Formulation comprising heat-treated lactoferrin for nutritional support in subjects in need thereof

Abstract

The present invention discloses a formulation for oral administration comprising a protein component, including heat-treated lactoferrin, whey protein hydrolysate, alpha-lactalbumin, and a naturally sourced milk. The combination of these components provides a synergistic effect for improved oral delivery of proteins, enhancing bioavailability and efficacy. The formulation 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

FORMULATION COMPRISING HEAT-TREATED LACTOFERRIN FORNUTRITIONAL SUPPORT IN SUBJECTS IN NEED THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims priority to U.S. Provisional Application No. 63 / 575.266 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. Pasteurization is often used to kill harmful bacteria and keep consumers safe. However, the components of these nutntional supplements are often sensitive to heat as well. In particular, lactoferrin is understood to be heat sensitive and degrades during pasteurization (Fernandez-Menendez et al., 2020, “Effect of Holder Pasteurization on Total Concentrations and Iron-Binding Profiles of Holo-lactoferrin Used as Fortifier in Donor Human Milk,’7International Dairy Journal, vol 100, 104564). To avoid degradation of lactoferrin during pasteurization, lactoferrin is commonly dry blended into nutritional supplements such as infant formulas, after pasteurization to avoid degradation. It is understood that the degradation of lactoferrin results in a reduction in bioactivity. The present disclosure is directed to a surprising and unexpected formulation comprising heat- treated lactoferrin wherein the formulation has a bioactivity approximately the same as a formulation including lactoferrin prior to heat-treatment.SUMMARY OF THE INVENTION

[0003] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) lactoferrin (b) whey protein hydrolysate; and (c) a naturally sourced milk; wherein the formulation is heat treated; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat- treated.

[0004] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) lactoferrin; and (b) a naturally sourced milk; wherein the formulation is heat treated; and wherein the formulation has a bioactivity' substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0005] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) lactoferrin (b) alpha-lactalbumin; and (c) a naturally sourced milk; wherein the formulation is heat treated; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0006] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) lactoferrin (b) whey protein hydrolysate; (c) alphalactalbumin; and (d) a naturally sourced milk; wherein the formulation is heat treated; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0007] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) heat-treated lactoferrin (b) whey protein hydrolysate; and (c) a naturally sourced milk; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0008] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) heat-treated lactoferrin; and (b) a naturally sourced milk; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0009] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) heat-treated lactoferrin (b) alpha-lactalbumin; and (c) a naturally sourced milk; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0010] In some aspects, the techniques described herein relate to a formulation for oral administration including: (a) heat-treated lactoferrin (b) whey protein hydrolysate; (c) alpha-lactalbumin; and (d) a naturally sourced milk: wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0011] In some aspects, the techniques described herein relate to a method of making a formulation for oral administration including heat treating lactoferrin and combining the heat-treated lactoferrin with: (a) whey protein hydrolysate: and (b) a naturally sourced milk component; wherein the formulation has a bioactivity substantially similar to the same formulation having lactoferrin that is not heat-treated.

[0012] In some aspects, the techniques described herein relate to a method of making a formulation for oral administration including: (a) lactoferrin (b) whey protein hydrolysate; and (c) a naturally sourced milk; wherein the formulation is heat-treated; andwherein the formulation has a bioactivity' substantially similar to the same formulation having lactoferrin that is not heat-treated.BRIEF DESCRIPTION OF DRAWINGS

[0013] Fig. 1 A demonstrates methods of isolating lactoferrin from milk.

[0014] Fig. IB demonstrates methods of isolating lactoferrin from milk.

[0015] Fig. 2A depicts protein detection by SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis).

[0016] Fig. 2B depicts protein detection by immunoblotting.

[0017] Fig. 2C depicts protein detection by immunoblotting.

[0018] Fig. 2D depicts protein detection by native PAGE (polyacrylamide gel electrophoresis).

[0019] Fig. 2E depicts protein detection by native immunoblotting.

[0020] Fig. 3 A shows the concentration of lactoferrin in infant formulas.

[0021] Fig. 3B shows the concentration of lactoferrin in infant formulas.

[0022] Fig. 4 shows the concentration of lipopolysaccharide in infant formulas.

[0023] Fig. 5A shows an immunoblot for lactoperoxidase in infant formulas.

[0024] Fig. 5B shows immunoblots for lactoperoxidase in infant formulas.

[0025] Fig. 6 depicts the iron binding capacity of infant formulas.

[0026] Fig. 7 shows binding of lactoferrin to the lactoferrin receptor.

[0027] Fig. 8 measures cellular proliferation.

[0028] Fig. 9 measures differentiation.

[0029] Fig. 10 measures transcription of the Transforming Growth Factor Beta 1 (TGF (31) in intestinal cells.

[0030] Fig. 11 measures the secretion of interleukin 18 (IL- 18) in intestinal cells.

[0031] Fig. 12A depicts lactoferrin in an SDS PAGE gel.

[0032] Fig. 12B depicts lactoferrin in a Western blot.

[0033] Fig. 13 depicts lactoferrin in a native PAGE gel (left) and a native western blot (right).

[0034] Fig. 14 measures iron concentrations in lactoferrin formulations.

[0035] Fig. 15 depicts images of lactoferrin binding and internalization in human intestinal epithelial cells (HIECs; Lf: green and LfR: red).

[0036] Fig. 16 measures effect of Lf formulations on proliferation of HIECs.

[0037] Fig. 17 measures effect of Lf formulations on differentiation of Caco-2 cells.

[0038] Fig. 18 measures effects of Lf formulations on transcription of the TGF (31 gene.

[0039] Fig. 19 measures effects of Lf formulations on transcription of the IL-18 secretion.DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. 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.

[0041] DEFINITIONS

[0042] 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.”

[0043] 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 of1or "consist of1the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0044] 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 thesingular 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.

[0045] 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 is intended, 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 BA

[0046] 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.

[0047] 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 be easily 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.

[0048] 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.

[0049] 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.

[0050] As used herein, the term “about” w hen 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.

[0051] The terms “administer,” “administering” or “administration” as used herein refer to directly administering a compound or a composition to a subject.

[0052] 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-J31) secretion by intestinal cells, modulates transforming growth factor |31 (TGF-|31 ) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability, increases expression of tight junction proteins, increases gut health, or combinations thereof. A heat-treated lactoferrin with a bioactivity substantially similar to a lactoferrin that is not heat-treated will be within a standard deviation of the lactoferrin that is not heat-treated.

[0053] As used herein, the term “effective amount” 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.

[0054] The term “heat-treated” as used herein refers to any intentional heating above 50°C for a sufficient time such that there is a reduction in the concentration of one or more microorganisms. The terms “heat-treated” and “pasteurized” as used herein are interchangeable. Examples of heat treatment are high-temperature short time pasteurization (72°C to 83°C for 15 seconds to 30 seconds) and ultra-high temperature (UHT) processing (135°C to 150°C for 1 second to 10 seconds).

[0055] The term “naturally sourced milk” as used herein refers to milk from any natural source. Naturally sourced milk may contain the same or similar ingredients as the formulations described herein, but these ingredients are not used in calculating the amount of each ingredient in the formulations described herein. A naturally sourced milk may be from a non-human source, such as a cow (bovine), sheep, goat, yak, water buffalo, horse, reindeer, or camel. A naturally sourced milk may be non-animal milk, such as soy, rice, hemp, pea, oat, almond or other nuts. A naturally sourced milk may be whole milk, reduced fat milk, low fat milk, and fat-free milk.

[0056] The term “nutritional support” as used herein refers to delivery of nutrition to maintain or restore nutritional status.

[0057] 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.

[0058] The term “subject” as use herein, refer to any recipient of the formulation 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.

[0059] The term “substantially free” as used herein refers to 1% or less of a substance.

[0060] The term “substantially similar” as used herein refers to having a shared property within 10% of its counterpart.

[0061] 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.

[0062] The term "micellar structure" as used herein refers to an aggregation of amphiphilic molecules in a solution, ty pically forming a spherical or cylindrical structure with a hydrophobic core and hydrophilic exterior. In the context of this disclosure, micellar structures may be used for encapsulation or delivery7of bioactive compounds.

[0063] As used herein, the term "pasteurized" may refer to a process of heat treatment applied to a formulation or component thereof to reduce the number of viable pathogens. Pasteurization may involve heating the formulation or component to a specific temperature for a predetermined time period, which may vary depending on the pasteurization method used. Examples of pasteurization methods may include, but are not limited to, high- temperature short-time (HTST) pasteurization, low-temperature long-time (LTLT) pasteurization, and ultra-high temperature (UHT) processing.

[0064] As used herein, the term "oral administration" may refer to a method of delivering a formulation or substance to a subject through the mouth. Oral administration may involve ingesting the formulation in various forms, such as liquids, powders, tablets, capsules, or other dosage forms that can be swallowed. The formulation may then pass through the digestive system, where it may be absorbed into the bloodstream or exert local effects in the gastrointestinal tract.

[0065] FORMULATIONS FOR NUTRITIONAL SUPPORT

[0066] The formulations for nutritional support described herein comprise a protein component, a fat component, a naturally sourced milk component, a carbohydrate component, or combinations thereof. In some embodiments, the formulation for nutritional support is pasteurized. In some embodiments, the formulation for nutritional support is heat-treated. In some embodiments, the protein component comprises heat-treated lactoferrin. In some embodiments, the formulation comprises lactoferrin, whey protein hydrolysate, alphalactalbumin. In some embodiments, the formulation comprises lactoferrin and whey protein hydrolysate. In some embodiments, the formulation comprises lactoferrin and alphalactalbumin. In some embodiments, the formulation comprises, whey protein hydrolysate, alpha-lactalbumin, and a naturally sourced milk. In some embodiments, the lactoferrin is heat- treated lactoferrin.

[0067] In some embodiments, the formulation has a bioactivity substantially similar to the same formulation having lactoferrin that is not heat-treated. In some embodiments, the formulation comprising heat-treated lactoferrin has a bioactivity substantially similar to the same formulation having lactoferrin that is not heat-treated. In some embodiments, the lactoferrin is heat-treated before addition to the formulation. In some embodiments, the formulation is heat-treated after the lactoferrin is added.

[0068] In some embodiments, the bioactive formulation 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 grow th factor |31 (TGF-01) 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, or combinations thereof.

[0069] 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-J31) secretion by intestinal cells, increasing intestinal immunity, lowering cell permeability, increasing expression of tight junction proteins, increasing gut health, or combinations thereof.

[0070] In some embodiments, the bioactive heat-treated 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, increasing intestinal immunity', lowering cell permeability’, increasing expression of tight junction proteins, increasing gut health, or combinations thereof.

[0071] In some embodiments, the formulation comprises about 5% to about 20% protein by weight of the formulation, about 15% to about 40% fat by weight of the formulation, about 1% to about 40% milk by weight of the protein component, about 40% to about 70% carbohydrate by weight of the formulation, or combinations thereof.

[0072] Protein Component

[0073] In some embodiments, the protein component comprises lactoferrin, heat- treated 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), or combinations thereof. In some embodiments, the protein component comprises nutrient carrier proteins comprising lactoferrin with iron, alpha-lactalbumin with calcium and zinc, (3-casein with calcium and phosphorous, folate binding protein with folate, haptocorrin with vitamin B12, or 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 basedmilks. In some embodiments, the protein component comprises a protein disclosed in the commercial product: "Whole Nutritional Infant Formula” available from ByHeart, of which the product label is incorporated herein by reference in its entirety. Further additional proteins are disclosed in Bardanzellu et al. ("'Omics' in human colostrum and mature milk: looking to old data with new ey es," Nutrients, 2017, vol. 9, no. 8, E843), which is incorporated herein by reference in its entirety.

[0074] In some embodiments, the protein component 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 hy drolysate (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 by7any 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 degree of 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.

[0075] In some embodiments, the protein component comprises proteins that are intact. In some embodiments, the protein component comprises proteins that are not hydrolyzed. For example, the protein component may comprise alpha-lactalbumin enriched whey protein concentrate or isolate ("alpha-lactalbumin" is used to denote the alphalactalbumin protein in the formulation provided by the concentrate or isolate). Alphalactalbumin is a small protein (14 kDa) that is nearly the size of a hydrolyzed protein. Alphalactalbumin 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 formulation. Further, peptides generated from proteolysis of alpha-lactalbumin have shown in vivo to have bactericidal, opioid agonist, and immunostimulating activity.

[0076] In some embodiments, the protein component is about 5% to about 20% by weight of the formulation. In some embodiments, the protein component is about 8% to about 16% or about 10% to about 15% by weight of the formulation. In other embodiments, the protein component is about 5% to about 10%, about 10% to about 15%, about 15% to about20% by weight of the formulation. 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 formulation, 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 formulation. In some embodiments, the protein component comprises about 10 g / L to about 25 g / L of the formulation. 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 formulation and ranges between any two of these values.

[0077] In some embodiments, the lactoferrin or heat-treated 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.

[0078] 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.

[0079] In some embodiments, the lactoferrin is about 0.1 % to about 30% by weight of the protein component. 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%, about0.8%, 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 formulation. In some embodiments, the lactoferrin is about 0.5 g / L to about 2 g / L of the formulation. 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 0.85 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 2.6 g / L, about 2.8 g / L, about 3 g / L of the formulation and ranges between any two of these values.

[0080] In some embodiments, the heat-treated lactoferrin is about 0.1% to about 30% by weight of the protein component. In some embodiments, the heat-treated lactoferrin is about 0.5% to about 6% by weight of the protein component. In some embodiments, the heat- treated lactoferrin is about 4% to about 30% by weight of the protein component. In some embodiments, the heat-treated 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 heat-treated 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 heat- treated lactoferrin is about 0.05 g / L to about 3 g / L. In some embodiments, the heat-treated lactoferrin is about 0.3 g / L to about 3 g / L of the formulation. In some embodiments, the heat- treated lactoferrin is about 0.5 g / L to about 2 g / L of the formulation. In some embodiments, the heat-treated lactoferrin is about 0. 1 g / L to about 1 g / L. In some embodiments, the heat- treated lactoferrin is about 0.05 g / L, about 0. 1 g / L, about 0. 12 g / L, about 0. 125 g / L, about 0. 126g / 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 0.85 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 2.6 g / L, about 2.8 g / L, about 3 g / L of the formulation and ranges between any two of these values.

[0081] In some embodiments, the formulation comprises whey protein hydrolysate. In some embodiments, the whey protein hydrolysate is about 10% to about 75% by weight of the protein component. 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 weight 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 formulation. In some embodiments, the whey protein hydrolysate comprises about 3 g / L to about 11 g / L of the formulation. 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, about4. 1 g / L, about 4.3 g / L, about 4.5 g / L, about 4.7 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, about9.1 g / L, about 9.3 g / L, about 9.5 g / L, about 9.7 g / L, about 9.9 g / L, about 10 g / L, about 11 g / L, about 12 g / L, about 13 g / L of the formulation, and ranges between any tw o of these values.

[0082] In some embodiments, the formulation comprises alpha-lactalbumin. In some embodiments, the alpha-lactalbumin comprises about 5% to about 25% by weight of the protein component. 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% byweight 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%, about 15%, 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 formulation. In some embodiments, the alpha-lactalbumin comprises about 1 g / L to about 4 g / L of the formulation. 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 formulation, and ranges between any two of these values.

[0083] In some embodiments, the formulation 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.

[0084] In some embodiment, the osteopontin comprises about 0. 1 % to about 4% by weight of the protein component. 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 formulation. In some embodiments, the osteopontin comprises about 0.08 g / L to about 0.3 g / L of the formulation. 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 0.11 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 0.2 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 formulation and ranges between any two of these values.

[0085] In some embodiments, the formulation comprises one or more digestion aiding proteins. In some embodiments, the protein component comprises one or more digestion aiding proteins. In some embodiments, the formulation comprises two or more digestion aidingproteins. In some embodiments, the formulation comprises three or more digestion aiding proteins. In some embodiments, the digestion aiding proteins are selected from whey protein hydrolysate, alpha-lactalbumin, K-casein, a-casein, |3-casein, bile-salt stimulated lipase, amylase, ou-antitrypsin, or 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. Formulations that are easy to digest are especially important for infants who may be relying on formulas as their primary or sole source of nutrition.

[0086] In some embodiments, the formulation comprises one or more than one immunoprotective proteins. In some embodiments, the protein component comprises one or more immunoprotective proteins. In some embodiments, the one or more immunoprotective proteins are selected from the group consisting of lactoferrin, heat-treated lactoferrin, osteopontin, K-casein, a-casein, p-casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin, lactoperoxidase, milk fat globule membrane (MF GM), or combinations thereof. In embodiments in which the formulation 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 formulation with greater immunoprotective properties as compared to infant formulas that are currently on the market.

[0087] In some embodiments, the formulation comprises a protein component comprising one or more digestion aiding proteins and one or more immunoprotective proteins. Such a formulation is designed to provide both comfort and immunity7, which is in contrast to many known 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.

[0088] In some embodiments, the formulation comprises a protein component comprising one or more digestion aiding proteins and one or more immunoprotective 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 moreimmunoprotective 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.

[0089] Fat Component

[0090] In some embodiments, the fat component 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, or combinations thereof. In some embodiments, the fat component comprises tocopherols, such as RRR-tocopherols concentrate (70% concentrate in edible vegetable oil), or combinations thereof. In certain embodiments, the oil comprises vegetable oil, soy oil, palm oil, avocado oil, or combinations thereof. Suitable fat or lipid sources for the fat component of any formulation 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 and nutritional functions, including supporting antiviral and antibacterial mechanisms that combat gut-derived infections.

[0091] In some embodiments, the fat component comprises a combination of one or more of: low erucic acid rapeseed oil (canola oil), sunflower oil. high oleic sunflower seedoil, coconut oil, an oil source for arachidonic acid (AA), and oil source for docosahexaenoic acid (DHA), and monoglycerides.

[0092] In some embodiments, the fat component is about 15% to about 40% by weight of the formulation. In some embodiments, the fat component is about 20% to about 35% by weight of the formulation. 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 formulation, and ranges between any two of these values.

[0093] In some embodiments, the fat component comprises about 25 g / L to about 50 g / L of the formulation. 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 formulation. 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 formulation and ranges between any two of these values.

[0094] In some embodiments, the fat component 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 formulation. 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 formulation 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 weightof 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 formulation. 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 formulation. 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 formulation and ranges between any two of these values.

[0095] In some embodiments, the fat component 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 weight 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 to about 0.3 g / L of the formulation. 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 formulation. 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 formulation and ranges between any two of these values. Insome 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 formulation. 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 formulation and ranges between any two of these values.

[0096] In some embodiments, the fat component 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%, about 25%, 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 formulation. 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 formulation. 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 1 1 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 formulation and ranges between any two of these values.

[0097] In some embodiments, the fat component 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 formulation. In some embodiments, the sunflower oil is in an amount of about 2 g / L to about 4 g / L of the formulation. 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 formulation 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%, about 33.5%, 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 formulation. 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 formulation. 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 13.2g / 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 formulation and ranges between any two of these values.

[0098] In some embodiments, the fat component 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 formulation. 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 formulation. 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, about 6.5 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, about 6.6 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 formulation and ranges between any two of these values.

[0099] In some embodiments, the fat component comprises monoglycerides, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate, monoglyceryl linoleate, or combinations thereof. In some embodiments, the monoglycerides are in an amount of about 0.1% to about 5% by weight of the fat component. In some embodiments, the monoglycendes 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 formulation. In some embodiments, the monoglycerides are in an amount of about 0.25 g / L to about 0.4 g / L of the formulation. 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 formulation and ranges between any two of these values.

[0100] In some embodiments, the fat component 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.

[0101] In some embodiments, the fat component 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.

[0102] In certain embodiments, the fat component 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 the fat 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 monoglycendes, such as monoglyceryl palmitate, monoglyceryl stearate, monoglyceryl oleate.monoglyceryl linoleate, or 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.

[0103] In certain embodiments, the fat component 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, or 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.

[0104] In certain embodiments, the fat component 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%, or about 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, monoglycer l stearate, monoglyceryl oleate, monoglycer 1 linoleate, or 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 about0.02%, 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.

[0105] Milk Component

[0106] In some embodiments, the naturally sourced milk 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 sourced milk is non-animal milk, such as soy, rice, hemp, pea, oat, almond or other nuts. In some embodiments, the naturally sourced milk comprises whole milk, reduced fat milk, low fat milk, and fat-free milk. In some embodiments, the naturally sourced milk comprises whole milk from a bovine source. Whole milk can help the formulation 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 sourced 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.

[0107] 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.

[0108] In some embodiments, the naturally sourced milk protein is about 1% to about 40% by weight of the protein component. In some embodiments, the naturally sourced 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 sourced 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 sourced 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.

[0109] In some embodiments, the naturally sourced milk fat is about 0% to about 20% by weight of the fat component. In some embodiments, the naturally sourced milk fat is about 5% to about 20% by weight of the fat component. In some embodiments, the naturally sourced 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 sourced milk fat 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 1 1.1%, about 11.2%, about 1 1.3%, 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.

[0110] Carbohydrate Component[OHl] In some embodiments, the carbohydrate component 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, waxy com, waxy rice starch, or combinations thereof. In some embodiments, the formulation comprises lactose. Lactose has important natural prebiotic benefits, and also provides a natural sweetness to the formulation, which avoids the need for artificial sweeteners. In some embodiments, the formulation comprises HMO. In some embodiments, the formulation comprises MOS. In some embodiments, the pre-biotic fibers are from HMO or MOS. In some embodiments, the formulation comprises GOS. GOS is a prebiotic derived from lactose that drives stool softening and other potential digestive benefits. In some embodiments, the formulation may comprise FOS, which is a prebiotic.

[0112] In some embodiment, the carbohydrate component is about 40% to about 70% by weight of the formulation. In some embodiments, the carbohydrate component is about 50% to about 60% by weight of the formulation. 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 formulation 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 formulation. In some embodiments, the carbohydrate component is about 70 g / L to about 80 g / L of the formulation. 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 formulation and ranges between any two of these values.

[0113] In some embodiments, the carbohydrate is GOS. In some embodiments, the GOS comprises about 2% to about 10% by weight of the carbohydrate component. 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 formulation. In some embodiments, the GOS comprises about 3 g / L to about 6 g / L of the formulation. 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 formulation and ranges between any two of these values.

[0114] In some embodiments, the formulation 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 formulation 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 formulation comprises no com syrup, gluten, GMO, maltodextrin, soy, hexane extracted DHA, artificial growth hormones, palm oil, or combinations thereof. In some embodiments, the formulation 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 formulation comprises trace amounts 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 formulation 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.

[0115] In some embodiments, lactoferrin in the formulation is protected from degradation by the protein matrix and antioxidants created by any formulation disclosed herein. In some embodiments, the lactoferrin is incorporated into a micellar structure prior to spray drying. In some embodiments, the lactoferrin comprises lactoperoxidase. Lactoperoxidase competes for iron-binding sites on lactoferrin receptors, such as lactoferrin receptors in the gut, and may reduce bioactivity of the lactoferrin. In some embodiments, any formulation described herein comprises no lactoperoxidase. In some embodiments, any formulation described herein comprises substantially no lactoperoxidase. In some embodiments, any formulation 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 formulations of the present invention compared to the same concentration of lactoferrin that has not been heat-treated. In some embodiments, the formulation comprises less than about 0.01% lactoperoxidase.

[0116] In some embodiments, the formulation 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 formulation, as one of skill in the art would recognize. For example, an infant may have different vitamin and mineral requirements than a child of age one to thirteen years old, or an adult. In some embodiments, the vitamins or minerals in the formulation may be provided, in part of or in full, from the naturally sourced milk. In certain embodiments, the vitamins or minerals in the formulation may be provided from non-milk sources known in the art in addition to, or instead of, from the naturally sourced milk.

[0117] In some embodiments, a formulation according to any of the embodiments described herein comprises one or more than one vitamin. In some embodiments, the vitamins comprise vitamin Bl (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, deoxyadenosylcobalamin, cyanocobalamin, hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid), vitamin A (retinol.retinyl acetate, retinyl palmitate, retinyl esters with other long-chain fatty acids, retinal, retinoic acid, retinol esters), vitamin D (calciferol, chol ecalciferol, vitamin D3, l,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, menaquinone-11, menaquinone-12, mena-quinone-13). choline, inositol. 6-carotene. and any derivative or combination thereof.

[0118] In some embodiments, a formulation 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 chlonde, potassium acetate, selenium, sulfur, sodium, docusate sodium, sodium chloride, sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate, and any derivative or combination thereof. Nonlimiting exemplary derivatives of mineral compounds include salts, alkaline salts, esters, and chelates of any mineral compound.

[0119] In any embodiment described herein, the formulation comprises ingredients wherein each ingredient has at least about 40% purity. In any embodiment described herein, the formulation 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.

[0120] The formulation according to any of the embodiments described herein may be in a dry form or a liquid form. In some embodiments, the formulation may be in a dry powder form. In some embodiments, the formulation may be in a liquid emulsion. In someembodiments, the formulation may be in a ready-to-feed formulation. Any formulation 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 formulations is a food, beverage, tablet, capsule, or powder.

[0121] In some embodiments, administration of any formulation 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.

[0122] In some embodiments, the formulation is in a form selected from the group consisting of pellets, beads, beadlets, granules, powder, or a combination thereof. In some embodiments, the formulation 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.

[0123] In some embodiments, the formulation 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.

[0124] In embodiments in which the formulation is prepared as a tablet, the formulation 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, carboxystarch sodium, carboxymethyl starch sodium, potato starch, wheat starch, com starch, rice starch, partly pregelatinized starch, and hydroxypropyl starch.

[0125] In some embodiments, the formulation comprises pharmaceutically acceptable additives comprising excipients, lubricants, pH adjusters, taste-masking 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.

[0126] 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.

[0127] 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.

[0128] In some embodiments, the formulation 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, PHI 02 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 chlonde, and talc. One or more diluents may be used in the dosage form.

[0129] In some embodiments the formulation 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.

[0130] When the formulations 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.

[0131] In some embodiments, the formulations 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, or combinations thereof.

[0132] METHODS OF MAKING FORMULATIONS FOR NUTRITIONAL SUPPORT

[0133] V arious embodiments of the present disclosure relate to methods of making any formulation as described herein. Some embodiments relate to a method of making a formulation comprising a protein component, a fat component, a naturally sourced milk, a carbohydrate component, or combinations thereof. Some embodiments relate to a method of making a formulation compnsing lactoferrin, wherein the formulation is heat-treated. Some embodiments relate to a method of making a formulation comprising a protein component comprises heat-treated lactoferrin. In some embodiments, the formulation comprises heat- treated lactoferrin, whey protein hydrolysate, and alpha-lactalbumin. Some embodiments relate to a method of making a formulation comprising heat-treated lactoferrin, whey protein hydrolysate, alpha-lactalbumin, and a naturally sourced milk including milk protein naturally occurring in cow's milk. Some embodiments relate to a method of making a formulation with a bioactivity approximately the same as a formulation including lactoferrin without heattreatment.

[0134] In some embodiments, the lactoferrin is added to the formulation prior to pasteurization. In some embodiments, the lactofenin is heat-treated using pasteurization according to any method known in the art. In some embodiments, the heat-treated lactoferrin has the same bioactivity as non-heat-treated lactoferrin. In some embodiments, a heat-treated formulation comprising lactoferrin has the same bioactivity as a formulation wherein the lactoferrin is added after heat-treatment.

[0135] In some embodiments, lactoferrin is added as part of an infant formula bulk feed solution prior to pasteurization and spray drying. By incorporating lactoferrin into the bulk feed, thus removing the need for a dry' blending step, reduces risk of microbial contamination such as cronobacter.

[0136] In some embodiments, lactoferrin in the formulation is protected from degradation by the protein matrix and antioxidants created by any formulation disclosed herein. In some embodiments, the lactoferrin is incorporated into a micellar structure prior to spray drying. Lactoperoxidase competes for iron-binding sites on lactoferrin receptors, such as lactoferrin receptors in the gut, and may reduce bioactivity of the lactoferrin. In some embodiments, any formulation described herein comprises no lactoperoxidase. In some embodiments, any formulation described herein comprises substantially no lactoperoxidase. In some embodiments, any formulation 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 formulations of the present invention compared to the same concentration of lactoferrin that has not been heat- treated.

[0137] In some embodiments, the techniques described herein relate to a method of making any formulation described herein, wherein the heat-treated lactoferrin is heat treated using High Temperature Short Time pasteurization (HTST), Holder pasteurization. Direct Steam Injection (DSI), or Aseptic sterilization.

[0138] One of ordinary' skill in the art would have expected any known heat treatment or pasteurization techniques to denature any one or more of the components of the formulation. However, it was surprisingly discovered that after heat treatment or pasteurization, the formulation, including lactoferrin, remains substantially similar in bioactivity to the same formulation not subjected to heat treatment or pasteurization.

[0139] Known heat treatment techniques such as described herein are not limiting in temperature or time domains. The following embodiments are merely illustrative.

[0140] In some embodiments, any formulation described herein is prepared by mixing the components of the formulation together. In some embodiments, essential nutrients and other components are compounded in a wet process and then spray dried, after which heatsensitive ingredients such as vitamins are dry-blended into the spray-dried powder. The dried powder product is packaged and sealed and held until it undergoes a final check for conformance to specifications and regulations, including testing for microbiological contaminants. In some embodiments, liquid ready-to-feed and concentrated liquid infant formula is processed as a low-acid food and must meet additional processing regulations.

[0141] In some embodiments, the whey7protein hydrolysate is provided from various sources, such as a commercially available whey protein hydrolysate that would be Generally Recognized as Safe (GRAS) (the same would be true for all the various ingredients envisioned in this application). For example, in some embodiments, WPH protein is provided in the formulation using Whey Protein Hydrolysate (DI3071). In certain embodiments, WPH protein is provided in the formulation using Whey Protein Hy drolysate 8350 which is derived from Hilmar Ingredients.

[0142] In some embodiments, the alpha-lactalbumin is provided in the formulation using Lacprodan® ALPHA-10 or ALPHA-20 in the formulation. Both ALPHA-10 and ALPHA-20 are a native whey protein isolate that contains alpha-lactalbumin among other components. ALPHA-10 comprises 43% alpha-lactalbumin, and ALPHA-20 comprises 60% alpha-lactalbumin.

[0143] In some embodiments, lactoferrin is provided in the formulation using Glanbia Nutritionals Inc.'s Bioferrin 1000® or Bioferrin 2000®. Bioferrin 2000® comprises 93% lactoferrin.

[0144] In some embodiments, osteopontin is provided by including Lacprodan® OPN-10 which comprises 86% osteopontin.

[0145] In some embodiments, alpha-lactalbumin, lactoferrin, and osteopontin are also provided in the formulation using whole milk, such as bovine whole milk.101461 METHODS OF PROVIDING NUTRITIONAL SUPPORT

[0147] Various embodiments of the present disclosure relate to methods of using any formulation as described herein for nutritional support of a subject. Some embodiments relate to a method of using a formulation for nutritional support comprising a protein component, a fat component, a naturally sourced milk, a carbohydrate component, or combinations thereof. Some embodiments relate to a method of using a formulation fornutritional support comprising a protein component comprises heat-treated lactoferrin. In some embodiments, the formulation comprises heat-treated lactoferrin, whey protein hydrolysate, and alpha-lactalbumin. Some embodiments relate to a method of using a formulation for nutritional support comprising heat-treated lactoferrin, whey protein hydrolysate, alphalactalbumin, and a naturally sourced milk including milk protein naturally occurring in cow's milk. Some embodiments relate to a method of using a formulation with a bioactivity approximately the same as a formulation including lactoferrin without heat-treatment.

[0148] In any embodiment described herein, the method comprises administering any formulation described herein.

[0149] In some embodiments, the method of providing nutritional support according to any formulation described herein results in easy digestion, immune support, brain heath, gut health, or combinations thereof.

[0150] In some embodiments, the method of providing nutritional support according to any formulation 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.

[0151] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in promoting proliferation of intestinal cells in a subject.

[0152] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in inducing proliferation of intestinal cells in a subject.

[0153] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in promoting differentiation of intestinal cells in a subject.

[0154] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in inducing differentiation of intestinal cells in a subject.

[0155] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in inhibiting grow th of enteropathogenic Escherichia coli in the digestive system of a subject.

[0156] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in inhibiting growth of enteropathogenic Escherichia coli in the digestive system of a subject.

[0157] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in increasing interleukin- 18 secretion by intestinal cells in a subject.

[0158] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in increasing intestinal immunity in a subj ect.

[0159] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in increasing gut health in a subject, the method comprising administering to the subject any formulation described herein.

[0160] In some embodiments, the method of providing nutritional support according to any formulation 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 pi (TGF- 1) secretion by intestinal cells, or combinations thereof.

[0161] In some embodiments, the method of providing nutritional support according to any formulation described herein, results in increasing intestinal immunity in a subject, the method comprising administering to the subject any formulation described herein.

[0162] In some embodiments, the method of providing nutritional support according to any formulation described herein, wherein increasing intestinal immunity includes modulating interleukin- 18 transforming growth factor pi (TGF-pi) secretion by intestinal cells.

[0163] In some embodiments, efficacy of the methods described herein can be determined by measuring an at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% improvement or change relative to a measurement in a control. In some embodiments, the efficacy of the formulation 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.

[0164] In some embodiments, efficacy of the methods described herein can be determined by measuring an at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, and ranges between any two of these values, improvement or change relative to a measurement in a competitor's formulation. In some embodiments, the efficacy of the formulation of the present invention may be determined by establishing a significant difference from a competitor’s formulation, in which p<0. 10, or p<0.05, or p<0.01 .

[0165] In some embodiments, the formulation is expelled directly into a subject's intestinal tract. In some embodiments, the formulation 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, or combinations thereof.

[0166] In some embodiments, the method comprises administering any formulation as described herein is an infant formula. In some embodiments, the formulation is delivered to an infant starting at birth. In some embodiments, delivery of the formulation continues from birth until about two years of age. In some embodiments, deliver of the formulation continues through about one month of age, about three months 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.

[0167] In some embodiments, any formulation as described herein is administered to infants bom prematurely. In some embodiments, the formulation is administered until a time that matches full-term gestation. In some embodiments, the formulation 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 formulation is delivered to a subject as long as is necessary to correct nutritional deficiencies.

[0168] In some embodiments, any formulation 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.

[0169] In some embodiments, any formulation as described herein is administered as a daily or multiple-times-a-day supplement.ADDITIONAL EMBODIMENTS

[0170] Clause 1. A formulation for oral administration comprising: (a) lactoferrin; (b) whey protein hydrolysate; (c) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0171] Clause 2. The formulation of clause 1. wherein the formulation comprises one or more digestion aiding proteins.

[0172] Clause 3. The formulation of clause 2, wherein the one or more digestion aiding proteins are selected from whey protein hydrolysate, alpha-lactalbumin, K-casein, a- casein, [3-casein. bile-salt stimulated lipase, amylase, al-antitrypsin, or combinations thereof.

[0173] Clause 4. The formulation of any one of clauses 1-3, wherein the formulation comprises one or more immunoprotective proteins.

[0174] Clause 5. The formulation of clause 4, wherein the one or more immunoprotective proteins are selected from lactoferrin, heat-treated lactoferrin, osteopontin, K-casein, a-casein, [3-casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin, lactoperoxidase, milk fat globule membrane (MFGM), or combinations thereof.

[0175] Clause 6. The formulation of clause 5, wherein the lactoferrin comprises apolactoferrin, hololactoferrin. or a combination of both.

[0176] Clause 7. The formulation of any one of clauses 1-6, wherein the formulation comprises a protein, a fat, and a carbohydrate.

[0177] Clause 8. The formulation of clause 7, wherein the formulation comprises about 5% to about 20% protein, about 15% to about 40% fat, and about 40% to about 70% carbohydrate.

[0178] Clause 9. The formulation of any one of clauses 7-8. wherein the protein is lactoferrin, heat-treated lactoferrin, whey protein hydrolysate, alpha-lactalbumin, osteopontin, P-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 (MF GM), or combinations thereof.

[0179] Clause 10. The formulation of any one of clauses 7-8. 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.

[0180] Clause 11. The formulation of any one of clauses 7-8, 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.

[0181] Clause 12. The formulation of any one of clauses 1-11, wherein the formulation does not comprise lactoperoxidase.

[0182] Clause 13. The formulation of any one of clauses 1-12, wherein the lactoferrin is incorporated into a micellar structure.

[0183] Clause 14. The formulation of any one of clauses 1-13, wherein the 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 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- i) secretion by intestinal cells, modulates transforming growth factor i (TGF- i) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability', increases expression of tight junction proteins, increases gut health, or combinations thereof.

[0184] Clause 15. The formulation of any one of clauses 1-13, wherein the formulation 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 growth factor (31 (TGF-(31) secretion by intestinal cells, increases intestinal immunity’, lowers cell permeability’, increases expression of tight junction proteins, increases gut health, or combinations thereof.

[0185] Clause 16. A method of manufacturing the formulation of any one of clauses 1-15, wherein the formulation is pasteurized.

[0186] Clause 17. A method of providing nutritional support to a subject, the method comprising administering to the subject a formulation according to any one of clauses 1 -15.

[0187] Clause 18. The method of clause 17, wherein the formulation provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

[0188] Clause 19. The method of clause 17, wherein the formulation 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.

[0189] Clause 20. A method of promoting proliferation of intestinal cells in a subj ect, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0190] Clause 21. A method of inducing proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to clause 1-15.

[0191] Clause 22. A method of promoting differentiation of intestinal cells in a subj ect, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0192] Clause 23. A method of inducing differentiation of intestinal cells in a subj ect, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0193] Clause 24. A method of inhibiting growth of enteropathogenic Escherichia coli in a digestive system of a subject, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0194] Clause 25. A method of inhibiting bacterial infection in an intestinal lumen of a subject, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0195] Clause 26. A method of increasing interleukin- 18 secretion by intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0196] Clause 27. A method of increasing intestinal immunity' in a subject, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0197] Clause 28. A method of increasing gut health in a subject, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0198] Clause 29. The method of clause 28, 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 grow th 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 (31 (TGF-|31) secretion by intestinal cells, or combinations thereof.

[0199] Clause 30. A method of increasing intestinal immunity7in a subject, the method comprising administering to the subject a formulation according to any one of clauses 1-15.

[0200] Clause 31. The method of clause 30, wherein increasing intestinal immunity comprises modulating interleukin- 18 transforming growth factor [31 (TGF-(31 ) secretion by intestinal cells.

[0201] Clause 32. A formulation for oral administration comprising: (a) heat- treated lactoferrin; (b) whey protein hydrolysate; (c) a naturally sourced milk; and wherein the formulation has a bioactivity7substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0202] Clause 33. The formulation of clause 32, wherein the formulation comprises one or more digestion aiding proteins.

[0203] Clause 34. The formulation of clause 33, wherein the one or more digestion aiding proteins are selected from whey protein hydrolysate, alpha-lactalbumin, K-casein, a- casein, [3-casein, bile-salt stimulated lipase, amylase, al-antitrypsin, or combinations thereof.

[0204] Clause 35. The formulation of any one of clauses 32-34, wherein the formulation comprises one or more immunoprotective proteins.

[0205] Clause 36. The formulation of clause 35, wherein the one or more immunoprotective proteins are selected from lactoferrin, heat-treated lactoferrin, osteopontin, K-casein, a-casein, [3-casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase,cytokines, lactadherin, lactoperoxidase, milk fat globule membrane (MFGM). or combinations thereof.

[0206] Clause 37. The formulation of any one of clauses 32-36, wherein the heat- treated lactoferrin comprises apolactoferrin, hololactoferrin, or a combination of both.

[0207] Clause 38. The formulation of any one of clauses 1-37, wherein the formulation comprises a protein, a fat, and a carbohydrate.

[0208] Clause 39. The formulation of clause 38, wherein the formulation comprises about 5% to about 20% protein, about 15% to about 40% fat, and about 40% to about 70% carbohydrate.

[0209] Clause 40. The formulation of any one of clauses 38-39, wherein the protein is lactoferrin, heat-treated 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.

[0210] Clause 41. The formulation of any one of clauses 38-39, 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.

[0211] Clause 42. The formulation of any one of clauses 38-39, 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.

[0212] Clause 43. The formulation of any one of clauses 32-42, wherein the formulation does not comprise lactoperoxidase.

[0213] Clause 44. The formulation of any one of clauses 32-43, wherein the heat- treated lactoferrin is incorporated into a micellar structure.

[0214] Clause 45. The formulation of any one of clauses 32-44, wherein the heat- treated 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 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 growth factor (31 (TGF-(31) secretion by intestinal cells, increases intestinal immunity’, lowers cell permeability’, increases expression of tight junction proteins, increases gut health, or combinations thereof.

[0215] Clause 46. The formulation of any one of clauses 32-44, wherein the formulation 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-pi) secretion by intestinal cells, modulates transforming growth factor pi (TGF-pi) secretion by intestinal cells, increases intestinal immunity’, lowers cell permeability, increases expression of tight junction proteins, increases gut health, or combinations thereof.

[0216] Clause 47. A method of manufacturing the formulation of any one of clauses 32-46, wherein the formulation is pasteurized.

[0217] Clause 48. The method of any one of clauses 32-46. wherein the heat-treated lactoferrin is pasteurized.

[0218] Clause 49. A method of making a formulation for oral administration comprising heat treating lactoferrin and combining the heat-treated lactoferrin with: (a) whey protein hydrolysate; and (b) a naturally sourced milk component; wherein the formulation has a bioactivity substantially similar to an equivalent formulation having lactoferrin that is not heat-treated.

[0219] Clause 50. The method of making a formulation of clause 49, wherein the heat-treated lactoferrin pasteurized.

[0220] Clause 51. A method of making a formulation for oral administration comprising: (a) lactoferrin; (b) whey protein hydrolysate; (d) a naturally sourced milk; and wherein the formulation is heat-treated; wherein the formulation has a bioactivity substantially similar to an equivalent formulation having lactoferrin that is not heat-treated.

[0221] Clause 52. The method of making a formulation of clause 51, wherein the formulation is pasteurized.

[0222] Clause 53. A method of providing nutritional support to a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0223] Clause 54. The method of clause 53, wherein the formulation provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

[0224] Clause 55. The method of clause 53, wherein the formulation 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.

[0225] Clause 56. A method of promoting proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0226] Clause 57. A method of inducing proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to clause 32-46.

[0227] Clause 58. A method of promoting differentiation of intestinal cells in a subj ect, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0228] Clause 59. A method of inducing differentiation of intestinal cells in a subj ect, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0229] Clause 60. A method of inhibiting growth of enteropathogemc Escherichia coli in a digestive system of a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0230] Clause 61. A method of inhibiting bacterial infection in an intestinal lumen of a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0231] Clause 62. A method of increasing interleukin- 18 secretion by intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0232] Clause 63. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0233] Clause 64. A method of increasing gut health in a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0234] Clause 65. The method of clause 64, 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- i) secretion by intestinal cells, or combinations thereof.

[0235] Clause 66. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a formulation according to any one of clauses 32-46.

[0236] Clause 67. The method of clause 66, wherein increasing intestinal immunity comprises modulating interleukin-18 transforming growth factor pi (TGF-pi) secretion by intestinal cells.

[0237] Clause 68. A formulation for oral administration comprising: (a) lactoferrin; (b) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0238] Clause 69. A formulation for oral administration comprising: (a) lactoferrin; (b) alpha-lactalbumin; (c) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0239] Clause 70. A formulation for oral administration comprising: (a) lactoferrin; (b) alpha-lactalbumin; (c) whey protein hydrolysate; (d) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0240] Clause 71. A formulation for oral administration comprising: (a) heat- treated lactoferrin; (b) a naturally sourced milk; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0241] Clause 72. A formulation for oral administration comprising: (a) heat- treated lactoferrin; (b) alpha-lactalbumin; (c) a naturally sourced milk; and wherein the formulation has a bioactivity' substantially similar to a same formulation having lactoferrin that is not heat-treated.

[0242] Clause 73. A formulation for oral administration comprising: (a) heat- treated lactoferrin; (b) alpha-lactalbumin; (c) whey protein hydrolysate; (d) a naturally sourced milk; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.EXAMPLES

[0243] 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.

[0244] Example 1: Effects of Heat Treating Lactoferrin in Infant Formula

[0245] Objective: Determine the effects of high-temperature short-time, direct steam injection, and aseptic processing on functionalities of lactoferrin.

[0246] Materials and Methods:

[0247] Lactoferrin was isolated from milk using the methods outlined in Figure 1 . Lactoferrin was sourced from commercial bovine lactoferrin (CbLf), bovine lactoferrin, and human lactoferrin (Fig. 1 A, Fig. IB). In all the experiments described in this example, the tester was blind to batch number and heat treatment. Samples used in the experiments outlined in this example are described in Table 1.

[0248] Table 1. Infant Formula Samples

[0249] Results:

[0250] Effects of pasteurization on integrity of lactoferrin using SDS-PAGE and immunoblotting. Samples were subjected Direct Steam Injection (DSI) pasteurization, High Temperature Short Time (HTST) pasteurization, aseptic pasteurization, or no pasteurization (see Table 1; Fig. 2A, Fig. 2B, Fig. 2C, Fig. 2D, Fig. 2E). Lactoferrin (approximately 80-90 kDa) was not detected by SDS PAGE (Fig. 2A) or immunoblotting for anti-bovine lactoferrin (Fig. 2B) and anti-human lactoferrin (Fig. 2C) in Sample S6, which was subjected to aseptic pasteurization. As expected, lactoferrin was detected in both samples that did not undergo pasteurization, but surprising was also detected in Sample 3, which underwent HTST pasteurization.

[0251] The concentration of lactoferrin (Fig. 3A, Fig. 3B) and lipopolysaccharide (LPS; Fig. 4) was measured in each sample.

[0252] Iron binding capacity was maintained in infant formula pasteurized using HTST but not DSI and aseptic processing (Fig. 6). Binding of lactoferrin to the lactoferrin receptor on cell membranes was maintained after pasteurization (Fig. 7).

[0253] Pasteurized lactoferrin promotes proliferation and differentiation in intestinal cells (Fig. 8 and Fig. 9) and promotes transcription of TGF-(31 (Fig. 10) and interleukin 18 (Fig. 11) from intestinal cells.

[0254] Conclusions: The majority of lactoferrin in infant formula remained intact after HTST pasteurization, whereas pasteurization of infant formula using DSI and aseptic processing resulted in formation of denatured lactoferrin peptides. However, the pasteurized formulations all retained bioactivity of the formulation as a whole as well as the lactoferrin.

[0255] Example 2: Detection of Lactoperoxidase in Infant Formulas

[0256] Lactoperoxidase (LPO) is an enzyme present in milk and is detected in unpasteurized formulas (Fig. 5 A and Fig. 5B; approximately 15 kDa). However, LPO was not present in heat-treated formulas (Fig. 5).

[0257] Example 3: Effects of Heat Treating Lactoferrin at Commercial Scale

[0258] Goal: The goal of this study was to determine if commercial scale formulations using heat-treated lactoferrin according to the embodiments disclosed herein retain bioactivity comparable to non-heat-treated lactoferrin.

[0259] Methods: Human and bovine lactoferrin (hLf and bLf, respectively) were isolated from human and bovine milk and served as positive controls in all experiments. Five infant formula samples were used in this study (Table 2), and the bovine Lf from these formulas was isolated using the same method as that used for bovine and human milk Lf.

[0260] Table 2: Lactoferrin Formulations

[0261] Results:

[0262] Effect of pasteurization on integrity of Lf:

[0263] Heat treatment did not degrade lactoferrin present in any of the formulations comprising heat-treated lactoferrin (Lf 2-Lf5; Fig. 12, SDS PAGE; Fig. 12B, Western blot of Lf; Fig. 13, Native PAGE and Western blot of Lf).

[0264] Evaluation of the iron-binding capacity of Lf:

[0265] Lf was digested with nitric acid (3 M) overnight at room temperature and iron saturation rate was evaluated using an iCETM 3300 AAS Atomic Absorption Spectrometer (Thermo Fisher Scientific). As shown in Fig. 14, Lf 2, Lf 3, and Lf 4 maintain a similar iron saturation compared to Lf 1 (results are shown as mean ± SD. Bars without a common letter are significantly different (n = 3, P<0.05).

[0266] Binding of Lf to the Lf Receptor (LfR) and Lf internalization by human intestinal epithelial cells:

[0267] HIECs grown on glass cover slips were treated with each Lf (50 pg / rnL) at 37 °C for 1 h, fixed, and then probed with primary antibodies (anti-Lf or anti-LfR antibodies) and then fluorescent secondary antibodies. As shown in Fig. 15, colocalization of Lf and the LfR is observed in all Lf samples, suggesting that all Lfs are capable of binding to the LfR and being internalized by HIECs (Lf (green), and LfR (red).

[0268] Effects of Lf on proliferation and differentiation of human intestinal epithelial cells:

[0269] Effects of Lf on intestinal proliferation were assessed using HIECs. After HIECs (-50% in 96-well plates) were treated with Lf (200 pg / mL) for 24 h, effects of these proteins on intestinal proliferation were evaluated by using a BrdU cell proliferation assay kit (Millipore). All the formulations significantly promote proliferation of HIECs (Fig. 16; results are shown as mean ± SD. Bars without a common letter are significantly different (n = 5, P<0.05).

[0270] To determine effects of Lf on cell differentiation, Caco-2 cells (90% confluence in 24- w ell plates) were incubated with Lf (50 pg / mL) at 37 °C for 72 h. Activities of alkaline phosphatase, an indicator of intestinal differentiation, in cell lysates were measured. As shown in Fig. 17, Lf 4 exhibits the greatest effect on increase differentiation of Caco-2 cells. However, all heat-treated Lf formulations had at least the same effect on Caco-2 differentiation as the non-heat-treated lactoferrin formulation, Lf 1 (results are shown as mean ± SD. Bars without a common letter are significantly different (n = 5, P<0.05).

[0271] Effects of Lf on transcription of the TGF-(31 gene:

[0272] After Caco-2 cells (day 8) on 6-well plates were treated with each Lf (50 pg / mL) at 37°C for 72 h, RNA was extracted to evaluate effects on transcription of the TGF- (31 gene using qRT-PCR. All heat-treated Lf formulations had at least the same effect on TGF- ( 1 expression as the non-heat-treated lactoferrin formulation, Lf 1 as well as bovine Lf and human Lf (Fig. 18; results are shown as mean fold change ± SD. Bars labeled with different letters are significantly different (n = 5, P < 0.05).

[0273] Effects of Lf on interleukin 18 (IL- 18) secretion

[0274] Caco-2 cells (day 14) were treated with Lf (400 pg / mL) for 48 and secreted interleukin 18 (IL-18) was then measure using an ELISA kit (R&D Systems). All heat-treated Lf formulations had at least the same effect on IL- 18 expression as the non-heat-treated lactoferrin formulation, Lf 1 as well as bovine Lf and human Lf (Fig. 19; results are shown as mean ± SD. Bars labeled with different letters are significantly different (n = 5, P < 0.05)).

[0275] Summary. The Lf in infant formulas remains intact after heat treatment, as compared to the non-heat treated Lf. In, addition heat treatment on Lf has equivalent or greater functionality of Lf as compared to the control in these formulas.

[0276] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identity7similar 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.

[0277] The disclosures of each and every patent, patent application, publication, and accession number cited herein are hereby incorporated herein by reference in their entirety7.

[0278] 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

CLAIMS)What is claimed is:

1. A formulation for oral administration comprising:(a) lactoferrin;(b) whey protein hydrolysate;(c) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioactivi ty substantially similar to a same formulation having lactofernn that is not heat-treated.

2. The formulation of claim 1, wherein the formulation comprises one or more digestion aiding proteins.

3. The formulation of claim 2, 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, ou-antitrypsin, or combinations thereof.

4. The formulation of any one of claims 1-3, wherein the formulation comprises one or more immunoprotective proteins.

5. The formulation of claim 4, wherein the one or more immunoprotective proteins are selected from lactoferrin, heat-treated lactoferrin, osteopontin, K-casein, a-casein, P-casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin, lactoperoxidase, milk fat globule membrane (MFGM), or combinations thereof.

6. The formulation of claim 5. wherein the lactoferrin comprises apolactoferrin, hololactoferrin, or a combination of both.

7. The formulation of any one of claims 1-6, wherein the formulation comprises a protein, a fat, and a carbohydrate.

8. The formulation of claim 7, wherein the formulation comprises about 5% to about 20% protein, about 15% to about 40% fat, and about 40% to about 70% carbohydrate.

9. The formulation of any one of claims 7-8. wherein the protein is lactoferrin, heat-treated lactoferrin, whey protein hydrolysate, alpha-lactalbumin, osteopontin, p-casein, a-casein, K- casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, amylase, ou- antitrypsin, folate-binding protein, insulin-like grow th factor-1, epidermal growth factor, cytokines, lactadherin, lactoperoxidase, milk glycans, milk fat globule membrane (MFGM), or combinations thereof.

10. The formulation of any one of claims 7-8, 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.

11. The formulation of any one of claims 7-8, wherein the carbohydrate is lactose, glucose, fructose, sucrose, galactooligosaccharide (GOS), fructooligosaccharide (FOS), inulin, com syrup solids, dextrin, maltodextrin, dextrose, poly dextrose, tapioca, starch, tapioca starch, rice syrup solids, waxy com, waxy rice starch, or combinations thereof.

12. The formulation of any one of claims 1-11, wherein the formulation does not comprise lactoperoxidase.

13. The formulation of any one of claims 1-12, wherein the lactoferrin is incorporated into a micellar structure.

14. The formulation of any one of claims 1-13, wherein the lactoferrin promotes proliferation of intestinal cells, induces proliferation of intestinal cells, promotes differentiation of intestinal cells, induces differentiation of intestinal cells, inhibits grow th 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- i) secretion by intestinal cells, modulates transforming growth factor pi (TGF-pi) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability', increases expression of tight junction proteins, increases gut health, or combinations thereof.

15. The formulation of any one of claims 1-13, wherein the formulation 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 anintestinal lumen, increases interleukin- 18 secretion by intestinal cells, modulates interleukin- 18 secretion by intestinal cells, increases transforming growth factor pi (TGF-pi) secretion by intestinal cells, modulates transforming growth factor pi (TGF-pi) secretion by intestinal cells, increases intestinal immunity, lowers cell permeability, increases expression of tight junction proteins, increases gut health, or combinations thereof.

16. A method of manufacturing the formulation of any one of claims 1-15, wherein the formulation is pasteurized.

17. A method of providing nutritional support to a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

18. The method of claim 17, wherein the formulation provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

19. The method of claim 17, wherein the formulation 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.

20. A method of promoting proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

21. A method of inducing proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to claim 1-15.

22. A method of promoting differentiation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

23. A method of inducing differentiation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

24. A method of inhibiting growth of enteropathogenic Escherichia coli in a digestive system of a subject, the method comprising administering to the subject a formulation according to anv one of claims 1-15.

25. A method of inhibiting bacterial infection in an intestinal lumen of a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

26. A method of increasing interleukin- 18 secretion by intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

27. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

28. A method of increasing gut health in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

29. The method of claim 28, 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 grow th 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.

30. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a formulation according to any one of claims 1-15.

31. The method of claim 30, wherein increasing intestinal immunity comprises modulating interleukin- 18 transforming growth factor pi (TGF-pi) secretion by intestinal cells.

32. A formulation for oral administration comprising:(a) heat-treated lactoferrin;(b) whey protein hydrolysate;(c) a naturally sourced milk; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

33. The formulation of claim 32. wherein the formulation comprises one or more digestion aiding proteins.

34. The formulation of claim 33, wherein the one or more digestion aiding proteins are selected from whey protein hydrolysate, alpha-lactalbumin, K-casein, a-casein, p-casein, bilesalt stimulated lipase, amylase, ai -antitrypsin, or combinations thereof.

35. The formulation of any one of claims 32-34, wherein the formulation comprises one or more immunoprotective proteins.

36. The formulation of claim 35, wherein the one or more immunoprotective proteins are selected from lactoferrin, heat-treated lactoferrin, osteopontin, K-casein, a-casein, p-casein, haptocorrin, lysozyme, secretory IgA, bile-salt stimulated lipase, cytokines, lactadherin, lactoperoxidase, milk fat globule membrane (MFGM), or combinations thereof.

37. The formulation of any one of claims 32-36, wherein the heat-treated lactoferrin comprises apolactoferrin, hololactoferrin, or a combination of both.

38. The formulation of any one of claims 1-37, wherein the formulation comprises a protein, a fat, and a carbohydrate.

39. The formulation of claim 38. wherein the formulation comprises about 5% to about 20% protein, about 15% to about 40% fat, and about 40% to about 70% carbohydrate.

40. The formulation of any one of claims 38-39, wherein the protein is lactoferrin, heat- treated lactoferrin, whey protein hydrolysate, alpha-lactalbumin, osteopontin, P-casein, a- casein, K- casein, haptocorrin, lysozy me, 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 (MFGM), or combinations thereof.

41. The formulation of any one of claims 38-39, 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.

42. The formulation of any one of claims 38-39, 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.

43. The formulation of any one of claims 32-42, wherein the formulation does not comprise lactoperoxidase.

44. The formulation of any one of claims 32-43. wherein the heat-treated lactoferrin is incorporated into a micellar structure.

45. The formulation of any one of claims 32-44, wherein the heat-treated 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 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 grow th 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, or combinations thereof.

46. The formulation of any one of claims 32-44, wherein the formulation 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 groyvth 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, or combinations thereof.

47. A method of manufacturing the formulation of any one of claims 32-46, wherein the formulation is pasteurized.

48. The method of any one of claims 32-46, wherein the heat-treated lactoferrin is pasteurized.

49. A method of making a formulation for oral administration comprising heat treating lactoferrin and combining the heat-treated lactoferrin with:(a) whey protein hydrolysate; and(b) a naturally sourced milk component;wherein the formulation has a bioactivity substantially similar to an equivalent formulation having lactoferrin that is not heat-treated.

50. The method of making a formulation of claim 49, wherein the heat-treated lactoferrin pasteurized.

51. A method of making a formulation for oral administration comprising:(a) lactoferrin;(b) whey protein hydrolysate;(d) a naturally sourced milk; and wherein the formulation is heat-treated; wherein the formulation has a bioactivity' substantially similar to an equivalent formulation having lactoferrin that is not heat-treated.

52. The method of making a formulation of claim 51, wherein the formulation is pasteurized.

53. A method of providing nutritional support to a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

54. The method of claim 53, wherein the formulation provides easy digestion, immune support, brain heath, gut health, or combinations thereof.

55. The method of claim 53, wherein the formulation 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.

56. A method of promoting proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

57. A method of inducing proliferation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to claim 32-46.

58. A method of promoting differentiation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

59. A method of inducing differentiation of intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

60. A method of inhibiting growth of enteropathogenic Escherichia coli in a digestive system of a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

61. A method of inhibiting bacterial infection in an intestinal lumen of a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

62. A method of increasing interleukin- 18 secretion by intestinal cells in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

63. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

64. A method of increasing gut health in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

65. The method of claim 64, 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 grow th 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.

66. A method of increasing intestinal immunity in a subject, the method comprising administering to the subject a formulation according to any one of claims 32-46.

67. The method of claim 66, wherein increasing intestinal immunity comprises modulating interleukin- 18 transforming growth factor pi (TGF-pi) secretion by intestinal cells.

68. A formulation for oral administration comprising:(a) lactoferrin;(b) a naturally sourced milk; and wherein the formulation is heat treated;wherein the formulation has a bioacti vity substantially similar to a same formulation having lactoferrin that is not heat-treated.

69. A formulation for oral administration comprising:(a) lactoferrin;(b) alpha-lactalbumin;(c) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioacti vity substantially similar to a same formulation having lactoferrin that is not heat-treated.

70. A formulation for oral administration comprising:(a) lactoferrin;(b) alpha-lactalbumin;(c) whey protein hydrolysate;(d) a naturally sourced milk; and wherein the formulation is heat treated; wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

71. A formulation for oral administration comprising:(a) heat-treated lactoferrin;(b) a naturally sourced milk; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

72. A formulation for oral administration comprising:(a) heat-treated lactoferrin;(b) alpha-lactalbumin;(c) a naturally sourced milk; andwherein the formulation has a bioacti vity substantially similar to a same formulation having lactoferrin that is not heat-treated.

73. A formulation for oral administration comprising:(a) heat-treated lactoferrin;(b) alpha-lactalbumin;(c) whey protein hydrolysate;(d) a naturally sourced milk; and wherein the formulation has a bioactivity substantially similar to a same formulation having lactoferrin that is not heat-treated.

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