Edible compositions comprising recombinant human secretory proteins

Abstract

Disclosed herein are recombinant human secretory proteins, as well as compositions comprising such proteins and methods of using such compositions. Aspects of the present disclosure include microbially produced human secretory proteins, as well as edible and drinkable compositions comprising the proteins, such as infant formulas comprising such proteins. Also disclosed are methods of using the disclosed edible and drinkable compositions to provide nutritional and other benefits.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing submitted in XML format, the entire contents of which are incorporated herein by reference. The XML copy was created on August 4, 2023, named 080201-80180201.xml, and is 7,577 bytes in size. background 1. Technical Field

[0003] Embodiments relate to human secreted proteins, compositions comprising such proteins, and their use in particular edible compositions, such as food, beverages, and supplement compositions, including infant formulas. 2. Background Technology

[0004] Secretory proteins play important roles in the body, including nutrition, protection, and lubrication. Specialized cellular machinery is required to modify early secretory proteins (such as glycosylation) and transport the newly secreted proteins to the cell membrane or outside the cell.

[0005] Although secretory proteins may be similar across taxa from a phylogenetic perspective, the range, quantity, and structure of secretory proteins are species-specific properties or characteristics. For example, in mammalian milk, the type of secretory proteins, the amount of secretory proteins, and the structure of secretory proteins (e.g., the nature of glycosylation and carbohydrate structure) vary and can differ significantly between species. Thus, for example, the secretory protein or glycosylation profile, pattern, or fingerprint of bovine milk differs from that of human milk.

[0006] (Secretionaries, serum, etc., containing secretory proteins, also contain a range of lipids and carbohydrates that may be unique to an organ, a life stage, etc. Thus, for example, human milk contains fat globules enclosed in a milk fat globule membrane (MFGM), which is a complex collection of lipids and proteins. The composition of the MFGM can vary as the infant ages. The content and amount of oligosaccharides (human milk oligosaccharides, HMOs) also vary between species. The HMOs in breast milk are not necessarily a source of energy for the infant; instead, they may help provide a favorable environment and food source for microorganisms in the infant's gut).

[0007] Due to the interspecies variability of secretory molecules, the goal of isolating and using secretory molecules for therapeutic or nutritional benefit in humans is difficult to achieve and is currently unattainable for the following reasons: obtaining commercial quantities of collected human secretory molecules is not feasible; and species differences translate into the use of non-human secretory molecules, resulting in products that are not fully compatible with human use. If existing commercial products lack molecules normally present in human milk and contain excessive amounts of compensatory non-human components, this can be burdensome to the subjects, among other things.

[0008] For example, many existing infant formulas are produced with food ingredients derived from conventional agricultural and manufacturing practices. In recent years, manufacturers have attempted to create formulas with components that more closely resemble human secretory proteins, such as breast milk. Microbially expressed HMOs are added to some brands of infant formula. This type of technology has been shown to be useful for producing high-value and highly nutritious food ingredients that are not available from conventional agricultural practices or natural resources.

[0009] Infant formula is typically based on cow's milk protein, which differs from human breast milk protein (e.g., in terms of total amino acid composition). To address the amino acid needs of human infants, the level of cow's milk protein in infant formula is typically increased. This provides a much higher total protein intake for formula-fed infants, which may cause metabolic stress on the infant.

[0010] Increased protein intake due to the high protein content of commercial formulas results in higher insulin levels in formula-fed infants. Between 3 and 6 months of age, formula-fed infants receive approximately 70% more protein than breast-fed infants (e.g., Heinig et al., Am J Clin Nutr, 1993; 58: 152-156; and Koletzko et al., Adv Exp Med Biol, 2005; 569: 69-79).

[0011] Differences in morbidity have been observed between breast-fed and formula-fed infants, with breast-fed infants having fewer respiratory infections, ear infections, and gastroenteritis than formula-fed infants (Wright et al., BMJ, 299, 946-949, 1989; Duncan et al., Pediatrics, 2003, 91(5): 867-873; Aniansson et al., Pediatr Infect Dis J, 1994, 13(3): 182-188; and Dewey et al., J Pediatr, 1992, 126(5) part 1: 695-702). One possible explanation is that human milk contains higher levels of immunomodulatory substances than cow's milk.

[0012] For example, approximately 70% of the proteins present in human milk are whey proteins. The major whey proteins are α-lactalbumin, lactoferrin (LF), and secretory IgA. These proteins are biologically active and have unique properties, such as antimicrobial activity, anti-inflammatory activity, and antioxidant properties. These proteins may also aid in the absorption of key vitamins and minerals.

[0013] Human milk whey protein for human consumption is currently unavailable for commercial use. Existing infant formulas rely solely on protein sources from cows, goats, or soy. There is no commercially available human milk whey protein that can be used in edible or drinkable compositions. Human newborns without access to human milk will not receive the health benefits of human secretory molecules. Adults also will not benefit from the advantages of human secretory molecules.

[0014] Due to the various health and nutritional benefits of human secretory molecules, there is interest in including human secretory molecules in a range of edible or drinkable compositions (e.g., drinks, infant formulas, bars, dairy products, beverages, etc.). Summary of the Invention

[0015] The present disclosure addresses the needs in the art by providing microbially expressed human secretory proteins (e.g., human milk proteins) and unique edible or drinkable compositions comprising such proteins (hereinafter referred to as "edible compositions") and methods for providing nutrition to a subject. Such compositions achieve the immunomodulatory and other benefits previously available only using natural human secretory proteins from non-human sources. Aspects of the present disclosure include combinations of ingredients that maximize the immunomodulatory impact of the formulation. In some aspects, in addition to one or more human secretory proteins, the compositions of the present disclosure also include one or more human secretory oligosaccharides (e.g., 2'-fucosyllactose), probiotics (e.g., Bifidobacterium, such as Bifidobacterium infantis), a lipid source (e.g., MFGM), and the like.

[0016] Thus, in some aspects, disclosed herein is an edible composition comprising lactose, one or more lipids, a protein, a human secretory oligosaccharide (e.g., human milk oligosaccharide (HMO)), one or more minerals, one or more vitamins, a probiotic organism, and a human secretory protein. In embodiments, the HMO is 2'-fucosyllactose, lactose-N-neotetraose, 3-fucosyllactose, 6'-sialyllactose, difucosyllactose, or lactose-N-tetraose. In embodiments, the HMO is 2'-fucosyllactose. In embodiments, the HMO is lactose-N-neotetraose. In embodiments, the edible composition comprises one or more of high oleic sunflower oil, mid-oleic sunflower oil, canola oil, safflower oil, coconut oil, rapeseed oil, sunflower oil, ascorbyl palmitate, mixed tocopherol concentrate, Mortierella alpina oil, algae oil, soybean oil, or palm oil. In embodiments, edible compositions include high oleic sunflower oil, coconut oil, rapeseed oil, canola oil, sunflower oil, ascorbyl palmitate, mixed tocopherol concentrate, Mortierella alpina oil and algae oil. In embodiments, one or more minerals include one or more of calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride (chloride), potassium, sodium or selenium. In embodiments, edible compositions include calcium phosphate, calcium citrate, magnesium chloride, potassium citrate, potassium chloride, sodium chloride, copper sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium selenite or potassium iodide. In embodiments, one or more vitamins include one or more of vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, nicotinamide, calcium pantothenate, folic acid, pantothenic acid, vitamin B12, vitamin C, biotin, choline, inositol and salts thereof. In an embodiment, the edible composition includes vitamin A palmitate, vitamin D3, D-alpha tocopherol, vitamin K1, thiamine hydrochloride, riboflavin, niacinamide, calcium pantothenate, pyridoxine hydrochloride, biotin, folic acid, cyanocobalamin, choline bitartrate, inositol, and ascorbic acid.

[0017] In an embodiment, the edible composition comprises one or more nucleotides. In an embodiment, the one or more nucleotides comprise one or more of cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, or guanosine 5'-monophosphate. In an embodiment, the edible composition comprises cytidine 5'-monophosphate, uridine 5'-monophosphate disodium, adenosine 5'-monophosphate, and guanosine 5'-monophosphate disodium.

[0018] In embodiments, the edible composition includes a whey protein-lipid concentrate, for example, the edible composition includes from about 3.0% to about 5.0% by weight of the whey protein-lipid concentrate.

[0019] In an embodiment, the edible composition includes milk fat globule membrane (MFGM).

[0020] In embodiments, the edible composition comprises casein, such as micellar casein. In embodiments, the edible composition comprises from about 5% to about 8% micellar casein by weight.

[0021] In an embodiment, the edible composition comprises from about 50% to about 55% by weight lactose powder. In an embodiment, the edible composition comprises whole milk powder. In an embodiment, the edible composition comprises from about 13% to about 18% by weight whole milk powder.

[0022] In an embodiment, the probiotic organism is a Bifidobacterium. In an embodiment, the Bifidobacterium is Bifidobacterium infantis.

[0023] In an embodiment, the edible composition comprises about 43% to about 70% by weight lactose, about 0.5% to about 6% by weight milk fat, about 9% to about 13% by weight protein, about 1.5% to about 3.0% by weight minerals, about 0.5% to about 2.0% by weight vitamins, about 0.5% to about 2.0% by weight 2'-fucosyllactose, about 5 x 10 8 cfu / 100g is about 1×10 11 cfu / 100 g of probiotic bacteria and about 0.02 wt% to about 0.3 wt% human secretory proteins.

[0024] In embodiments, the edible composition includes from about 0.01% to about 0.06% by weight nucleotides.

[0025] In an embodiment, the edible composition includes from about 0.06% to about 0.4% by weight arachidonic acid (ARA).

[0026] In an embodiment, the edible composition includes from about 0.03% to about 0.3% by weight docosahexaenoic acid (DHA).

[0027] In an embodiment, the edible composition includes from about 0.00002% to about 0.00009% lutein by weight.

[0028] In an embodiment, the edible composition includes from about 0.025% to about 0.07% by weight taurine.

[0029] In an embodiment, the edible composition includes from about 0.002% to about 0.015% by weight L-carnitine.

[0030] In embodiments, an edible composition is also disclosed, comprising lactose, one or more lipids, a protein, human milk oligosaccharides (HMOs), one or more minerals, one or more vitamins, a probiotic microorganism, and recombinant human lactoferrin (rhLF), wherein the recombinant human lactoferrin may include one or more human-like glycans, i.e., N-glycans present in human proteins. In embodiments, an edible composition is further described, comprising a recombinant human secretory protein from a non-mammalian cell, wherein the recombinant human secretory protein includes one or more human-like glycans, i.e., N-glycans present on human glycoproteins.

[0031] In embodiments, the human secretory protein is a recombinant protein. In embodiments, the human secretory protein is from a mammalian cell. In embodiments, the human secretory protein is from a non-mammalian cell. In embodiments, the human secretory protein is from a bacterium. In embodiments, the human secretory protein is from a fungal cell. In embodiments, the human secretory protein is from a yeast cell. In embodiments, the yeast cell is of the genus Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiel In some embodiments, the human secretory protein is secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin. In an embodiment, the human secretory protein is human lactoferrin. In an embodiment, the human secretory protein is human whey protein.

[0032] In embodiments, human secretory proteins include human-like glycans, i.e., N-glycans present in human glycoproteins. In embodiments, human secretory proteins include hybrid N-glycans. In embodiments, human secretory proteins include complex N-glycans. In embodiments, human secretory proteins include bi-antennary, tri-antennary, or tetra-antennary N-glycans. In embodiments, human secretory proteins include glycans comprising sialic acid, galactose, N-acetylgalactosamine, or fucose. In embodiments, human secretory proteins include high mannose N-glycans. In embodiments, human secretory proteins include glycans having less than 10 mannose residues. In embodiments, human secretory proteins include glycans having less than 4 mannose residues. In embodiments, human secretory proteins include glycans having 4 or more mannose residues.

[0033] In an embodiment, a method for providing nutrition to a subject comprises feeding the subject an edible composition of interest as described herein.

[0034] Also described is a method for producing an edible composition comprising generating a mixture disclosed herein, e.g., a mixture comprising a protein and a human secretory protein (e.g., a recombinant human milk protein, such as recombinant human lactoferrin, which may have human-like glycans), and optionally comprising one or more of lactose, one or more lipids, a protein, a human secretory oligosaccharide (e.g., human milk oligosaccharide (HMO)), one or more minerals, one or more vitamins, and a probiotic microorganism.

[0035] In an embodiment, edible compositions include foods such as bars, dairy products (e.g., yogurt or ice cream), nutritional powders, infant formula powders, candies (e.g., gummy), etc. Edible compositions of interest also include drinkable compositions, such as beverages, smoothies, shakes, ready-to-use infant formulas, liquid nutritional products, drinkable nutritional compositions, etc.

[0036] In a first embodiment, the present invention relates to an infant formula comprising lactose, one or more lipids, protein, human milk oligosaccharides (HMOs), one or more minerals, one or more vitamins, probiotic microorganisms and human secretory proteins.

[0037] In a second embodiment, the infant formula of the first embodiment comprises secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin (LF) (e.g., human LF (hLF)), lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

[0038] In a third embodiment, the human secretory protein of the second embodiment is human lactoferrin.

[0039] In a fourth embodiment, in the infant formula of the first embodiment, the human secretory protein is human whey protein.

[0040] In a fifth embodiment, the human secreted protein of any one of the formulations of embodiments 1-4 comprises hybrid N-glycans.

[0041] In a sixth embodiment, the human secretory protein of any one of the formulations of embodiments 1-4 comprises complex N-glycans.

[0042] In a seventh embodiment, the human secreted protein of the sixth embodiment comprises biantennary, triantennary, or tetraantennary N-glycans.

[0043] In an eighth embodiment, the human secreted protein of any one of the formulations of embodiments 1-4 comprises high mannose N-glycans.

[0044] In a ninth embodiment, the human secretory protein of any one of the formulations of embodiments 1-4 comprises sialic acid, galactose, N-acetylgalactosamine, or fucose.

[0045] In a tenth embodiment, the human secreted protein of any one of the formulations of embodiments 1-4 comprises glycans having fewer than 10 mannose residues.

[0046] In an eleventh embodiment, the human secreted protein of any one of the formulations of embodiments 1-4 comprises glycans having fewer than 4 mannose residues.

[0047] In a twelfth embodiment, the human secreted protein of any one of the formulations of embodiments 1-4 comprises glycans having 4 or more mannose residues.

[0048] In a thirteenth embodiment, the formulation of any one of the preceding embodiments, wherein the HMO is 2'-fucosyllactose, lacto-N-neotetraose, 3-fucosyllactose, 6'-sialyllactose, difucosyllactose, or lacto-N-tetraose.

[0049] In a fourteenth embodiment, the formulation of embodiment 13 comprises 2'-fucosyllactose.

[0050] In a fifteenth embodiment, the formulation of embodiment 13 comprises lactose-N-neotetraose.

[0051] In a sixteenth embodiment, the formulation of any of the preceding embodiments comprises one or more of high oleic sunflower oil, mid-oleic sunflower oil, safflower oil, coconut oil, canola oil, rapeseed oil, sunflower oil, ascorbyl palmitate, mixed tocopherol concentrate, Mortierella alpina oil, algae oil, soybean oil, and palm oil.

[0052] In a seventeenth embodiment, the formulation of the sixteenth embodiment comprises high oleic sunflower oil, coconut oil, canola oil, rapeseed oil, sunflower oil, ascorbyl palmitate, mixed tocopherol concentrate, Mortierella alpina oil, and algae oil.

[0053] In an eighteenth embodiment, the formula of any of the preceding embodiments comprises one or more of calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, and selenium.

[0054] In a nineteenth embodiment, the formulation of the eighteenth embodiment comprises calcium phosphate, calcium citrate, magnesium chloride, potassium citrate, potassium chloride, sodium chloride, copper sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium selenite, and potassium iodide.

[0055] In a twentieth embodiment, the formulation of any of the preceding embodiments comprises one or more of vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, niacinamide, calcium pantothenate, folic acid, pantothenic acid, vitamin B12, vitamin C, biotin, choline, inositol, and salts thereof.

[0056] In a twenty-first embodiment, the formulation of the twentieth embodiment comprises vitamin A palmitate, vitamin D3, D-alpha-tocopherol, vitamin K1, thiamine hydrochloride, riboflavin, niacinamide, calcium pantothenate, pyridoxine hydrochloride, biotin, folic acid, cyanocobalamin, choline bitartrate, inositol, and ascorbic acid.

[0057] In a twenty-second embodiment, the formulation of any of the preceding embodiments comprises one or more nucleotides.

[0058] In a twenty-third embodiment, the formulation of the twenty-second embodiment, wherein the one or more nucleotides include cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, and guanosine 5'-monophosphate.

[0059] In a twenty-fourth embodiment, the formulation of the twenty-third embodiment comprises cytidine 5'-monophosphate, uridine 5'-monophosphate disodium, adenosine 5'-monophosphate, and guanosine 5'-monophosphate disodium.

[0060] In a twenty-fifth embodiment, the formula of any one of the preceding embodiments comprises a whey protein-lipid concentrate.

[0061] In a twenty-sixth embodiment, the formula of the twenty-fifth embodiment comprises from about 3.0% to about 5.0% whey protein-lipid concentrate.

[0062] In a twenty-seventh embodiment, the formula of any one of the preceding embodiments comprises milk fat globule membrane (MFGM).

[0063] In a twenty-eighth embodiment, the formula of any of the preceding embodiments comprises casein.

[0064] In a twenty-ninth embodiment, the formulation of the twenty-eighth embodiment comprises from about 5% to about 8% micellar casein.

[0065] In a thirtieth embodiment, the formulation of any of the twenty-fifth to twenty-ninth embodiments comprises from about 50% to about 55% by weight lactose powder.

[0066] In a thirty-first embodiment, the formula of any of the first to twenty-fourth embodiments comprises whole milk powder.

[0067] In a thirty-second embodiment, the formula of any of the preceding embodiments comprises from about 13% to about 18% whole milk powder.

[0068] In a thirty-third embodiment, the formula of any of the preceding embodiments comprises Bifidobacterium.

[0069] In the thirty-fourth embodiment, the formula of the thirty-third embodiment comprises Bifidobacterium infantis.

[0070] In a thirty-fifth embodiment, the human secretory protein of any of the formulations described in the preceding embodiments is a recombinant protein.

[0071] In a thirty-sixth embodiment, the human secreted protein in the formulation of the thirty-fifth embodiment is derived from mammalian cells.

[0072] In a thirty-seventh embodiment, the human secreted protein in the formulation of the thirty-fifth embodiment is derived from non-mammalian cells.

[0073] In a thirty-eighth embodiment, the human secreted protein in the formulation of the thirty-fifth embodiment is derived from bacteria.

[0074] In a thirty-ninth embodiment, the human secreted protein of the formulation of the thirty-fifth embodiment is derived from a fungal cell.

[0075] In a 40th embodiment, the human secreted protein of the formulation of the 35th embodiment is derived from yeast cells.

[0076] In a forty-first embodiment, the human secretory protein of the formulation of the fortieth embodiment is derived from a yeast cell of the genus Akzois, Aspergillus, Schizochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodacryozyme, Komagata, Leucosporidium, Lipomyces, Mortierella, Ogata, Pichia, Protothecoides, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickhamella, or Yarrowia.

[0077] In a 42nd embodiment, the formula of any of the preceding embodiments comprises about 43% to about 70% by weight lactose, about 0.5% to about 6% by weight milk fat, about 9% to about 13% by weight protein, about 1.5% to about 3.0% by weight minerals, about 0.5% to about 2.0% by weight vitamins, about 0.5% to about 2.0% by weight 2'-fucosyllactose, about 5×10 8 cfu / 100g is about 1×10 11 cfu / 100 g of probiotic organisms and from about 0.02 wt% to about 0.3 wt% human milk protein.

[0078] In a forty-third embodiment, the formulation of any one of the preceding embodiments comprises about 0.01 wt% to about 0.06 wt% nucleotides.

[0079] In a forty-fourth embodiment, the formulation of any of the preceding embodiments comprises from about 0.06% to about 0.4% by weight arachidonic acid.

[0080] In a forty-fifth embodiment, the formulation of any of the preceding embodiments comprises from about 0.03% to about 0.3% by weight of docosahexaenoic acid.

[0081] In a forty-sixth embodiment, the formulation of any of the preceding embodiments comprises from about 0.00002% to about 0.00009% by weight lutein.

[0082] In a forty-seventh embodiment, the formulation of any of the preceding embodiments comprises about 0.025% to about 0.07% by weight taurine.

[0083] In a forty-eighth embodiment, the formulation of any of the preceding embodiments comprises about 0.002% to about 0.015% by weight L-carnitine.

[0084] A forty-ninth embodiment is directed to an infant formula comprising lactose, one or more lipids, protein, human milk oligosaccharides (HMOs), one or more minerals, one or more vitamins, probiotic microorganisms, and recombinant human lactoferrin comprising one or more human-like glycans.

[0085] In a fiftieth embodiment, the formulation of the forty-ninth embodiment, wherein the recombinant human lactoferrin is from non-mammalian cells.

[0086] In the fifty-first embodiment, the formulation of the forty-ninth or fiftieth embodiment, wherein the recombinant human lactoferrin is from yeast cells.

[0087] A fifty-second embodiment is directed to a method for providing nutrition to an infant, comprising feeding the infant the infant formula of any one of the preceding embodiments.

[0088] A fifty-third embodiment is directed to an infant formula comprising a recombinant human secretory protein from a non-mammalian cell, wherein the recombinant human secretory protein comprises one or more human-like glycans.

[0089] In the fifty-fourth embodiment, in the infant formula of the fifty-third embodiment, the human secretory protein is secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

[0090] In the fifty-fifth embodiment, in the infant formula of the fifty-third embodiment, the human secretory protein is human lactoferrin (hLF).

[0091] In a fifty-sixth embodiment, in the infant formula of the fifty-third embodiment, the human secretory protein is human whey protein.

[0092] In a fifty-seventh embodiment, the human secretory protein of any of the fifty-third to fifty-sixth embodiments comprises a glycan comprising sialic acid, galactose, N-acetylgalactosamine, or fucose.

[0093] In a fifty-eighth embodiment, the human secretory protein of any of the fifty-third to fifty-sixth embodiments comprises complex N-glycans.

[0094] In a fifty-ninth embodiment, the human secretory protein of the fifty-eighth embodiment comprises biantennary, triantennary, or tetraantennary N-glycans.

[0095] In a sixtieth embodiment, the human secretory protein of the fifty-third to fifty-sixth embodiments comprises hybrid N-glycans.

[0096] In the sixty-first embodiment, the human secreted protein of the fifty-third embodiment is from bacteria.

[0097] In the sixty-second embodiment, the human secreted protein of the fifty-third embodiment is from a fungal cell.

[0098] In the sixty-third embodiment, the human secreted protein of the fifty-third embodiment is from a yeast cell.

[0099] In the sixty-fourth embodiment, the yeast cell of the sixty-third embodiment is of the genus Akzois, Aspergillus, Schizochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodacryocystis, Komagata Pseudomonas, Leucosporidium, Lipomyces, Mortierella, Ogata, Pichia, Protothecoides, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickhamella, or Yarrowia.

[0100] A sixty-fifth embodiment is directed to a method for providing nutrition to an infant, comprising feeding the infant formula of any one of the fifty-third to sixty-fourth embodiments to the infant.

[0101] The sixty-sixth embodiment is directed to a method for producing an infant formula comprising generating a mixture comprising lactose, one or more lipids, bovine protein, human milk oligosaccharides (HMOs), one or more minerals, one or more vitamins, probiotic microorganisms, and human secretory proteins.

[0102] In the sixty-seventh embodiment, in the method of the sixty-sixth embodiment, the human secretory protein is from a mammalian cell.

[0103] In the sixty-eighth embodiment, in the method of the sixty-sixth embodiment, the human secretory protein is from a non-mammalian cell.

[0104] In a sixty-ninth embodiment, in the method of the sixty-eighth embodiment, the cell is a yeast cell.

[0105] In the seventieth embodiment, in the method described in the sixty-ninth embodiment, the yeast cell is of the following genus: Akzois, Aspergillus, Schizochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodacryocystis, Komagata Pseudomonas, Leucosporidium, Lipomyces, Mortierella, Ogata Pseudomonas, Pichia, Protothecoides, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickham's yeast or Yarrowia.

[0106] In the seventy-first embodiment, in the method of any of the sixty-sixth to seventieth embodiments, the human secretory protein is secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

[0107] In the seventy-second embodiment, in the method of the seventy-first embodiment, the human secretory protein is human lactoferrin.

[0108] In the seventy-third embodiment, in the method of any of the sixty-sixth to seventieth embodiments, the human secretory protein is human whey protein.

[0109] In the seventy-fourth embodiment, in the method of the sixty-sixth to seventy-third embodiments, the human secretory protein comprises a glycan comprising sialic acid, fucose, galactose, or N-acetylgalactosamine.

[0110] In the seventy-fifth embodiment, in the method of the seventy-fourth embodiment, the human secretory protein comprises complex N-glycans.

[0111] In the seventy-sixth embodiment, in the method of the seventy-fifth embodiment, the human secretory protein comprises biantennary, triantennary, or tetraantennary N-glycans.

[0112] In the seventy-seventh embodiment, in the method of any of the sixty-sixth to seventy-third embodiments, the human secretory protein comprises hybrid N-glycans.

[0113] In the seventy-eighth embodiment, in the method of any of the sixty-sixth to seventy-third embodiments, the human secretory protein comprises high mannose N-glycans.

[0114] It should be contemplated that any embodiment discussed herein can be implemented using any method or edible composition of embodiments of the present disclosure, and vice versa. Additionally, compositions of certain embodiments can be used to implement methods of certain embodiments.

[0115] Other objects, features and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, as various changes and modifications will be apparent to those skilled in the art based on this detailed description. DETAILED DESCRIPTION

[0116] Aspects of the present disclosure address the lack or scarcity of human secretory proteins in the commercial market by including a source of recombinant human secretory proteins (e.g., lactoferrin) in edible compositions, such as infant formulas, for use as food ingredients or foods. This provides consumers with the antimicrobial, anti-inflammatory, and antioxidant properties of these proteins, which were previously only practically available in non-human milk, despite species differences in the milk of different mammals or in human milk. In addition to one or more human secretory proteins, the edible compositions also include customized combinations of other ingredients with similar biological activities to maximize the immunomodulatory and nutritional impact of the target formulation. Also disclosed is an edible composition comprising one or more human secretory proteins and one or more human oligosaccharides, such as human milk oligosaccharides (HMOs) with probiotic microorganisms (e.g., Bifidobacterium, such as Bifidobacterium infantis).

[0117] I. Definition

[0118] "Infant formula" is any composition comprising components that are used as a mimetic of human milk or as a complete or partial substitute for human milk. In some aspects, "infant formula" is a composition as defined in 21 U.S.C. § 321. The infant formula of the present disclosure can be a solid composition (e.g., a powder). The infant formula of the present invention can be a liquid composition. The liquid composition can be a concentrate or a ready-to-use form.

[0119] "Nutritional composition" or "edible composition" according to the present invention is, for example, an "infant formula" and relates to a food intended for use as a nutritional supplement for infants from birth and during the first 6-12 months of life and to meet the nutritional needs of infants and young children. Infant formula can be intended to be used as the sole source of nutrition from birth to about 6 months of age. The formula used in the first six months of life can be referred to as a "started formula".

[0120] The term "preterm formula" refers to an infant formula intended for premature infants.

[0121] "Milk fortifier" refers to a liquid or solid edible and nutritional composition suitable for mixing with breast milk (or human milk in the case of human milk fortifiers) or infant formula. Milk fortifiers can be used to increase the calories, protein content, mineral content, and vitamins in breast milk fed to premature or low birth weight infants. "Breast milk" refers not only to milk, but also to the mother's colostrum, or donor milk or donor colostrum.

[0122] In addition, edible compositions according to the present invention can be intended as " subsequent formula " (i.e. supplement) or as a part of gradually diversified diet, wherein formula feeding for example starts when the baby is about 4 months old, and the feeding of formula is up to the baby's first birthday as a part of the baby's diet. Between 4-6 months (this is the period when most babies start to eat solids), or at least when the nutritional intake of the baby of breastfeeding is supplemented with solids from 6 months until 12 months, the baby can begin to include formula according to the present invention. Subsequent formula is the formula that is intended to be used from about 6 months during the weaning period, as the supplement of the solids introduced when about 6 months.

[0123] Infant formula may be mixed with cereals to form an "infant cereal composition", ie a food product intended for specific nutritional use by infants or children (eg toddlers) during the first year of life.

[0124] The edible composition may be a powder concentrate or a liquid concentrate intended to be mixed with water before use, or a liquid ready-to-use product.

[0125] As used herein, unless otherwise indicated, the term "ready-to-use product" or equivalent terms (e.g., ready-to-feed, ready-to-drink, or ready-to-consume) refers to a liquid formula suitable for oral administration to an infant directly from the package, wherein the formula is a ready-to-feed liquid, a reconstituted powder, or a diluted liquid concentrate.

[0126] As used herein, an "infant" is a child under 12 months of age.

[0127] "Isolated" is used to describe a part of an organism that has been removed from its normal environment. Isolation is a form of "purification" in that foreign material is removed from the target of interest. Thus, a gene can be excised from a chromosome to remove it from upstream and downstream elements normally associated with an open reading frame. Similarly, for example, a protein can be removed from the soup of the intracellular environment, or it can be dissociated from intracellular structures or from membranes. External intervention removes an entity from its normal environment to isolate it.

[0128] In biotechnology or molecular biology, the term "recombinant" refers to the joining of biomolecular fragments that would not normally be found together to form a new, artificially synthesized, complex molecule. Thus, nucleic acid fragments from different sources are joined to create a new, non-natural, single nucleic acid that would not otherwise be found in nature. Similarly, proteins, polypeptides, or their domains can be joined to form new molecular entities with complex or novel functions that would not otherwise be found in nature. A recombinant is artificial. A synonym in this field is "engineered" or its grammatical form.

[0129] "N-glycans" are N-linked oligosaccharides, ie, sugars attached to the nitrogen of an asparagine residue of a polypeptide through an asparagine-N-acetylglucosamine linkage.

[0130] "Hybrid" N-glycans are glycans (also called polysaccharides) having both substituted (GlcNAc linkages) and unsubstituted mannose residues.

[0131] "Complex" N-glycans are glycans that have at least one GlcNAc attached to a 1,3-mannose arm of a trimannosyl core and at least one GlcNAc attached to a 1,6-mannose arm of a trimannosyl core. In some cases, complex N-glycans have at least one branch terminated with an oligosaccharide, such as NeuNAc-, NeuAca2-6GalNAca1-, NeuAca2-3Galb1-3GalNAca1-, or

[0132] NeuAca2-3 / 6Galb1-4GlcNAcbl-. Complex N-glycans can also have intrachain substitutions that include a "bisecting" GlcNAc and a core fucose ("Fuc").

[0133] "High mannose" glycans are glycans having at least 4 mannose residues. In some aspects, high mannose glycans have 4, 5, 6, 7, 8, 9 or more mannose (Man) residues.

[0134] The "milk fat globule membrane" (MFGM) comprises approximately 120 different proteins in the phospholipid bilayer surrounding the fat droplets in milk. The major phospholipids of MFGM are sphingomyelin, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Butyrophilin, MUC1, PAS6 / 7 (lactamin), CD14, TLR1 and TLR4 are examples of MFGM proteins that have antimicrobial effects (Spitsberg, JD Dairy Sci, 2005, 88:2289-2294; Reinhardt & Lippolis, J Dairy Res, 2006, 73(4)406-416; Brink & Lonnerdahl, J Nutr Biochem, 86:108465, 2020; Wang et al., Fron Nutr 8:807284, 2022; and Chai et al., Food Sci Anim Res 42(3)351-371, 2022).

[0135] Lacprodan MFGM-10 (Arla Foods, Viby J, Denmark) or similar raw materials from other suppliers are rich in MFGM and can be used as the phospholipid-enriched whey protein concentrate solid or lipid source in the intended edible composition.

[0136] As mentioned above, "recombinant" refers to cells, nucleic acids, proteins or vectors that are modified due to the introduction of exogenous nucleic acids or by artificial manipulation or intervention to change natural nucleic acids. The resulting cells, nucleic acids, proteins or vectors are considered to be recombinants, and their progeny, offspring, duplications or replicas are also considered to be recombinants. Therefore, for example, recombinant cells can express genes that do not exist in the natural (non-recombinant) form of the cell, or express natural genes in a manner different from that in which non-recombinant cells express those same genes. Recombinant cells can include but are not limited to recombinant nucleic acids that encode gene products or inhibitory elements, such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the level of active gene products in the cell. "Recombinant nucleic acids" are derived from nucleic acids initially formed in vitro, typically by exogenous, human or human-mediated nucleic acid manipulations (e.g., using polymerases, ligases, exonucleases and endonucleases) or other means to form artificial forms that are typically not present in nature. Once a recombinant nucleic acid is prepared and introduced into a host cell or organism, the nucleic acid produced by such artificial synthesis can be replicated using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced by recombinant methods, are still considered recombinant (for the purposes of this disclosure) despite subsequent intracellular replication. Furthermore, a recombinant nucleic acid refers to a nucleotide sequence that includes both endogenous and exogenous nucleotide sequences; thus, an endogenous gene that has undergone recombination with an exogenous promoter is a recombinant nucleic acid. A "recombinant protein" is a protein produced using recombinant technology (e.g., by expressing a recombinant nucleic acid).

[0137] "Individual," "infant," "subject," and "patient" are used interchangeably herein and may refer to a human or non-human, except for age, from the perspective of the consumer of the intended edible composition.

[0138] Throughout this application, "about" is used to indicate that a numerical value includes the inherent variation of error in the measurement or quantitative method. Such variations may be up to 10% above and below a value, i.e., (0.9×q) to q to (1.1×q).

[0139] The use of "a" or "an" can mean "one," but is also consistent with "one or more," "at least one," and "one or more than one."

[0140] "And / or" means "and" or "or." For example, A, B, and / or C includes: A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B, and C. In other words, "and / or" acts as an inclusive "or."

[0141] The terms “comprising” (and any grammatical form of comprising, e.g., “comprise” and “comprises”), “having” (and any grammatical form of having, e.g., “have” and “has”), “including” (and any grammatical form of including, e.g., “includes” and “include”), or “containing” (and any grammatical form of containing, e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0142] Compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed throughout the specification. Compositions and methods that "consist essentially of" disclosed ingredients or steps limit the scope of the claims to the specified materials or steps that materially affect the basic and novel characteristics of the claimed embodiments.

[0143] The focus of the present invention is a "food product" that relates to a composition that is consumed. A consumable food product includes a composition that is edible or used to produce a food product, ie, an edible food-grade ingredient.

[0144] "Food grade" refers to materials used in the manufacture of food or permitted to come into direct contact with food intended for human consumption. Food grade generally refers to materials that meet FDA standards and comply with FDA food guidelines and regulations. These FDA guidelines and regulations define under what circumstances foods are generally recognized as safe (GRAS) for consumption.

[0145] Foods can be manufactured and processed under local and federal guidelines and regulations to provide food that is safe for human consumption, such as in accordance with ISO 9001 or 22000 (International Organization of Standardization, Geneva, CH) and various sections (e.g., 117) of the GMP guidelines (e.g., 21 CFR) provided by the FDA.

[0146] As used herein, "edible" refers to a composition that can be consumed by a subject. Edible compositions can be solid or liquid. As is well known, solids have a defined volume and a defined shape, such as powders, sticks, gummies, pastes, etc. Liquids, such as beverages, syrups, emulsions, supplements, etc., have a volume but no shape.

[0147] The edible food product can be a consumer packaged good (CPG), loose or bulk commodity, etc., consumer product and its packaging found in, for example, grocery stores, shops, supermarkets, convenience stores, service stations, markets, vending machines, etc., where the edible composition is sold, marketed, or dispensed.

[0148] As used herein, "flavoring agent" is any ingredient in an edible composition that imparts flavor or taste to the composition. Flavoring agents may include compounds and compositions used in the food and pharmaceutical fields to impart taste or flavor, such as essences, compounds (e.g., butyric acid), etc., which have no significant or essential nutritional value, but as used herein, flavoring agents also include any ingredient, compound, or composition used in the edible composition of interest to impart flavor or taste to the edible composition, such as fruit juice, milk, milk solids, ascorbic acid, corn syrup, lactose, sugar, nuts (which may be ground), fruits (which may be dried, macerated, etc.), roots, vegetables, etc.

[0149] Pichia pastoris is a yeast commonly used in the commercial production of recombinant proteins. Recently, the genus Pichia was renamed Komagataella, and the species was renamed Komagataella Pastoris. Other studies have shown that many strains, cultures, and strains identified as P. pastoris used in laboratories around the world include at least two species: K. pastoris and K. phaffii. For the purposes of this document, because the genetic differences between Pichia pastoris and Pichia spp. are minimal and negligible; phenotypically, the two strains may not be easily distinguished; and for all intents and purposes, despite the different nomenclature, Pichia pastoris, Pichia pastoris, and Pichia spp. are essentially equivalent, unless specifically noted herein as specifically directed to only one species, a name may be used to refer to the other, and use of a name also includes the other two names. One name may be substituted for the other two names.

[0150] "Probiotics" describe microbial cells or components of microbial cells that have a beneficial effect on the health or well-being of the host. Probiotics may be of the genera known to have probiotic activity: Lactobacillus, Bifidobacterium, and Bacillus. Probiotic compositions may include fungi or yeast.

[0151] Probiotic microorganisms may be present in the compositions of any aspect or embodiment of the invention, for example, in an amount of ≥5 million, ≥10 million, ≥15 million, ≥20 million, ≥25 million, ≥30 million, ≥35 million, ≥45 million, ≥50 million, ≥75 million, ≥100 million, ≥250 million, ≥500 million, ≥750 million, ≥1 billion or ≥2 billion bacteria per serving. For example, the probiotics can be present in amounts of 5 million to 2.5 billion, 10 million to 2.5 billion, 30 million to 2.5 billion, 50 million to 2.5 billion, 50 million to 1 billion, 75 million to 2.5 billion, 75 million to 1 billion, 100 million to 2.5 billion, 100 million to 1 billion, 250 million to 2.5 billion, 250 million to 1 billion, 500 million to 2.5 billion, 500 million to 1 billion, 750 million to 2.5 billion, or 750 million to 1 billion, 1 billion to 2.5 billion, 1.5 billion to 2.5 billion, or more microorganisms per serving.

[0152] The composition of the invention may be formulated to provide the following daily doses of probiotic microorganisms: for example 10 3 to 10 14 , 10 4 to 10 12 , 10 5 to 10 12 , 10 6 to 10 12 , 10 7 to 10 11 , about 10 7 to about 10 10 or more colony forming units (cfu), microorganisms, or other units of measurement.

[0153] Probiotic microorganisms can be cultured according to any suitable method and prepared by known techniques (e.g., freeze drying or spray drying) for addition to edible compositions. Probiotic microorganism preparations can be purchased from, for example, MorinagaMilk, Institut Rosell, Christian Hansen, etc.

[0154] "Prebiotic" molecules or moieties are compounds that promote, encourage, support, enhance, etc., the growth of beneficial microorganisms in the body (e.g., in the intestines), thereby providing a beneficial microbial population. Typically, prebiotic moieties are indigestible in the stomach and are fermented by microorganisms, stimulating their growth. Compounds considered prebiotics include fiber, beta-glucans, inulin, oligosaccharides, pectin, HMOs, fructans, xylans, and the like.

[0155] The ingredients used to prepare the intended edible compositions are known and commercially available as food-grade or pharmaceutical-grade ingredients, or can be prepared or obtained as taught in the art. For example, some of the ingredients taught herein are available from Hilmar Cheese, Hilmar, CA (lactose, whey protein isolate, whey protein hydrolysate, whey protein concentrate, and milk protein isolate); Foremost Farms, Middleton, WI (lactose); Adams Group, Arbuckle, CA (vegetable oil blend and lecithin); AAK USA Inc., Edison, NJ (Sn2 palmitate and vegetable oil blend); Fonterra, Chicago, IL (whole milk powder and milk protein isolate); Leprino Nutrition, Denver, CO (whey protein concentrate); Arla Foods, Basking Ridge, NJ (MFGM and α-lactalbumin enriched whey protein); DSM Foods USA, Germantown, WI (mineral blend, 2'-fucosyllactose, lutein, DHA oil, and ARA-DHA blend); Balchem, Montvale, NJ (choline bitartrate); Univar, Baltimore, MD (potassium bicarbonate); Chr. Hansen, New Berlin, WI (2'-fucosyllactose and probiotics); Morinaga Milk, Irvine, CA (probiotics); Ingredion, Westchester, IL (corn syrup, resistant starch, pea protein concentrate, and maltodextrin); ADM, Decatur, IL (soy protein isolate, flavoring, fructose, and sodium citrate); DuPont, Wilmington, DE (carrageenan and cellulose); Idaho Milk Products, Jerome, ID (casein and milk protein concentrate); Sensus, Rye Brook, NJ (fructooligosaccharides); Beneo, Troy, NJ (fructooligosaccharides); ICL Food Specialties, Creve Coeur, ID (tricalcium phosphate); Lipotech, SA=Ingredients: Pharmacy Inc., Lewisville, TX (magnesium chloride); Cargill, Minneapolis, MN (lecithin, canola oil, prebiotics, citric acid, caramel, and potassium chloride); Jungbunzlauer, Des Plaines, IL (sodium citrate, zinc citrate, and potassium citrate); Belle Chemical, Belle, WV (sodium hydroxide); Spectrum Chemical, Brunswick, NJ (magnesium phosphate, high oleic safflower oil, and potassium phosphate); Prinova, Hanover Park, IL (flavoring); CellMark Ingredients, Naugatuck, RI (choline chloride, vitamin A, taurine, and carnitine); CP Kelco, Atlanta, GA (gellan gum); Univar, Baltimore, MD (potassium iodide); Milkfood Ltd, Punjab, India (lactic casein); Farbest, Park Ridge, NJ (fructose and vitamin D); Penta Mfg. Co., Livingston, NJ (myo-inositol, tapioca starch, casein, and maltitol); Foodchem Intl Coop, City of Industry, CA (maltitol, carnitine, sucralose, cocoa powder, and myo-inositol); Northwest Naturals, Bothell, WA (juice); Saputo, Montreal, Canada (skim milk powder); Virginia Date, Brooklyn, NY (flavoring); Firmenich, Plainsboro, NJ (flavoring); Exberry, Mierlo, Netherlands (color); United Sugars, Edina, MN (sugar); Graham Chem Corp, Barrington, IL (sucralose and sugar alcohol); Vyse, Schiller Park, IL (gelatin); Wilmar, Pearland, TX (glucose syrup); Georg Lemke, Berlin, Germany (almonds); Royal Ridge Fruits, Royal City, WA (dried fruit); Orient Resources Co., Hong Kong (pumpkin seeds); Golden Barrel, Honey Brook, PA (invert sugar); AG Commodities, Oxford, PA (brown rice syrup and glycerin); Martin Bauer, Secaucus, NJ (broad bean protein concentrate); Puratos, Pennsauken, NJ (chocolate syrup).

[0156] II. Protein

[0157] "Protein" or "polypeptide" refers to a molecule comprising at least five amino acid residues. "Wild-type" refers to the endogenous form of a molecule naturally occurring in wild organisms. The wild-type form is generally more prevalent in a random mating population. A common form can be, for example, a gene, protein, trait, etc.

[0158] In embodiments, wild-type forms of proteins or polypeptides are employed, however, in embodiments of the present disclosure, modified proteins or polypeptides may be employed. The above terms may be used interchangeably. A "modified protein" or "modified polypeptide," "mutant," "altered," or "variant" refers to a protein or polypeptide whose chemical structure (particularly its amino acid sequence) is altered relative to the wild-type protein or polypeptide; or a variant carries one or more other elements that are linked to the wild-type polypeptide. In embodiments, the modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). A modified / variant protein or polypeptide may be altered in one activity or function but retain the wild-type activity or function in other respects.

[0159] Where a protein is specifically mentioned herein, this refers to a naturally occurring (wild-type) or recombinant (modified) protein, e.g., an early secretory protein from which any attached signal sequence has been removed. The protein can be isolated directly from the organism in which it naturally occurs, produced by recombinant DNA / exogenous expression methods, produced by solid phase peptide synthesis (SPPS), or other in vitro methods. In embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides.

[0160] In embodiments, the size of a protein or polypeptide (wild type or modified) can include, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 65 0, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or more amino acid residues, or any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that polypeptides can be mutated by truncation to render them shorter than their corresponding wild-type or modified forms. Polypeptides can also be altered by fusing or conjugating thereto or therein heterologous protein or polypeptide sequences having a specific function (e.g., for targeting or localization, for purification purposes, etc.).

[0161] As used herein, a "domain" known in the art refers to a unit or part of a protein or polypeptide having different functions or structures, and generally refers to an amino acid sequence having a structure or function recognizable by those skilled in the art, which is associated with a protein having one or more structures or functions.

[0162] "Polynucleotide" refers to a nucleic acid molecule that is recombinant or has been separated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, etc. Polynucleotides include regulatory sequences that are substantially separated from relevant or related naturally occurring genes or protein coding sequences (e.g., control sequences). Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, synthetic, etc.), analogs thereof, or combinations thereof. Additional coding sequences or non-coding sequences can, but need not be, present in a polynucleotide. Polynucleotides can be modified for desired purposes, such as codon optimization, stability, etc., as known in the art.

[0163] "Gene," "polynucleotide," or "nucleic acid" is used to refer to a nucleic acid encoding a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization, such as control sequences). As will be understood by those skilled in the art, the term includes genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be modified to be suitable for expressing proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may comprise a contiguous nucleic acid sequence encoding all or part of such a polypeptide. It should also be considered that a particular polypeptide may be encoded by a nucleic acid that contains variations (having slightly different nucleic acid sequences) but still encodes the same or substantially similar protein and, therefore, may be identified herein as a modified polynucleotide.

[0164] In embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those polynucleotide variants include at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity compared to the polynucleotide sequences herein provided using the methods described herein (e.g., BLAST analysis using standard parameters), including all values ​​and ranges therebetween. In some aspects, an isolated polynucleotide will include a nucleotide sequence encoding a polypeptide having at least 90%, and in some cases 95% or higher identity to an amino acid sequence described herein over the full length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0165] Regardless of the length of the coding sequence, the open reading frame of the nucleic acid segment can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, other control and expression regions, etc., so that the total length can vary greatly. The nucleic acid can be of any length. The nucleic acid of interest can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 nucleotides in length or more and / or can include one or more additional sequences (e.g., regulatory sequences), and / or be part of a larger nucleic acid (e.g., a vector). Therefore, it should be contemplated that nucleic acid fragments of virtually any length can be used, and the total length is preferably limited by the ease of preparation, manipulation, and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification, transport, secretion, post-translational modification of the polypeptide, or to allow for therapeutic benefits (e.g., targeting or therapeutic efficacy). As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is different from the modified polypeptide.

[0166] The polypeptides, proteins or polynucleotides encoding such polypeptides or proteins of the present disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 (or any range derivable therein) or more variant amino acid or nucleic acid substitutions; or, for example, may be substituted with SEQ ID NO: 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 4, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids (or any range derivable therein), at least 60%, 61%, 62%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) similar, identical or homologous.

[0167] The nucleotide and protein, polypeptide and peptide sequences of various genes have been previously published and can be found in recognized computerized databases. Two commonly used databases are Genbank and GenPept (ncbi.nlm.nih.gov / ) of the National Center for Biotechnology Information and the Universal Protein Resource (UniProt; uniprot.org). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or techniques known to those skilled in the art.

[0168] It is contemplated that compositions of the present disclosure may contain from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per milliliter. The concentration of protein in the composition may be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0169] A. Human milk protein

[0170] Aspects of the present disclosure include human milk proteins, as well as compositions comprising such proteins and methods of use thereof. As used herein, "human milk protein" describes any protein present in human breast milk. Human milk proteins include proteins derived from (e.g., isolated from) human breast milk, as well as any protein having the amino acid sequence of a protein present in human breast milk (i.e., wild-type form) produced by other methods (e.g., recombinant expression, chemical synthesis, etc.). Various human milk proteins are well-known in the art and are contemplated herein, including but not limited to secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin (LF), lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, and α-lactalbumin. In an embodiment, the human milk protein of the present disclosure is human whey protein. In an embodiment, the human milk protein of the present disclosure is a recombinant human milk protein (e.g., produced by non-mammalian cells such as yeast cells).

[0171] Certain aspects of the present disclosure relate to human milk proteins having "human-like" glycans. Human-like glycans (also referred to as "human-like glycan structures") are glycans having structures found in human glycoproteins. These glycans include, for example, hybrid N-glycans, complex N-glycans, biantennary, triantennary, and tetraantennary N-glycans, high mannose glycans, and glycans comprising sialic acid, galactose, N-acetylgalactosamine, or fucose. Human-like glycans include those having a Man3GlcNAc2 core structure. Thus, human milk proteins of the present disclosure include those proteins having one or more human-like glycans (e.g., hybrid N-glycans, complex N-glycans, high mannose glycans, biantennary, triantennary, tetraantennary, and combinations thereof).

[0172] Thus, in embodiments, disclosed are recombinant (r) human (rh) milk proteins (e.g., recombinant human lactoferrin, rhLF) comprising one or more human-like glycans. Such recombinant proteins include, for example, recombinant proteins produced by engineered mammalian, fungal, yeast, bacterial, or other cells (including the engineered cells described herein). In some aspects, such recombinant proteins have a glycan pattern that is different from the glycan pattern of the corresponding native or naturally occurring wild-type human milk protein. For example, in embodiments, disclosed are recombinant human lactoferrin comprising one or more human-like glycans, wherein the lactoferrin has a glycan pattern that is different from the glycan pattern of any naturally occurring human lactoferrin (e.g., human lactoferrin in human breast milk). Also disclosed are compositions comprising a plurality of recombinant human lactoferrin proteins, wherein the glycan pattern of all recombinant human lactoferrin proteins may be different from the glycan pattern of all naturally occurring human lactoferrin proteins in human secretions (e.g., breast milk).

[0173] Recombinant glycoproteins typically have a glycosylation profile, pattern, or fingerprint that is different from that present on, in, or of a corresponding wild-type or naturally occurring glycoprotein. However, the different carbohydrates may not negatively impact glycoprotein function, such as that observed with rhLF, the subject of the present invention, such as iron binding. Furthermore, carbohydrates on recombinant glycoproteins may not be allergenic or immunogenic like wild-type, or may be immunogenic or allergenic (Almond et al., Toxicology 301:50-57, 2012).

[0174] B. Lactoferrin

[0175] Aspects of the present disclosure relate to lactoferrin (LF), and compositions comprising lactoferrin, including edible compositions, such as infant formula compositions. Lactoferrin (also known as "lactotransferrin") is a whey protein found in exocrine fluids (e.g., breast milk) and encoded by the LTF gene. LF binds iron (the holo form), while the apo form does not. Without wishing to be bound by theory, lactoferrin is understood to have antimicrobial and anti-inflammatory properties. Aspects of the present disclosure relate to human lactoferrin (hLF) (UniProtKB / Swiss-Prot Accession No. P02788), including its isoforms. The complete sequence of human lactoferrin (including the signal peptide) is provided as SEQ ID NO: 1. The sequence of mature human lactoferrin (after cleavage of the signal peptide) is provided as SEQ ID NO: 2.

[0176]

[0177]

[0178] In some aspects, the human lactoferrin of the present disclosure is recombinant human lactoferrin (rhlactoferrin). In some aspects, the recombinant human lactoferrin of the present disclosure is obtained from mammals, fungi, yeast, bacteria or other cells. In some aspects, the recombinant human lactoferrin of the present disclosure is not obtained from mammalian cells. In some aspects, the recombinant human lactoferrin of the present disclosure is obtained from yeast cells. The yeast cells can, for example, be from the following genus: Akzoys, Aspergillus, Schizochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodakella, Komagata, White Winter Spore, Lipomyces, Mortierella, Ogata, Pichia, Protothecoides, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickham's yeast or Yarrowia. In some aspects, the yeast cell is a Komagataella cell (e.g., Komagataella phaffii, Pichia pastoris, Komagataella pastoris, Komagataella pseudopastoris). Additional yeast species suitable for recombinant protein production are recognized in the art and are contemplated herein. In some aspects, the recombinant human lactoferrin of the present disclosure is obtained from bacteria. In some aspects, the human lactoferrin of the present disclosure is isolated from a natural source.

[0179] Aspects of the present disclosure relate to human lactoferrin having at least one hybrid or complex N-glycan. In some aspects, the human lactoferrin comprises glycans comprising one or more of sialic acid, galactose, N-acetylgalactosamine, or fucose. In some aspects, the human lactoferrin comprises biantennary, triantennary, or tetraantennary N-glycans. As disclosed herein, human lactoferrin having one or more hybrid, complex, biantennary, triantennary, or tetraantennary N-glycans can be used, for example, in infant formula or other edible or nutritional compositions or supplements.

[0180] Recombinant technology provides a virtually unlimited supply of human secretory proteins in quantities that are unattainable from natural sources due to the need for human tissue or secretions, the low amounts of human secretory proteins in fluids, etc. Furthermore, human tissue sources require rigorous testing and specialized purification methods to produce safe and reliable, but limited, quantities of naturally occurring secretory proteins.

[0181] On the other hand, the target recombinant human secretory protein has at least three advantages over naturally occurring secretory proteins. Because the present method involves a unique non-human source of secretory proteins, once the target bioprocess is approved by the FDA, the transformed microorganism will be able to provide unlimited amounts of recombinant secretory proteins (e.g., rhLF, which has lower immunogenicity and allergenicity than wild-type). The present invention enables the commercial development of edible compositions comprising human secretory proteins, thereby allowing everyone to benefit from the benefits of human secretory proteins.

[0182] C.α-lactalbumin

[0183] Aspects of the present disclosure relate to α-lactalbumin (alpha-lac, α-lac, etc.), and compositions comprising α-lactalbumin, including infant formula compositions. α-lactalbumin is a whey protein found in breast milk and encoded by the LALBA gene. Aspects of the present disclosure relate to human α-lactalbumin (UniProtKB / Swiss-Prot Accession No. P00709), including its isoforms. The complete sequence of human α-lactalbumin (including the signal peptide) is provided as SEQ ID NO: 3. The sequence of mature human α-lactalbumin (after cleavage of the signal peptide) is provided as SEQ ID NO: 4.

[0184]

[0185]

[0186] In some aspects, the human α-lactalbumin of the present disclosure is recombinant human α-lactalbumin. In some aspects, the recombinant human α-lactalbumin of the present disclosure is obtained from mammals, fungi, yeast, bacteria or other cells. In some aspects, the recombinant human α-lactalbumin of the present disclosure is not obtained from mammalian cells. In some aspects, the recombinant human α-lactalbumin of the present disclosure is obtained from yeast cells. The yeast cells can be, for example, of the following genera: Akzoys, Aspergillus, Schizochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodakella, Komagata, Leucospora, Lipomyces, Mortierella, Ogata, Pichia, Protothecoides, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickhamella or Yarrowia. In some aspects, the yeast cell is a Saccharomyces cerevisiae cell (e.g., Pichia pastoris, Pichia pastoris, Pseudomonas aeruginosa). Additional yeasts suitable for recombinant protein production are well known in the art and are contemplated herein. In some aspects, the recombinant human α-lactalbumin of the present disclosure is obtained from bacteria. In some aspects, the human α-lactalbumin of the present disclosure is isolated from a natural source.

[0187] Particular aspects of the present disclosure relate to human α-lactalbumin having at least one hybrid or complex N-glycan. In some aspects, human α-lactalbumin comprises glycans comprising one or more of sialic acid, galactose, N-acetylgalactosamine, or fucose. In some aspects, human lactoferrin comprises biantennary, triantennary, or tetraantennary N-glycans. As disclosed herein, human α-lactalbumin having one or more hybrid, complex, biantennary, triantennary, or tetraantennary N-glycans can be used, for example, in infant formula or other nutritional or edible compositions or supplements.

[0188] D. Additional human milk proteins

[0189] Additional human milk proteins contemplated in the compositions (e.g., edible compositions) and methods of the present disclosure include, but are not limited to, those described above for rhLF and rhαLac, and applicable to secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, osteopontin, bile salt-stimulated lipase (BSSL), and lysozyme. Any one or more of these human milk proteins (the coding sequences of which are available in the art) can be included in the edible compositions (e.g., edible or drinkable compositions) of the present disclosure. Any one or more of these human milk proteins can be excluded from the embodiments.

[0190] III. Edible Compositions and Infant Formulas

[0191] Aspects of the present disclosure relate to edible compositions for human consumption (e.g., infant formulas), and methods for providing nutrition to a subject (e.g., an infant). The descriptions below and above also relate to preparing edible compositions of interest, including nutritional compositions, such as candies, bars, yogurts, ice creams, beverages, and the like. Thus, compositions comprising lactose and a recombinant secretory protein of interest can include dairy products, such as ice creams, milk-based nutritional beverages, and the like. Any discussion herein relating to infant formulas also applies to any edible composition.

[0192] Infant formula disclosed herein can be in any form that can be consumed or can be dissolved, combined or diluted for consumption. In embodiments, infant formula can be in a ready-to-consume form. In embodiments, infant formula is in a solid concentrated form (e.g., as a powder). In embodiments, infant formula is in a liquid concentrated form. Infant formula of the present disclosure may include one or more components that can be used to provide nutrition to an infant. Such components may include, but are not limited to, lactose (e.g., lactose from lactose powder), lipids (e.g., lipids from one or more oils, such as sunflower oil, safflower oil, coconut oil, canola oil, low rapeseed erucic acid rapeseed oil, etc.). oil), proteins (e.g., α-lactalbumin, whey protein concentrate, etc.), casein, a human milk oligosaccharide (HMO) (e.g., 2'-fucosyllactose, lactose-N-neotetraose, 3-fucosyllactose, 6'-sialyllactose, difucosyllactose, lactose-N-tetraose), minerals (e.g., calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, and salts thereof), vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, niacinamide, calcium pantothenate, vitamin B6, biotin, folic acid), , pantothenic acid, vitamin B12, vitamin C, choline, inositol, L-carnitine, taurine and its salts), probiotics, nucleotides (e.g., cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate), arachidonic acid (ARA), docosahexaenoic acid (DHA), milk fat globule membrane (MFGM) and human milk proteins (e.g., secretory IgA, human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme or α-lactalbumin). Any one or more or all of the foregoing components may be included in the edible composition of the present invention. Any one or more of these components may be excluded from the embodiment.

[0193] As described herein, infant formulas comprising human milk proteins address various needs by providing formulas with specific benefits previously associated only with breast milk, including anti-inflammatory and antimicrobial benefits. Accordingly, aspects of the present disclosure relate to infant formula compositions comprising human secretory proteins (in some cases human milk proteins), comprising one or more human-like glycans, and methods of using the same for providing nutrition to infants.

[0194] In embodiments, infant formula includes lactose. In embodiments, infant formula includes at least, at most, about or just 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70% or more lactose by weight of dry composition, including any range or value that can be derived therefrom. In embodiments, infant formula includes at least 45% lactose by dry weight. In embodiments, infant formula includes at least 70% lactose by dry weight. Infant formula may include lactose from any source, including, for example, edible lactose powder. In some aspects, infant formula includes less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01% lactose by dry weight. In some aspects, infant formula does not include lactose.

[0195] In an embodiment, the infant formula includes one or more lipids. The infant formula may include one or more lipids from any source, including, for example, one or more of high oleic sunflower oil, mid-oleic sunflower oil, safflower oil, coconut oil, canola oil, rapeseed oil, sunflower oil, Mortierella alpina oil, algae oil (e.g., from Schizochytrium), soybean oil, and palm oil. Any one or more or all of the foregoing components may be included in the infant formula of the present disclosure. Any one or more of these components may be excluded from the embodiment. In an embodiment, the infant formula comprises at least, at most, about, or exactly 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7 , 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 or 12% of any one or more of oil, including any range or value derivable therein.

[0196] In an embodiment, the infant formula includes protein. The infant formula may include protein from any one or more sources, including, for example, whey protein lipid concentrate, whole milk powder, whey protein concentrate, skim milk powder, milk protein concentrate or isolate, whey protein hydrolysate, isolated protein (e.g., isolated α-lactalbumin), or any other protein source. Any one or more or all of the foregoing components may be included in the infant formula of the present disclosure. Any one or more of these components may be excluded from the embodiment. In an embodiment, the infant formula includes, by dry weight, at least, at most, about, or exactly 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 , 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 or 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9 or 13% protein, including any range or value derivable therein.

[0197] In embodiments, the infant formula includes one or more human milk oligosaccharides (HMOs). The HMO can be, for example, 2'-fucosyllactose or lactose-N-neotetraose. Various additional HMOs are well known in the art and are contemplated herein. In embodiments, the infant formula includes at least, at most, about, or exactly 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3% HMOs by dry weight, including any range or value derivable therein.

[0198] In embodiments, infant formula includes one or more mineral substances. Various mineral substances are generally recognized in the art and are considered in this article. In embodiments, infant formula includes one or more of the following: calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, including any salt thereof. For example, infant formula may include one or more (or all) of the following: calcium phosphate, calcium citrate, magnesium chloride, potassium citrate, potassium chloride, sodium chloride, copper sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium selenite, calcium chloride, magnesium phosphate, sodium citrate, potassium phosphate and potassium iodide. Any one or more or all of the aforementioned components may be included in the infant formula of the present disclosure. Any one or more of these components may be excluded from embodiments. In embodiments, infant formula comprises at least, at most, about, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3% of any one or more minerals by dry weight, including any range or value derivable therein. In embodiments, infant formula comprises at least 0.1% of any one or more minerals by dry weight. In embodiments, infant formula comprises up to 3% of any one or more minerals by dry weight.

[0199] In embodiments, infant formula includes one or more vitamins. Various vitamins are generally recognized in the art and are considered herein. In embodiments, infant formula includes one or more of the following: vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, nicotinamide, calcium pantothenate, pyridoxine hydrochloride, biotin, folic acid, pantothenic acid, cyanocobalamin, vitamin C, choline, inositol, L-carnitine, inositol and taurine, including any salt thereof. For example, in embodiments, infant formula includes one or more (or all) of the following: vitamin A palmitate, vitamin D3, D-alpha tocopherol, vitamin K1, thiamine hydrochloride, riboflavin, nicotinamide, calcium pantothenate, pyridoxine hydrochloride, biotin, folic acid, cyanocobalamin, choline bitartrate, inositol and ascorbic acid. Any one or more or all of the aforementioned components may be included in the infant formula of the present disclosure. Any one or more of these components may be excluded from embodiments. In embodiments, infant formula includes at least, at most, about or exactly 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, or 1% by weight of any one or more vitamins, including any range or value derivable therein. In embodiments, infant formula includes at least 0.000001% by weight (and sometimes by volume for liquid agents) of any one or more vitamins. In embodiments, infant formula includes at most 1% by weight of any one or more vitamins.

[0200] In embodiments, the infant formula disclosed herein includes one or more probiotic microorganisms. As used herein, "probiotic" microorganisms as described above include microorganisms, such as bacteria or other microorganisms that are known or suspected to have a beneficial effect on the health or well-being of an individual. Various probiotic microorganisms are well-known in the art and are considered herein. Probiotic microorganisms that can be used in the disclosed compositions and methods include, for example, probiotic Lactobacillus and probiotic Bifidobacterium organisms. In an embodiment, the probiotic microorganism is Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus helveticus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, or Bifidobacterium infantis.

[0201] The infant formula of the present disclosure may include at least, at most, or exactly 1, 2, 3, 4, or 5 different probiotic microorganisms. In an embodiment, the infant formula does not include a probiotic organism. In an embodiment, the infant formula includes Bifidobacterium infantis, such as Bifidobacterium infantis M-63. Additional beneficial probiotic microorganisms are disclosed, for example, in U.S. Patent No. 9,226,521, the entire contents of which are incorporated herein by reference. The infant formula of the present disclosure may include at least, at most, about, or exactly 10 probiotic organisms per 100 grams. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15or more colony forming units (cfu), particles, or other measure of probiotic microorganisms (eg, Bifidobacterium infantis), including any range or value derivable therein.

[0202] In embodiments, infant formula includes one or more nucleotides. Various nucleotides, including nucleotides that can be used as supplements for infant formula or other food compositions, are well-known in the art and are considered herein. In embodiments, infant formula includes one or more (or all) of cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, or guanosine 5'-monophosphate. Any one or more or all of the aforementioned components may be included in the infant formula of the present disclosure. Any one or more of these components may be excluded from embodiments.

[0203] In an embodiment, the infant formula includes one or more additional components including, but not limited to, arachidonic acid, docosahexaenoic acid, prebiotics, casein (eg, micellar casein), and MFGM.

[0204] In embodiments, an infant formula is disclosed that includes, in addition to one or more additional components described herein, a human secretory protein. The infant formula may include any human secretory protein, including human milk proteins disclosed herein (e.g., recombinant human milk proteins), human milk proteins including hybrid, complex, or other human-like glycans, human whey proteins, and any one or more specific human milk proteins, such as lactoferrin (e.g., recombinant lactoferrin). In embodiments, the infant formula includes a human secretory protein of natural origin. In embodiments, the infant formula includes a recombinant human secretory protein. In embodiments, the infant formula includes a recombinant human secretory protein having a glycan pattern that is different from the glycan pattern of the corresponding natural human secretory protein.

[0205] 31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0. 71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96 , 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1. 47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00% or more of one or more human secretory proteins (e.g., sIgA, human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin), including any ranges or values ​​derivable therein.

[0206] In an embodiment, the infant formula comprises about or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55 .49, 0.5% or more human lactoferrin (e.g., recombinant human lactoferrin), or any range or value derivable therein.

[0207] In an embodiment, the infant formula comprises, by dry weight, about or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0. 98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23 , 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1 .49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74 , 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.100% or more of human alpha-lactalbumin, or any range or value derivable therein.

[0208] IV. Genetic Engineering

[0209] Aspects of the present disclosure include recombinant protein products (e.g., human secretory proteins) produced by engineered microorganisms. In some aspects, recombinant human secretory proteins (e.g., recombinant lactoferrin) produced and secreted by engineered microorganisms (e.g., engineered eukaryotic cells, fungal cells, yeast cells, bacteria, or other cells) are disclosed. Vectors for transforming microorganisms according to the present disclosure can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. Vectors typically comprise one or more genes, each of which encodes expression of a desired product (gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a specific location in the recombinant cell.

[0210] Exogenous nucleic acid sequence (comprising the nucleotide sequence of such as encoding fusion protein, the nucleotide sequence of encoding wild type or mutant protein etc.) can be introduced into different host cells.As further described herein, the nucleotide sequence that can be configured to promote the genetic mutation in gene can also be introduced into various host cells.Suitable host cell is the microbial host that can be widely present in Fungi Section.The example of suitable host strain includes but is not limited to fungi or yeast species, such as following genus: Aksu yeast, Aspergillus, Schizochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Hansenula, Kluyveromyces, Komagata yeast, white winter spore yeast, oil yeast, Mortierella, Ogata yeast, Pichia, Protothecoides, Rhizopus, red winter spore yeast, red yeast, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon and Yeshiva.Various fungal organisms are generally recognized in the art and are considered in this article.In embodiments, the host cell of present disclosure is Komagata yeast cell. In embodiments, the host cell of the present disclosure is Komagataella phaffii. In embodiments, the host cell of the present disclosure is Komagataella phaffii or Pichia pastoris. In embodiments, the host cell of the present disclosure is Komagataella pseudopasteur. Additional host cells include non-fungal eukaryotic cells and bacteria.

[0211] Microbial expression systems and expression vectors are known to those skilled in the art. Any such expression vector can be used to introduce the instant gene and nucleic acid sequence into an organism. The nucleic acid sequence can be introduced into an appropriate microorganism by transformation techniques. For example, the nucleic acid sequence can be cloned into a suitable plasmid, and the resulting plasmid can be used to transform the parent cell. The plasmid is not particularly limited, as long as the plasmid makes the desired nucleic acid sequence heritable to the offspring of the microorganism.

[0212] The vector or box for transforming suitable host cells are generally recognized in the art. Typically, the vector or box comprises a gene, a sequence (including a promoter) for directing related gene transcription and translation, a selectable marker, and a sequence that allows autonomous replication or chromosomal integration. Suitable vectors include the 5' region (5' untranslated region, 5'UTR) of the gene carrying a promoter and other transcription initiation control elements and the 3' region (3'UTR) of the DNA fragment that controls transcription termination.

[0213] Promoters, cDNAs, and 3'UTRs, as well as other elements of the vector, can be generated by cloning techniques using fragments isolated from natural sources (Green & Sambrook, "Molecular Cloning: A Laboratory Manual," (4th ed., 2012); and U.S. Pat. No. 4,683,202; both of which are incorporated herein by reference in their entirety). Alternatively, the elements can be generated synthetically using known methods (Gene 164:49-53 (1995)).

[0214] A. Vectors and Vector Components

[0215] According to the present disclosure, vectors for transforming microorganisms can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. A vector generally comprises one or more genes, each of which encodes a desired product for expression (gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a specific location in the recombinant cell.

[0216] 1. Control Sequence

[0217] A control sequence is a nucleic acid sequence that regulates the expression of a coding sequence or directs a gene product to a specific location within or outside the cell. Control sequences that regulate expression include, for example, promoters that regulate the transcription of a coding sequence and terminators that terminate the transcription of a coding sequence. Another control sequence is a 3' non-translated sequence located downstream of the coding sequence that encodes a polyadenylation signal. Control sequences that direct gene products to specific locations, such as those encoding signal peptides, direct proteins attached thereto to specific locations within the cell.

[0218] Thus, exemplary vector designs for expressing genes in microorganisms include coding sequences for desired gene products (e.g., selectable markers, enzymes, fusion proteins, etc.) operably linked to promoters active in the host yeast cells. Alternatively, if the vector does not contain a promoter operably linked to the desired coding sequence, the coding sequence can be transformed into the cell such that the open reading frame is operably linked to an endogenous promoter at the site of vector integration. Examples of promoters contemplated herein include, but are not limited to, the known and publicly available AOX1, GAP, TEF1, TPI1, DAS1, DAS2, CAT1, and FMD promoters.

[0219] The promoter used to express a gene may be the promoter naturally associated with the gene, or may be a different promoter of a different gene.

[0220] Promoters can generally be characterized as constitutive or inducible. Constitutive promoters are generally active or function to drive expression at the same level all the time (or at some time during the cell life cycle). Inducible promoters are active (or become inactive) only in response to a stimulus, or significantly raise or lower the expression. Both types of promoters are applicable herein. Useful inducible promoters include those that mediate transcription of operably linked genes in response to a stimulus (such as exogenously provided small molecules (such as methanol), temperature (hot or cold), lack of nitrogen in the culture medium, etc.). Suitable promoters can activate transcription of substantially silent genes, or raise the transcription of operably linked genes that are transcribed at a low level.

[0221] The inclusion of termination region control sequences is optional. The termination region may be native with the transcription initiation region (promoter), native with the DNA sequence of interest, or may be obtained from other sources (see, e.g., Chen & Orozco, Nucleic Acids Research 16:8411 (1988)).

[0222] In some cases, the entire nucleotide sequence of a promoter is not necessary to drive transcription, and a sequence shorter than the full-length nucleotide sequence of a promoter can drive transcription of an operably linked gene. The smallest portion of a promoter (called a core promoter) includes a transcription start site, a binding site for RNA polymerase, and a binding site for transcription factors.

[0223] The promoter and target can be linked by introducing them into a nucleic acid molecule (e.g., a vector). The vector can be introduced into a cell, thereby expressing the promoter and target. In one embodiment, the promoter and target are linked by introducing the promoter into the DNA of the cell (e.g., by homologous recombination), thereby integrating the promoter into the genome of the cell.

[0224] B. Gene and Codon Optimization

[0225] In another embodiment, the present invention relates to a gene that is expressed in a host cell and is derived from a vector comprising a promoter, a coding sequence, and a termination control sequence. Typically, a gene comprises a promoter, a coding sequence, and a termination control sequence. When assembled by recombinant DNA technology, a gene can be referred to as an expression cassette, and flanks can be restriction sites, so that insertion is used to introduce the recombinant gene into the vector of the host cell. The flanks of an expression cassette can be DNA sequences from genome or other nucleic acid targets, to promote the stably integration of the expression cassette into the genome by homologous recombination. Alternatively, carrier and expression cassette can keep unintegrated state (for example, episome), and in this case, carrier generally comprises a replication origin, which can ensure the duplication of the vector DNA.

[0226] A common gene present on a vector is a gene encoding a protein whose expression allows differentiation between recombinant cells containing the protein and cells that do not express the protein. Such genes and their corresponding gene products are referred to as selectable markers or selection markers. Any of a variety of selectable markers can be used in transgenic (exogenous gene, recombinant gene or gene of interest) constructs that can be used to transform the organisms encompassed in the disclosed embodiments.

[0227] In order to achieve optimal expression of recombinant proteins, it may be beneficial to employ coding sequences that produce mRNAs with codons that are optimally used by the cells to be transformed. Thus, correct or maximal expression of a transgene may require that the codon usage of the transgene be matched to the specific codon preferences of the organism in which the transgene is expressed. The precise mechanisms behind this type of effect are numerous, but include a proper balance between the available pool of aminoacylated tRNAs and the proteins synthesized in the cell, and more efficient translation of the transgene messenger RNA (mRNA) when this requirement is met. When codon usage in a transgene is not optimized, the available tRNA pool may be insufficient to allow efficient translation of the transgene mRNA, leading to ribosome arrest and termination, and possible instability of the transgene mRNA.

[0228] The coding sequences of the present disclosure can be codon-optimized for a specific host cell by replacing one or more rare codons with one or more codons that are more frequently present in the host cell. Rare codons in a host cell are codons that are present in less than 5%, less than 10%, or less than 20% of the coding sequence in the host cell. Rare codons can be identified using methods known to those skilled in the art.

[0229] Aspects of the present disclosure include using nucleic acid sequences comprising protein-coding genes to transform microorganisms. The gene may be natural to the cell or may be from different species. The gene may be derived from different species, but may be modified (e.g., codon optimized) to achieve optimal expression in the microorganism. In embodiments, the gene may be inherited in the offspring of the transformed cell. In embodiments, the gene may be heritable by being present on a plasmid. In embodiments, the gene may be heritable by being integrated into the genome of the transformed cell.

[0230] Other aspects of the present disclosure may include transforming a microorganism with a nucleic acid sequence configured to produce mutations in the microorganism's genes. For example, aspects of the present disclosure may include transforming a microorganism with a nucleic acid sequence comprising upstream and downstream sequences of a gene (e.g., the OCH1 gene encoding a yeast 1,6-mannosyltransferase) to promote reduced or absent gene expression through homologous recombination. Various methods for producing mutations in microbial genes (including deletion or knockout mutations, as well as mutations that reduce gene expression) are well-known in the art and are contemplated herein. Microorganisms with gene deletion or knockout mutations do not produce functional copies of proteins. For example, recombinant yeast cells of the present disclosure may include a deletion of the endogenous OCH1 gene such that the recombinant yeast cells do not express endogenous, functional OCH1 protein. Microorganisms with reduced gene or protein expression may produce functional copies of proteins, but at reduced levels compared to a wild-type (i.e., non-recombinant or non-genetically modified) microorganism of the same species. Methods for reducing protein expression are well-known in the art and include, for example, replacing endogenous promoters and / or modifying one or more regulatory elements.

[0231] C. Transformation

[0232] Cells can be transformed by any suitable technique, including, for example, use of a gene gun device, electroporation, glass bead transformation, and silicon carbide whisker transformation, etc. Any convenient technique for introducing a transgene into a microorganism can be employed in the embodiments disclosed herein.

[0233] The vector for transforming microorganisms can be prepared by known techniques familiar to those skilled in the art. In one embodiment, the exemplary vector design for expressing genes in microorganisms comprises a gene encoding an enzyme, which is operably connected to an active promoter in the microorganism. Alternatively, if the vector does not comprise a promoter operably connected to the target gene, the gene can be transformed into the cell to be operably connected to the natural promoter at the vector integration site. The vector can also comprise a second gene encoding a protein. Alternatively, one or two genes are followed by a 3' non-translated sequence comprising a polyadenylation signal. The expression cassette encoding these two genes can be physically connected in the vector or be present on a separate vector. The co-transformation of microorganisms can also be used, in which different vector molecules are used for transforming cells simultaneously (Protist 155:381-93,2004). Under conditions where the cell lacking a resistance box can not grow or breed, the cell transformed can be optionally selected according to the ability to grow in the presence of antibiotics or other selectable markers.

[0234] D. Genetically engineered cells

[0235] Various aspects of the present disclosure include genetically engineered cells (also referred to as "engineered cells" and "recombinant cells") and methods for making and using such cells. In embodiments, disclosed are recombinant cells comprising one or more exogenous nucleic acid sequences. Also disclosed are methods for generating such recombinant cells, comprising introducing one or more exogenous nucleic acid sequences into a host cell. Further described are methods for collecting one or more products (e.g., mammalian glycoproteins) from such recombinant cells, comprising culturing the cells and collecting the products.

[0236] In embodiments, the recombinant cell is a prokaryotic cell, such as a bacterium. In embodiments, the recombinant cell is a eukaryotic cell, such as a mammalian cell, a yeast cell, a filamentous fungal cell, a protist cell, an algae cell, an avian cell, a plant cell, or an insect cell. In embodiments, the recombinant cell is not a mammalian cell. In embodiments, the host cell is a yeast cell. Those skilled in the art will recognize that many forms of filamentous fungi produce yeast-like growth, and the definition of yeast herein includes such cells. The recombinant cell of the present disclosure can be selected from algae, bacteria, mold, fungi, plants, and yeast. In embodiments, the recombinant cell of the present disclosure is a bacteria (e.g., Escherichia coli), a fungal cell, or a yeast cell (e.g., Saccharomyces cerevisiae).

[0237] In embodiments, the recombinant cell of the present disclosure is a recombinant yeast cell. The recombinant yeast cell can be any suitable yeast cell generally recognized in the art. In some aspects, the yeast cell is an Akzo yeast, an Aspergillus, a Schizochytrium, a Candida, a Claviceps, a Cryptococcus, a Cunninghamella, a Geotrichum, a Hansenula, a Kluyveromyces, a Kodak yeast, a Komamoto yeast, a White Winter Spore Yeast, a Lipozyme, a Mortierella, a Ogata yeast, a Pichia pastoris, a Protothecoides, a Rhizopus, a Red Winter Spore Yeast, a Rhodotorula, a Saccharomyces, a Schizosaccharomyces, a Tremella, a Trichosporon, a Wickham yeast, or a Yarrowia cell. In embodiments, the yeast cell is Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha), Kluyveromyces lactis, Komagataella phaffii, Komagataella pastoris, Komagataellapseudopastoris, Kluyveromyces marxianus, Kodamaeaohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces staryistarkeyi), Lipomycetes stetrasporus, Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae cerevisiae), Schizosaccharomyces pombe, Tremella enchepala, Trichosporon cutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii, or Yarrowia lipolytica.

[0238] In embodiments, the yeast cell is a Kluyveromyces cell. In embodiments, the yeast cell is Kluyveromyces phaffii, Pichia pastoris, Kluyveromyces pastoris, or Kluyveromyces pseudopasteur. In embodiments, the yeast cell is Kluyveromyces phaffii.

[0239] V. Manufacturing

[0240] Edible compositions according to the present invention can be prepared by any known or other suitable means. For example, infant formula can be obtained by blending protein with human secretory protein and, optionally, a carbohydrate source and a lipid source in appropriate proportions. An emulsifier may be included. Vitamins and minerals may be added, possibly later in the manufacturing process to prevent thermal degradation. Water (e.g., purified water, such as reverse osmosis or deionized water) may then be added and mixed to form a liquid mixture. The mixing temperature may be room temperature, but may be higher to facilitate suspension and mixing, but below temperatures that would otherwise damage the ingredients.

[0241] If necessary, the liquid mixture is dried in a suitable drying apparatus (eg a spray dryer or freeze dryer) and can be converted or transformed into a powder.

[0242] Powdered infant formula can be produced using a variety of methods, for example, dry mixing dehydrated ingredients to form a uniform mixture, or hydration and wet mixing ingredients (for example, fat, protein and carbohydrate components) mixture, then evaporation or spray drying the resulting mixture. A combination of various methods can be used, wherein first by wet mixing and spray drying all or some large amounts of ingredients (macro-ingredients) to produce a basic powder, then dry mixing the remaining ingredients (including carbohydrates, minerals and vitamins and other micronutrients), to produce a final composition. Liquid formula can be obtained in the form of instant or concentrated liquid (which needs to be diluted with water, for example 1: 1).

[0243] To produce liquid infant formula, the homogenous mixture is filled into suitable containers (preferably aseptically). However, liquid compositions may be retorted in suitable containers, filled and retorted by suitable means using commercially available machines.

[0244] As mentioned above, the present invention relates to ready-to-drink formulations and methods for preparing ready-to-drink formulations.The liquid food product may be a beverage, such as a nutritional drink, an energy drink, a milkshake, a performance or nutritional product (such as a protein shake), or the like.

[0245] Steps are implemented during the production process to eliminate or limit microbial growth. Typically, oil-in-water (O / W) emulsions are preserved, for example, by dehydration for powdered products or sterilization for ready-to-feed or concentrated liquid products. The liquid mixture can then be heat-treated or sterilized by another method to reduce the risk of spoilage. The mixture can then be homogenized.

[0246] The manufacture of the present invention may involve high temperature processing of about 110°C or above.

[0247] During ultra-high temperature processing (UHT), the edible composition is heated sufficiently to sterilize the liquid composition, which can extend the shelf life of the composition or formulation.

[0248] The high temperature treatment may be performed at about 110° C. to about 150° C. For example, the high temperature treatment may be performed at the following temperatures: about 110° C. to about 145° C., about 110° C. to about 140° C., about 110° C. to about 135° C., about 110° C. to about 130° C., about 110° C. to about 125° C., about 110° C. to about 120° C., about 110° C. to about 115° C., about 115° C. to about 150° C., about 115° C. to about 145° C., about 115° C. to about 140° C., about 115° C. to about 135° C., about 115° C. to about 130° C., about 115° C. to about 125° C., about 115° C. to about 120° C., about 120° C. to about 150° C., about 120° C. to about 145° C., about 115° C. to about 140° C. ℃, about 120 ℃ to about 135 ℃, about 120 ℃ to about 130 ℃, about 120 ℃ to about 125 ℃, about 125 ℃ to about 150 ℃, about 125 ℃ to about 145 ℃, about 125 ℃ to about 140 ℃, about 125 ℃ to about 135 ℃, about 125 ℃ to about 130 ℃, about 130 ℃ to about 150 ℃, about 130 ℃ to about 145 ℃, about 130 ℃ to about 140 ℃, about 130 ℃ to about 135 ℃, about 135 ℃ to about 150 ℃, about 135 ℃ to about 145 ℃, about 135 ℃ to about 140 ℃, about 140 ℃ to about 150 ℃, about 140 ℃ to about 145 ℃, or about 145 ℃ to about 150 ℃.

[0249] The high temperature treatment may be performed for about 5 seconds to about 60 minutes, about 5 seconds to about 45 minutes, about 5 seconds to about 30 minutes, about 5 seconds to about 15 minutes, about 5 seconds to about 5 minutes, about 5 seconds to about 120 seconds, about 5 seconds to about 60 seconds, about 5 seconds to about 30 seconds, about 30 seconds to about 60 minutes, about 30 seconds to about 45 minutes, about 30 seconds to about 30 minutes, about 30 seconds to about 15 minutes, about 30 seconds to about 5 minutes, about 30 seconds to about 120 seconds, about 30 seconds to about 60 seconds, about 60 seconds to about 45 minutes, about 60 seconds to about 30 minutes, about 60 seconds to about 15 minutes. minutes, about 60 seconds to about 5 minutes, about 60 seconds to about 120 seconds, about 120 seconds to about 60 minutes, about 120 seconds to about 45 minutes, about 120 seconds to about 30 minutes, about 120 seconds to about 15 minutes, about 120 seconds to about 5 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 15 minutes, about 15 minutes to about 60 minutes, about 15 minutes to about 45 minutes, about 15 minutes to about 30 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 45 minutes, or about 45 minutes to about 60 minutes.

[0250] The high temperature treatment can be carried out by direct or indirect heating. For example, the high temperature treatment can be carried out by steam injection, heat exchange or use of a still.

[0251] Edible compositions can be treated with high pressure prior to packaging and distribution, which enhances safety and extends shelf life. High pressure processing (HPP) is known and requires pressures of about 6000 bar or higher. Pressures of 50 MPa to about 1000 MPa can be used at room temperature or lower, see, for example, U.S. Patent Nos. 5,213,029; 5,316,745; 5,683,735; and 6,033,717; and U.S. Publication No. 20080050507.

[0252] The edible composition of interest is aseptically filled into suitable packaging (e.g., lined cans, pouches, package wrappings, powder containers, bottles, etc.), sealed, optionally treated for quality control and shelf stability, and distributed for sale.

[0253] All references cited herein are hereby incorporated by reference in their entirety.

[0254] The present disclosure will now be illustrated in the following non-limiting examples. It should be understood that the examples, although indicating embodiments of the present invention, are given by way of illustration only. From the above discussion and examples, those skilled in the art can ascertain the essential characteristics of the present invention and, without departing from the spirit and scope of the present invention, can make various changes and modifications to the present invention to adapt it to various uses and conditions.

[0255] Herein, the percentages of dry ingredients are based on weight. For liquid compositions, the percentages can be based on weight, since water is typically the primary medium with a density of 1 g / cc, or on a weight / volume basis. The weights of liquid ingredients in dry formulations can be based on weight. The ratios of ingredients in dry formulations can be used to determine the percentages of agents in similar liquid formulations based on the target solids level in the similar liquid formulation.

[0256] Example

[0257] Example 1

[0258] A batch of milk-based infant formula was prepared according to the formula in the table below. Lactose was hydrated in 150-160°F water at 45-55% solids, and then protein was added. The vegetable oil blend, ARA-DHA blend, and fat-soluble vitamin premix were mixed into the lactose. Minerals and trace minerals were added, along with other minerals, vitamins, and nutrients. During mixing, the mixture was maintained at 140-145°F with high agitation, then homogenized at 2000-3500 PSI and cooled at 40°F. The nucleotide premix was added to the cooled mixture and stirred, followed by pasteurization at 170-180°F for 60 seconds and then spray-dried to a moisture content of 1.5-3.5%. Probiotic microorganisms and human lactoferrin were added to the base formula by dry mixing.

[0259] Mix the powder with water at a ratio of 1 tablespoon to 2 ounces of water to produce a drinkable infant formula.

[0260] Table 1

[0261]

[0262]

[0263] Example 2

[0264] A batch of milk-based infant formula containing a lower amount of lactose was prepared as provided in Example 1 using the formula provided in the table below.

[0265] Table 2

[0266] Element Product Code %scope corn syrup solids - 50-55％ Vegetable oil blend - 22-27％ Milk protein isolate - 4.0-7.0％ Whey protein concentrate 80% - 4.0-7.0％ Whey protein enriched with α-Lac - 1.5-4.0％ Mineral premix XR73983000 1.0-3.5％ 2'-fucosyllactose (HMO) - 1.5-2.0％ ARA-DHA blend - 0.5-1.0％ Choline bitartrate - 0.2-0.5％ Vitamin premix XR74360000 0.2-0.6％ potassium hydroxide - 0.1-0.3％ ascorbic acid - 0.1-0.3％ potassium bicarbonate - 0.05-0.12％ Trace mineral premix XR73965000 0.02-0.07％ Nucleotide premix XR73202000 0.01-0.05％ Liquid fat-soluble premix XR74558000 0.01-0.05％ Human lactoferrin - 0.4-1.0％ Probiotic microorganisms - 0.05-0.2％ Moisture - 1.5-3.5％

[0267] Example 3

[0268] A batch of soy-based infant formula was prepared as provided in Example 1 using the formula provided in the table below.

[0269] Table 3

[0270] Element Product Code %scope corn syrup solids - 52-57％ Vegetable oil blend - 22-27％ soy protein isolate - 11-14％ Mineral premix XR73983000 1.0-3.5％ 2'-fucosyllactose (HMO) - 1.5-2.0％ ARA-DHA blend - 0.5-1.0％ Choline bitartrate - 0.2-0.5％ Vitamin premix XR74360000 0.2-0.6％ potassium hydroxide - 0.1-0.3％ ascorbic acid - 0.1-0.3％ potassium bicarbonate - 0.05-0.12％ Trace mineral premix XR73965000 0.02-0.07％ Liquid fat-soluble premix XR74558000 0.01-0.05％ Human lactoferrin - 0.4-1.0％ Probiotic microorganisms - 0.05-0.2％ Moisture - 1.5-3.5％

[0271] Example 4

[0272] A batch of milk-based infant formula comprising partially hydrolysed whey was prepared as provided in Example 1 using the formula shown in the table below.

[0273] Table 4

[0274]

[0275]

[0276] Example 5

[0277] Using the formula provided in Table 5, a batch of milk-based ready-to-feed infant formula was prepared as follows.

[0278] Reverse osmosis (RO) water, accounting for 50% of the total volume, is heated to 150-160°F. High shear agitation is used to hydrate the protein and carrageenan. While maintaining high agitation, the vegetable oil blend, ARA-DHA blend, and fat-soluble vitamin premix are added, followed by lactose. Minerals and trace minerals are added, along with other minerals, vitamins, and nutrients. During mixing, the mixture is maintained at 140-145°F with high agitation, then pasteurized at 185-190°F for 30 seconds, homogenized at 2000-3500 PSI, and cooled at 40°F. The nucleotide premix and human lactoferrin are added to the cooled mixture along with the remaining RO water to bring the total solids to 12.8-13.2% w / w. The mixture is UHT treated and aseptically filled into 8-ounce plastic bottles.

[0279] Table 5

[0280]

[0281]

[0282] Example 6

[0283] Prepare a batch of ready-to-use liquid nutritional beverages according to the recipe in Table 6. Reverse osmosis (RO) water accounting for 50% of the total amount is heated to 150-160°F. Use high shear stirring to hydrate the proteins (milk protein concentrate and soy protein isolate), cellulose gel and gum and carrageenan. While maintaining high agitation, add oil (high oleic safflower oil and canola oil), lecithin and fat-soluble vitamin premix, then add maltodextrin, sugar and oligofructose. Add individual minerals, trace mineral premix, water-soluble vitamin premix and other nutrients. During mixing, the mixture is maintained at 140-145°F and highly agitated, then the mixture is pasteurized at 185-190°F for 30 seconds, homogenized at 2000-3500PSI, and then cooled at 40°F. A 5% solution of vitamin C, a portion of potassium hydroxide, a 10% solution of human lactoferrin, flavoring, and the remaining RO water were added to the cooled mixture to bring the total solids to 22.8-24.2% w / w. The mixture was UHT treated and aseptically filled into 8 oz plastic bottles.

[0284] Table 6

[0285]

[0286]

[0287] Example 7

[0288] A batch of ready-to-use liquid nutritional low glycemic index beverage was prepared as provided in Example 6 using the recipe of Table 7.

[0289] Table 7

[0290]

[0291]

[0292] Example 8

[0293] A batch of nutritional beverage powder was prepared using the recipe in Table 8. The proteins (milk protein concentrate and soy protein isolate) were hydrated in 145-150°F water at 45-55% solids. Oils (high oleic safflower and canola), lecithin, and a fat-soluble vitamin premix were then added, followed by maltodextrin, sugar, and oligofructose. Minerals and trace minerals were added, along with vitamins and nutrients. During mixing, the mixture was maintained at 140-145°F with high agitation, then homogenized at 2000-3500 PSI and cooled at 40°F. A 5% solution of vitamin C, a portion of potassium hydroxide, and a 10% solution of human lactoferrin were added to the cooled mixture, along with N&A flavoring. The mixture was pasteurized at 170-180°F for 60 seconds and then spray-dried to a moisture content of 2.5-4.0%.

[0294] Optionally, human lactoferrin is added to the base formula by dry blending.

[0295] Table 8

[0296]

[0297]

[0298] Example 9

[0299] A stable juice-based protein beverage is prepared by mixing juice with 0.1-10.0% protein (e.g., casein, lactalbumin, serum albumin, glycomacropeptide, soy protein, rice protein, pea protein, whey protein, canola protein, wheat protein, zein, flax protein, egg white protein, ovalbumin, gelatin, and combinations thereof) using the recipe of Table 9. The pH of the mixture is adjusted to 2-3.4. Optionally, one or more defoamers, nutrients, calcium, herbal supplements, flavorings, sweeteners, coloring agents, preservatives, and energy-generating additives (selected from caffeine, magnesium, and citrulline malate) are added to the mixture. Microorganisms are inactivated by high pressure processing (HPP). The protein beverage is packaged in a container that can be stored without refrigeration for more than one year before use by the consumer of the protein beverage.

[0300] Table 9

[0301] Element %scope Juice Blends 80-98％ protein 0.1-10％ Prebiotics 0.1-5.0％ Human lactoferrin 0.1-3.0％ Zinc citrate 0.1-0.3％ Vitamin D 0.1-0.3％

[0302] Example 10

[0303] A carbonated milk beverage with enhanced storage stability was prepared based on the recipe provided in Table 10. First, a skim milk powder solution was heated at least 85-138° C. for 5 seconds. The liquid dairy product was subjected to ultra-heat treatment at least 150° C. and a pressure of 700 kPa. In a mixing tank, the other ingredients were added and homogenized at 2,000-5,000 psi for 5 minutes. The liquid dairy product was cooled to a temperature below 10° C. The cooled liquid dairy product was subjected to pressurized carbon dioxide treatment so that at least 3-8 volumes of carbon dioxide were dissolved in the liquid dairy product. The carbonated liquid dairy product was packaged in a closed container that was stable for storage and did not require refrigeration.

[0304] Table 10

[0305]

[0306]

[0307] Example 11

[0308] The recipe of protein-fortified frozen dessert is prepared using the recipe of record in Table 11. These ingredients are mixed at a speed of 5,000-7,000 rpm to prepare the sweet mixture. The sweet mixture is preheated at 30-60° C. The preheated sweet mixture is pasteurized by heating at 69° C. for 30 minutes (in batches) or at 80° C. for 25 seconds (continuously). The sweet mixture is homogenized at 2000-3000 psi (one-stage homogenization) or first at 2000-2500 psi and then at 500 psi (two-stage homogenization). The homogenized sweet mixture is cooled to 3-5° C. (continuously 4-6 hours) to produce a stale sweet mixture, and the stale mixture is frozen to prepare the frozen dessert of protein fortification.

[0309] Table 11

[0310] Element %scope water 50-65％ sugar 10-20％ milk solids 10-15％ Fat 0.1-20％ flavoring 0.1-10％ Protein components 0.1-11％ Human lactoferrin 0.1-2.0％

[0311] Example 12

[0312] A protein-fortified spreadable yogurt product is prepared by mixing a casein-containing ingredient with milk to prepare yogurt according to the recipe provided in Table 12. The yogurt culture is added to the yogurt and allowed to ferment. The resulting yogurt mixture is separated into a yogurt retentate and a yogurt permeate. The yogurt retentate is used as a spreadable protein-fortified yogurt. The spreadable yogurt can be flavored and then packaged.

[0313] Table 12

[0314]

[0315]

[0316] Example 13

[0317] The recipe in the table below is used to produce a high-protein soft candy snack. A mixture is formed by mixing water, glucose syrup, a first sugar alcohol, and a second sugar alcohol. The mixture is cooked at 230-285°F for 10-60 minutes to form a sugar mixture, which is then cooled to 60-80°F. An acid solution (malic acid) is added to form a sugar-acid mixture with a pH of 2-4. The sugar-acid mixture is mixed with gelatin for 4-12 minutes to form a sugar-gelatin mixture. The sugar-gelatin mixture is mixed with protein to form a gel mixture. The gel mixture is then processed by a machine to form it.

[0318] Table 13

[0319] Element %scope Whey protein concentrate 10-50％ water 5-35％ Sugar alcohol 1 5-30％ Sugar alcohol 2 5-20％ gelatin 0.1-15％ glucose syrup 0.1-15％ Human lactoferrin 0.1-3.0％

[0320] Example 14

[0321] A high-protein snack food was produced using the recipe provided in Table 14. Any of a variety of protein particles (e.g., chicken meal, pea protein powder, etc.) was extruded to form protein pieces. The pieces or crisps were combined with the remaining ingredients, and the mixture was used to form bars, which were processed at 160°F until an internal temperature of 140°F was reached.

[0322] Table 14

[0323]

[0324]

[0325] Example 15

[0326] A batch of nutritional plant-based beverage powder was prepared according to the recipe in Table 15. Proteins (pea protein concentrate and faba bean protein isolate) were hydrated in 145-150°F water at 45-55% solids. Oils (high oleic safflower and canola), lecithin, and a fat-soluble vitamin premix were then added, followed by maltodextrin, sugar, and oligofructose. Minerals and trace minerals were added, along with vitamins and nutrients. During mixing, the mixture was maintained at 140-145°F with high agitation, then homogenized at 2000-3500 PSI and cooled to 40°F. A 5% solution of vitamin C, a portion of potassium hydroxide, a 10% solution of human lactoferrin, and N&A flavoring were added to the cooled mixture. The mixture was pasteurized at 170-180°F for 60 seconds and then spray-dried to a moisture content of 2.5-4.0%.

[0327] Human lactoferrin may be added by dry mixing.

[0328] Table 15

[0329]

[0330]

[0331] Example 16

[0332] An immunity shot beverage containing human lactoferrin was prepared using the recipe in Table 16. Human lactoferrin was mixed with water and stirred for 10 minutes. A high protein juice (e.g., guava, orange, blackberry, banana, peach, kiwi, etc.) was selected, or a protein source (e.g., whey protein) could be included. The elderberry juice-based matrix was mixed with the human lactoferrin solution for several minutes and transferred to a plastic bottle (approximately 60 mL). The blended sample was cooled to approximately 4° C. and subjected to high pressure processing (HPP) at approximately 87,000 psi.

[0333] The bottles were tested for storage stability.

[0334] Table 16

[0335]

[0336]

[0337] Example 17

[0338] Prepare a batch of liquid nutritional (ready-to-feed) compositions using the recipe in Table 17. Heat reverse osmosis (RO) water to 150-160°F. Hydrate the pea protein isolate, cellulose gel and gum, and tapioca starch using high shear agitation. While maintaining high agitation, add oils (high oleic safflower and canola), lecithin, and a fat-soluble vitamin premix, followed by maltodextrin and sucralose. Add individual minerals, a trace mineral premix, a water-soluble vitamin premix, and other nutrients. During mixing, maintain the mixture at 140-145°F with high agitation, then pasteurize at 185-190°F for 30 seconds, homogenize at 2000-3500 PSI, and cool to 40°F. Add a 5% solution of vitamin C, a portion of the potassium hydroxide, a 10% solution of human lactoferrin, along with N&A flavoring and the remaining RO water to the cooled mixture to bring the total solids to 20-30% w / w. The blend is UHT treated and aseptically filled into 8 oz plastic bottles.

[0339] The composition is suitable for a FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) diet.

[0340] Table 17

[0341]

[0342]

[0343] Example 18

[0344] A batch of milk-based infant formula was prepared according to the recipe in Table 18. Lactose was hydrated in 140-145°F water at 45-55% solids, followed by the addition of protein, followed by the vegetable oil blend, ARA-DHA blend, and fat-soluble vitamin premix. Minerals and trace minerals, as well as vitamins and nutrients, were added to the mixture. During mixing, the mixture was maintained at 117-127°F with high agitation, then homogenized at 2000-3500 PSI and pasteurized at 170-180°F for 60 seconds. The mixture was cooled to 122-131°F and then spray-dried to a moisture content of 1.5-3.5%. Probiotics and human lactoferrin were added to the base formula by dry blending.

[0345] The resulting infant formula powder is packaged and added to water to form a drinkable infant formula.

[0346] Table 18

[0347]

[0348]

[0349] Example 19

[0350] A nutritional bar fortified with human milk oligosaccharides (HMOs) and human lactoferrin was prepared using the recipe in Table 19. The powdered ingredients, including HMO powder, human lactoferrin, and a vitamin premix were mixed. The liquid components of the binder syrup (maltitol syrup, chocolate syrup) were mixed and heated to 150°F. The dry components of the binder (e.g., cocoa powder) were mixed and heated to 180°F. The binder material was added to the mixed powdered ingredients and stirred with a spatula. Approximately 160 grams of the mixed sample was placed in a bar press mold and refrigerated for 5-10 minutes.

[0351] Table 19

[0352]

[0353]

[0354] Example 20

[0355] A batch of milk-based infant formula containing OPO was prepared according to the formula in Table 20. Lactose was hydrated in 150-160°F water at 45-55% solids, then protein was added, followed by a vegetable oil blend consisting of Sn2 palmitate fat (OPO), coconut oil, high oleic safflower oil or sunflower oil, soybean oil, and sunflower or canola lecithin.

[0356] ARA-DHA blend and fat-soluble vitamin premix are added. Minerals and trace minerals are added, along with vitamins and nutrients. During mixing, the mixture is maintained at 140-145°F with high agitation, then homogenized at 2000-3500 PSI and cooled at 40°F. The nucleotide premix is ​​added to the cooled mixture and stirred, then pasteurized at 170-180°F for 60 seconds and spray-dried to a moisture content of 1.5-3.5%. Probiotics and human lactoferrin are added to the base formula via dry blending.

[0357] Table 20

[0358]

[0359]

[0360] Example 21

[0361] A batch of prenatal formula powder (containing DHA and choline) was prepared according to the recipe in Table 21. The proteins (milk protein concentrate and soy protein isolate) were hydrated in 145-150°F water at 45-55% solids. Oils (high oleic safflower and canola), lecithin, DHA oil, and a fat-soluble vitamin premix were then added, followed by maltodextrin, sugar, and oligofructose. Minerals and trace minerals, as well as other vitamins and nutrients, were added. The mixture was maintained at 140-145°F with high agitation during mixing, then homogenized at 2000-3500 PSI and cooled at 40°F. A 5% solution of vitamin C, a portion of potassium hydroxide, a 10% solution of human lactoferrin (human lactoferrin can be added at another stage by dry mixing), and N&A flavoring were added to the cooled mixture. The mixture was pasteurized at 170-180°F for 60 seconds and then spray dried to a resulting moisture content of 2.5-4.0%.

[0362] Table 21

[0363]

[0364]

[0365] Example 22

[0366] Recombinant human lactoferrin (rhLF) produced from the yeast Pichia pastoris (Philips fulvidraco) was tested to determine the survivability of the oral administration form through the digestive system. Apo and holo forms were tested to determine the effect, if any, of iron loading. Native apo and holo forms of human and bovine lactoferrin served as controls.

[0367] Prepare simulated saliva at a pH of 7. Add lactoferrin sample and mix and incubate the mixture at 37°C for 2 minutes. Remove samples for SDS gel and Western blot analysis to determine the presence of LF.

[0368] Simulated gastric chyme and gastric fluid were prepared at a pH of 3. Porcine pepsin (Sigma) (2000 U / ml) was added. Simulated oral cavity samples were introduced into the simulated gastric fluid, and the mixture was mixed and incubated at 37°C for 2 hours. Samples were collected for SDS gel and Western blot analysis to determine the presence of LF.

[0369] Prepare simulated intestinal fluid. Add bile salts (Sigma) (10 mM), 100 U / ml porcine trypsin (Sigma), and 25 U / ml porcine chymotrypsin (Sigma). The pH of the mixture is 7. Simulated gastric fluid samples are introduced into the simulated intestinal fluid, mixed, and incubated at 37°C for 2 hours. Samples are collected for SDS gel and Western blot analysis to determine the presence of LF.

[0370] The assay showed the presence of intact protein and LF peptides in both apo and holo forms of the enzyme after the gastric phase. LF peptides were detected after intestinal digestion. The holo form produced slightly more peptides.

[0371] Recombinant human lactoferrin behaves similarly to human and bovine apo and holo forms of lactoferrin.

[0372] Example 23

[0373] Almond et al. (1996) suggested that allergens such as bovine β-lactoglobulin are pepsin-resistant. Therefore, the sensitivity of rhLF to pepsin was tested.

[0374] Simulated gastric fluid (SGF) at pH 1-2 and 1600 U / ml porcine pepsin (Sigma) were prepared. Native bovine and human LF as well as rhLF were tested in both holo (iron loaded) and apo forms.

[0375] After LF was added to SGF, the mixture was incubated for 60 minutes at 37° C. Samples were taken at 0, 0.5, 2, 5, 10, 30, and 60 minutes, and the digestion progress (if any) was monitored by SDS gel electrophoresis and Western blotting.

[0376] Pepsin was present in all samples over the 60-minute period, indicating the absence of auto- or self-digestion.

[0377] All LF samples were completely digested within 2 min.

[0378] Example 24

[0379] The immunogenicity of rhLF produced from Pichia pastoris (Philips fuscae) was tested. Cord blood mononuclear cells (StemCell) from infant girls were exposed to rhLF and natural bovine LF (Sigma) and human LF obtained from human breast milk. Samples were tested with or without LPS (Sigma) stimulation. The levels of IL-6, IL-1β, IL-8, TNF-α, IL-10, IL12p70, MCP1 / CCL-2, MIP-1α / CCL-3 and MIP-1β / CCL-4 were determined. All samples did not contain endotoxin.

[0380] Neither native human LF nor rhLF stimulated cytokine production in the presence or absence of LPS. On the other hand, human immune cells responded to the bovine protein.

[0381] In view of the present disclosure, all materials and methods disclosed and claimed herein can be made and performed without undue experimentation. The ingredients or reagents of the edible compositions disclosed herein can be commercially available in the form of food grade or pharmaceutical grade compounds, or can be prepared as provided in the art. Although the compositions and methods of the present disclosure have been described according to certain embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps or the sequence of steps described herein without departing from the concept, spirit and scope of the present disclosure. More specifically, certain chemically and physiologically related reagents can obviously replace the reagents described herein while achieving the same or similar results. All of these similar substitutes and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure.

Claims

1. An edible or drinkable composition comprising a protein and a recombinant human secretory protein.

2. The edible or drinkable composition of claim 1, wherein the secretory protein comprises a human milk protein.

3. The edible or drinkable composition of claim 1, wherein the recombinant human secretory protein comprises secretory IgA (sIgA), serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

4. The edible or drinkable composition of claim 1, wherein the recombinant human secretory protein comprises lactoferrin.

5. The edible or drinkable composition of claim 1, wherein the secreted protein comprises one or more of fucose, hybrid N-glycans, complex N-glycans, N-acetylgalactosamine, mannose, or sialic acid.

6. The edible or drinkable composition of claim 1, further comprising 2'-fucosyllactose, lacto-N-neotetraose, 3-fucosyllactose, 6'-sialyllactose, difucosyllactose or lacto-N-tetraose.

7. The edible or drinkable composition of claim 1, further comprising one or more lipids.

8. The edible or drinkable composition of claim 1, further comprising one or more minerals.

9. The edible or drinkable composition of claim 1 further comprising one or more vitamins.

10. The edible or drinkable composition of claim 1, further comprising one or more nucleotides.

11. The edible or drinkable composition of claim 1 , further comprising whey protein lipid concentrate, whole milk powder, whey protein concentrate, skim milk powder, milk protein concentrate or isolate, whey protein hydrolysate, or isolated protein.

12. The edible or drinkable composition of claim 1 , further comprising high oleic sunflower oil, mid oleic sunflower oil, safflower oil, coconut oil, canola oil, sunflower oil, ascorbyl palmitate, mixed tocopherol concentrate, Mortierella alpina oil, algae oil, soybean oil, or palm oil.

13. The edible or drinkable composition of claim 1, further comprising one or more of the following: calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, or selenium.

14. The edible or drinkable composition of claim 1, further comprising one or more of the following: vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, niacinamide, calcium pantothenate, vitamin B6, biotin, folic acid, pantothenic acid, vitamin B12, vitamin C, choline, inositol, and salts thereof.

15. The edible or drinkable composition of claim 1, further comprising a probiotic or a prebiotic.

16. The edible or drinkable composition of claim 1, wherein the recombinant human secretory protein is derived from mammalian cells, bacteria, or fungal cells.

17. The edible or drinkable composition of claim 1, wherein the recombinant human secretory protein is from yeast cells.

18. The edible or drinkable composition of claim 1, wherein the recombinant human secretory protein is derived from a cell of the genus Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidium, The yeast may be selected from the genera Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia.

19. The edible or drinkable composition of claim 1, wherein the recombinant human secretory protein comprises one or more N-glycans present on human proteins.

20. The edible or drinkable composition of claim 1, comprising from about 0.01% to about 2% by weight of the recombinant human secretory protein.

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