against in vitro simulated gastrointestinal digestion. Lf alone was minimally resistant to in vitro gastrointestinal digestion, but by formingprotein complexes with a-Lac and proteins and peptides from WPH and NFMP, a higher percentage of Lf survived digestion, suggesting that Lf in protein complexes may exert more potent beneficial effects on the intestine in early life. In agreement with the findings from this current study, Lf complexed with OPN or blended with OPN, a-Lac, and WPH exhibited higher resistance to gastrointestinal digestion than Lf alone

[0028] .

[0242] Forming complexes did not influence binding of Lf to the LfR or internalization by intestinal epithelial cells, but affected various bioactivities of Lf. As shown in FIG. 3, Lf alone and in three protein blends were able to bind to the LfR on the cell membrane of HIECs. Lf exerts multiple functions by binding to the LfR, followed by initiation of cell signaling pathways or translocation to the nucleus to regulate gene expression [6], Lf promotes proliferation and differentiation of intestinal epithelial cells, lowers cell permeability of intestinal epithelial cells by upregulating tight junction proteins, regulates immune functions and immune responses, inhibits grow th of EPEC and regulates immune responses to EPEC infection. Lf and protein complexes varied in their effects on proliferation, differentiation, and transcription of TGF-01. The differing total concentrations of protein(s) in these preparations may have affected results in specific assays. NFMP and WPH exhibit many bioactivities similar to Lf, but they did not promote transcription of the TGF-01 gene. NFMP and WPH consist of diverse protein components, such as P-lactoglobulin, immunoglobulins, lactoperoxidase [29-31], Lf may have a more potent effect on TGF-J31 gene transcription than other bioactive components in whey proteins.

[0243] a-Lac is relatively resistant to in vitro simulated gastrointestinal digestion and exhibits beneficial effects on intestinal epithelial cells. Both a-Lac alone and a-Lac in protein complexes are partly resistant to in vitro simulated gastrointestinal digestion. a-Lac and Lf as well as peptides / proteins from NFMP form protein complexes, as shown in FIG. 1 and FIG. 2. The complex form of a-Lac in blends (Blends 3) that include Lf, a-Lac, WPH, and NFMP was more resistant to in vitro digestion than were the Lf-a-Lac complexes. Studies on bioactivities of a-Lac have been comparatively limited and largely focused on its peptides, but we show that a-Lac exhibits multiple effects on intestinal epithelial cells, including promotion of cell proliferation and differentiation, decreasing cell permeability, increasing expression of a tight junction protein, inhibition of growth of EPEC, regulation of immunity of intestinal epithelial cells. Our results are consistent with some in a previous study showing that a-Lac increased TEER in differentiated Caco-2 cells

[0032] and effects on IL-1 beta and TNF-alpha secretion

[0014] ,

[0244] In summary, Lf forms complexes with a-Lac and other milk proteins / peptides from WPH and NFMP. Compared to Lf alone, Lf in complex forms is more resistant to in vitro simulated gastrointestinal digestion, suggesting that Lf in complex forms may play important roles in intestinal development. Forming protein complexes does not influence binding of Lf to LfR and uptake of Lf by intestinal epithelial cells and Holder pasteurization does not cause degradation of Lf affecting its bioactivities. Lf, a-Lac, and protein blends exhibit beneficial effects on intestinal epithelial cells including promotion of proliferation and differentiation, lowering cell permeability by increasing expression of tight junction proteins, regulating immune functions by regulating IL-18 and TGF-|H, and inhibiting growth of EPEC as well as modifying immune responses to EPEC infection. Our finding that a-Lac stimulated cell proliferation and differentiation, TEER and occludin as well as IL- 18 expression was somewhat unexpected. It is known, however, that breast-fed infants have a well-developed and differentiated small intestine and it is thus possible that the high concentration of a-Lac in breast milk is in part responsible for this. These activities of a-Lac merit further studies.

[0245] References:

[0246] 1. Lonnerdal, B.; Iyer, S. Lactoferrin: Molecular Structure and BiologicalFunction. Annu Rev Nutr 1995, 15, 93-110, doi: 10.1146 / annurev.nu.l5.070195.000521.

[0247] 2. Rai, D.; Adelman, A S.; Zhuang, W.; Rai, G.P.; Boettcher, J.; Lonnerdal,B. Longitudinal Changes in Lactoferrin Concentrations in Human Milk: A Global Systematic Review. Cnt Rev Food Sci Nutr 2014, 54, 1539-1547, doi: 10. 1080 / 10408398.201 1.642422.

[0248] 3. Sanchez, L.; Aranda, P.; Perez, M.D.; Calvo, M. Concentration ofLactoferrin and Transferrin throughout Lactation in Cow’s Colostrum and Milk. Biol Chem Hoppe Seyler 1988, 369. 1005-1008, doi: 10. 1515 / bchm3. 1988.369.2. 1005.

[0249] 4. Lonnerdal, B. Nutritional Roles of Lactoferrin. Curr Opin Clin NutrMetab Care 2009, 12, 293-297, doi: 10.1097 / MCO.0b013e328328dl3e.

[0250] 5. Suzuki, Y.A.; Lopez, V.; Lonnerdal, B. Mammalian LactoferrinReceptors: Structure and Function. Cell Mol Life Sci 2005, 62, 2560-2575, doi : 10. 1007 / S00018-005 -5371 - 1.

[0251] 6. Liao, Y .; Jiang, R.; Lonnerdal, B. Biochemical and Molecular Impacts ofLactoferrin on Small Intestinal Grow th and Development during Early Life. Biochem Cell Biol 2012, 90. 476-484, doi: 10. 1139 / ol 1-075.

[0252] 7. Layman, D.K.; Lonnerdal. B.; Femstrom, J.D. Applications for a-Lactalbumin in Human Nutrition. Nutr Rev 2018, 76, 444-460, doi: 10. 1093 / nutrit / nuy004.

[0253] 8. Ma, J.; Li, Q.; Li, Y.; Wen, X.; Li, Z.; Zhang, Z.; Zhang, J.; Yu, Z.; Li,N. Expression of Recombinant Human a-Lactalbumin in Milk of Transgenic Cloned Pigs Is Sufficient to Enhance Intestinal Growth and Weight Gain of Suckling Piglets. Gene 2016, 584, 7-16, doi: 10.1016 / j.gene.2016.02.024.

[0254] 9. Corrochano, A.R.; Sarifay, Y.; Arranz, E.; Kelly, P.M.; Buckin, V.;Giblin, L. Comparison of Antioxidant Activities of Bovine Whey Proteins before and after Simulated Gastrointestinal Digestion. J Dairy Sci 2019. 102. 54-67. doi: 10.3168 / jds.2018- 14581.

[0255] 10. Pei, W.; Cai, L.; Gong, X.; Zhang, L.; Zhang, J.; Zhu, P.; Jiang, H.;Wang, C.; Wang, S.; Chen, J. Drug-Loaded Oleic-Acid Grafted Mesoporous Silica Nanoparticles Conjugated with a-Lactalbumin Resembling BAMLET-like Anticancer Agent with Improved Biocompatibility and Therapeutic Efficacy. Mater Today Bio 2022, 15, 100272, doi : 10. 1016 / j .mtbio.2022. 100272.

[0256] 11. Li, H.Y.; Li, P.; Yang. H.G.; Wang, Y.Z.; Huang, G.X.; Wang, J.Q.;Zheng, N. Investigation and Comparison of the Anti-Tumor Activities of Lactoferrin, a- Lactalbumin. and [3-Lactoglobulin in A549, HT29, HepG2, and MDA231-LM2 Tumor Models. J Dauy Sci 2019, 102, 9586-9597, doi:10.3168 / jds.2019-16429.

[0257] 12. Lai, X.; Yu, Y.; Xian, W.; Ye, F.; Ju, X.; Luo, Y.; Dong, H.; Zhou, Y -H.; Tan, W.; Zhuang, H.; et al. Identified Human Breast Milk Compositions Effectively Inhibit SARS-CoV-2 and Variants Infection and Replication. iScience 2022. 25, 104136, doi: 10. 1016 / j.isci.2022. 104136.

[0258] 13. Pellegrini, A.; Thomas, U.; Bramaz, N.; Hunziker, P.; von Fellenberg,R. Isolation and Identification of Three Bactericidal Domains in the Bovine AlphaLactalbumin Molecule. Biochim Biophys Acta 1999. 1426, 439-448. doi: 10.1016 / s0304- 4165(98)00165-2.

[0259] 14. Yamaguchi, M.; Takai, S.; Hosono, A.; Seki, T. Bovine Milk-Derived a-Lactalbumin Inhibits Colon Inflammation and Carcinogenesis in Azoxymethane and Dextran Sodium Sulfate-Treated Mice. Biosci Biotechnol Biochem 2014, 78, 672-679, doi: 10. 1080 / 09168451.2014.890034.

[0260] 15. Yamaguchi, M.; Uchida, M. Alpha-Lactalbumin SuppressesInterleukin-6 Release after Intestinal Ischemia / Reperfusion via Nitric Oxide in Rats. Inflammopharmacology 2007, 15, 43-47, doi:10.1007 / sl0787-006-1558-9.

[0261] 16. Yang. W.; Qu, X ; Deng, C.; Dai, L.; Zhou. H.; Xu, G.; Li, B.; Yulia,N.; Liu, C. Heat Sensitive Protein-Heat Stable Protein Interaction: Synergistic Enhancement inthe Thermal Co-Aggregation and Gelation of Lactoferrin and a-Lactalbumin. Food Res Int 2021, 142, 110179, doi: 10. 1016 / j.foodres.202L 110179.

[0262] 17. Yamniuk, A.P.; Burling, H.; Vogel, H.J. ThermodynamicCharacterization of the Interactions between the Immunoregulatory Proteins Osteopontin and Lactoferrin. Mol Immunol 2009, 46, 2395-2402, doi: 10.1016 / j.molimm.2009.04.024.

[0263] 18. Flanagan, S.E.; Malanowski, A J.; Kizilay, E .; Seeman, D.; Dubin, P.L.;Donato-Capel. L.; Bovetto, L.; Schmitt, C. Complex Equilibria. Speciation, and Heteroprotein Coacervation of Lactoferrin and P-Lactoglobulin. Langmuir 2015, 31, 1776-1783, doi: 10. 1021 / 1 a504020e.

[0264] 19. Yan, Y.; Kizilay, E.; Seeman, D.; Flanagan, S.; Dubin, P.L.; Bovetto,L.; Donato. L.; Schmitt, C. Heteroprotein Complex Coacervation: Bovine P-Lactoglobulin and Lactoferrin. Langmuir 2013, 29, 15614-15623, doi: 10.1021 / la4027464.

[0265] 20. Liu, L.; Jiang, R.; Liu, J.; Lonnerdal, B. The Bovine Lactoferrin-Osteopontin Complex Increases Proliferation of Human Intestinal Epithelial Cells by Activating the PI3K / Akt Signaling Pathway. Food Chem 2020, 310, 125919, doi: 10. 1016 / j.foodchem.2019. 125919.

[0266] 21. D’Auria, E.; Salvatore, S.; Acunzo, M.; Peroni, D.; Pendezza, E.; DiProfio, E.; Fiore, G.; Zuccotti, G.V.; Verduci, E. Hydrolysed Formulas in the Management of Cow's Milk Allergy: New Insights, Pitfalls and Tips. Nutrients 2021, 13, 2762, doi: 10.3390 / nul3082762.

[0267] 22. Perreault, N.; Beaulieu, J.F Use of the Dissociating EnzymeThermolysin to Generate Viable Human Normal Intestinal Epithelial Cell Cultures. Exp Cell Res 1996, 224, 354-364, doi: 10.1006 / excr, 1996.0145.

[0268] 23. Delie. F.; Rubas, W. A Human Colonic Cell Line Sharing Similarities with Enterocytes as a Model to Examine Oral Absorption: Advantages and Limitations of the Caco-2 Model. Crit Rev Ther Drug Carrier Syst 1997, 14, 221-286.

[0269] 24. Lonnerdal, B.; Jiang, R.; Du, X. Bovine Lactoferrin Can Be Taken up by the Human Intestinal Lactoferrin Receptor and Exert Bioactivities. J Pediatr Gastroenterol Nutr 2011. 53, 606-614. doi: 10.1097 / MPG.0b013e318230a419.

[0270] 25. Jiang, R.; Lopez, V.; Kelleher, S.L.; Lonnerdal, B. Apo- and Holo-Lactoferrin Are Both Internalized by Lactoferrin Receptor via Clathrin-Mediated Endocytosis but Differentially Affect ERK-Signaling and Cell Proliferation in Caco-2 Cells. J Cell Physiol 2011, 226, 3022-3031, doi: 10.1002 / jcp.22650.

[0271] 26. Buccigrossi, V.; de Marco, G.: Bruzzese, E.; Ombrato, L.; Bracale, I.;Polito, G.; Guarino, A. Lactoferrin Induces Concentration-Dependent Functional Modulation of Intestinal Proliferation and Differentiation. Pediatr Res 2007, 61, 410-414, doi: 10.1203 / pdr.0b013e3180332c8d.

[0272] 27. Hinnebusch, B.F.; Siddique, A.; Henderson, J.W.; Malo, M.S.; Zhang,W.; Athaide, C.P.; Abedrapo, M.A.; Chen, X.; Yang, V.W.; Hodin, R.A. Enterocyte Differentiation Marker Intestinal Alkaline Phosphatase Is a Target Gene of the Gut- Enriched Kruppel-like Factor. Am J Physiol Gastrointest Liver Physiol 2004, 286, G23-30, doi: 10. 1152 / ajpgi.00203.2003.

[0273] 28. Jiang, R.; Liu, L.; Du, X.; Lonnerdal, B. Evaluation of Bioactivities of the Bovine Milk Lactoferrin-Osteopontin Complex in Infant Formulas. J Agric Food Chem 2020, 68, 6104-6111, doi: 10.1021 / acs.jafc.9b07988.

[0274] 29. Lin, T.; Meletharayil, G.; Kapoor, R.; Abbaspourrad, A. Bioactives inBovine Milk: Chemistry, Technology', and Applications. Nutr Rev 2021, 79, 48-69, doi: 10. 1093 / nutrit / nuab099.

[0275] 30. Ng. T.B.; Cheung. R.C.F.: Wong. J.H.; Wang. Y.; Ip. D.T.M.; Wan.D.C.C.; Xia, J. Antiviral Activities of Whey Proteins. Appl Microbiol Biotechnol 2015, 99, 6997-7008, doi: 10. 1007 / s00253-015-6818-4.

[0276] 31. Giblin, L.; Yalcin. A S.; Biqim, G.; Kramer. A.C.; Chen, Z.; Callanan,M.J.; Arranz, E.i Davies, M.J. Whey Proteins: Targets of Oxidation, or Mediators of Redox Protection. Free Radic Res 2019, 53, 1136-1 152, doi : 10. 1080 / 10715762.2019. 1632445.

[0277] 32. Kobayashi, C.; Inagaki, M.; Nohara, M.; Fukuoka, M.; Xijier, null;Yabe, T.; Kanamarua, Y. The Effects of Denatured Major Bovine Whey Proteins on the Digestive Tract, Assessed by Caco-2 Cell Differentiation and on Viability of Suckling Mice. J Dairy Res 2021, 88, 221-225, doi: 10.1017 / S0022029921000376.

[0278] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged,substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0279] The disclosures of each and even7patent, patent application, publication, and accession number cited herein are hereby incorporated herein by reference in their entirety.

[0280] While present disclosure has been disclosed with reference to various embodiments, it is apparent that other embodiments and variations of these may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS1. A protein complex for nutritional supplements, the protein complex comprising lactoferrin and alpha-lactalbumin.

2. A protein complex for nutritional supplements, the protein complex comprising lactoferrin and whey protein hydrolysate proteins.

3. A protein complex for nutritional supplements, the protein complex comprising lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.

4. The protein complex of any one of claims 1-3, wherein the protein complex further comprises proteins from naturally occurring milk.

5. The protein complex of claim 4, wherein the naturally occurring milk is nonfat milk.

6. The protein complex of claim 4, wherein the naturally occurring milk is whole milk.

7. The protein complex of any one of claims 1-6, wherein the protein complex is encapsulated within a lipid-based delivery' system.

8. The protein complex of claim 7, wherein the lipid-based delivery system enhances stability7or bioavailability of the protein complex.

9. The protein complex of any one of claims 1-6, wherein the protein complex is encapsulated in a protective matrix.

10. The protein complex of claim 9, wherein the protective matrix enhances stability or bioavailability of the protein complex.

11. The protein complex of any one of claims 1-10, wherein the protein complex is resistant to gastrointestinal digestion and is capable of binding to a lactoferrin receptor on a cell membrane of intestinal epithelial cells.

12. A nutritional supplement, the nutritional supplement comprising a protein complex comprising lactoferrin and alpha-lactalbumin.

13. A nutritional supplement, the nutritional supplement comprising a protein complex comprising lactoferrin and whey protein hydrolysate proteins.

14. A nutritional supplement, the nutritional supplement comprising a protein complex comprising lactoferrin, alpha-lactalbumin, and whey protein hydrolysate proteins.

15. The nutritional supplement of any one of claims 12-14, wherein the protein complex further comprises proteins from naturally occurring milk.

16. The nutritional supplement of claim 15, wherein the naturally occurring milk is nonfat milk.

17. The nutritional supplement of claim 15, wherein the naturally occurring milk is whole milk.

18. The nutritional supplement of any one of claims 12-17, wherein the protein complex is encapsulated within a lipid-based delivery system to enhance absorption.

19. The nutritional supplement of claim 18, wherein the lipid-based delivery system enhances stability or bioavailability of the protein complex.

20. The nutritional supplement of any one of claims 12-17, wherein the protein complex is encapsulated in a protective matrix.

21. The nutritional supplement of claim 20, wherein the protective matrix enhances stability or bioavailability of the protein complex.

22. The nutritional supplement of any one of claims 12-21, wherein the protein complex is resistant to gastrointestinal digestion and is capable of binding to a lactoferrin receptor on a cell membrane of intestinal epithelial cells.

23. The nutritional supplement of any one of claims 12 to 22, wherein the nutritional supplement comprises one or more digestion aiding proteins.

24. The nutritional supplement of claim 23, wherein the one or more digestion aiding proteins are selected from whey protein hydrolysate, alpha-lactalbumin, K-casein, a-casein, P-casein, bile-salt stimulated lipase, amylase, al -antitrypsin, and combinations thereof.

25. The nutritional supplement of any one of claims 12-22, wherein the nutritional supplement comprises one or more immunoprotective proteins.

26. The nutritional supplement of claim 25, wherein the one or more immunoprotective proteins are selected from lactoferrin, osteopontin, K-casein, a-casein, p-casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin. lactoperoxidase, milk fat globule membrane (MFGM), and combinations thereof.

27. The nutritional supplement of claim 26, wherein the lactoferrin comprises apolactoferrin, hololactoferrin, or a combination of both.

28. The nutritional supplement of any one of claims 12-27, wherein the nutritional supplement comprises about 5% to about 20% protein, about 15% to about 40% fat, and about 40% to about 70% carbohydrate.

29. The nutritional supplement of claim 28, wherein the protein is lactoferrin, whey protein hydrolysate, alpha-lactalbumin, osteopontin, 0-casein, a-casein, K- casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, amylase, al -antitrypsin, folate-binding protein, insulin-like growth factor- 1, epidermal growth factor, cytokines, lactadherin, lactoperoxidase, milk glycans, milk fat globule membrane (MFGM), or combinations thereof.

30. The nutritional supplement of claim 28, wherein the fat is innate milk fat globule membrane (MFGM), added MFGM, phospholipids, cholesterol, oil, non-hexane extracted docosahexaenoic acid (DHA), hexane extracted arachidonic acid (AA). non-hexane extracted AA, ascorbyl palmitate, or combinations thereof.

31. The nutritional supplement of claim 28, wherein the carbohydrate is lactose, glucose, fructose, sucrose, galactooligosaccharide (GOS), fructooligosaccharide (FOS), inulin, com syrup solids, dextrin, maltodextrin, dextrose, polydextrose, tapioca, starch, tapioca starch, rice syrup solids, waxy com, waxy rice starch, or combinations thereof.

32. The nutritional supplement of any one of claims 12-31, wherein the nutritional supplement does not comprise lactoperoxidase.

33. The nutritional supplement of any one of claims 12-32, wherein the lactoferrin is incorporated into a micellar structure.

34. The nutritional supplement of any one of claims 12-33, wherein the protein complex promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promotes differentiation of intestinal cells, induces differentiation of intestinal cells, inhibits growth of enteropathogenic Escherichia coli in a digestive system, inhibits bacterial infection in an intestinal lumen, increases interleukin- 18 secretion by intestinal cells, modulates interleukin- 18 secretion by intestinal cells, increases transforming growth factor 01 (TGF-01) secretion by intestinal cells, modulates transforming growth factor 01 (TGF-01) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability, increases expression of tight junction proteins, increases gut health, and combinations thereof.

35. The nutritional supplement of any one of claims 12-33, wherein the nutritional supplement promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promotes differentiation of intestinal cells, induces differentiation of intestinal cells, inhibits growth of enteropathogenic Escherichia coli in a digestive system, inhibits bacterial infection in an intestinal lumen, increases interleukin- 18 secretion by intestinal cells, modulates interleukin- 18 secretion by intestinal cells, increases transforming growth factor pi (TGF-(31) secretion by intestinal cells, modulates transforming growth factor pi (TGF- i) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability, increases expression of tight junction proteins, increases gut health, and combinations thereof.

36. The nutritional supplement of any one of claims 12-33, wherein the nutritional supplement provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

37. The nutritional supplement of any one of claims 12-33, wherein the nutritional supplement provides less spit up, softer stools, more efficient w eight gain, longer time between nighttime feeds, enhanced nutrient absorption, brain development, eye development, immune health, gut health, or combinations thereof38. The nutritional supplement of any one of claims 12-37, wherein the nutritional supplement is for infants.

39. The nutritional supplement of claim 38, wherein the nutritional supplement is formulated to enhance cognitive development.

40. The nutritional supplement of claim 39, wherein the nutritional supplement is formulated to facilitate healthy intestinal and immune development in early childhood.

41. A method of providing nutritional support to a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

42. The method of claim 41, w herein the nutritional supplement provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

43. The method of claim 41, wherein the nutritional supplement results in less spit up, softer stools, more efficient weight gain, longer time between nighttime feeds, enhanced nutrient absorption, brain development, eye development, immune health, gut health, or combinations thereof.

44. A method of promoting proliferation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

45. A method of inducing proliferation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to claim 12-33.

46. A method of promoting differentiation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

47. A method of inducing differentiation of intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

48. A method of inhibiting growth of enteropathogenic Escherichia coli in a digestive system of a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

49. A method of inhibiting bacterial infection in an intestinal lumen of a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

50. A method of increasing interleukin- 18 secretion by intestinal cells in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

51. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

52. A method of increasing gut health in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

53. The method of claim 52, wherein increasing gut health comprises promoting or inducing proliferation of intestinal cells, promoting or inducing differentiation of intestinal cells, preventing of inhibiting growth of enteropathogenic Escherichia coli in a digestive system, preventing or inhibiting bacterial growth in an intestinal lumen, modifying an immune response to an infection by enteropathogenic Escherichia coli, lowering cell permeability, increasing expression of tight junction proteins, modulating interleukin- 18 secretion (IL-18)by intestinal cells, modulating transforming growth factor pi (TGF-pi) secretion by intestinal cells, or combinations thereof.

54. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a nutritional supplement according to any one of claims 12-33.

55. The method of claim 54, wherein increasing intestinal immunity comprises modulating interleukin- 18 transforming grow th factor pi (TGF-pi) secretion by intestinal cells.

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