Compositions comprising casein and methods of producing the same

Abstract

To provide a dairy substitute or composition that has desirable flavor and performance characteristics.SOLUTION: Disclosed herein are compositions including casein, and methods for making these compositions.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 040,393, filed Aug. 21, 2014, the entire contents of which are incorporated by reference.

[0002] The present invention is directed to dairy substitutes, methods for making same, and compositions comprising animal-derived-free milk fat and protein for application in food products such as milk, butter, cheese, yogurt, and cream. [Background technology]

[0003] The global dairy market is estimated to be $500 billion with an average annual growth rate of 4%. A significant portion of the market is from cow's milk, but plant-based alternatives also account for $1 billion in the United States, with an estimated $700 million from lactose-free milk. Cow's milk is known to have four specialized caseins: α-s1-casein, α-s2-casein, β-casein, and κ-casein. Mammalian milk or mammalian produced milk is a highly complex fluid containing thousands of components (e.g., when all triglycerides are identified). Mammalian milk or mammalian produced milk contains water, a wide variety of different lipids, sugars, a variety of different proteins, and a variety of different inorganic salts and compounds. (See, e.g., Boland and Thompson (eds.), Milk Proteins from Expression to Food, Academic Press, 2014.) While mammalian produced milk, such as cow's milk, is considered by many to be an ideal source of nutrition, there is also a demand for a variety of milk alternatives to mammalian or mammalian produced milk (e.g., cow's milk), such as plant- or nut-based milks, e.g., soy milk, almond milk, or coconut milk, for reasons related to the allergenicity of mammalian or mammalian produced milk, lactose intolerance due to certain components, personal preferences, and the environmental benefits that may be gained by reducing dairy production.

[0004] For example, environmental impacts from wastewater from dairy production can result in significant levels of nitrates that can potentially contaminate groundwater. Groundwater is the primary water supply for many urban areas and farms where surface water supplies are limited. In the United States, half of the population relies completely or partially on groundwater, and similar figures are available for Europe. (See, for example, The Victoria State Government Department of Environment and Primary Industries website, www.depi.vic.gov.au / agriculture-and-food / dairy / managing-effluent / dairy-effluent-protecting-groundwater.) The presence of foodborne pathogens in milk results from direct contact with contaminated sources within the dairy farm environment and from udder secretions from infected animals. Disease outbreaks in humans have been traced to the ingestion of unpasteurized and also pasteurized milk. The major contaminants encountered in milk and dairy products typically include pesticide residues, heavy metals, and aflatoxin M1 (Awasthi et al., Indian J. Public Health, 56:95-99, 2012).

[0005] Existing dairy milk substitutes, such as soy milk, almond milk, or coconut milk, are inferior in both flavor and functionality; moreover, most of the industrial and cultural importance of dairy milk comes from its usefulness in derived products such as cheese, yogurt, cream, or butter. Although non-dairy plant-based milks address environmental and health concerns (and provide suitable flavor for a small percentage of the population), they almost always cannot be subjected to the same processes used for dairy milk to obtain such derived products. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Boland and Thompson (eds.), Milk Proteins from Expression to Food, Academic Press, 2014. [Non-Patent Document 2] The Victorian Government Department of Environment and Primary Industries website: www.depi.vic.gov.au / agriculture-and-food / dairy / managing-effluent / dairy-effluent-protecting-groundwater [Non-Patent Document 3] Awasthi et al., Indian J.Public Health, 56:95-99, 2012 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, what is needed is a dairy substitute or composition that has desirable flavor and efficacy characteristics, for example, a composition that replicates the flavor of dairy products, retains the ability to be used for dairy milk derivatives or downstream applications, and provides a nutritional profile similar to mammalian milk or mammalian produced milk while minimizing food borne pathogens and having a low environmental impact during production. [Means for solving the problem]

[0008] Summary of the Invention The present invention is based on the discovery that a composition can be created that has a similar flavor, similar appearance, similar nutritional value, similar aroma, and similar mouthfeel to mammalian produced milk using only a subset of the ingredients in mammalian produced milk.

[0009] Provided herein are compositions containing about 0.3 g / L to about 1.1 g / L of κ-casein protein; about 1.25 g / L to about 4.9 g / L of β-casein protein; one or more lipids in a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; one or more sweeteners in a total final concentration of about 0.1% to about 6% by weight; and ash in a total final concentration of about 0.15% to about 1.5% by weight, wherein the compositions are free of animal-derived components.

[0010] Also provided are compositions comprising about 0.3 g / L to about 1.1 g / L of κ-casein protein, about 1.25 g / L to about 4.9 g / L of β-casein protein, one or more lipids at a total final concentration of about 0% to about 45% by weight, one or more flavor compounds at a total final concentration of about 0.01% to about 6% by weight, one or more sweeteners at a total final concentration of about 0.1% to about 6% by weight, and ash at a total final concentration of about 0.15% to about 1.5% by weight, and which do not include at least one component found in mammalian produced milk, include at least one component not present in mammalian produced milk, and / or include at least one component at a higher or lower concentration compared to the concentration of the at least one component in mammalian produced milk. In some embodiments of these compositions, the composition includes at least one component selected from the group of calcium, phosphate, vitamin B complex, vitamin A, vitamin D, vitamin E, and vitamin K at a higher concentration compared to the concentration of the one or more components in mammalian produced milk. In some embodiments of these compositions, the composition does not contain at least one component found in mammalian produced milk, which is selected from the group consisting of lactose, bacteria, mycobacteria, allergens, viruses, prions, yeast, growth hormones, white blood cells, antibiotics, heavy metals, immunoglobulins, lactoferrin, lactoperoxidase, and lipase.In some embodiments of these compositions, the composition contains at least one component not present in mammalian produced milk, which is selected from the group consisting of artificial sweeteners, lipids of plant origin, β-casein protein with unglycosylated or non-mammalian glycosylation pattern, and κ-casein protein with unglycosylated or non-mammalian glycosylation pattern.

[0011] Also provided is a composition comprising: about 0.3 g / L to about 1.1 g / L of κ-casein protein, the κ-casein protein being unglycosylated or having a non-mammalian glycosylation pattern; about 1.25 g / L to about 4.9 g / L of β-casein protein, the β-casein protein being unglycosylated or having a non-mammalian glycosylation pattern; one or more lipids in a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; one or more sweeteners in a total final concentration of about 0.1% to about 6% by weight; and an ash content of about 0.15% to about 1.5% by weight.

[0012] Also provided are compositions comprising micelles containing κ-casein protein and β-casein protein, the micelles having diameters of about 50 nm to about 350 nm, and the κ-casein protein and β-casein protein being non-glycosylated or having a non-mammalian glycosylation pattern. In some embodiments of these methods, the compositions comprise micelles at a final concentration of about 2.0% to about 6% by weight. In some embodiments of these compositions, the ratio of β-casein protein to κ-casein protein in the micelles is about 3.5:1 to about 5.5:1 (e.g., about 4:1 to about 5:1). In some embodiments of these methods, the composition further comprises one or more lipids in a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; one or more sweeteners in a total final concentration of about 0.1% to about 6% by weight; and ash in a total final concentration of about 0.15% to about 1.5% by weight.

[0013] In some embodiments of any of the compositions described herein, the composition comprises about 0.27% to about 0.75% by weight of κ-casein protein and about 1.23% to about 3.27% by weight of β-casein. In some embodiments of any of the compositions described herein, the total final concentration of one or more lipids is about 0% to about 4.5% by weight.

[0014] In some embodiments of any of the compositions described herein, one or more lipids are selected from the group consisting of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acid, and phospholipid.In some embodiments of any of the compositions described herein, the composition comprises one or more of the following: sunflower oil with a final concentration of about 1% to about 28% by weight; coconut oil with a final concentration of about 0.5% to about 14% by weight; tributyrin with a final concentration of about 0.05% to about 1.0% by weight; mono- and diglycerides with a total final concentration of about 0.08% to about 1.2% by weight; free fatty acid with a total final concentration of about 0.02% to about 0.28% by weight; and phospholipid with a total final concentration of about 0.02% to about 0.3% by weight. In some embodiments of any of the compositions described herein, the free fatty acid comprises at least one fatty acid selected from the group consisting of butyric acid, caproic acid, caprylic acid, and capric acid.In some embodiments of any of the compositions described herein, the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid.In some embodiments of any of the compositions described herein, the monoglyceride and diglyceride are plant-derived monoglycerides and diglycerides, or bacterial-derived monoglycerides and diglycerides.

[0015] In some embodiments of any of the compositions described herein, the flavor compound comprises at least one flavor compound selected from the group consisting of δ-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, and δ-undecalactone.In some embodiments of any of the compositions described herein, one or more sweeteners are saccharides.In some embodiments of any of the compositions described herein, the saccharides are selected from the group consisting of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose.In some embodiments of any of the compositions described herein, one or more sweeteners are artificial sweeteners. In some embodiments of any of the compositions described herein, the artificial sweetener is selected from the group of stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000.

[0016] In some embodiments of any of the compositions described herein, the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride.In some embodiments of any of the compositions described herein, the ash comprises one or more (e.g., one, two, or three) of CaCl2, KH2PO4, and Na3 citrate.In some embodiments of any of the compositions described herein, the final concentration of CaCl2 is about 0.05 g / L to about 0.2 g / L; the final concentration of KH2PO4 is about 0.2 g / L to about 0.4 g / L; the final concentration of Na3 citrate is about 0.1 g / L to about 0.3 g / L.

[0017] In some embodiments of any of the compositions described herein, the κ-casein protein is from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth κ-casein protein. In some embodiments of any of the compositions described herein, the β-casein protein is from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth β-casein protein.

[0018] In some embodiments of any of the compositions described herein, the composition further comprises alpha-lactalbumin protein at a final concentration of about 0.4% to about 2.5% by weight; and / or β-lactoglobulin protein at a final concentration of about 2.5% to about 4.5% by weight. In some embodiments of any of the methods described herein, the alpha-lactalbumin protein is cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth alpha-lactalbumin protein. In some embodiments of any of the compositions described herein, the β-lactoglobulin protein is a β-lactoglobulin protein from a cow, a human, a sheep, a goat, a buffalo, a camel, a horse, a donkey, a lemur, a panda, a guinea pig, a squirrel, a bear, a macaque, a gorilla, a chimpanzee, a mountain goat, a monkey, an ape, a cat, a dog, a wallaby, a rat, a mouse, an elephant, a possum, a rabbit, a whale, a baboon, a gibbon, an orangutan, a mandrill, a pig, a wolf, a fox, a lion, a tiger, an echidna, or a woolly mammoth.

[0019] In some embodiments of any of the compositions described herein, the composition further comprises α-S1-casein protein at a final concentration of about 11% to about 16% by weight; and / or α-S2-casein protein at a final concentration of about 2% to about 5% by weight. In some embodiments of any of the compositions described herein, the α-S1-casein protein is selected from the group consisting of α-S1 casein protein from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. and / or the α-S2-casein protein is an α-S2-casein protein from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

[0020] In some embodiments of any of the compositions described herein, the composition further comprises one or more of serum albumin, lactoferrin, and transferrin. In some embodiments of any of the compositions described herein, the serum albumin protein is bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth serum albumin; lactoferrin is bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat , monkey, ape, cat, dog, wallaby, rat, mouse, elephant, pouch, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth lactoferrin; and / or the transferrin is a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, pouch, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth transferrin protein.

[0021] Some embodiments of any of the compositions described herein further comprise one or more color correcting agents. In some embodiments of any of the compositions described herein, the one or more color correcting agents are β-carotene or annatto. In some embodiments of any of the compositions described herein, the pH of the composition is about 6.2 to about 7.2 (e.g., about 6.2 to about 6.8).

[0022] Also provided are compositions comprising mammalian produced milk or processed mammalian produced milk; and one or both of κ-casein protein with a glycosylation pattern that is not glycosylated or that is not mammalian, and β-casein protein with a glycosylation pattern that is not glycosylated or that is not mammalian. In some embodiments of these methods, the final concentration of κ-casein protein with a glycosylation pattern that is not glycosylated or that is not mammalian in the composition is 0.02% to about 3.0% by weight. In some embodiments of these methods, the final concentration of β-casein protein with a glycosylation pattern that is not glycosylated or that is not mammalian in the composition is 0.02% to about 3.0% by weight. In some embodiments of these methods, the final concentration of κ-casein protein that is unglycosylated and / or has a non-mammalian glycosylation pattern in the composition is from about 0.02% to about 0.6% by weight; the final concentration of β-casein protein that is unglycosylated and / or has a non-mammalian glycosylation pattern in the composition is from about 0.02% to about 2.5% by weight.

[0023] Also provided are powder compositions comprising κ-casein protein at a final concentration of about 3.6% to about 5.4% by weight; β-casein protein at a final concentration of about 16.3% to about 24.5% by weight; a sweetener at a final concentration of about 35% to about 40% by weight; one or more lipids at a final concentration of about 25% to about 30% by weight; ash at a final concentration of about 5% to about 7% by weight; and water at a final concentration of about 2% to about 5% by weight, wherein the κ-casein protein is unglycosylated and / or has a non-mammalian glycosylation pattern and / or the β-casein protein is unglycosylated and / or has a non-mammalian glycosylation pattern.

[0024] Also provided are nucleic acids comprising a promoter; a sequence encoding a signal sequence; a sequence encoding a milk protein; and a yeast termination sequence, the promoter being operably linked to the signal sequence, the signal sequence being operably linked to the sequence encoding the milk protein, and the termination sequence being operably linked to the sequence encoding the milk protein. In some embodiments of these nucleic acids, the promoter is a constitutive promoter. In some embodiments of these nucleic acids, the promoter is an inducible promoter. In some embodiments of these nucleic acids, the signal sequence is a signal sequence from the encoded milk protein or a different milk protein, or is a signal sequence from a yeast mating factor. In some embodiments of these nucleic acids, the encoded milk protein is selected from the group consisting of β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin, or transferrin. In some embodiments of these nucleic acids, the nucleic acid comprises a bacterial origin of replication. In some embodiments of these nucleic acids, the nucleic acid further comprises a selection marker. In some embodiments of these nucleic acids, the selection marker is an antibiotic resistance gene.

[0025] Some embodiments of these nucleic acids further comprise an additional promoter sequence; an additional sequence encoding a signal sequence; a sequence encoding an additional milk protein; and an additional yeast termination sequence, where the additional promoter sequence is operably linked to the additional sequence encoding a signal sequence, where the sequence encoding the signal sequence is operably linked to the sequence encoding the additional milk protein, where the sequence encoding the additional milk protein is operably linked to the additional yeast termination sequence.

[0026] Also provided are host cells comprising any of the nucleic acids described herein. In some embodiments of these host cells, the host cell is a yeast strain (e.g., Kluyveromyces sp., Pichia sp., Saccharomyces sp., Tetrahymena sp., Yarrowia sp., Hansenula sp., Blastobotrys sp., Candida sp., Zygosaccharomyces sp., or Debaryomyces sp.).

[0027] Also provided herein is a method of making a recombinant milk protein that is unglycosylated or has a non-mammalian glycosylation pattern, the method comprising culturing any of the host cells described herein in a culture medium under conditions sufficient to allow secretion of the milk protein that is unglycosylated or has a non-mammalian glycosylation pattern, and recovering the milk protein that is unglycosylated or has a non-mammalian glycosylation pattern from the culture medium.

[0028] Also provided is a method of making micelles comprising β-casein which is unglycosylated or has a non-mammalian glycosylation pattern and κ-casein which is unglycosylated or has a non-mammalian glycosylation pattern, comprising culturing any of the host cells provided herein in a culture medium under conditions sufficient to allow release of the micelles from the host cells, wherein the host cells comprise nucleic acids comprising sequences encoding β-casein and sequences encoding κ-casein.

[0029] Also provided is a method of supplementing mammalian produced milk comprising the steps of providing mammalian produced milk or processed mammalian produced milk; and mixing with the milk at least one of: β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern; κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern; and micelles comprising β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern and κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern.

[0030] Also provided is a method of making a composition, comprising the steps of: sonicating a liquid containing a protein mixture containing β-casein protein and casein κ-protein, or containing micelles containing β-casein protein and κ-casein protein; mixing ash into the liquid; adding a mixture of one or more lipids, one or more flavor compounds, and one or more color correctors to the liquid, and sonicating the liquid; and adding one or more sweeteners to the liquid, thereby making the composition. In some embodiments of these methods, the β-casein protein is non-glycosylated or has a non-mammalian glycosylation pattern, and / or the κ-casein protein is non-glycosylated or has a non-mammalian glycosylation pattern. In some embodiments of these methods, the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride. In some embodiments of these methods, the added ash comprises one or more (e.g., one, two, or three) of CaCl2, KH2PO4, and Na3 citrate. In some embodiments of these methods, the one or more lipids comprise at least one of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids. In some embodiments of these methods, the free fatty acids comprise at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid. In some embodiments of these methods, the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid. In some embodiments of these methods, the mono- and diglycerides are plant-derived mono- and diglycerides or bacterial-derived mono- and diglycerides. In some embodiments of these methods, the flavor compound comprises at least one flavor compound selected from the group consisting of delta-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, gamma-undecalactone, and delta-undecalactone.In some embodiments of these methods, the one or more color correcting agents are beta-carotene or annatto. In some embodiments of these methods, the one or more sweeteners are saccharides. In some embodiments of these methods, the saccharides are selected from the group consisting of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose. In some embodiments of these methods, the one or more sweeteners are artificial sweeteners. In some embodiments of these methods, the artificial sweetener is selected from the group consisting of stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000. In some embodiments of these methods, the pH of the liquid is between about 6.2 and about 7.4 (e.g., about 6.4 to about 6.8).In some embodiments of these methods, the β-casein protein is from a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth β-casein protein. and / or the κ-casein protein is bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth κ-casein protein. In some embodiments of these methods, the protein mixture further comprises one or more proteins selected from the group of α-lactalbumin, β-lactoglobulin, α-S1-casein, α-S2-casein, lactoferrin, transferrin, and serum albumin.

[0031] Also provided are compositions made by any of the methods described herein.

[0032] Also provided is a method of making butter, cheese, caseinate, or yogurt, comprising providing any of the compositions described herein; and using any of the compositions described herein as a starting material to make butter, cheese, caseinate, or yogurt.

[0033] Also provided are kits that include (a) a mixture of one or more milk proteins, one or more fats, and one or more flavor compounds; and (b) a mixture of ash and at least one sweetener. In some embodiments of these kits, the one or more milk proteins are selected from the group of β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, α-S1-casein, α-S2-casein, lactoferrin, transferrin, and serum albumin. In some embodiments of these kits, the one or more milk proteins are milk proteins of cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. In some embodiments of these kits, the one or more fats are selected from the group consisting of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids. In some embodiments of these kits, the free fatty acid comprises at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid. In some embodiments of these kits, the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid. In some embodiments of these kits, the mono- and diglycerides are plant-derived mono- and diglycerides, or bacterial-derived mono- and diglycerides. In some embodiments of these kits, the flavor compound comprises at least one flavor compound selected from the group consisting of δ-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, and δ-undecalactone. In some embodiments of these kits, the mixture in (a) further comprises one or more color correctors.In some embodiments of these kits, the one or more color correcting agents are β-carotene or annatto. In some embodiments of these kits, the one or more sweetening agents are saccharides (e.g., saccharides selected from the group of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose). In some embodiments of these kits, the one or more sweetening agents are artificial sweeteners (e.g., artificial sweeteners selected from the group consisting of stevia, aspartame, cyclamate, saccharin, sucralose, mogrosides, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000). In some embodiments of any of these kits, the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride. In some embodiments of these kits, the ash comprises one or more (e.g., one, two, or three) of CaCl2, KH2PO4, and Nacitrate3. Some embodiments of these kits further comprise instructions for making any of the compositions described herein.

[0034] Also provided are kits that include at least one of the nucleic acids described herein.

[0035] Also provided herein are dairy substitute foods comprising one or more isolated milk protein components, fat, carbohydrates, and ash. In some embodiments of these dairy substitute foods, the food is of non-animal origin. In some embodiments of these substitute foods, the food comprises milk, butter, cheese, caseinate, yogurt, and cream. In some embodiments of these dairy substitute foods, the isolated milk protein components comprise casein and whey protein. In some embodiments of these dairy substitute foods, the casein protein further comprises alpha-s1-casein, alpha-s2-casein, beta-casein, and kappa-casein. In some embodiments of these dairy substitute foods, the casein protein further comprises alpha-s1-casein, beta-casein, and kappa-casein. In some embodiments of these dairy substitute foods, the casein protein further comprises components for micelle formation. In some embodiments of these dairy substitute foods, the casein protein exhibits coagulation properties at pH 4.0-6.0. In some embodiments of these dairy substitute foods, the casein protein is 2.5% (w / v) or more and 10% (w / v) or less. In some embodiments of these dairy substitute foods, the whey protein further comprises beta-lactoglobulin and alpha-lactalbumin. In some embodiments of these dairy substitute foods, the whey protein forms a polymer matrix gel. In some embodiments of these dairy substitute foods, the whey protein is at least 0.1% (w / v) and 1% (w / v) or less. In some embodiments of these dairy substitute foods, the one or more milk protein components are isolated from a microorganism. In some embodiments of any of these dairy substitute foods, the one or more milk protein components are isolated from a recombinant microorganism. In some embodiments of these dairy substitute foods, the one or more milk protein components are synthesized in a eukaryotic microorganism.In some embodiments of these dairy substitute food products, the eukaryotic microorganism comprises a yeast. In some embodiments of these dairy substitute food products, the yeast comprises Kluyveromyces spp., Pichia spp., Saccharomyces spp., and Tetrahymena spp.

[0036] In some embodiments of these dairy substitute foods, the fat comprises a triglyceride. In some embodiments of these dairy substitute foods, the fat comprises a high oleic oil. In some embodiments of these dairy substitute foods, the high oleic oil further comprises one or more of monounsaturated oil, oleic oil, linoleic oil, linolenic oil, and saturated oil. In some embodiments of these dairy substitute foods, the fat comprises a short chain fatty acid. In some embodiments of these dairy substitute foods, the short chain fatty acid comprises butanoic acid, hexanoic acid, octanoic acid, and decanoic acid. In some embodiments of these dairy substitute foods, one or more of the fats comprise transesterified fatty acids. In some embodiments of these dairy substitute foods, one or more of the fats are isolated from a plant. In some embodiments of these dairy substitute foods, the fat isolated from plants is selected from one or more of the following: sunflower oil, corn oil, olive oil, soybean oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, canola oil, safflower oil, flax oil, date oil, date kernel oil, date fruit oil, coconut oil, babassu oil, shea butter oil, mango butter oil, cocoa butter oil, wheat germ oil and rice bran oil. In some embodiments of these dairy substitute foods, the sugar comprises galactose, sucrose, glucose, fructose and maltose. In some embodiments of these dairy substitute foods, the dairy substitute foods are essentially free of lactose. In some embodiments of these dairy substitute foods, the ash comprises minerals. In some embodiments of these dairy substitute foods, the minerals further comprise one or more of the following: sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc, chloride, manganese, selenium, iodine, retinol, carotene, vitamins, vitamin D, vitamin E, vitamin B12, thiamine, and riboflavin. In some embodiments of these dairy substitute foods, the ash comprises anions.In some embodiments of these dairy substitutes, the minerals further comprise one or more of the following: phosphate, citrate, sulfate, carbonate, and chloride.

[0037] Also provided are methods of making a dairy substitute comprising contacting one or more isolated milk protein components, interesterified fat, carbohydrates and ash. Some embodiments of these methods further comprise isolating one or more milk protein components from a lower eukaryote.

[0038] Also provided are methods of modifying the flavor profile of a dairy substitute product, comprising modulating a combination of fatty acids in a mixture comprising milk protein components, carbohydrates, and ash. In some embodiments of these methods, the modulating step comprises a triglyceride comprising three oleic acids and a short-chain triglyceride comprising butyric acid, one hexanoic acid, and one octanoic acid. In some embodiments of these methods, the modulating step comprises increasing or decreasing one or more fatty acids comprising butyric acid, caproic acid, caprylic acid, and capric acid. In some embodiments of these methods, the flavor profile of the dairy substitute product mimics the flavor profile of one or more dairy products. In some embodiments of these methods, the flavor profile of one or more dairy foods comprises cow's milk, goat's milk, soy milk, almond milk, and coconut milk. In some embodiments of these methods, the flavor profile comprises one or more sensory impressions selected from buttery, nutty, sweet, sour, fruity, floral, bitter, woody, earthy, beany, spicy, metallic, sweet, musty, oily and vinegary.

[0039] Disclosed herein are methods and compositions for making dairy substitutes. In some embodiments, methods and compositions for dairy substitute foods are provided, comprising one or more isolated milk protein components, fat, carbohydrates and ash. In certain embodiments, methods and compositions for dairy substitute compositions are provided, comprising casein protein and whey protein, essentially free of animal products, and with a preferred (w / v) ratio of casein protein to whey protein. In certain other embodiments, methods are provided for modulating the flavor profile of dairy substitute foods, comprising modulating the fatty acid content in a mixture comprising milk protein components, fat, carbohydrates and ash. The preferred modulating step comprises increasing or decreasing one or more fatty acids, including butyric acid, caproic acid, caprylic acid and capric acid. In further embodiments, the methods and compositions of the present invention provide milk protein components and fats in a desired (w / v) ratio.

[0040] In various aspects, the methods and compositions of the present invention provide dairy substitutes that still retain their functional characteristics and organoleptic properties. In some embodiments, the core function can be, but is not limited to, achieving a similar nutritional profile as traditional dairy products and replicating one or more, if not all, of this core function.

[0041] In other embodiments, the core function may be, but is not limited to, replicating the same or similar sensory characteristics of traditional dairy-based products, including, but not limited to, taste, appearance, handling and mouthfeel, desired density, structure, texture, elasticity, springiness, coagulation, binding, leavening, aeration, foaming, creaminess, and emulsification.

[0042] The preferred methods and compositions provide dairy substitute products such as milk, butter, cheese, yogurt, and cream. Presented herein is a formula for a non-dairy milk substitute, comprising one or more isolated milk protein components (3.3%), fat (4.0%), carbohydrates (2.4%), and ash (0.7%) (w / v). Varying the fat content through modulating triglyceride levels and fatty acid composition of triglycerides enhances the flavor profile of the non-dairy milk substitute.

[0043] Advantages of the methods and dairy substitute compositions include the reduction or elimination of antibiotic residues, heavy metals, bacteria and adulterations commonly found in natural dairy-containing products, as well as reduced environmental impact.

[0044] Thus, certain aspects of the present invention provide animal-derived-free dairy substitutes that have desirable flavor characteristics, e.g., replicate dairy flavor and preserve downstream applications of dairy milk, while minimizing food-borne pathogens and having a low environmental impact.

[0045] In this specification, unless otherwise specified, the scientific and technical terms used in the context of the present invention shall have the meanings commonly understood by those skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include singularities.In general, the terminology used in the context of dairy processing, biochemistry, enzymology, molecular cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, and the techniques therefor, described herein, are well-known terminology and techniques and are commonly used in the art.

[0046] Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0047] All publications, patents, patent applications, sequences, database entries, and other references mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, patent application, sequence, database entry, or other reference was specifically and individually indicated to be incorporated by reference. In the event of a conflict of interest, the present specification, including definitions, shall control.

[0048] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0049] The terminology and descriptions used herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" may be intended to include the plural forms as well, unless the context indicates otherwise. The terms "including," "including," "having," "having," "with," or variations thereof, are intended to be inclusive, as is the term "including."

[0050] An "isolated" RNA, DNA or mixed polymer is a polymer that has been substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, such as ribosomes, polymerases, and genomic sequences with which it is naturally associated.

[0051] As used herein, an "isolated" organic molecule (e.g., a fatty acid or SCFA) is an organic molecule that is substantially separated from the intracellular components (membrane lipids, chromosomes, proteins) of the host cell from which it is derived. The term "isolated" as used herein with respect to a protein indicates that a preparation of the protein is at least 60% pure, e.g., greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. While certain isolated biomolecules may be purified to near homogeneity, the term does not require that the biomolecule be separated from all other chemicals.

[0052] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA that contain non-natural nucleotide analogs, non-natural internucleotide linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, or circular.

[0053] The term "SCFA" is an abbreviation for short chain fatty acids, the term "HOSO" is an abbreviation for high oleic sunflower oil, and "SCTG" is an abbreviation for short chain triglycerides.

[0054] The term "milk protein components" refers to proteins or protein equivalents and variants, including their subunits, derived from various sources and found in milk, such as casein, whey or a combination of casein and whey, as further defined herein.

[0055] The term "milk protein" refers to a protein found in mammalian produced milk or a protein having a sequence at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the sequence of a protein found in mammalian produced milk. Non-limiting examples of milk proteins include β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin, transferrin, and serum albumin. Additional milk proteins are known in the art.

[0056] The term "casein protein" is known in the art and refers to a family of proteins present in mammalian produced milk that can self-assemble with other proteins in the family to form micelles and / or precipitate from aqueous solutions at acidic pH. Non-limiting examples of casein proteins include β-casein, κ-casein, α-S1-casein, and α-S2-casein. Non-limiting examples of casein protein sequences from different mammals are presented herein. Additional sequences of other mammalian caseins are known in the art.

[0057] The term "mammalian produced milk" is known in the art and means milk produced by a mammal.

[0058] The term "processed mammalian produced milk" means mammalian produced milk that has been processed using one or more steps known in the dairy manufacturing industry (e.g., homogenization, pasteurization, irradiation, or supplementation).

[0059] The term "mammal-derived component" refers to a molecule or compound (e.g., a protein, lipid, or nucleic acid) obtained from within a mammal or from a fluid or solid produced by a mammal.

[0060] The term "milk component" or "milk component" refers to a molecule, compound, element, or ion that is present in milk produced by a mammal.

[0061] The term "non-mammalian glycosylation pattern" refers to a difference in one or more glycosylation sites in a protein and / or a difference in the amount and / or type of glycosylation at one or more sites of a protein produced and post-translationally modified in a non-mammalian cell (e.g., a yeast cell, an insect cell, or a bacterial cell) compared to the same protein produced and post-translationally modified in a reference protein (e.g., a mammalian cell, e.g., a CHO cell, a MEK cell, or a mammalian breast cell).

[0062] The term "lipid" refers to one or more molecules (e.g., biomolecules) that contain fatty acyl groups (e.g., saturated or unsaturated acyl chains). For example, the term lipid includes oils, phospholipids, free fatty acids, phospholipids, monoglycerides, diglycerides, and triglycerides. Non-limiting examples of lipids are described herein. Further examples of lipids are known in the art.

[0063] The term "plant-derived lipid" refers to lipids obtained from and / or produced by plants (eg, monocotyledonous or dicotyledonous plants).

[0064] The term "sweetening agent" refers to a saccharide (e.g., a monosaccharide, disaccharide, or polysaccharide) or an artificial sweetener (e.g., a small molecule artificial sweetener or a protein-based artificial sweetener) that, when added to a composition, provides a sweet taste when ingested by a mammal, such as a human. Non-limiting examples of sweetening agents are described herein. Additional examples of sweetening agents are known in the art.

[0065] The term "ash" is a term known in the art and refers to one or more ions, elements, minerals, and / or compounds that may be found in mammalian produced milk. Non-limiting ions, elements, minerals, and compounds that may be found in mammalian produced milk are described herein. Additional ions, elements, minerals, and compounds that may be found in mammalian produced milk are also known in the art.

[0066] The term "color correcting agent" or "colorant" refers to an agent added to a composition to modulate the color of the composition, e.g., to make the color of the composition appear more similar to mammalian produced milk. Non-limiting examples of color correcting agents or colorants include beta-carotene and annatto. Other examples of color correcting agents are known in the art. Color correcting agents or colorants can be made or obtained from plants.

[0067] The term "micelle" refers to a generally (or roughly) spherical supramolecular structure present as a dispersion in a composition. A micelle may, for example, have a surface consisting of a charged outer layer. A micelle may encapsulate one or more biomolecules. For example, a micelle may encapsulate two or more proteins (e.g., β-casein protein and κ-casein protein). The diameter of the micelles can be between about 10 nm and about 350 nm. Further aspects and characteristics of micelles are known in the art.

[0068] The phrase "concentration of a component in milk produced by a mammal" means the concentration of the component in milk produced by a mammal or the average concentration of the component in milk produced by a population of mammals of the same species.

[0069] The term "attenuating..." as used herein generally refers to functional deletions, including mutations, partial or complete deletions, insertions, or other mutations made to a gene sequence or a sequence controlling the transcription of a gene sequence that reduces or inhibits the production of a gene product or renders the gene product non-functional. In some cases, functional deletions are described as knockout mutations. Attenuation also includes altering the amino acid sequence by modifying the nucleic acid sequence, placing the gene under the control of a promoter with low activity, downregulation, expressing an interfering RNA, ribozymes or antisense sequences targeting the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or reactant (non-pathway specific feedback) is reduced so that the enzyme activity is not affected by the presence of a compound. In other cases, an enzyme that is altered to have reduced activity may be referred to as attenuated.

[0070] Deletion: The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, such that the flanking regions are joined together.

[0071] Knockout: A gene whose expression or activity level is reduced to zero.In some cases, a gene is knocked out through the deletion of part or all of its coding sequence.In other cases, a gene is knocked out through the introduction of one or more nucleotides into its open reading frame, which results in the translation of a nonsense or otherwise non-functional protein product.

[0072] The term "synthetic milk substitute" refers to a composition that is similar to, similar to, equivalent to, or nearly identical to dairy milk.

[0073] The term "flavor" refers to the taste and / or aroma of a food or beverage.

[0074] The term "recombinant" is a term known in the art. When referring to a nucleic acid (e.g., a gene), the term "recombinant" can be used to describe, for example, a nucleic acid that has been removed from its natural environment, a nucleic acid that is not associated with all or a portion of a nucleic acid that is adjacent or proximal to the nucleic acid as it is found in nature, a nucleic acid that is operably linked to a nucleic acid with which it is not linked in nature, or a nucleic acid that does not occur in nature. The term "recombinant" can be used to describe, for example, a cloned DNA isolate, or a nucleic acid that contains chemically synthesized nucleotide analogs. When "recombinant" is used to describe a protein, it can refer, for example, to a protein that is produced in a different species or type of cell compared to the species or type of cell that naturally produces the protein.

[0075] As used herein, an endogenous nucleic acid sequence (or the encoded protein product of that sequence) within the genome of an organism is considered "recombinant" herein when a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence such that expression of the endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, whether the heterologous sequence is itself endogenous (originating from the same host cell or its descendants) or exogenous (originating from a different host cell or its descendants). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that the expression pattern of this gene is altered. The gene is now "recombinant" because it is separated from at least some of the sequences that naturally flank it.

[0076] A nucleic acid is also considered "recombinant" if it contains any modification that does not occur naturally in the corresponding nucleic acid in a genome. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation that has been artificially introduced, e.g., by human intervention. "Recombinant nucleic acid" also includes nucleic acids integrated into a host cell chromosome at a heterologous site, and nucleic acid constructs that exist as episomes.

[0077] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues that are the same in two sequences when sequenced for maximum correspondence. The length of sequence identity comparison can span a stretch of at least about 9 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. Many different algorithms are known in the art and can be used to measure the identity of nucleotide sequences. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, a program in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignment and percent sequence identity of the best overlap region between the query sequence and the search sequence. See, e.g., Pearson, Methods Enzymol., 183:63-98, 1990, which is incorporated by reference in its entirety. For example, percent sequence identity between nucleic acid sequences may be determined using FASTA with its default parameters (using a word size of 6 and NOPAM coefficients in the scoring matrix), or may be determined using Gap with its default parameters as provided by GCG Version 6.1, which is incorporated by reference herein.Alternatively, sequences can be compared using the computer programs BLAST (Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish and States, Nature Genet., 3:266-272, 1993; Madden et al., Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; Zhang and Madden, Genome Res., 7:649-656, 1997), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997).

[0078] The terms "substantial homology" or "substantial similarity" when referring to a nucleic acid or a fragment thereof, indicates that when sequenced with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is at least about 76%, 80%, 85%, preferably at least about 90% nucleotide sequence identity, and more preferably at least about 95%, 96%, 97%, 98% or 99% nucleotide sequence identity, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST or Gap, discussed above.

[0079] Alternatively, substantial homology or similarity exists when a nucleic acid or a fragment thereof hybridizes with another nucleic acid, another nucleic acid strand, or its complementary strand under stringent hybridization conditions. In the context of nucleic acid hybridization experiments, "stringent hybridization conditions" and "stringent washing conditions" depend on many different physical parameters. Nucleic acid hybridization is influenced by conditions such as salt concentration, temperature, solvent, base composition of hybridizing molecular species, length of complementary region, and number of nucleotide base mismatches between hybridizing nucleic acids, as can be easily appreciated by those skilled in the art. Those skilled in the art are familiar with how to vary these parameters to achieve a particular stringency of hybridization.

[0080] In general, "stringent hybridization" is performed at about 25°C below the melting temperature (Tm) of a specific DNA hybrid under a particular set of conditions. "Stringent washing" is performed at about 5°C below the Tm of a specific DNA hybrid under a particular set of conditions. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 9.51, 1989, which is incorporated herein by reference. For purposes herein, "stringent conditions" are defined as, for solution phase hybridization, aqueous hybridization (i.e., containing no formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS for 20 minutes at 65° C. One of skill in the art will appreciate that hybridization at 65° C. will occur at different rates depending on a number of factors, including the length and percent identity of the hybridizing sequences.

[0081] The nucleic acids (also referred to as polynucleotides) of the present invention can include both the sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the above. They can be chemically or biochemically modified and can contain non-natural or derivatized nucleotide bases, as can be easily recognized by those skilled in the art. Such modifications include, for example, labels, methylation, replacement of one or more of natural nucleotides with analogs, internucleotide modifications such as non-charged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidites, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalating agents (e.g., acridines, psoralens, etc.), chelating agents, alkylating agents, and linkage modifications (e.g., alpha-anomeric nucleic acids, etc.). Examples of modified nucleotides are described in Malyshev et al., Nature, 509:385-388, 2014; and Li et al., J.Am.Chem.Soc., 136:826-829, 2014. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to designated sequences via hydrogen bonds and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide bonds replace phosphate bonds in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structures such as the modifications found in "locked" nucleic acids.

[0082] The term "mutated" as applied to a nucleic acid sequence means that nucleotides within a nucleic acid sequence can be inserted, deleted, or changed compared to a reference nucleic acid sequence. A single change can be made at a locus (point mutation), or multiple nucleotides can be inserted, deleted, or changed at a single locus. In addition, one or more changes can be made at any number of loci within a nucleic acid sequence. Nucleic acid sequences may be mutated by any method known in the art, including, but not limited to, mutagenesis methods such as "error-prone PCR" (a process for performing PCR under conditions in which the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15, 1989, and Caldwell and Joyce, PCR Methods Applic., 2:28-33, 1992); and "oligonucleotide-directed mutagenesis" (a process that allows for the generation of site-specific mutations within any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science, 241:53-57, 1988).

[0083] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules, such as those resulting from amplification by polymerase chain reaction (PCR) or treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Still further, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0084] Promoters useful for expressing the recombinant genes described herein include both constitutive promoters and inducible / repressible promoters. An example of an inducible / repressible promoter includes the galactose-inducible promoter (e.g., PLAC4-PBI). When multiple recombinant genes are expressed in an engineered yeast, the different genes may be controlled by different promoters, may be controlled by the same promoter in separate operons, or the expression of two or more genes may be controlled by a single promoter as part of an operon.

[0085] The term "operably linked" expression control sequence refers to a linkage in which the expression control sequence is adjacent to a gene of interest and controls the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0086] The terms "expression control sequence" or "regulatory sequence" are used interchangeably and, as used herein, refer to polynucleotide sequences necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences that control the transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences, and enhancer sequences; signals for efficient RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0087] The terms "transfect", "transfection", "transfecting" and the like refer to the introduction of heterologous nucleic acid into eukaryotic cells, both higher and lower eukaryotic cells. Historically, the term "transformation" has been used to describe the introduction of nucleic acid into yeast or fungal cells, but herein the term "transfection" is used to refer to the introduction of nucleic acid into any eukaryotic cell, including yeast and fungal cells.

[0088] The term "recombinant host cell" ("expression host cell", "expression host system", "expression system" or simply "host cell") as used herein is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications occur in successive generations due to mutations or environmental influences, such progeny cells may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell, a cell line grown in culture, or a cell present in a living tissue or organism. Preferred host cells are yeast and fungi.

[0089] The term "yeasts and filamentous fungi" includes any Kluyveromyces species, such as Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces species, such as Saccharomyces cerevisiae, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta), Pichia lindneri (Pichia lindneri), and the like. Pichia species such as Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Hansenula polymorpha, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp.), including, but not limited to, Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0090] The term "predominantly" or variations thereof as used herein is understood to mean, for example, a) in the context of fat, the amount of a particular fatty acid composition relative to the total amount of fatty acid compositions; b) in the context of protein, the amount of a particular protein composition (e.g., β-casein) relative to the total amount of protein compositions (e.g., α-casein, β-casein, and κ-casein).

[0091] The terms "about," "approximately," or "similar to" mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined and may depend on the limitations of the measurement system. It should be understood that all ranges and amounts set forth below are approximate and are not intended to limit the invention. When ranges and numbers are used, they may be approximate to include statistical ranges or measurement errors or variations. In some embodiments, for example, measurements may be ±10%.

[0092] The phrase "essentially free of" is used to indicate that the indicated component, if present, is present in an amount that does not contribute, or contributes only minimally, to the properties of the composition. In various embodiments where the composition is essentially free of a particular component, the component is present in less than a functional amount. In various embodiments, the component may be present in trace amounts. The specific limit varies depending on the nature of the component, but may be selected, for example, from less than 10% by weight, less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, or less than 0.5% by weight.

[0093] As used herein, the term "essentially free of" a particular carbohydrate, such as lactose, is used to indicate that the food composition is substantially devoid of carbohydrate residues. When expressed in the context of purity, "essentially free of..." means that the amount of carbohydrate residues does not exceed 10%, preferably below 5%, more preferably below 1%, and most preferably below 0.5%, where percentages are weight percent or mole percent. Thus, substantially all of the carbohydrate residues in the food composition according to the present invention are free of, for example, lactose.

[0094] Unless otherwise indicated, percentages (%) of contents refer to total weight %.

[0095] Unless otherwise indicated, and by way of example for all sequences described herein under the general format of "SEQ ID NO:", "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid, at least a portion of which has (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The choice between the two is determined by the context. For example, when the nucleic acid is used as a probe, the choice between the two is determined by the need for the probe to be complementary to the desired target. [Brief description of the drawings]

[0096] [Figure 1] FIG. 1 depicts a flow chart illustrating an exemplary process for making a synthetic milk substitute. [Figure 2A] FIG. 1 depicts a photograph depicting precipitation of exemplary milk protein components. [Figure 2B] FIG. 1 represents a photograph depicting pellets of an exemplary milk protein ingredient. [Diagram 3] FIG. 1 depicts an image of a silver stained SDS-PAGE gel visualizing milk protein components. [Figure 4] FIG. 1 shows a SYPRO Ruby stained SDS-PAGE gel showing the secretion levels of α-lactalbumin mediated by the OST signal sequence, the native α-lactalbumin signal sequence and the α-mating factor signal sequence as described in Example 6. [Diagram 5] FIG. 1 shows the level of α-lactalbumin secretion by wild-type or α-lactalbumin-expressing yeast cells using the native α-lactalbumin signal peptide or the OST1 signal peptide (determined by ELISA assay, as described in Example 6). [Figure 6] FIG. 1 shows the levels of β-lactoglobulin secretion by wild-type yeast cells and vector-containing yeast cells as described in Example 6 (using SDS-PAGE). [Figure 7] FIG. 1 shows a Western blot showing the levels of secretion of β-lactoglobulin from wild-type yeast cells and vector-containing yeast cells, as described in Example 6. [Figure 8] FIG. 1 shows a graph depicting the levels of β-casein secretion and the levels of α-S1-casein secretion produced by wild-type yeast cells and yeast cells containing the vector, as described in Example 6. [Figure 9] FIG. 1 is a schematic showing steps within the process described in Example 7. [Figure 10]FIG. 1 shows images of compositions made by the methods described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] The present invention is based on the discovery that only a few components present in mammalian produced milk provide the texture and taste of mammalian produced milk, and the development of compositions that have similar taste, aroma, and mouthfeel compared to mammalian produced milk. In light of this discovery, presented herein are such compositions, methods of making the compositions, and kits that include these compositions and mixtures useful for making these compositions.

[0098] The compositions provided herein have similar taste, texture, aroma, and nutritional value compared to mammalian produced milk, but lack one or more of the components of mammalian produced milk that may be considered undesirable (e.g., allergens, lactose, antibiotics, hormones (e.g., stress hormones and / or growth hormones), heavy metals, bacteria (e.g., E. coli), viruses, and prions). The compositions provided herein also have an improved shelf life compared to mammalian produced milk, and may also have an improved aroma profile compared to mammalian produced milk.

[0099] Also provided herein are methods and compositions for dairy substitutes that include one or more isolated milk protein components, fat, carbohydrates and ash.In certain embodiments, the methods and compositions include milk or milk-like protein equivalents. Preferably, the milk protein component is essentially free of impurities. In some embodiments, the milk protein component comprises microbially derived or microbially produced casein, whey or combinations thereof. More preferably, a method is presented for introducing engineered nucleic acid sequences encoding one or more milk protein components. Even more preferably, the milk protein component is not of animal origin. In other preferred embodiments, the recombinant milk protein component is modified to express the same phosphate groups or to lack phosphate and / or carbohydrate groups conjugated to casein proteins. By having recombinant β-casein and κ-casein with the same phosphate groups as the same proteins present in mammalian produced milk, recombinant β-casein and recombinant κ-casein are capable of forming micelles.

[0100] The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art and described in various general and more specific references, unless otherwise indicated, cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992, and supplements until 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990; Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press, 2003; Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry: Section A Proteins, Vol. I, CRC Press, 1976; Handbook of Biochemistry: Section A Proteins, Vol. II, CRC Press, 1976; Essentials of Glycobiology, Cold Spring Harbor Laboratory See, Press, 1999.

[0101] Exemplary materials and methods for use in any of the methods and compositions are described below, but may be used in any combination. Additional materials and methods that may be used in any of the methods and compositions are also known in the art.

[0102] Casein Protein Casein proteins include a variety of different proteins found in mammalian milk. Non-limiting examples of casein proteins include β-casein, κ-casein, α-S2-casein, and α-S1-casein.

[0103] As an alternative to obtaining casein protein from mammals or mammal-produced milk for use in the manufacture of dairy products, the present invention provides methods and compositions for producing recombinant casein protein. In various aspects of the present invention, methods and compositions are provided for non-animal derived casein with similar solubility and similar turbidity, as well as heat stability suitable for incorporation into various food products. Preferably, the non-animal derived casein has excellent solubility and similar turbidity, as well as heat stability suitable for incorporation into various dairy substitute products. In addition, further characterization of the protein includes low or no aggregation or precipitation upon such heat treatment, and suitability for procedures such as pasteurization, concentration, etc.

[0104] The functional differences in non-animal caseins in milk can be characterized in relation to the viscosity of the liquid; the ability of the protein to withstand heat; the ability of the protein to form micelles; and the ability of the protein to retain different minerals and vitamins.

[0105] β-Casein The primary structure of human β-casein, determined by Greenberg et al. (J. Biol. Chem., 259:5132-5138, 1984), showed it to be a phosphorylated protein, with phosphorylation sites at unusual seryl and threonyl residues located near the amino terminus. Comparison of human and bovine β-casein showed 47% identity.

[0106] Non-limiting examples of β-casein proteins are SEQ ID NOs: 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, and 46. Non-limiting examples of nucleic acid sequences encoding β-casein proteins are SEQ ID NOs: 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 47, and 144. The β-casein protein can be from any mammalian species, for example, cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth β-casein protein. Additional sequences of different β-casein proteins and nucleic acids encoding different β-casein proteins are known in the art.

[0107] The β-casein protein may also be a protein that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type β-casein protein (e.g., SEQ ID NOs: 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, or 46). A nucleic acid encoding a β-casein protein may encode a protein that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type β-casein protein (e.g., SEQ ID NO: 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, or 46).

[0108] Known methods for isolating β-casein from genetically engineered bacterial cells typically involve precipitating β-casein from the supernatant derived from lysed or fractionated cells. For example, Simons et al., Protein Eng., 6:763-770 (1993) used genetically engineered E. coli to express bovine β-casein. The protein accumulated in the periplasmic space of the bacteria was released into the cell suspension by osmotic shock. After centrifugation of the suspension, the β-casein in the pellet was resuspended in cold water, washed, and centrifuged again. The β-casein present in the supernatant was precipitated by acidification with acetic acid, filtered, and further purified by HPLC. Similarly, Hansson et al., Protein Express. Purif. 4:373-381, 1993, also used genetically engineered E. coli to express β-casein. β-casein present in the cell lysate was precipitated with ammonium sulfate, dissolved in ethanolamine and 6M urea, and further purified by ion exchange chromatography, see, e.g., U.S. Patent No. 6,121,421.

[0109] Additionally, methods are also known for isolating recombinantly produced β-casein in yeast that are simpler and more efficient than known techniques (Choi et al., J. Agric. Food Chem., 49(4):1761-1766, 2001). Expression and purification of glycosylated bovine β-casein (L70S / P71S) in Pichia pastoris resulted in the observation that the majority of bovine β-casein was not highly glycosylated in P. pastoris, with an estimated molecular weight of 33.6 kD. Glycosylated bovine β-casein was usually phosphorylated to the same extent as native bovine β-casein.

[0110] Kappa-casein Kappa-casein is both phosphorylated and glycosylated. The sequence of human κ-casein was determined by Brignon et al. (Fed. Eur. Biol. Soc. Lett., 188:48-54, 1985). See, e.g., U.S. Patent No. 5,710,044.

[0111] Non-limiting examples of κ-casein proteins are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. Non-limiting examples of nucleic acid sequences encoding κ-casein proteins are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 148. The κ-casein protein can be from any mammalian species, for example, cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. Further sequences of different κ-casein proteins and nucleic acids encoding different κ-casein proteins are known in the art.

[0112] The κ-casein protein may also be a protein that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type κ-casein protein (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23). A nucleic acid encoding a κ-casein protein may encode a protein that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type κ-casein protein (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23).

[0113] α-S1-Casein Non-limiting examples of α-S1-casein proteins are SEQ ID NOs: 48, 50, 52, 54, 56, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, and 76. Non-limiting examples of nucleic acid sequences encoding α-S1-casein proteins are SEQ ID NOs: 49, 51, 53, 55, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, and 147. The α-S1-casein protein can be from any mammalian species, for example, cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. Further sequences of different α-S1-casein proteins and nucleic acids encoding different α-S1-casein proteins are known in the art.

[0114] The α-S1-casein protein may also be a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type α-S1-casein protein (e.g., SEQ ID NOs: 48, 50, 52, 54, 56, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, or 76). A nucleic acid encoding an α-S1-casein protein may encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type α-S1-casein protein (e.g., SEQ ID NO: 48, 50, 52, 54, 56, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, or 76).

[0115] α-S2-Casein Non-limiting examples of α-S2-casein proteins are SEQ ID NOs: 78, 80, 82, 84, 86, 88, and 90. Non-limiting examples of nucleic acid sequences encoding α-S2-casein proteins are SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 145, and 146. The α-S2-casein protein can be from any mammalian species, for example, cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. Further sequences of different α-S2-casein proteins and nucleic acids encoding different α-S2-casein proteins are known in the art.

[0116] The α-S2-casein protein may also be a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type α-S2-casein protein (e.g., SEQ ID NO: 78, 80, 82, 84, 86, 88, or 90). A nucleic acid encoding an α-S2-casein protein may encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to the wild-type α-S2-casein protein (e.g., SEQ ID NO: 78, 80, 82, 84, 86, 88, or 90).

[0117] Micelles containing casein proteins In milk, casein or casein micelles typically make up 2.5% of the total mixture in suspension. If there is not enough casein, the micelles, which are crucial for optimal milk behavior, do not form. Too much protein does not dissolve properly, resulting in an undesirable mixture. Casein micelles may contain water and salts (mainly calcium and phosphorus). They are easily separated and removed by centrifugation. Separation from whey is easily achieved by precipitating the casein with acid and lowering the pH to about 4.6.

[0118] In some embodiments, the micelles may comprise β-casein protein (e.g., any of the β-casein proteins described herein) and κ-casein protein (e.g., any of the κ-casein proteins described herein). In some examples, the ratio of β-casein protein to κ-casein protein in the micelles is from about 2.0:1 to about 5.5:1, 2.0:1 to about 5.0:1, 2.0:1 to about 4.5:1, from about 2.0:1 to about 4.0:1, from about 2.0:1 to about 3.5:1, from about 2.0:1 to about 3.0:1, from about 2.0:1 to about 2.5:1, from about 2.5:1 to about 5.0:1, from about 2.5:1 to about 4.5:1, from about 2.5:1 to about 4. 0:1, about 2.5:1 to about 3.5:1, about 2.5:1 to about 3.0:1, 3.0:1 to about 5.0:1, about 3.0:1 to about 4.5:1, about 3.0:1 to about 4.0:1, about 3.0:1 to about 3.5:1, about 3.5:1 to about 5.0:1, about 3.5:1 to about 4.5:1, about 3.5:1 to about 4.0:1, about 4.0:1 to about 5.0:1, about 4.0:1 to about 4.5:1, or about 4.5:1 to about 5.0:1.

[0119] In some examples, the diameter of a micelle (or the average diameter of a population of micelles) is from about 20 nm to about 350 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, about 180 nm, about 160 nm, about 140 nm, about 120 nm, about 100 nm, about 80 nm, about 60 nm, or about 40 nm; nm, about 140 nm, about 120 nm, about 100 nm, about 80 nm, or about 60 nm; about 60 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, about 180 nm, about 160 nm, about 140 nm, about 120 nm, or about 100 nm; about 80 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, about 180 nm, about 160 nm, about 140 nm, about 120 nm, or about 100 nm; about 100 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, about 180 nm, about 160 nm, about 140 nm, or about 120 nm; about 120 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, about 180 nm, about 160 nm, or about 140 nm; about 140 nm to about 35 0 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, about 180 nm, or about 160 nm; about 160 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, about 200 nm, or about 180 nm; about 180 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, about 220 nm, or about 200 nm;about 200 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, about 240 nm, or about 220 nm; about 220 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, about 260 nm, or about 240 nm; about 240 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, about 280 nm, or about 260 nm; about 260 nm to about 350 nm, about 340 nm, about 320 nm, about 300 nm, or about 280 nm; about 280 nm to about 350 nm, about 340 nm, about 320 nm, or about 300 nm; about 300 nm to about 350 nm or about 325 nm; or about 325 nm to about 350 nm.

[0120] Whey Protein Whey is generally known as a by-product of cheese and is also known to be one cause of milk allergies. A typical whey composition includes a mixture of β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, lactoferrin, and transferrin. Whey proteins do not contain phosphorus and are still soluble at low pH, whereas casein proteins are insoluble. In one embodiment, a select combination of whey proteins, including β-lactoglobulin and α-lactalbumin, is used as the main component or at least part of the milk protein component or composition. Non-limiting examples of different whey proteins are presented below.

[0121] Alpha-lactalbumin Non-limiting examples of alpha-lactalbumin proteins are SEQ ID NOs: 92, 94, 96, and 98. Non-limiting examples of nucleic acid sequences encoding alpha-lactalbumin proteins are SEQ ID NOs: 93, 95, 97, 99, and 157. The alpha-lactalbumin protein can be from any mammalian species, for example, cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth alpha-lactalbumin protein. Further sequences of different alpha-lactalbumin proteins and nucleic acids encoding different alpha-lactalbumin proteins are known in the art.

[0122] The alpha-lactalbumin protein may also be at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to the wild-type alpha-lactalbumin protein (e.g., SEQ ID NO: 92, 94, 96, or 98). The nucleic acid encoding the alpha-lactalbumin protein may encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to the wild-type alpha-lactalbumin protein (e.g., SEQ ID NO: 92, 94, 96, or 98).

[0123] β-Lactoglobulin Non-limiting examples of β-lactoglobulin proteins are SEQ ID NOs: 100, 102, 104, and 106. Non-limiting examples of nucleic acid sequences encoding β-lactoglobulin proteins are SEQ ID NOs: 101, 103, 105, 107, and 143. The β-lactoglobulin protein can be a β-lactoglobulin protein from any mammalian species, such as a β-lactoglobulin protein from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. Further sequences of different β-lactoglobulin proteins and nucleic acids encoding different β-lactoglobulin proteins are known in the art.

[0124] A β-lactoglobulin protein may also be a protein that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type β-lactoglobulin protein (e.g. SEQ ID NO: 100, 102, 104, or 106). A nucleic acid encoding a β-lactoglobulin protein may encode a protein that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type β-lactoglobulin protein (e.g. SEQ ID NO: 100, 102, 104, or 106).

[0125] Lactoferrin Non-limiting examples of lactoferrin proteins are SEQ ID NOs: 108, 110, 112, and 114. Non-limiting examples of nucleic acid sequences encoding lactoferrin proteins are SEQ ID NOs: 109, 111, 113, and 115. The lactoferrin protein can be from any mammalian species, such as cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth lactoferrin protein. Further sequences of different lactoferrin proteins and nucleic acids encoding different lactoferrin proteins are known in the art.

[0126] The lactoferrin protein can also be a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type lactoferrin protein (e.g., SEQ ID NO: 108, 110, 112, or 114). The nucleic acid encoding the lactoferrin protein can encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type lactoferrin protein (e.g., SEQ ID NO: 108, 110, 112, or 114).

[0127] Transferrin Non-limiting examples of transferrin proteins are SEQ ID NOs:116 and 118. Non-limiting examples of nucleic acid sequences encoding transferrin proteins are SEQ ID NOs: 117 and 119. The transferrin protein can be from any mammalian species, such as a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth transferrin protein. Additional sequences of different transferrin proteins and nucleic acids encoding different transferrin proteins are known in the art.

[0128] A transferrin protein can also be a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type transferrin protein (e.g., SEQ ID NO: 116 or 118). A nucleic acid encoding a transferrin protein can encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type transferrin protein (e.g., SEQ ID NO: 116 or 118).

[0129] Serum albumin Non-limiting examples of serum albumin proteins are SEQ ID NOs: 120, 122, 124, and 126. Non-limiting examples of nucleic acid sequences encoding serum albumin proteins are SEQ ID NOs: 121, 123, 125, and 127. The serum albumin protein can be from any mammalian species, such as cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth serum albumin protein. Additional sequences of different serum albumin proteins and nucleic acids encoding different serum albumin proteins are known in the art.

[0130] The serum albumin protein can also be a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type serum albumin protein (e.g., SEQ ID NO: 20, 122, 124, or 126). The nucleic acid encoding the serum albumin protein can encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wild-type serum albumin protein (e.g., SEQ ID NO: 20, 122, 124, or 126).

[0131] Lipids in Produced Mammalian Milk Milk fat contains about 400 different fatty acids, making it the most complex of all natural fats.Milk fatty acids are derived almost equally from two sources, cow feed and microbial activity in the cow's rumen, and lipids in milk are primarily present as oil-in-water emulsions in milk fat globules.Fats are present in all natural dairy products and are crucial for sensory characteristics such as flavor, mouthfeel and consistency.In addition, fats provide nutritional and health benefits.

[0132] Milk fat consists mainly of triglycerides (about 98%), but other milk lipids are diacylglycerols (about 2% of the lipid fraction), cholesterol (less than 0.5%), phospholipids (about 1%) and free fatty acids (FFA) (about 0.1%) (Jensen RG, Newburg DS, Bovine milk lipids, Handbook of milk composition, Jensen RG London: Academic Press; 1995, 543-75). In addition, traces of ether lipids, hydrocarbons, fat-soluble vitamins, flavour compounds and compounds introduced by the feed are also included (Lindmark Mansson H., Food & Nutrition Research 2008, DOI:10.3402 / fnr.v52i0.1821).

[0133] Milk fat triglycerides are synthesized from over 400 different fatty acids, making them the most complex of all natural fats. Nearly all fatty acids in milk are present in trace amounts, with only about 15 acids at levels above 1%. Many factors are associated with the variability in the amount and fatty acid composition of milk lipids. They can be of animal origin, i.e., related to genetics (breed and selection), lactation stage, mastitis and fermentation in the rumen, or they can be feed-related factors, i.e., dietary fiber and energy intake, dietary fat, as well as seasonal and regional effects. The fatty acids in milk fat are arranged in triglycerides according to a pattern that is considered universal among ruminants. The percentage of unsaturated fatty acids (e.g., oleic and linoleic acids) in goats does not differ from the average found for cow's milk. The main difference between goat milk fat and cow milk fat is the percentage distribution among the specific short-chain fatty acids. Goats have appreciably higher proportions of capric, caprylic and caproic acids. High amounts of these specialized fatty acids contribute to the characteristic flavor and odor associated with goat's milk (John C. Bruhn, FST, UC Davis, Davis, CA 95616-8598; see www.drinc.ucdavis.edu / goat1.htm; www.ncbi.nlm.nih.gov / pmc / articles / PMC2596709 / #_ffn_sectitle; Food Nutr Res. 2008;52:10.3402 / fnr.v52i0.1821, published online June 11, 2008, doi:10.3402 / fnr.v52i0.1821).

[0134] Milk fatty acids are derived approximately equally from two sources: the cow's diet and microbial activity in the cow's rumen. The fatty acid synthesis system in the cow's mammary gland produces fatty acids with an even number of carbons, from 4 to 16 carbons in length, which account for approximately 60 and 45% of the fatty acids on a molar and weight basis, respectively. This de novo synthesis in the mammary gland is the synthesis of 4:0 to 14:0 acids, with approximately half of the 16:0 acids derived from acetate and β-hydroxybutyrate. Acetate and butyrate are produced in the rumen by fermentation of feed ingredients. Butyrate is converted to β-hydroxybutyrate upon absorption through the ruminal epithelium.

[0135] Predominantly the 18:0 acids, but medium and long chain fatty acids can be desaturated in the mammary gland to form the corresponding monosaturated acids.

[0136] Fatty acids are not randomly esterified at the three positions of the triacylglycerol molecule (MacGibbon AHK, Taylor MW, Composition and structure of bovine milk lipids Advanced dairy chemistry, Fox PFMcSweeney PLH New York: Springer; 2006, 1-42). The short chain acids butyric acid (4:0) and caproic acid (6:0) are almost completely esterified at sn-3. The medium chain fatty acids (8:0-14:0), as well as 16:0, are preferentially esterified at the sn-1 and sn-2 positions. Stearic acid (18:0) is preferentially placed at the sn-1 position, whereas oleic acid (18:1) shows a preference for the sn-1 and sn-3 positions (Lindmark, 2008).

[0137] Milk replacers exist in which the fat components are formulated to have a selected fatty acid profile, but such triglycerides are not interesterified to long chain monounsaturated triglycerides such as those found in vegetable oils.US Patent Application No. 20140147548 discloses milk replacers for young animals by adding medium chain triglycerides, specifically the fatty acids caproic, caprylic, capric and lauric fatty acids or combinations thereof.

[0138] Lipids in the Composition The lipid in any of the compositions or used in any of the methods described herein can comprise one or more fats, one or more oils, one or more monoglycerides, diglycerides, and / or triglycerides, one or more free fatty acids, and one or more phospholipids.Exemplary oils, monoglycerides, diglycerides, free fatty acids, and phospholipids are described below.Further examples of fats, oils, monoglycerides, diglycerides, triglycerides, free fatty acids, and phospholipids are known in the art.

[0139] oil The oil used in the composition or method can include, for example, oil of plant origin.Non-limiting examples of plant-based oil include sunflower oil, coconut oil, peanut oil, corn oil, cottonseed oil, olive oil, date oil, rapeseed oil, safflower oil, sesame oil, soybean oil, almond oil, beech nut oil, Brazil nut oil, cashew nut oil, hazelnut oil, macadamia nut oil, mongo nut oil, pecan oil, pine nut oil, pistachio nut oil, walnut oil and avocado oil.

[0140] Monoglycerides and diglycerides The monoglycerides and diglycerides that can be used in the present invention can be plant-derived monoglycerides and diglycerides.For example, the monoglycerides and diglycerides can be derived from sunflower, coconut, peanut, cottonseed, olive, date palm, rapeseed, safflower, sesame, soybean, almond, beech nut, Brazil nut, cashew nut, hazelnut, macadamia nut, mongo nut, pecan, pine nut, pistachio, walnut and avocado.The monoglycerides and diglycerides can contain any acyl chain of the free fatty acids listed herein.More examples of monoglycerides and diglycerides are known in the art.

[0141] free fatty acids The compositions described herein can include the use of one or more free fatty acids, and the methods described herein can include the use of one or more free fatty acids. Non-limiting examples of free fatty acids include butyric acid, caproic acid, caprylic acid, and capric acid. Further examples of fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, omega-3 fatty acid, and omega-6 fatty acid. In some examples, the free fatty acid is a saturated fatty acid. In some examples, the free fatty acid is an unsaturated fatty acid. In some embodiments, the free fatty acid is not derived from or produced by a mammal. Further examples of free fatty acids are known in the art.

[0142] Phospholipids The compositions described herein and the methods described herein may include the use of one or more phospholipids.Non-limiting examples of phospholipids include lecithin phospholipids (e.g., soybean lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, rapeseed lecithin phospholipids, rice bran lecithin phospholipids, and corn lecithin phospholipids).In some embodiments, the phospholipids are not derived from or produced by mammals.Further aspects of phospholipids are known in the art.

[0143] flavor compounds Any of the compositions or methods described herein may include or include the use of one or more different flavor compounds.Non-limiting examples of flavor compounds include δ-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, and δ-undecalactone.Further examples of flavor compounds include artificial flavors, such as chocolate flavoring, coffee flavoring, strawberry flavoring, almond flavoring, hazelnut flavoring, vanilla flavoring, green tea flavoring, Irish cream flavoring, and coconut flavoring.Further examples of flavor compounds are known in the art.

[0144] ash Any of the compositions or methods described herein may include or include the use of ash. Ash may include, for example, one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) of calcium, phosphorus, potassium, sodium, citrate, chloride, phosphate, magnesium, iron, molybdenum, manganese, copper, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), vitamin B6 (pyridoxine), vitamin B12 (cobalamin), vitamin C, folate, vitamin A, vitamin D, vitamin E, and vitamin K. In some examples, ash includes one or more (2 or 3) of CaCl2, KH2PO4, and Na3 citrate. The ash may be provided as a powder or as a solution. Additional components in and aspects of the ash are known in the art. In some embodiments, the ash is not derived from or produced by a mammal.

[0145] Color Corrector A variety of different color correction agents are known in the art. For example, the color correction agent can be a compound obtained from a plant (e.g., a monocotyledonous or dicotyledonous plant). In some cases, the color correction agent is a synthetic compound. In some cases, the color correction agent is not obtained from or produced by a mammal or mammalian cell. Non-limiting examples of color correction agents include beta-carotene and annatto.

[0146] Sweetener The sweetener may be a saccharide (e.g., a monosaccharide, disaccharide, or polysaccharide) or an artificial sweetener. Non-limiting examples of sweeteners that are saccharides include glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose. Further examples of saccharides that may be used as sweeteners in any of the compositions or methods described herein are known in the art.

[0147] Non-limiting examples of sweeteners that are artificial sweeteners include stevia, aspartame, cyclamate, saccharin, sucralose, mogrosides, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000. Additional artificial sweeteners that may be used as sweeteners in any of the compositions or methods described herein are known in the art.

[0148] composition As used herein, the term "aqueous solubility limiting agent" refers to an aqueous solubility limiting agent that is an aqueous solubility limiting agent that is a ... / L, about 0.4g / L to about 0.5g / L, about 0.5g / L to about 1.1g / L, about 0.5g / L to about 1.0g / L, about 0.5g / L to about 0.9g / L, about 0.5g / L to about 0.8g / L, about 0.5g / L to about 0.7g / L, about 0.5g / L to about 0.6g / L, about 0.6g / L to about 1.1g / L, about 0.6g / L to about 1.0g / L, about 0.6g / L to about 0.9g / L, about 0.6g / L to about 0.8g / L, about 0.6g / L to about 0.7g / L, about 0.7g / L to about 1.1g / L, about 0.7g / L to about 1.0g / L, about 0.7g / L from about 0.9 g / L, from about 0.7 g / L to about 0.8 g / L, from about 0.8 g / L to about 1.1 g / L, from about 0.8 g / L to about 1.0 g / L, from about 0.8 g / L to about 0.9 g / L, from about 0.9 g / L to about 1.1 g / L, from about 0.9 g / L to about 1.0 g / L, from about 1.0 g / L to about 1.1 g / L, or from about 0.27% to about 0.75% by weight of κ-casein protein (e.g., of any of the κ-casein proteins described herein); from about 1.25 g / L to about 4.9 g / L (e.g., from about 1.25 g / L to about 4.6 g / L, from about 1.25 g / L to about 4.9 g / L, / L to about 4.4g / L, about 1.25g / L to about 4.2g / L, about 1.25g / L to about 4.0g / L, about 1.25g / L to about 3.8g / L, about 1.25g / L to about 3.6g / L, about 1.25g / L to about 3.4g / L, about 1.25g / L to about 3.2g / L, about 1.25g / L to about 3.0g / L, about 1.25g / L to about 2.8g / L, about 1.25g / L to about 2.6g / L, about 1.25g / L to about 2.4g / L, about 1.25g / L to about 2.2g / L, about 1.25g / L to about 2.0g / L, about 1.25g / L to about 1.8g / L, about 1.25g / L to about 1.6g / L, about 1.25g / L to about 1.4g / L, about 1.4g / L to about 4.9g / L, about 1.4g / L to about 4.6g / L, about 1.4g / L to about 4.4g / L, about 1.4g / L to about 4.2g / L, about 1.4g / L to about 4.0g / L, about 1.4g / L to about 3.8g / L, about 1.4g / L to about 3.6g / L, about 1.4g / L to about 3.4g / L, about 1.4g / L to about 3.2g / L, about 1.4g / L to about 3.0g / L, about 1.4g / L to about 2.8g / L, about 1.4g / L to about 2.6g / L, about 1.4g / L to about 2. 4g / L, about 1.4g / L to about 2.2g / L, about 1.4g / L to about 2.0g / L, about 1.4g / L to about 1.8g / L, about 1.4g / L to about 1.6g / L, about 1.6g / L to about 4.9g / L, about 1.6g / L to about 4.6g / L, about 1.6g / L to about 4.4g / L, about 1.6g / L to about 4.2g / L, about 1.6g / L to about 4.0g / L, about 1.6g / L to about 3.8g / L, about 1.6g / L to about 3.6g / L, about 1.6g / L to about 3.4g / L, about 1.6g / L to about 3.2g / L, about 1.6g / L to about 3 ...8g / L, about 1.6g / L to about 3.6g / L, about from about 1.6g / L to about 2.6g / L, from about 1.6g / L to about 2.4g / L, from about 1.6g / L to about 2.2g / L, from about 1.6g / L to about 2.0g / L, from about 1.6g / L to about 1.8g / L, from about 1.8g / L to about 4.9g / L, from about 1.8g / L to about 4.6g / L, from about 1.8g / L to about 4.4g / L, from about 1.8g / L to about 4.2g / L, from about 1.8g / L to about 4.0g / L, from about 1.8g / L to about 3.8g / L, from about 1.8g / L to about 3.6g / L, from about 1.8g / L to about 3.4g / L, from about 1.8g / L to about 3.2g / L, about 1.8g / L to about 3.0g / L, about 1.8g / L to about 2.8g / L, about 1.8g / L to about 2.6g / L, about 1.8g / L to about 2.4g / L, about 1.8g / L to about 2.2g / L, about 1.8g / L to about 2.0g / L, about 2.0g / L to about 4.9g / L, about 2.0g / L to about 4.6g / L, about 2.0g / L to about 4.4g / L, about 2.0g / L to about 4.2g / L, about 2.0g / L to about 4.0g / L, about 2.0g / L to about 3.8g / L, about 2.0g / L to about 3.6g / L, about 2.0g / L to about 3.4g / L, about 2.0g / L to about 3.2g / L, from about 2.0g / L to about 3.0g / L, from about 2.0g / L to about 2.8g / L, from about 2.0g / L to about 2.6g / L, from about 2.0g / L to about 2.4g / L, from about 2.0g / L to about 2.2g / L, from about 2.2g / L to about 4.9g / L, from about 2.2g / L to about 4.6g / L, from about 2.2g / L to about 4.4g / L, from about 2.2g / L to about 4.2g / L, from about 2.2g / L to about 4.0g / L, from about 2.2g / L to about 3.8g / L, from about 2.2g / L to about 3.6g / L, from about 2.2g / L to about 3.4g / L, from about 2.2g / L to about 3.2g / L, from about 2.2g / L to about 3.0g / L, about 2.2g / L to about 2.8g / L, about 2.2g / L to about 2.6g / L, about 2.2g / L to about 2.4g / L, about 2.4g / L to about 4.9g / L, about 2.4g / L to about 4.6g / L, about 2.4g / L to about 4.4g / L, about 2.4g / L to about 4.2g / L, about 2.4g / L to about 4.0g / L, about 2.4g / L to about 3.8g / L, about 2.4g / L to about 3.6g / L, about 2.4g / L to about 3.4g / L, about 2.4g / L to about 3.2g / L, about 2.4g / L to about 3.0g / L, about 2.4g / L to about 2.8g / L , about 2.4g / L to about 2.6g / L, about 2.6g / L to about 4.9g / L, about 2.6g / L to about 4.6g / L, about 2.6g / L to about 4.4g / L, about 2.6g / L to about 4.2g / L, about 2.6g / L to about 4.0g / L, about 2.6g / L to about 3.8g / L, about 2.6g / L to about 3.6g / L, about 2.6g / L to about 3.4g / L, about 2.6g / L to about 3.2g / L, about 2.6g / L to about 3.0g / L, about 2.6g / L to about 2.8g / L, about 2.8g / L to about 4.9g / L, about 2.8g / L to about 4.6 ... about 4.4g / L, about 2.8g / L to about 4.2g / L, about 2.8g / L to about 4.0g / L, about 2.8g / L to about 3.8g / L, about 2.8g / L to about 3.6g / L, about 2.8g / L to about 3.4g / L, about 2.8g / L to about 3.2g / L, about 2.8g / L to about 3.0g / L, about 3.0g / L to about 4.9g / L, about 3.0g / L to about 4.6g / L, about 3.0g / L to about 4.4g / L, about 3.0g / L to about 4.2g / L, about 3.0g / L to about 4.0g / L, about 3.0g / L to about 3.8g / L, about 3.0g / L to about 3.6g / L, about 3.0g / L to about 3.4g / L, about 3.0g / L to about 3.2g / L, about 3.2g / L to about 4.9g / L, about 3.2g / L to about 4.6g / L, about 3.2g / L to about 4.4g / L, about 3.2g / L to about 4.2g / L, about 3.2g / L to about 4.0g / L, about 3.2g / L to about 3.8g / L, about 3.2g / L to about 3.6g / L, about 3.2g / L to about 3.4g / L, about 3.4g / L to about 4.9g / L, about 3.4g / L to about 4.6g / L, about 3.4g / L to about 4.4g / L L, about 3.4g / L to about 4.2g / L, about 3.4g / L to about 4.0g / L, about 3.4g / L to about 3.8g / L, about 3.4g / L to about 3.6g / L, about 3.6g / L to about 4.9g / L, about 3.6g / L to about 4.6g / L, about 3.6g / L to about 4.4g / L, about 3.6g / L to about 4.2g / L, about 3.6g / L to about 4.0g / L, about 3.6g / L to about 3.8g / L, about 3.8g / L to about 4.9g / L, about 3.8g / L to about 4.6g / L, about 3.8g / L to about 4. 0.4g / L, about 3.8g / L to about 4.2g / L, about 3.8g / L to about 4.0g / L, about 4.0g / L to about 4.9g / L, about 4.0g / L to about 4.6g / L, about 4.0g / L to about 4.4g / L, about 4.0g / L to about 4.2g / L, about 4.2g / L to about 4.9g / L, about 4.2g / L to about 4.6g / L, about 4.2g / L to about 4.4g / L, about 4.4g / L to about 4.9g / L, about 4.4g / L to about 4.6g / L, about 4.6g / L to about 4.9g / L, or about 1.2 3% to about 3.27% by weight of β-casein protein (e.g., any of the β-casein proteins described herein); a total final concentration of about 0% to about 45% by weight (e.g., 0% by weight; about 0% to about 4.5% by weight; about 0.5% to about 40% by weight, about 35% by weight, about 30% by weight, about 25% by weight, about 20% by weight, about 15% by weight, about 10% by weight, about 8% by weight, about 6% by weight, about 5% by weight, about 4% by weight, about 3% by weight, about 2% by weight, or about 1% by weight;0% to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 8%, about 6%, about 5%, about 4%, about 3%, or about 2% by weight; about 2% to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 8%, about 6%, about 5%, about 4%, or about 3% by weight; about 3% to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 8%, about 6%, about 5%, or about 4% by weight; about 4% to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 8%, about 6%, or about 5% by weight; about 5% to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 8%, or about 6% by weight; about 6% to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 8% by weight; about 8% to about 40% by weight. %, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% by weight; about 10% to about 40%, about 35%, about 30%, about 25%, about 20%, or about 15% by weight; about 15% to about 40%, about 35%, about 30%, about 25%, or about 20% by weight; about 20% to about 40%, about 35%, about 30%, or about 25% by weight; about 25% to about 40%, about 35%, or about 30% by weight; about 30% to about 40%, or about 35% by weight; or about 35% to about 40% by weight of one or more lipids (e.g., any of one or more lipids described herein); a total final concentration of about 0.01% to about 6% by weight (e.g., about 0.1% to about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, about 3.0% by weight, about 2.5% by weight, about 2.0% by weight, about 1.5% by weight, about 1.0% by weight, or about 0.5% by weight;0% by weight, about 2.5% by weight, about 2.0% by weight, about 1.5% by weight, or about 1.0% by weight; about 1.0% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, about 3.0% by weight, about 2.5% by weight,. about 2.0% by weight, or about 1.5% by weight; about 1.5% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, about 3.0% by weight, about 2.5% by weight, or about 2.0% by weight; about 2.0% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, about 3.0% by weight, or about 2.5% by weight; about 2.5% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, or about 3.0% by weight % to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, about 4.0 wt%, or about 3.5 wt%; about 3.5 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, or about 4.0 wt%; about 4.0 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%; about 4.5 wt% to about 6.0 wt%, about 5.5 wt%, or about 5.0 wt%; about 5.0 wt% to about 6.0 wt%, or about 5.5 wt%; or about 5.5 wt% to about 6.0 wt%. flavor compounds (e.g., any of one or more flavor compounds described herein); a total final concentration of about 0.1% to about 6% by weight (e.g., about 0.1% to about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, about 3.0% by weight, about 2.5% by weight, about 2.0% by weight, about 1.5% by weight, about 1.0% by weight, or about 0.5% by weight; , about 1.5 wt%, or about 1.0 wt%; about 1.0 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, about 4.0 wt%, about 3.5 wt%, about 3.0 wt%, about 2.5 wt%, about 2.0 wt%, or about 1.5 wt%; about 1.5 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, about 4.0 wt%, about 3.5 wt%, about 3.0 wt%, about 2.5 wt%, or about 2.0 wt%; about 2.0 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, about 4.0 wt%, about 3.5% by weight, about 3.0% by weight, or about 2.5% by weight; about 2.5% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, or about 3.0% by weight; about 3.0% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, or about 3.5% by weight; about 3.5% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, or about 4.0% by weight; about 4.0% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, or about 4. 5%, about 4.5% to about 6.0%, about 5.5%, or about 5.0%; about 5.0% to about 6.0%, or about 5.5%; or about 5.5% to about 6.0% by weight of one or more sweetening agents (e.g., any of one or more sweetening agents described herein); and a total final concentration of about 0.15% to about 1.5% by weight (e.g., about 0.15% to about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.6%, about 0.5% %, about 0.4%, about 0.3%, or about 0.2% by weight; about 0.2% to about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.6%, about 0.5%, about 0.4%, or about 0.3% by weight; about 0.3% to about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.6%, about 0.5%, or about 0.4% by weight; about 0.4% to about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.6%, or about 0.5% by weight; about 0.5% to about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, or about 0.6% by weight; about 0.6% to about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, or about 0.8% by weight; about 0.8% by weight to about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, or about 0.9% by weight; about 0.9% by weight to about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, or about 1.0% by weight; about 1.0% by weight to about 1.5%, about 1.4%, about 1.3%, about 1.2%, or about 1. Compositions are presented that contain an ash content of 1% by weight; about 1.1% to about 1.5% by weight, about 1.4% by weight, about 1.3% by weight, or about 1.2% by weight; about 1.2% to about 1.5% by weight, about 1.4% by weight, or about 1.3% by weight; about 1.3% to about 1.5% by weight, or about 1.4% by weight; or about 1.4% to about 1.5% by weight.

[0149] Also included are κ-casein protein (e.g., any of the κ-casein proteins described herein) at about 0.3 g / L to about 1.1 g / L (e.g., any of the subranges of about 0.3 g / L to about 1.1 g / L described in the paragraph above); β-casein protein (e.g., any of the β-casein proteins described herein) at about 1.25 g / L to about 4.9 g / L (e.g., any of the subranges of about 1.25 g / L to about 4.9 g / L described in the paragraph above); one or more lipids (e.g., any of the one or more lipids described herein) to a total final concentration of about 0% to about 45% by weight (e.g., any of the subranges of about 0% to about 45% by weight described in the paragraph above); or one or more lipids (e.g., any of the lipids described herein) to a total final concentration of about 0.01% to about 6% by weight (e.g., any of the subranges of about 0.01% to about 6% by weight described in the paragraph above). Also provided are compositions that include one or more flavor compounds (e.g., any of one or more flavor compounds described herein) in a total final concentration of about 0.1% to about 6% by weight (e.g., any of the subranges of about 0.1% to about 6% by weight described in this section); one or more sweeteners (e.g., any of one or more sweeteners described herein) in a total final concentration of about 0.1% to about 6% by weight (e.g., any of the subranges of about 0.1% to about 6% by weight described in this section); and an ash (e.g., any of the exemplary ash described herein) in a total final concentration of about 0.15% to about 1.5% by weight (e.g., any of the subranges of about 0.15% to about 1.5% by weight described in the paragraph above), and that do not include at least one component found in mammalian produced milk; include at least one component that is not present in mammalian produced milk; and / or include at least one component at a higher or lower concentration compared to the concentration of the at least one component in mammalian produced milk. In some examples of these compositions, the composition comprises at least one component selected from the group of calcium, phosphate, vitamin B complex, vitamin A, vitamin D, vitamin E, and vitamin K at an elevated concentration compared to the concentration of the one or more components in mammalian produced milk.In some embodiments of these compositions, the composition does not contain at least one component found in mammalian produced milk, which is selected from the group consisting of lactose, bacteria, mycobacteria, allergens, viruses, prions, yeast, growth hormones, white blood cells, antibiotics, heavy metals, immunoglobulins, lactoferrin, lactoperoxidase, and lipase.In some examples of these compositions, the composition contains at least one component not present in mammalian produced milk, which is selected from the group consisting of artificial sweeteners, lipids of plant origin, β-casein protein with unglycosylated or non-mammalian glycosylation pattern, and κ-casein protein with unglycosylated or non-mammalian glycosylation pattern.

[0150] Also included are κ-casein proteins at about 0.3 g / L to about 1.1 g / L (e.g., any of the subranges of about 0.3 g / L to about 1.1 g / L described in this section) that are unglycosylated or have a non-mammalian glycosylation pattern (e.g., any of the κ-casein proteins described herein); β-casein proteins at about 1.25 g / L to about 4.9 g / L (e.g., any of the subranges of about 1.25 g / L to about 4.9 g / L described in this section) that are unglycosylated or have a non-mammalian glycosylation pattern (e.g., any of the β-casein proteins described herein); and β-casein proteins at a total final concentration of about 0% to about 45% by weight (e.g., any of the subranges of about 0% to about 45% by weight described in this section). Also provided are compositions comprising one or more lipids (e.g., any one or more lipids described herein) in a total final concentration of about 0.01% to about 6% by weight (e.g., any one or more flavor compounds described herein) in a total final concentration of about 0.01% to about 6% by weight (e.g., any one or more subranges of about 0.01% to about 6% by weight described in this section); one or more sweeteners (e.g., any one or more sweeteners described herein) in a total final concentration of about 0.1% to about 6% by weight (e.g., any one or more subranges of about 0.1% to about 6% by weight described in this section); and an ash (e.g., any one of the ash compositions described herein) in a total final concentration of about 0.15% to about 1.5% by weight (e.g., any one of the subranges of about 0.15% to about 1.5% by weight described in this section).

[0151] Also provided are compositions comprising micelles comprising κ-casein protein (e.g., any of the κ-casein proteins described herein) and β-casein protein (e.g., any of the β-casein proteins described herein), wherein the micelles have diameters from about 50 nm to about 350 nm (e.g., any of the subranges of micelle diameters described herein), and wherein the κ-casein protein and β-casein protein are unglycosylated or have a non-mammalian glycosylation pattern. In some embodiments, the composition is formulated to a final concentration of about 2.0% to about 6% by weight (e.g., about 2.0% to about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, about 3.0% by weight, or about 2.5% by weight; about 2.5% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, about 3.5% by weight, or about 3.0% by weight; about 3.0% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 4.5% by weight, about 4.0% by weight, or about 3.5% by weight; about 3.5% by weight to about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 5.0% by weight, about 5.0% by weight, about 5.0% by weight, about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 5.0% by weight, about 6.0% by weight, about 5.5% by weight, about 5.0% by weight, about 6 ... %, about 4.5 wt%, about 4.0 wt%, or about 3.5 wt%; about 3.5 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, or about 4.0 wt%; about 4.5 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, about 4.5 wt%, or about 4.0 wt%; about 4.0 wt% to about 6.0 wt%, about 5.5 wt%, about 5.0 wt%, or about 4.5 wt%; about 4.5 wt% to about 5.5 wt%, or about 5.0 wt%; about 5.0 wt% to about 6.0 wt% or about 5.5 wt%; or about 5.5 wt% to about 6.0 wt%. In some embodiments of these compositions, the ratio of β-casein protein to κ-casein protein in the micelles is from about 2.0:1 to about 5.5:1 (e.g., any of the subranges of about 2.0:1 to about 5.5:1 described for micelles herein).In some embodiments, these compositions further comprise one or more lipids (e.g., any of one or more lipids described herein) at a total final concentration of about 0% to about 45% by weight (e.g., any of the subranges of about 0% to about 45% by weight described in this section); one or more flavor compounds (e.g., any of one or more flavor compounds described herein) at a total final concentration of about 0.01% to about 6% by weight (e.g., any of the subranges of 0.01% to about 6% by weight described in this section); one or more sweeteners (e.g., any of the sweeteners described herein) at a total final concentration of about 0.1% to about 6% by weight (e.g., any of the subranges of about 0.1% to about 6% by weight described in this section); and an ash (e.g., any of the ash described herein) at a total final concentration of about 0.15% to about 1.5% by weight (e.g., any of the subranges of about 0.15% to about 1.5% by weight described in this section).

[0152] In some embodiments of any of the compositions described herein, the one or more lipids are selected from the group consisting of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids. Some examples of any of the compositions described herein include lipids having a final concentration of about 1% to about 28% by weight (e.g., about 1% to about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 8%, about 6%, about 4%, or about 2% by weight; about 2% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12 ... %, about 10%, about 8%, about 6%, or about 4% by weight; about 4% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 8%, or about 6% by weight; about 6% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, or about 8% by weight; about 8% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, or about 10% by weight; about 10% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, or about 12% by weight; about 12% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, or about 14% by weight; about 14% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, or about 14% by weight. % to about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, or about 16% by weight; about 16% to about 28%, about 26%, about 24%, about 22%, about 20%, about 18% by weight; about 18% to about 28%, about 26%, about 24%, about 22%, or about 20% by weight; about 20% to about 28%, about 26%, about 24%, about 22% by weight; about 22% to about 28%, about 26%, about 24% by weight;sunflower oil at a final concentration of about 24% to about 28% by weight, or about 26% by weight; or about 28% to about 30% by weight; sunflower oil at a final concentration of about 0.5% to about 14% by weight (e.g., about 0.5% to about 12% by weight, about 10% by weight, about 8% by weight, about 6% by weight, about 4% by weight, about 2% by weight, or about 1% by weight; about 1% to about 14% by weight, about 12% by weight, about 10% by weight, about 8% by weight, about 6% by weight, about 4% by weight, or about 2% by weight; about 2% to about 12% by weight, about 10% by weight, about 8% by weight, about 6% by weight, or about 4% by weight; about 12%, about 10%, about 8%, or about 6% by weight; about 6% to about 14%, about 12%, about 10%, or about 8% by weight; about 8% to about 14%, about 12%, or about 10% by weight; about 10% to about 14% or about 12% by weight; or about 12% to about 14% by weight; coconut oil to a final concentration of about 0.05% to about 1.0% by weight (e.g., about 0.05% to about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, or about between 0.2% by weight; 0.1% to about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, or about 0.2% by weight; from about 0.2% to about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, or about 0.4% by weight; from about 0.4% to about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, or about 0.5% by weight; from about 0.5% to about 1.0%, about 0.9%, about 0.8%, about 0.7%, or about 0.6% by weight; about 0.6% to about 1.0%, about 0.9%, about 0.8%, or about 0.7% by weight; about 0.7% to about 1.0%, about 0.9%, or about 0.8% by weight; about 0.8% to about 1.0%, about 0.9%, or about 0.8% by weight; about 0.8% to about 1.0%, or about 0.9% by weight; or about 0.9% to about 1.0% by weight; a total final concentration of about 0.08% to about 1.2% by weight (e.g., 0.08% to about 1.0%, about 0.8%, about 0.6%, about 0.4%, or about 0.2% by weight;about 0.2% to about 1.2%, about 1.0%, about 0.8%, about 0.6%, or about 0.4% by weight; about 0.4% to about 1.2%, about 1.0%, about 0.8%, or about 0.6% by weight; about 0.6% to about 1.2%, about 1.0%, or about 0.8% by weight; about 0.8% to about 1.2% or about 1.0% by weight; or about 1.0% to about 1.2% by weight of monoglycerides and diglycerides (e.g., any of one or more monoglycerides or diglycerides described herein); and free fatty acids to a total final concentration of about 0.02% to about 0.28% by weight; and free fatty acids to a total final concentration of about 0.02% to about 0.3% by weight. 0.25wt%, about 0.20wt%, about 0.15wt%, or about 0.10wt%; about 0.05wt% to about 0.3wt%, about 0.25wt%, about 0.20wt%, about 0.15wt%, or about 0.10wt%; about 0.10wt% to about 0.30wt%, about 0.25wt%, about 0.20wt%, or about 0.15wt%; about 0.15wt% to about 0.30wt%, about 0.25wt%, or about 0.20wt%; about 0.20wt% to about 0.30wt% or about 0.25wt%; or about 0.25wt% to about 0.30wt%;

[0153] In some embodiments of any of the compositions, the free fatty acid comprises at least one (e.g., two, three, or four) fatty acid selected from the group consisting of butyric acid, caproic acid, caprylic acid, and capric acid. In some embodiments of any of the compositions, the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid. In some examples of any of the compositions described herein, the flavor compound comprises at least one flavor compound selected from the group consisting of δ-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, and δ-undecalactone. In some embodiments of any of the compositions described herein, one or more sweeteners are saccharides (e.g., glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, or tagatose). In some examples of any of the compositions described herein, the one or more sweetening agents are artificial sweeteners (e.g., stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, or P-4000).

[0154] In some examples of any of the compositions described herein, the ash includes one or more (e.g., two, three, four, five, or six) of calcium, phosphorus, potassium, sodium, citrate, and chloride. In some embodiments of any of the compositions described herein, the ash includes one or more (e.g., two or three) of CaCl2, KH2PO4, and Na3 citrate. Some embodiments of the compositions described herein include CaCl to a final concentration of about 0.05 g / L to about 0.2 g / L (e.g., about 0.05 g / L to about 0.15 g / L, about 0.05 g / L to about 0.10 g / L, about 0.10 g / L to about 0.20 g / L, about 0.10 g / L to about 0.15 g / L, or about 0.15 g / L to about 0.2 g / L); and / or a final concentration of about 0.1 g / L to about 0.3 g / L (e.g., 0.1 g / L to about 0.25 g / L, about 0.1 g / L to about 0.20 g / L, about 0.1 g / L to about 0.40 g / L, or about 0.30 g / L to about 0.35 g / L, or about 0.35 g / L to about 0.40 g / L); ... from about 0.15 g / L, from about 0.15 g / L to about 0.30 g / L, from about 0.15 g / L to about 0.25 g / L, from about 0.15 g / L to about 0.20 g / L, from about 0.20 g / L to about 0.30 g / L, from about 0.20 g / L to about 0.25 g / L, or from about 0.25 g / L to about 0.30 g / L.

[0155] In any of the compositions described herein, the κ-casein protein can be cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth κ-casein protein. In any of the compositions described herein, the β-casein protein can be cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth β-casein protein.

[0156] In some examples of any of the compositions described herein, the composition may further comprise a final concentration of about 0.4% to about 2.5% by weight (e.g., about 0.4% to about 2.0% by weight, about 1.5% by weight, or about 1.0% by weight; about 1.0% to about 2.5% by weight, about 2.0% by weight, or about 1.5% by weight; about 1.5% to about 2.5% by weight or 2.0% by weight; or about 2.0% to about 2.5% by weight of alpha-lactalbumin protein (e.g., any of the alpha-lactalbumin proteins described herein)) and / or a final concentration of about 2.5% to about 4.5% by weight of beta-lactoglobulin protein (e.g., any of the beta-lactoglobulin proteins described herein). In some embodiments of any of the compositions described herein, the alpha-lactalbumin protein can be from a cow, a human, a sheep, a goat, a buffalo, a camel, a horse, a donkey, a lemur, a panda, a guinea pig, a squirrel, a bear, a macaque, a gorilla, a chimpanzee, a mountain goat, a monkey, an ape, a cat, a dog, a wallaby, a rat, a mouse, an elephant, a possum, a rabbit, a whale, a baboon, a gibbon, an orangutan, a mandrill, a pig, a wolf, a fox, a lion, a tiger, an echidna, or a woolly mammoth. In some embodiments of any of the compositions described herein, the β-lactoglobulin protein can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth β-lactoglobulin protein.

[0157] Some embodiments of any of the compositions described herein include α-S1-casein protein (e.g., any of the α-S1-casein proteins described herein) at a final concentration of about 11% to about 16% by weight (e.g., about 11% to about 15%, about 14%, about 13%, or about 12%; about 12% to about 16%, about 15%, about 14%, or about 13%; about 13% to about 16%, about 15%, or about 14%; about 14% to about 16% or 15%; or about 15% to about 16% by weight). For example, the α-S2-casein protein may be from about 2% to about 4.5%, about 4.0%, about 3.5%, about 3.0%, or about 2.5% by weight; from about 2.5% to about 5.0%, about 4.5%, about 4.0%, about 3.5%, or about 3.0% by weight; from about 3.0% to about 5.0% by weight, about 4.5%, about 4.0%, or about 3.5% by weight; from about 3.5% to about 5%, about 4.5%, or about 4.0% by weight; from about 4.0% to about 5.0% or about 4.5% by weight; or from about 4.5% to about 5.0% by weight (e.g., any of the α-S2-casein proteins described herein).

[0158] In some examples of any of the compositions described herein, the α-S1-casein protein is selected from the group consisting of α-S1 from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth. -casein protein; and / or the α-S2-casein protein may be α-S2-casein protein from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

[0159] Some examples of any of the compositions described herein further include one or more (e.g., two or three) serum albumin (e.g., any of the serum albumin proteins described herein), lactoferrin (e.g., any of the lactoferrin proteins described herein), and transferrin (e.g., any of the transferrin proteins described herein). In some examples of any of the compositions described herein, the serum albumin can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth serum albumin; lactoferrin can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth serum albumin; The lactoferrin may be from an ape, cat, dog, wallaby, rat, mouse, elephant, pouch, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth; and / or the transferrin may be from a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, pouch, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth transferrin protein.

[0160] In some examples of any of the compositions described herein, the composition further comprises one or more color correcting agents (e.g., any of the colorants described herein, such as beta-carotene or annatto).

[0161] The pH of any of the compositions described herein can be from about 6.2 to about 7.2 (e.g., from about 6.2 to about 7.0, from about 6.2 to about 6.8, from about 6.2 to about 6.6, from about 6.2 to about 6.4, from about 6.4 to about 7.2, from about 6.4 to about 7.0, from about 6.4 to about 6.8, from about 6.4 to about 6.6, from about 6.6 to about 7.2, from about 6.6 to about 7.0, from about 6.6 to about 6.8, from about 6.8 to about 7.2, from about 6.8 to about 7.0, or from about 7.0 to about 7.2).

[0162] In various embodiments, the milk protein component comprises about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6% milk protein by dry weight or total weight. In some embodiments, the composition may comprise about 0.5-2.5%, about 1-2%, about 2-3%, or about 4-10% protein by dry weight or total weight. In certain embodiments, the composition may comprise about 10-15% protein by dry weight or total weight.

[0163] A wide range of caseins, including caseins with substantial homology to wild-type casein, casein variants and mutants, have been expressed and incorporated as components of milk proteins.

[0164] dry composition Also, the final concentration may be from about 3.6% to about 5.4% by weight (e.g., from about 3.6% to about 5.2% by weight, about 5.0% by weight, about 4.8% by weight, about 4.6% by weight, about 4.4% by weight, about 4.2% by weight, about 4.0% by weight, or about 3.8% by weight; from about 3.8% to about 5.4% by weight, about 5.2% by weight, about 5.0% by weight, about 4.8% by weight, about 4.6% by weight, about 4.4% by weight, about 4.2% by weight, or about 4.0% by weight; from about 4.0% to about 5.4% by weight, about 5.2% by weight, about 5.0% by weight, about 4.8% by weight, about 4.6% by weight, about 4.4% by weight, or about 4.2% by weight; from about 4.2% to about 5.2% by weight, about 5.2% by weight, about 5.0% by weight, about 4.8% by weight, , 4.6%, or about 4.4% by weight; about 4.8% to about 5.4%, about 5.2%, or about 5.0% by weight; about 5.0% to about 5.4% or about 5.2% by weight; or about 5.2% to about 5.4% by weight of κ-casein protein (e.g., any of the α-casein proteins described herein); to a final concentration of about 16.3% to about 24.5% by weight; 16.3% to about 22% by weight, about 20% by weight, or about 18% by weight; about 18% to about 24.5% by weight, about 22% by weight, or about 20% by weight; about 20% to about 24.5% by weight to about 22% by weight; or about 22% to about 24.β-casein protein (e.g., any of the β-casein proteins described herein) to a final concentration of about 35% to about 40% by weight (e.g., about 35% to about 39%, about 38%, about 37%, or about 36% by weight; about 36% to about 40% by weight, about 39%, about 38%, or about 37% by weight; about 37% to about 40% by weight, about 39% by weight, or about 38% by weight; about 38% to about 40% by weight or 39% by weight; or about 39% by weight % to about 40% by weight of a sweetening agent (e.g., any of one or more sweetening agents described herein); a final concentration of about 25% to about 30% by weight (e.g., about 25% to about 29%, about 28%, about 27%, or about 26% by weight; about 26% to about 30% by weight, about 29%, about 28%, or about 27% by weight; about 27% to about 30% by weight, about 29%, or about 28% by weight; about 28% to about 30% by weight or about 29% by weight; or about 29% to about 30% by weight. one or more lipids (e.g., any of one or more lipids described herein) to a final concentration of about 30% by weight; an ash content (e.g., any of the ash content described herein) to a final concentration of about 5% to about 7% by weight (e.g., about 5% to about 6.5% by weight, about 6.0% by weight, or about 5.5% by weight; about 5.5% to about 7.0% by weight, about 6.5% by weight, or about 6.0% by weight; about 6.0% to about 7.0% by weight or about 6.5% by weight; or about 6.5% to about 7.0% by weight). and water to a final concentration of about 2% to about 5% by weight (e.g., about 2% to about 4% or about 3%; about 3% to about 5% or about 4%; or about 4% to about 5% by weight), wherein the κ-casein protein is unglycosylated and / or has a non-mammalian glycosylation pattern, and / or the β-casein protein is unglycosylated and / or has a non-mammalian glycosylation pattern.

[0165] Any of the powder compositions may contain any of the ingredients in any of the compositions described herein (e.g., any one or more of the color matching agents, α-S1-casein protein, α-S2-casein protein, α-lactalbumin protein, β-lactoglobulin protein, lactoferrin protein, transferrin protein, and serum albumin protein described herein, in any of the concentrations described herein for each ingredient).

[0166] Supplemented milk composition Also provided herein are compositions comprising mammalian produced milk or processed mammalian produced milk; and one or more (e.g., two or three) κ-casein proteins having an unglycosylated or non-mammalian glycosylation pattern; β-casein proteins having an unglycosylated or non-mammalian glycosylation pattern; or micelles comprising κ-casein proteins having an unglycosylated or non-mammalian glycosylation pattern and β-casein proteins having an unglycosylated or non-mammalian glycosylation pattern.

[0167] In some examples, the composition comprises mammalian produced milk or processed mammalian produced milk and a κ-casein protein having an unglycosylated or non-mammalian glycosylation pattern. In some examples, the composition comprises mammalian produced milk or processed mammalian produced milk and a β-casein protein having an unglycosylated or non-mammalian glycosylation pattern. In other examples, the composition comprises mammalian produced milk or processed mammalian produced milk and micelles comprising a κ-casein protein having an unglycosylated or non-mammalian glycosylation pattern and a β-casein protein having an unglycosylated or non-mammalian glycosylation pattern.

[0168] In some examples, the final concentration of κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern or the final concentration of β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern in the composition is from 0.02% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, about 0.5% by weight, about 0.6% by weight, about 0.8 ... 0.2% by weight, or about 0.1% by weight; from about 0.1% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, or about 0.2% by weight; from about 0.2% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8%, about 0.6%, or about 0.4% by weight; about 0.8% to about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, about 1.6%, about 1.4%, about 1.2%, or about 1.0% by weight; about 1.0% to about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, about 1.6%, about 1.4%, or about 1.2% by weight; about 1.2% to about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, about 1.6%, or about 1.4% by weight; about 1.4% to about 3.0% by weight, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, or about 1.6% by weight; about 1.6% to about 3.0% by weight, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, or about 1.8% by weight; about 1.8% to about 3.0% by weight, about 2.8%, about 2.6%, about 2.4%, about 2.2% by weight, or about 2.0% by weight; about 2.0% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, or about 2.2% by weight; about 2.2% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, or about 2.4% by weight; about 2.4% by weight to about 3.0% by weight, about 2.8% by weight, or about 2.6% by weight; about 2.6% by weight to about 3.0% by weight or about 2.8% by weight; or about 2.8% by weight to about 3.0% by weight (based on the final composition).

[0169] In some compositions, the final concentration of κ-casein protein that is unglycosylated and / or has a non-mammalian glycosylation pattern in the composition is from about 0.02% to about 0.6% by weight (e.g., from about 0.02% to about 0.5% by weight, from about 0.02% to about 0.4% by weight, from about 0.02% to about 0.3% by weight, from about 0.02% to about 0.2% by weight, from about 0.02% to about 0.1% by weight, from about 0.1% to about 0.5% by weight, from about 0.1% to about 0.4% by weight, from about 0.1% to about 0.3% by weight, from about 0.1% to about 0. 2% by weight, about 0.2% by weight to about 0.5% by weight, about 0.2% by weight to about 0.4% by weight, about 0.2% by weight to about 0.3% by weight, about 0.3% by weight to about 0.5% by weight, about 0.3% by weight to about 0.4% by weight, about 0.2% by weight to about 0.5% by weight, about 0.2% by weight to about 0.4% by weight, about 0.2% by weight to about 0.3% by weight, about 0.3% by weight to about 0.5% by weight, about 0.3% by weight to about 0.4% by weight, or about 0.4% by weight to about 0.5% by weight; , about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, or about 0.2% by weight; about 0.2% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, about 0. 6% by weight, or about 0.4% by weight; about 0.4% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, or about 0.6% by weight; about 0.6% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, or about 0.8% by weight; about 0.8% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, or about 1.0% by weight; about 1.0% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3. 0.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, or about 1.2% by weight; about 1.2% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, or about 1.4% by weight; about 1.4% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, or about 1.6% by weight; about 1.6% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, or about 1.8% by weight; about 1.8% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, or about 2.0% by weight; about 1.8% to about 4.0%, about 3.8%, about 3.6%, about 3.4%, about 3.2%, about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, or about 2.0% by weight; about 2.0% to about 4.0%, about 3.8%, about 3.6%, about 3.4%, about 3.2%, about 3.0%, about 2.8%, about 2.6%, about 2.4%, or about 2.2% by weight; about 2.2% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, or about 2.4% by weight; about 2.4% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, about 2.8% by weight, or about 2.6% by weight; about 2.6% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, or about 2.8% by weight; about 2.8% by weight to about 4.0% by weight, about 3.8% by weight, about 3.6% by weight, about 3.4% by weight, about 3.2% by weight, about 3.0% by weight, or about 2.8% by weight about 4.0%, about 3.8%, about 3.6%, about 3.4%, about 3.2%, or about 3.0% by weight; about 3.0% to about 4.0%, about 3.8%, about 3.6%, about 3.4%, or about 3.2% by weight; about 3.2% to about 4.0%, about 3.8%, about 3.6%, or about 3.4% by weight; about 3.4% to about 4.0%, about 3.8%, or about 3.6% by weight; about 3.6% to about 4.0% or about 3.8% by weight; or about 3.8% to about 4.0% by weight.

[0170] In some examples, the final concentration of micelles comprising κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern and β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern in the composition is from 0.02% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, about 0.5% by weight, about 0.6% by weight, about 0.8% by weight, about 0.6% by weight, about 0.8% by weight, about 0.4% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, about 0.6 ... 2% by weight, or about 0.1% by weight; about 0.1% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8% by weight, about 0.6% by weight, about 0.4% by weight, or about 0.2% by weight; about 0.2% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, about 2.2% by weight, about 2.0% by weight, about 1.8% by weight, about 1.6% by weight, about 1.4% by weight, about 1.2% by weight, about 1.0% by weight, about 0.8%, about 0.6%, or about 0.4% by weight; about 0.8% to about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, about 1.6%, about 1.4%, about 1.2%, or about 1.0% by weight; about 1.0% to about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, about 1.6%, about 1.4%, or about 1.2% by weight; about 1.2% to about 3.0%, about 2.8%, about 2. .6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, about 1.6%, or about 1.4% by weight; about 1.4% to about 3.0% by weight, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, about 1.8%, or about 1.6% by weight; about 1.6% to about 3.0% by weight, about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.0%, or about 1.8% by weight; about 1.8% to about 3.0% by weight, about 2.8%, about 2.6%, about 2.4%, about 2.2% by weight, or about 2.0% by weight; about 2.0% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, about 2.4% by weight, or about 2.2% by weight; about 2.2% by weight to about 3.0% by weight, about 2.8% by weight, about 2.6% by weight, or about 2.4% by weight; about 2.4% by weight to about 3.0% by weight, about 2.8% by weight, or about 2.6% by weight; about 2.6% by weight to about 3.0% by weight or about 2.8% by weight; or about 2.8% by weight to about 3.0% by weight (based on the final composition).

[0171] Nucleic acids and vectors Also provided are nucleic acids (e.g., vectors) that include a promoter (e.g., a yeast promoter, a bacterial promoter, or a mammalian promoter); a sequence encoding a signal sequence; a sequence encoding a milk protein (e.g., any of the exemplary sequences described herein); and a yeast termination sequence, where the promoter is operably linked to the signal sequence, the signal sequence is operably linked to the sequence encoding the milk protein, and the termination sequence is operably linked to the sequence encoding the milk protein. In some examples of these nucleic acids, the promoter is a constitutive promoter or an inducible promoter. Non-limiting examples of promoters are described herein. Additional promoters that may be used in these nucleic acids are known in the art.

[0172] The signal sequence in any of the vectors described herein may be a signal sequence from the encoded milk protein or a different milk protein, or a signal sequence from a yeast mating factor (e.g., any alpha mating factor). In some examples, the encoded milk protein is selected from the group of β-casein (e.g., any of the β-casein proteins described herein), κ-casein (e.g., any of the κ-casein proteins described herein), α-S1-casein (e.g., any of the α-S1-casein proteins described herein), α-S2-casein (e.g., any of the α-S2-casein proteins described herein), α-lactalbumin (e.g., any of the α-lactalbumin proteins described herein), β-lactoglobulin (e.g., any of the β-lactoglobulin proteins described herein), lactoferrin (e.g., any of the lactoferrin proteins described herein), or transferrin (e.g., any of the transferrin proteins described herein). Additional signal sequences that can be used in the present vectors are known in the art.

[0173] Any of the nucleic acids described herein may further comprise a bacterial origin of replication. Any of the nucleic acids described herein may further comprise a selection marker (e.g., an antibiotic resistance gene). The sequence of a bacterial origin of replication is known in the art. Non-limiting examples of antibiotic resistance genes are described herein. Further examples of resistance genes are known in the art.

[0174] Non-limiting examples of termination sequences are described herein. Further examples of termination sequences are known in the art.

[0175] Some embodiments of the nucleic acids presented herein further comprise an additional promoter sequence (e.g., any of the exemplary promoters described herein); an additional sequence encoding a signal sequence (e.g., any of the exemplary signal sequences described herein); a sequence encoding an additional milk protein (e.g., any of the exemplary sequences encoding a milk protein described herein); and an additional yeast termination sequence (e.g., any of the exemplary yeast termination sequences described herein), where the additional promoter sequence is operably linked to the additional sequence encoding a signal sequence, which sequence encoding the signal sequence is operably linked to the sequence encoding the additional milk protein, which sequence encoding the additional milk protein is operably linked to the additional yeast termination sequence. The additional milk protein may be, for example, β-casein (e.g., any of the β-casein proteins described herein), κ-casein (e.g., any of the κ-casein proteins described herein), α-S1-casein (e.g., any of the α-S1-casein proteins described herein), α-S2-casein (e.g., any of the α-S2-casein proteins described herein), α-lactalbumin (e.g., any of the α-lactalbumin proteins described herein), β-lactoglobulin (e.g., any of the β-lactoglobulin proteins described herein), lactoferrin (e.g., any of the lactoferrin proteins described herein), or transferrin (e.g., any of the transferrin proteins described herein). In some embodiments, the nucleic acid comprises a sequence encoding β-casein and a sequence encoding κ-casein. The promoter and the additional promoter may be the same or different. The yeast termination sequence and the further yeast termination sequence may be the same or different. The signal sequence and the further signal sequence may be the same or different.

[0176] The present invention also encompasses a vector containing an isolated DNA sequence encoding a casein or whey polypeptide and a host cell comprising the vector. The vector may further comprise an isolated DNA sequence encoding casein, the nucleotide sequence comprising a nucleotide sequence operably linked to a promoter, an alpha mating factor or variant thereof, a nucleotide sequence encoding a bacterial resistance marker and a transcription terminator. One or more of the suitable promoters used for expression of the gene encoding the casein or whey protein may be any promoter that is functional in the host cell and capable of inducing expression of the product encoded by the gene. Suitable promoters include, for example, P LAC4-PBI , T7, Ptac, Pgal, λPL, λPR, bla, spa, Adh, CYC, TDH3, ADH1 and CLB1.

[0177] Introduction of nucleic acids into cells The method of introducing nucleic acid (e.g., any of the nucleic acids described herein) into cells to generate host cells is well known in the art.Non-limiting examples of techniques that can be used to introduce nucleic acid into cells include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, electroporation, cell squeezing, sonoporation, phototransfection, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, and viral transduction.

[0178] Those skilled in the art can select one or more suitable techniques for introducing nucleic acid into cells based on the knowledge in the art that a certain technique for introducing nucleic acid into cells works better for different types of host cells.Exemplary methods for introducing nucleic acid into yeast cells are described in Kawai et al., Bioeng.Bugs, 1:395-403, 2010.

[0179] host cell Also provided herein is a host cell comprising any of the nucleic acids (e.g., vectors) described herein.In some instances, the nucleic acids described herein are stably integrated into the genome (e.g., chromosome) of the host cell.In other instances, the nucleic acids described herein are not stably integrated into the genome of the host cell.

[0180] In some embodiments, the host cell is a yeast strain or a bacterial strain. In some embodiments, the host cell may be a yeast strain selected from the group of, for example, Kluyveromyces species, Pichia species, Saccharomyces species, Tetrahymena species, Yarrowia species, Hansenula species, Blastobotrys species, Candida species, Zygosaccharomyces species, and Debaryomyces species. Further non-limiting examples of yeast strains that can be used as host cells are Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris. Further species of yeast strains that can be used as host cells are known in the art.

[0181] In some cases, the host cell may be a protozoan, such as, for example, Tetrahymena thermophila, T. hegewischi, T. hyperangularis, T. malaccensis, T. pigmentosa, T. pyriformis, and T. vorax.

[0182] It is an object of the present invention to isolate milk protein components by recombinantly expressing them in any of the host cells presented herein.

[0183] Methods for Producing Recombinant Milk Proteins and Making Micelles Also provided are methods of making a recombinant milk protein that is unglycosylated or has a non-mammalian glycosylation pattern (e.g., any of one or more milk proteins described herein), comprising culturing any of the host cells described herein in a culture medium under conditions sufficient to allow for secretion of the milk protein that is unglycosylated or has a non-mammalian glycosylation pattern; and harvesting the milk protein that is unglycosylated or has a non-mammalian glycosylation pattern from the culture medium. Culture media suitable for use in these methods are known in the art. Culture conditions sufficient to allow for secretion of the milk protein are also known in the art. The host cells used in these methods can be any of the host cells described herein. The host cells can comprise any of the nucleic acids described herein. The recombinant milk protein produced may be one or more of β-casein (e.g., any of the β-casein proteins described herein), κ-casein (e.g., any of the κ-casein proteins described herein), α-S1-casein (e.g., any of the α-S1 caseins described herein), α-S2-casein (e.g., any of the α-S2-caseins described herein), α-lactalbumin (e.g., any of the α-lactalbumin proteins described herein), β-lactoglobulin (e.g., any of the β-lactoglobulin proteins described herein), lactoferrin (e.g., any of the lactoferrin proteins described herein), transferrin (e.g., any of the transferrin proteins described herein), and serum albumin (e.g., any of the serum albumin proteins described herein). Some of these methods further include isolating (e.g., purifying) the recombinant milk protein from the culture medium.Methods for isolating (e.g., purifying) recombinant milk proteins from liquids are well known in the art. An exemplary method for isolating (e.g., purifying) recombinant milk proteins is described in Imafidon et al., Crit. Rev. Food Sci. Nutrition, 37:663-669, 1997.

[0184] Also provided is a method of making micelles comprising β-casein (e.g., any of the β-casein proteins described herein) that is unglycosylated or has a non-mammalian glycosylation pattern and κ-casein (e.g., any of the κ-casein proteins described herein) that is unglycosylated or has a non-mammalian glycosylation pattern, comprising culturing any of the host cells described herein in a culture medium under conditions sufficient to allow release of micelles from the host cells, the host cells comprising a nucleic acid comprising a sequence encoding β-casein and a sequence encoding κ-casein; and harvesting the micelles from the culture medium. Culture media suitable for use in these methods are known in the art. The host cells used in these methods can be any of the host cells described herein. The host cells can comprise any of the nucleic acids described herein. The micelles made can be any of the micelles described herein. (And may have any of the physical characteristics of micelles described herein.) Some of these methods further include isolating (e.g., purifying) the micelles from the culture medium. Methods for isolating (e.g., purifying) micelles from a liquid are well known in the art (e.g., ultracentrifugation).

[0185] Exemplary details for culturing yeast host cells are described in Idiris et al., Appl. Microbiol. Biotechnol. 86:403-417, 2010; Zhang et al., Biotechnol. Bioprocess. Eng., 5:275-287, 2000; Zhu, Biotechnol. Adv., 30:1158-1170, 2012; Li et al., MAbs, 2:466-477, 2010.

[0186] It is an object of the present invention to express one or more different forms of casein for application in a wide variety of dairy substitute products. Casein subunits such as α-s1-casein, α-s2-casein, β-casein and κ-casein differ by one or more amino acid changes. In certain embodiments, the methods and compositions include the incorporation of bovine casein such as α-s1-casein, α-s2-casein, β-casein and κ-casein. In other embodiments, the methods and compositions include the incorporation of human casein such as β-casein and κ-casein. See U.S. Patent No. 5,942,274. In alternative embodiments, the casein is selected from one or more of the following sources, including, but not limited to, cow, human, buffalo, camel, goat, sheep, horse, dolphin, whale, mountain goat and pig.

[0187] Also provided is a method for producing a milk protein component, which may include using a plasmid or construct of the invention, for example as described in Example 1. The method includes the steps of preparing a plasmid of interest, inserting the plasmid into a suitable host cell, culturing the host cell for a suitable time and under suitable conditions so that the protein of interest is expressed, and then purifying the protein.

[0188] Proteins can be separated based on their molecular weight, for example, by size exclusion chromatography, ultrafiltration through membranes, or density centrifugation. In some embodiments, proteins can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Proteins can also be separated based on their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, detergent, detergent, or solvent extraction. Proteins can also be separated by their affinity to other molecules, for example, using hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography can also include using antibodies with specific binding affinity to proteins, nickel-NTA for His-tagged recombinant proteins, lectins that bind to sugar moieties on glycoproteins, or other molecules that specifically bind to proteins.

[0189] Generally, centrifugation at the optimal pH results in a purification efficiency of >95%. The isoelectric points of native casein and whey proteins are known. In nature, the pH is 4.91 for bovine α-s1-casein, pH 4.1 for bovine α-s2-casein, pH 4.5 for bovine β-casein, pH 4.1 for bovine κ-casein, pH 4.2 for bovine α-lactalbumin, and pH 5.2 for bovine β-lactoglobulin. Recombinantly produced casein and whey may differ in the relationship of their phosphate and sugar groups. Other methods for protein purification include membrane filtration to remove any potential bacteria or contaminants, followed by lyophilization to isolate the protein.

[0190] Preferably, the methods and compositions provide production costs of no more than or less than $1,000, $500, $10, $1.0, $0.10, $0.010 or $0.0010 per kg of milk protein component. In more preferred embodiments, the costs are less than $0.009, $0.007, $0.006, $0.005 per kg of milk protein component.

[0191] Method for supplementing mammalian produced milk Also provided herein is a method of supplementing mammalian produced milk, the method comprising the steps of providing mammalian produced milk or processed mammalian produced milk; and mixing with the milk at least one of: a β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern (e.g., any of the β-casein proteins described herein); a κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern (e.g., any of the κ-casein proteins described herein); and micelles (e.g., any of the micelles described herein) comprising a β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern (e.g., any of the β-casein proteins described herein), and a κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern (e.g., any of the casein proteins described herein).

[0192] One or more of β-casein protein, κ-casein protein, and micelles can be mixed into milk to achieve any of the exemplary final concentrations of β-casein protein, κ-casein protein, and micelles in the composition described in the section entitled "Supplemented Milk Compositions" herein. Methods of mixing are well known in the art. As one skilled in the art can appreciate, additional ingredients described herein can also be mixed into milk (e.g., without limitation, any of the ingredients described herein).

[0193] Method of Making the Composition Also provided is a method of making a composition, comprising the steps of sonicating a liquid comprising a protein mixture comprising β-casein protein (e.g., any of the β-casein proteins described herein) and casein κ protein (e.g., any of the κ-casein proteins described herein), or comprising micelles comprising β-casein protein (e.g., any of the β-casein proteins described herein) and κ-casein protein (e.g., any of the κ-casein proteins described herein); Also provided is a method comprising the steps of: mixing a mixture of one or more lipids (e.g., any of one or more liquids described herein), one or more flavor compounds (e.g., any of one or more flavor compounds described herein), and one or more color correctors (e.g., any of one or more color correctors described herein) to the liquid and sonicating the liquid; and adding one or more sweeteners (e.g., any of one or more sweeteners described herein) to the liquid, thereby producing the composition.

[0194] In some examples of these methods, the β-casein protein is non-glycosylated or has a non-mammalian glycosylation pattern, and / or the κ-casein protein is non-glycosylated or has a non-mammalian glycosylation pattern. In some examples of these methods, the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride. In some examples of any of these methods, the added ash comprises one or more (e.g., two or three) of CaCl2, KH2PO4, and Na3 citrate.

[0195] In some examples of these methods, the one or more lipids include at least one (e.g., 2, 3, 4, 5, 6, or 7) of sunflower oil, coconut oil, tributyrin, monoglycerides and diglycerides, free fatty acids, and phospholipids. In some examples of these methods, the free fatty acids include at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid. In some examples of these methods, the phospholipids are soybean lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, or rapeseed lecithin phospholipids. In some embodiments of these methods, the flavor compounds include at least one (e.g., 2, 3, 4, 5, or 6) flavor compounds selected from the group of δ-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, and δ-undecalactone.

[0196] In some examples of these methods, one or more color correctors are β-carotene or annatto.In some embodiments of these methods, one or more sweeteners are saccharides (e.g., glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, or tagatose) or artificial sweeteners (e.g., stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentasine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, or P-4000).

[0197] The pH of the resulting composition can be between about pH 6.2 and about pH 7.4 (e.g., about 6.2 to about 7.2; about 6.2 to about 7.0, about 6.2 to about 6.8, about 6.2 to about 6.6, about 6.2 to about 6.4, about 6.4 to about 7.2, about 6.4 to about 7.0, about 6.4 to about 6.8, about 6.4 to about 6.6, about 6.6 to about 7.2, about 6.6 to about 7.0, about 6.6 to about 6.8, about 6.8 to about 7.2, about 6.8 to about 7.0, or about 7.0 to about 7.2).

[0198] In any of these methods, the β-casein protein is selected from the group consisting of cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, and woolly mammoth β-casein protein. and / or the κ-casein protein can be bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth κ-casein protein.

[0199] In some embodiments of these methods, the protein mixture further comprises one or more proteins selected from the group of α-lactalbumin (e.g., any of the α-lactalbumin proteins described herein), β-lactoglobulin (e.g., any of the β-lactoglobulin proteins described herein), α-S1-casein (e.g., any of the α-S1-casein proteins described herein), α-S2-casein (e.g., any of the α-S2-casein proteins described herein), lactoferrin (e.g., any of the lactoferrin proteins described herein), transferrin (e.g., any of the transferrin proteins described herein), and serum albumin (e.g., any of the serum albumin proteins described herein).

[0200] As one of ordinary skill in the art would appreciate, the amounts of each component used in these methods can be calculated to produce any of the compositions described herein.

[0201] How to Make Butter, Cheese, Caseinate, or Yogurt Also provided herein is a method of making butter, cheese, caseinate, or yogurt, the method comprising the steps of providing any of the compositions provided herein; and using any of the compositions provided herein as a starting material to make butter, cheese, caseinate, or yogurt.

[0202] Methods for making butter, cheese, caseinate, or yogurt are well known in the art. See, for example, Scott, "Cheesemaking Practice", Kluwer Academic / Plenum Publishers, New York, NY, 1998; U.S. Patent No. 4,360,535 (which describes a method for making cream); U.S. Patent No. 285,878 (which described a method for making butter).

[0203] kit Also provided are kits that include (a) a mixture of one or more milk proteins (e.g., any of the milk proteins described herein, including any of the β-casein protein, κ-casein protein, α-S1-protein, α-S2-protein, α-lactalbumin protein, β-lactoglobulin protein, lactoferrin protein, transferrin protein, and serum albumin protein described herein), one or more lipids (e.g., any of the one or more lipids described herein), and one or more flavor compounds (e.g., any of the one or more flavor compounds described herein); and (b) a mixture of ash (e.g., any of the ash described herein) and at least one sweetener (e.g., any of the one or more sweeteners described herein). In some examples of these kits, the one or more milk proteins are milk proteins from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

[0204] In some examples of these kits, the one or more fats are selected from the group of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids. The fatty acids present in the kit may include at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid. The phospholipids in the kit may be soybean lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, or rapeseed lecithin phospholipids.

[0205] The flavour compounds in the kit may comprise at least one flavour compound selected from the group consisting of delta-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, gamma-undecalactone, and delta-undecalactone.

[0206] In some embodiments of the kit, the mixture in (a) further comprises one or more color correcting agents (e.g., any of the color correcting agents described herein, e.g., β-carotene or annatto).

[0207] In some examples of the kit, the one or more sweetening agents are saccharides (e.g., glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, or tagatose) or artificial sweeteners (e.g., stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentasine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, or P-4000).

[0208] The kit may include an ash that includes one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride. In some examples, the ash in the kit includes one or more (e.g., two or three) of CaCl2, KH2PO4, and NaCitrate.

[0209] In some embodiments of the kit, the mixture in (a) is provided in a light-tight and airtight package (e.g., a metal foil, e.g., aluminum foil), and / or the mixture in (b) is provided in an airtight package (e.g., a sealed plastic bag).

[0210] Some examples of kits further include instructions for making any of the compositions described herein.

[0211] Also provided herein are kits that include at least one of the nucleic acids described herein.

[0212] Modulating the flavor profile Sensory impressions like "feed", "barny" or "filthy" are described as descriptions of flavors absorbed from the food ingested by the cow and the odors in its surrounding environment. Other sensory impressions are caused by microbial action resulting from the growth of large numbers of bacteria. Chemical changes can also occur through the action of enzymes, contact with metals (such as copper), or exposure to sunlight or strong fluorescent light. Quality control personnel are constantly striving to avoid off-flavors in milk and other dairy-based foods. It is therefore an object of the present invention to reduce, eliminate or mask undesirable flavors and odors in various dairy-based products.

[0213] In certain preferred aspects of the present invention, the flavor and odor of various dairy substitute products can be modified by changing fat content. For example, increasing butyric acid content can change the flavor profile of non-dairy cheese to a flavor profile similar to that of Parmesan cheese. In other embodiments, modulating triglycerides such as caproic acid, capric acid, and / or caprylic acid results in a flavor profile similar to that of goat cheese. Thus, modulating triglycerides through the ratio of fatty acid components results in different flavor profiles that can be fine-tuned to resemble the flavor profile of various desired dairy foods.

[0214] Similarly, the methods and compositions minimize one or more undesirable flavors by modulating the various triglycerides that are incorporated into the dairy substitute product.

[0215] In certain embodiments, the flavor profile is modulated by incorporating synthetic short chain triglycerides in combination with a plant-based oil, such as sunflower oil, in a desired combination. For example, a mixture of [C18 C18 C6] and [C18 C6 C18] will yield a different flavor profile than a mixture of [C18 C4 C4] and [C18 C10 C10].

[0216] Dairy substitute products Using the methods and compositions of the present invention, a wide variety of dairy substitute products may be made, including, but not limited to, milk, whole milk, buttermilk, skim milk, infant formula, condensed milk, milk powder, evaporated milk, butter, clarified butter, cream, and various types of cheese.

[0217] Dairy substitute products may also be incorporated into a variety of food applications as replacements for dairy-containing products, including ice cream, frozen custard, frozen yogurt, cookies, cakes, cottage cheese, cream cheese, creme fraiche, curds and yogurt.

[0218] In certain aspects, the present invention provides for one or more subunits of casein selected from α-s1-casein, α-s2-casein, β-casein and κ-casein for the milk protein component in the dairy substitute product. A select combination of casein subunits is used as the majority or at least part of the milk protein component. In a preferred embodiment, the casein composition comprises the following amounts of casein subunits: about 12-15 g / L α-s1-casein, about 3-4 g / L α-s2-casein, about 9-11 g / L β-casein and about 2-4 g / L κ-casein, representing the total casein in the synthetic dairy product.

[0219] In various embodiments, the casein composition may comprise about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6% protein by dry weight or total weight. In some embodiments, the casein composition may comprise about 0.5-2.5%, about 1-2%, about 2-3%, or about 4-10% casein protein by dry weight or total weight. In certain embodiments, the casein composition may comprise about 1.5-10% protein by dry weight or total weight.

[0220] In certain embodiments, the methods and compositions for dairy substitute products are essentially free of one or more serum proteins. Serum proteins typically include, among other proteins, enzymes, hormones, growth factors, nutrient transporters and disease resistance factors. In further embodiments, the methods and compositions for dairy substitute products are essentially free of one or more immunoglobulins that may induce undesirable immune responses.

[0221] In some embodiments, the whey composition may comprise about 0.001%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4% whey protein by dry weight or total weight. In some embodiments, the composition may comprise about 0.1-1%, about 1-2%, about 2-3%, or about 0.1-2.3% protein by dry weight or total weight. In certain embodiments, the composition may comprise about 10-15% protein by dry weight or total weight.

[0222] In various embodiments, carbohydrates are incorporated into the dairy substitute product. These carbohydrates provide a mild sweetness to the flavor profile of the product, and also act as a fast-acting energy and nutritional source. Carbohydrates include, but are not limited to, sugars such as galactose, sucrose, glucose, fructose, and maltose. Non-dairy sources of sugar include, but are not limited to, sugarcane and other plants such as celery, basil, honey, cherry, corn, spinach, plum, kiwi, and pea.

[0223] Lactose intolerance is common among many milk consumers. Thus, in a preferred embodiment, carbohydrates such as lactose are excluded from the dairy substitute composition. In a preferred embodiment, the method and composition for the dairy substitute composition is essentially lactose-free.

[0224] In some embodiments, the carbohydrate composition may comprise about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5% carbohydrate by dry weight or total weight. In some embodiments, the composition may comprise about 1-3%, about 2-4%, or about 10-30% carbohydrate by dry weight or total weight. In certain embodiments, the composition may comprise about 2-5% carbohydrate by dry weight or total weight.

[0225] Ash contributes to the structure and stability of the casein micelles. Ash is important for holding together the emulsion that is milk or cream. The calcium and phosphate present in the ash interact with the fat globules and the casein micelles to maintain the emulsified mixture.

[0226] Ash also influences the sensory characteristics of milk such as mouthfeel, consistency and, to a certain extent, the flavor of milk.

[0227] In some embodiments, the ash composition may comprise about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, or about 3% ash by dry weight or total weight. In some embodiments, the composition may comprise about 0.1-0.3%, about 0.5-0.7%, about 0.7-1%, or about 1-2% ash by dry weight or total weight. In certain embodiments, the composition may comprise about 0.6-0.8% protein by dry weight or total weight.

[0228] Further ingredients for various animal-origin free dairy products include vitamins, flavorings, natural or artificial sweeteners, colorants, salts, pH adjusters, binders, buffers, stabilizers, essential amino acids, anti-caking agents, anti-foaming agents, and mixtures thereof.

[0229] In some embodiments, the remaining ingredients of the composition may include about 0%, about 0.01%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% of the additive by dry weight or total weight. In some embodiments, the composition may include about 0.001-0.01%, about 0.01-1%, about 0.01-2%, or about 1-5% of the additive by dry weight or total weight. In certain embodiments, the composition may include about 0-10% of the additive by dry weight or total weight.

[0230] In some aspects, the present invention provides methods and compositions for dairy substitutes with fat, comprising varying the level of triglyceride content. In a preferred embodiment, triglycerides isolated from various plant sources are incorporated with milk protein components, carbohydrates and ash. It is an object of the present invention to modulate the fatty acids isolated in plants and interesterified in dairy substitutes to resemble the percentage of fatty acids found in natural dairy products, and / or develop novel flavor profiles with improved flavors not found in natural occurring products. In some embodiments, modulating special short- to medium-chain fatty acids, including but not limited to butyric acid, capric acid, caprylic acid, caproic acid and lauric acid, results in the desired flavor profile in dairy substitutes.

[0231] In some embodiments, the fat composition in the synthetic milk comprises about 0%, about 1%, about 2%, about 3%, about 3.5%, about 4% fat by dry weight or total weight. In some embodiments, the composition may comprise about 1-2%, about 2-3%, about 3-4% fat by dry weight or total weight. In certain embodiments, the composition may comprise about 3-4% fat by dry weight or total weight. In alternative embodiments, the fat composition in the cream may comprise about 10%, about 20%, about 30%, about 40%, about 50% or even 60%. Preferably, the fat composition in the cream is typically about 40 to about 50%.

[0232] In some embodiments, short-chain triglycerides are combined with long-chain oils to produce transesterified fatty acid esters.Preferably, the long-chain oils are selected from sunflower oil, corn oil, olive oil, soybean oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, canola oil, safflower oil, flax oil, date oil, date kernel oil, date fruit oil, coconut oil, babassu oil, shea butter oil, mango butter oil, cocoa butter oil, wheat germ oil and rice bran oil.More preferably, the long-chain oils comprise the long-chain oils from engineered sunflower varieties that overexpress oleic acid by 400%.

[0233] Long chain oils also contribute to the flavor profile, for example reducing or even removing sharpness and richness from the overall flavor profile of the desired end product.

[0234] In some embodiments, the fat component of the dairy substitute comprises select triglycerides interesterified to long-chain oils such as high oleic sunflower oil (Example 2). It is assumed that the same four short-chain fatty acids give derived products such as milk and cheese their particular flavors, such as robustness and richness. Various combinations of triglycerides and long-chain oils are incorporated to create a number of different flavor profiles. In one embodiment, a triglyceride with three oleic acids and a synthetic short-chain triglyceride, in this case with one butyric acid, one hexanoic acid, and one octanoic acid, results in the desired synthetic "milk fat" triglyceride. Further embodiments include incorporating a variety of short-chain triglycerides, such as a short-chain triglyceride with hexanoic acid; a short-chain triglyceride with hexanoic acid and butyric acid; a short-chain triglyceride with hexanoic acid and decanoic acid, to tune slightly different flavor profiles. Thus, the methods and compositions provide various combinations of synthetic short chain triglycerides with sunflower oil triglycerides that result in different flavor profiles.

[0235] Synthetic milk An exemplary embodiment of the synthetic milk formulation containing microbial-derived proteins of the present invention is illustrated in Example 4. For example, the formulation incorporates all four subunits of bovine casein: α-s1-casein, α-s2-casein, β-casein and κ-casein, and two whey proteins, α-lactalbumin and β-lactoglobulin, as the main milk protein components in the formulation. The exemplary synthetic milk formulation further includes a plant-based interesterified fat, as shown in Figure 1. Further components include carbohydrates and ash. The resulting milk substitute exhibits characteristics that look, function, taste, smell and feel like natural milk. As one of the key aspects of the present invention, modifying the formulation for synthetic milk can exhibit different sensory impressions, such as flavoring, by modulating the oil content, i.e., the type of triglycerides added to mimic different flavored milk.

[0236] As described by Young W. Park in "Bioactive Components in Milk and Dairy Products", Technology & Engineering, p. 60, 2009, sterols are a minor fraction of the total lipids in milk, with the major sterol being cholesterol (300 mg per 100 g fat, equivalent to 10 mg per 100 mL milk) (Park et al., Small Rumin. Res., 68:88-113, 2007). Goat milk has been shown to contain less cholesterol than other milks, but generally contains a higher amount of total fat than cow's milk. See Posati et al., 1976, "Composition of Foods," Agric. Handbook, No. 8-1, ARS, USDA, Washington, DC, 1976; Jenness, J. Dairy Sci., 63:1605-1630, 1980; and Juarez et al., Intl. Dairy Fed. Bull., No. 202, pp. 54-67, 1986, show that the palmitic and oleic acid fractions of goat's milk are greater than those of cow's milk. Cholesterol content varies widely among different breeds, with most of the cholesterol in goat's milk being in the free state, with only a small percentage, 52 mg per 100 g of fat, being in the ester form. See, e.g., Arora et al., Ind. J. Dairy Sci., 29:191.

[0237] In certain embodiments, the methods and compositions of the present invention provide synthetic dairy products with less cholesterol, cholesterol-free or the same cholesterol content as dairy milk by modulating the oil content, i.e., triglyceride type. In other embodiments, the amount of saturated and unsaturated fat in the dairy substitute is also modulated to at least less or the same amount of fat as dairy milk. In a preferred embodiment, the synthetic dairy product of the present invention has a very low amount of saturated fat but smells and tastes like dairy milk. Long chain fatty acids that are typically saturated fatty acids in milk are instead monounsaturated acids such as oleic acid in a preferred embodiment of the present invention.

[0238] The present invention may not require pasteurization, or at least minimizes it, as each component may be individually sterilized prior to combining via the blending process. In other embodiments of the present invention, the compounded dairy product of the present invention may undergo pasteurization.

[0239] As with natural milk, homogenization is optional for the methods and compositions of the present invention. When sold as a stand-alone liquid beverage, the synthetic dairy products of the present invention may be sold in homogenized form.

[0240] The differences between the milk replacer of the present invention and dairy-containing milks include flavor, nutritional value and storage stability. Flavoring can be adjusted based on triglycerides as well as other natural or artificial flavors or different aromas such as cow, goat, coconut, almond or soy, which can impart mildness or sharpness, to obtain the desired sensory impression.

[0241] Artificial cheese In another embodiment of the present invention, methods and compositions are provided that include one or more isolated milk protein components, fats, carbohydrates and ash to make various types of cheese products. Generally, cheese is made from the milk protein components of the present invention. One or more sensory impressions are incorporated into the cheese product through modulating triglycerides. Thus, cheeses with desired organoleptic characteristics, along with significantly different appearances, aromas, tastes and textures, can be made. For some cheese varieties, in addition to modulating triglycerides, one or more bacteria are utilized in the cheese making process for fermentation, which produces fermentation products and by-products such as lactic acid, carbon dioxide, alcohol, aldehydes and ketones. Cheese types include whey cheeses such as ricotta and mozzarella, semi-soft cheeses including Havarti and Munster, medium-hard cheeses such as Swiss and Jarlsberg, hard cheeses such as Cheddar, and ripened soft cheeses such as Brie and Camembert.

[0242] Synthetic cream The directly usable cream substitute preferably comprises about 50 to 90% by weight water, and more preferably about 65 to 80% by weight water, which is the base to be dispersed in water. The base for the substitute cream advantageously contains about 22 to 87% by weight carbohydrate (more preferably about 30 to 64%), about 12 to 70% by weight particulate fat (most preferably about 28 to 60%), and about 0.4 to 8% by weight of the selected emulsifier or emulsifiers (most preferably about 1 to 4%) (all percentages are calculated using the total weight of the base as 100%).

[0243] In a preferred embodiment, the products of the invention are stable in aqueous emulsions. As used herein, a dry, liquid, fat-containing non-dairy food product is said to be "stable" if the following minimum criteria are met: reconstituted emulsion stability, whitening capacity, oiling or oil separation, feathering-sedimentation. See U.S. Patent No. 4,310,561.

[0244] Synthetic butter Commercially available butter is 80-82% milk fat, 16-17% water, and 1-2% non-fat milk solids (sometimes called curds).

[0245] Advantages of the dairy substitute products or compositions presented herein A desired advantage of the present invention is environmental in nature, such that it is eight times more energy efficient and 260 times more water efficient than conventional dairy products. Other environmental benefits include less water use than conventional milk production, estimated at about 1000 L per liter, and reduced land use for conventional milk production, which typically requires arable land, pasture, and the potential to reduce the 600 billion kg of carbon dioxide emissions per year from conventional milk production. The present invention also results in reduced or eliminated feed, work, labor, animal, and marketing costs. A substantial eight-fold reduction in feed costs is preferred.

[0246] Food safety benefits include the reduction or elimination of antibiotic residues, heavy metals, bacteria, and adulterants. Thus, certain aspects of the present invention provide animal-derived-free milk that is bacteria-free and does not require pasteurization or cold shipping, yet has a long shelf life and exhibits a number of characteristics, such as taste, appearance, handling, and mouthfeel characteristics, that are the same or at least similar to their traditional dairy counterparts. Preferably, the dairy substitute product is essentially free of bacteria such as Brucella, Campylobacter, Listeria, Mycobacterium, Salmonella, Shigella, Yersinia, Giardia, and Norovirus, and thus is safe for consumption. Further benefits include minimal or no pasteurization and / or homogenization. More preferably, the dairy substitute is stable for production and sale over a relatively long period of storage (e.g., at least three weeks and preferably longer). Even more preferably, the dairy substitute product has a low environmental impact.

[0247] In the following, some aspects of the present invention will be described with reference to the application of examples for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a complete understanding of the present invention. However, those skilled in the art will readily recognize that the present invention may be practiced without one or more of the specific details, or may be practiced in other ways. EXAMPLES

[0248] [Example 1] vector The protein sequences of bovine α-S1 casein (UniProt accession number P02662), bovine α-2 casein (UniProt accession number P02663), bovine β-casein (UniProt accession number P02666), bovine κ-casein (UniProt accession number P02668), bovine α-lactalbumin (UniProt accession number B6V3I5) and bovine β-lactoglobulin (UniProt accession number P02754) were obtained at Uniprot.org and modified with the following changes: removal of the 15 or 21 residue signal peptide from the N-terminus; addition of a XhoI (CTC GAG) endonuclease recognition sequence and a KEX endopeptidase recognition sequence (AAA AGA) to the 5' end of the DNA; and addition of a SalI (GTC GAC) endonuclease recognition sequence to the 3' end of the DNA. The four casein sequences were combined in the order shown above to give the following DNA phrase:

[0249] [ka] Further combinatorial sequences were generated by isolating each sequence by: [In the sequence, the non-underlined segments encode the (GS)6 linker sequence for proper post-translational spacing and accessibility to the KEX protease, and the underlined segments encode the KEX endopeptidase sequence, which cleaves the protein after translation.] As above, the entire cassette is flanked by XhoI at the 5' end and SalI at the 3' end for ligation into pKLAC2 (New England Biolabs, Beverly, MA). DNA was synthesized by Gen9, Inc. (Cambridge, MA) or IDT (Coralville, IA). The plasmids used had, among other things, a multiple cloning site, a Lac promoter, an acetamide-based reporter gene, and an alpha-mating factor gene used as a fusion protein to secrete exogenous proteins.

[0250] Yeast transfection Yeast transfection was accomplished by thawing a 0.5 mL tube of competent cells containing 25% glycerol on ice and adding 0.62 mL of yeast transfection reagent. The mixture was then heated at 30° C. for 30 minutes and heat shocked at 37° C. for 1 hour. The cells were then pelleted at 7000 rpm and washed twice with 1.0 mL of YPGal medium. The cell mixture was then transferred to a sterile culture tube and incubated at 30° C. for 3 hours with constant shaking at 300 rpm. The cell mixture was then transferred to a sterile 1.5 mL microcentrifuge tube, the cells were pelleted at 7000 rpm for 2 minutes, and resuspended in 1 mL of sterile 1× PBS. 10, 50, and 100 μL of the cell suspension were placed in separate, unused, sterile 1.5 mL microcentrifuge tubes, each containing 50 μL of sterile demineralized water. The tubes were mixed briefly and plated onto individual yeast carbon-based agar (YCB Agar) plates containing 5 mM acetamide for selection. The plates were then incubated inverted at 30° C. for 4 days until colonies formed. 15 individual colonies were then streaked onto unused YCB Agar plates containing 5 mM acetamide and incubated at 30° C. for 2 days.

[0251] DNA encoding two key whey proteins, alpha-lactalbumin and beta-lactoglobulin, was designed in-house, commissioned for synthesis by IDT, and transfected into competent K. lactis cells using a New England Biolabs kit (catalog no. E1000S) following the protocol supplied by the vendor.

[0252] High-throughput transfected cell selection Once colonies from each YCB Agar plate had grown sufficiently, each of the 30 plates was checked for successful integration of the vector plasmid, followed by PCR analysis in which each plate was checked for specific cells carrying multiple components of the vector. Once isolated, the highest producing individual cultures were used for scale-up. This process can be repeated with gradually increasing concentrations of selection pressure to drive our engineered plasmid to high copy number in the colonies.

[0253] Five transfection events were performed and plated on five separate plates of nitrogen-free yeast carbon-based medium. (Any growth observed on these plates therefore indicates successful uptake of the plasmid, if not the exogenous DNA itself.) Of these five plates, 100% showed positive growth. Thirty separate colonies from the five plates were selected for scale-up. Each colony was grown on a separate YCB agar plate to create homozygous culture plates that allowed for easy characterization and management. After a three-day growth period, a single colony from each plate was first added to a 10-ml glass culture tube containing 2 ml of YPGal medium to check for protein expression. After a two-day growth period, the cells were pelleted and the supernatant was run on an SDS PAGE gel to check for protein expression. The strain that gave the best protein expression was scaled up to 10ml, 100ml, 500ml culture vessels and finally to 1L culture vessel. From each whey protein, 2 liter cultures were grown. Approximately 1 gram of protein was harvested from the total amount, suggesting a suboptimal yield / productivity of 0.5g / L.

[0254] Scale-up to 1 L shake flask culture The culture is scaled up and inoculated into 1 L shake flasks with a passage ratio of at least 1:10. Prior to inoculation, the contents of the inoculum flask are grown for 24 hours in production medium without acetamide supplementation. On the first day of the fed-batch production trial, the reactor is charged to 90% of the target starting volume and heated to the trial temperature. For the time being, the temperature is set at 30° C. to save on the energy costs associated with heating the reactor. Further parameters can be explored in the process optimization phase. Once the reactor reaches 30° C., the contents of the inoculum flask are added dropwise to the reactor using a peristaltic pump. The reactor is maintained at the target pH using software provided by the vendor. The reactor broth is sampled twice daily to quantify the amount of glucose and electrolyte usage by the cells as a double check for reactor pH and dissolved gas measurements. After each measurement, glucose is initially added by bolus to maintain the target glucose concentration at 10%, but this can also be modified during process development. Once the cells reach maximum density, protein production is induced by adding galactose, which induces the promoter for our pKLAC2 plasmid. Galactose is replenished until the end of the run. The optimal length of the run can also be determined during process development, but is set as a 5-day fed-batch culture. After the entire run, the yeast cells are removed from the reactor and the protein is purified as discussed below.

[0255] Casein Protein Purification The following casein proteins: α-s1 casein, α-s2 casein, and β-casein are hydrophobic in nature and are secreted from yeast and precipitate on contact with water. Purification from the reactor medium involves recovery of the protein from the surface of the medium followed by drying to isolate the pure protein. Kappa-casein is hydrophilic in nature and purification of κ-casein involves changing the pH of the solution to 4.6 followed by centrifugation at 10,000 rcf. Combinations of casein cassettes also work similarly to κ-casein.

[0256] Whey Protein Purification Alpha-lactalbumin: The isoelectric point of alpha-lactalbumin is 4.2. The pH of the bioreactor broth is lowered to 4.2, where the protein is at its lowest in solubility. This pulls the protein out of solution and allows it to be recovered by centrifugation. Beta-lactoglobulin: Similar to the purification of alpha-lactalbumin, the pH of the solution is lowered to 5.2, the isoelectric point of beta-lactoglobulin. This neutralizes the charge of the protein and allows it to be recovered by centrifugation at 14,000 rcf.

[0257] Protein purification To pellet the yeast cells, 2 L of culture medium is spun at 3,000 g in a floor centrifuge. The pellet is discarded, the supernatant is transferred to a new vessel, and the pH of the solution is lowered to 4.2 for alpha-lactalbumin and to 5.2 for beta-lactoglobulin (Figure 2A). This is followed by incubation of the supernatant in a shake flask at 35°C for 30 minutes and centrifugation at 14,000 g in a floor centrifuge to pellet the protein mixture (Figure 2B).

[0258] Protein characterization After separating the proteins by centrifugation, the solid pellet and the supernatant solution are run on a 14% SDA-PAGE gel to check for protein expression.Positive bands are observed at 14 kDa and 18 kDa (Figure 3), which correlate with the sizes of bovine-derived alpha-lactalbumin and beta-lactoglobulin, respectively.Further characterization is performed to confirm the equivalence in terms of primary sequence, glycosylation and phosphorylation.

[0259] [Example 2] Triglyceride synthesis The milk fat triglycerides were prepared by mixing short chain triglycerides from a custom engineered sunflower variant in the following ratios as listed in Table 1:

[0260] [Table 1] www.sunflowernsa.com / uploads / resources / 51 / warner_.pdf The oil was then transesterified into high oleic sunflower oil, an oil derived from a mutant expressing fatty acid esters of

[0261] Preparation of short chain triglycerides The short chain fatty acids responsible for the rich flavor in milk and cream are molecules with an even number of carbons between 4 and 10, and are in the following ratios as set out in Table 2:

[0262] [Table 2] www.ncbi.nlm.nih.gov / pmc / articles / PMC2596709 / pdf / FNR-52-1821.pdf Mix by.

[0263] [Table 3]

[0264] The fractions in Table 3 are based on the relative abundance of these species in cow's milk, but can be modified during process development to both design a better tasting product and to design the milk of other species such as buffalo or goat. Short chain fatty acids in the mass ratios shown above are combined with toluene, paratoluenesulfonic acid, and glycerol in a Dean-Stark water trap, commonly used for esterification reactions, to remove the water produced in the condensation reaction. The reaction is run in a fume hood for several hours until the level of water entering the water trap is observed to remain unchanged for more than 30 minutes. The vessel is cooled and the mixture is removed from the reaction flask. The mixture is washed twice with 5% sodium carbonate solution and five times with fresh water. Brine (10% NaCl solution in water) is added periodically to break the emulsion that forms in the separatory funnel. To minimize the possibility of food contamination, the washed mixture of short chain triglycerides, water, toluene, and impurities is dried in a rotary evaporator at 90° C. and 54 mbar for 1 hour until sufficiently over-dried.

[0265] Transesterification The short chain triglyceride mixture is combined with high oleic sunflower oil in a volume ratio of 1:8. To catalyze the transesterification, a mass of sodium methoxide equal to 1% of the mass of the oil mixture is added, and the reactor is heated to 65°C under inert argon atmosphere for 6 hours with constant stirring. To quench the reaction, a 5% acetic acid mixture is added, and the oil is then washed 5 times with demineralized water and dried in a rotary evaporator at >90°C for 1 hour. The final milk fat is autoclaved to ensure sterility, making it suitable for use in milk or cream as described above.

[0266] [Example 3] Milk blend One non-limiting milk composition formulation is described below.

[0267] [Table 4]

[0268] According to Table 4, the milk formulation is achieved via the following procedure per liter of milk: 26 grams of casein, 3.5 grams of whey and 5 grams of ash are combined and mixed thoroughly. 40 mL of triglycerides are melted and heated to 55° C. The protein mixture is slowly poured into the triglycerides and vortexed at high speed for 5 minutes. During this time, 3.5 grams of whey and 24 grams of galactose are added to 850 mL of demineralized water and the mixture is heated to 37° C. The triglyceride / protein / ash mixture is transferred to a Waring commercial blender and blended at low speed. The whey / galactose / water mixture is slowly poured into the blender and the blender is capped. The mixture is blended at high speed for 10 minutes. Demineralized water is added to a final volume of 1000 mL. The milk can be optionally homogenized using existing methods. The above protocol can be modified by altering the solids ratio depending on the cream or any milk formulation, however, preliminary research studies by the inventors suggest that the presence of ash in the protein mixture and a significant proportion of whey separation can have a significant impact on the quality of the emulsion.

[0269] [Example 4] Synthetic Milk Formula As a preliminary proof of concept, dried food grade purified casein and research grade whey were purchased to determine whether key milk components could be engineered to form milk. Irish cream was obtained from a local source and pure fat was isolated from the cream by centrifuging the cream at 14,000 g. Finally, all minerals used were purchased from Sigma Aldrich. term: C Rou = A roux made by mixing casein protein and fat together while maintaining the temperature of the mixture at 37°C.

[0270] W Roux = A roux made by mixing whey protein and fat together while maintaining the temperature of the mixture at 37°C.

[0271] CW Rou = A roux made by first mixing casein and whey proteins together in a mixture, adding fat and mixing at 37°C.

[0272] [Table 5]

[0273] It is hypothesized that the poor mouthfeel (eg, wateriness) was due to a lack of whey proteins.

[0274] [Table 6]

[0275] It is hypothesized that the poor mouthfeel is due to poor micelle formation by casein and that the addition of Ca allows the micelles to reform.

[0276] [Table 7]

[0277] To improve the taste, different sugars were added to the above mixture in different concentrations.

[0278] [Table 8]

[0279] All the additional ions found in milk were incorporated to recreate the ionic environment found in nature.

[0280] Source: R.Rosmaninho, LFMelo, Journal of Food Engineering, 73(2006)379-387

[0281] [Table 9]

[0282] The end result was a bright white liquid, likely because the ionic environment prevented the solids present in the milk from sticking together and increased the overall refractive index of the solution. The taste was excellent, but the mouthfeel was average. (e.g., a certain amount of chuckiness was observed in the liquid.) The exact mineral composition, listed in Table 9, can result in an excellent mouthfeel.

[0283] Milk fat synthesis Synthetic milk fat is made by transesterifying short chain fatty acids with the long chain fatty acids present in high oleic sunflower triglycerides. The four short chains used were: 40% C4: Butyric acid: Found in milk, especially goat, sheep and buffalo milk, butter, Parmesan cheese and as a product of anaerobic fermentation (including colonic fermentation and body odor). Butyric acid has an unpleasant odor and a pungent taste with a slightly sweet aftertaste (similar to ether). Butyric acid is present in human vomit and is its main characteristic odor; 26% C6: Caproic Acid: A colorless, oily liquid with a fatty, cheesy, waxy odor and an odor similar to that of goat or other barnyard animals; 11% C8: Caprylic Acid: An oily liquid. Minimally soluble in water with a slightly unpleasant rancid odor and taste; 22% C10: Capric acid: Not much is said about the flavor, but the longer the carbon chain, the weaker the flavor you get. Capric acid is present in coconut oil, so it doesn't have as much of a milk fat flavor as the other fatty acids. It was.

[0284] The repeat contains lauric acid (C12), which is present in 2.9% of the total fatty acid content in milk (Beare-Rogers, J., Dieffenbacher, A., Holm, J. V. (2001), "Lexicon of lipid nutrition (IUPAC Technical Report)", Pure and Applied Chemistry, 73(4):685-744, doi:10.1351 / pac200173040685).

[0285] The following procedure described in Yu et al., "The modification an analysis of vegetable oil for cheese making," J. Am. Oil Chem. Soc., 77:911 (2000) was followed at one-quarter the amounts specified below.

[0286] A mixture of butyric, caproic, caprylic, and capric acids (Sigma Chemical Co., St. Louis, MO) in the same ratios found for the milk fat samples [see above] and a total of 7.26 moles, 21.42 g of p-toluenesulfonic acid (Sigma Chemical Co.), 2.305 moles of glycerol (Sigma Chemical Co.), and 458 mL of toluene (Fisher Scientific) was perfused through the Dean-Stark water trap for 6 hours. The reaction was considered complete when water stopped dripping into the trap. The SCTG was washed once with 5% sodium carbonate solution and multiple times with water. The SCTG was then heated at 85° C. in a rotary evaporator to remove water and toluene.

[0287] To produce fat with the same percentage of SCFA as that of milk fat, SCTG from both commercial and natural sources is interesterified with HOSO (Trisun 80, RBD; AC Humko, Memphis, TN) at a SCTG / HOSO ratio of 1:8.82. SCTG from commercial sources is also interesterified at a SCTG / HOSO ratio of 1:7.19 to produce fat with SCFA levels equivalent to 120% of those in milk fat. Sodium methoxide (Aldrich Chemical Company, St., Louis, MO) is used as a catalyst at 0.5% of the total oil weight. The reaction was carried out under nitrogen at 65°C with stirring for 6 hours. 5% acetic acid (Fisher Scientific) was then added to neutralize the catalyst, and the oil was then washed several times with distilled water and dried on a rotary evaporator at 90°C for 30 minutes.

[0288] A pilot-scale continuous deodorizer similar to that described by Smouse (Smouse, TH, "A Laboratory Continuous Deodorizer", inform, 8:1176-1181 (1997)) is used to deodorize the interesterified oil. The oil flow rate is 600 mL / hr, the column temperature is 180° C., the pressure is 0.5 Torr, and the steam rate is 12.6 mL / hr. Each batch of deodorized oil is tasted to verify flavor. The deodorized oil is stored at 4° C. until it is used to make cheese.

[0289] [Example 5] Fatty Acid Modulation Sunflower oil triglyceride with three oleic acids is transesterified with four short chain fatty acids, including one butyric acid, one hexanoic acid, and one octanoic acid, as part of the fat composition in the synthetic dairy mixture. This array or combination of fats is expected to result in a synthetic dairy fat that contributes to its rich flavor compared to natural dairy milk. The ability to control the composition of one or more triglycerides is likely to enhance or change the flavor profile of the synthetic dairy product. Thus, a matrix of long and short chains can contribute to the flavor profile, including but not limited to multiple aromatic compounds associated with the sensory impressions of buttery, nutty, sweet, sour, fruity, floral, bitter, woody, earthy, beany, spicy, metallic, sweet, moldy, oily, and vinegary. In addition, it may exhibit increased texture such as creaminess, improved melting characteristics or melting resistance, and increased stretching ability compared to the corresponding dairy product.

[0290] [Example 6] Recombinant production of milk proteins Alpha-lactalbumin, beta-lactoglobulin, alpha-S1-casein, alpha-S2-casein, beta-casein, and kappa-casein were produced in a recombinant yeast strain (Pichia pastoris). Because the glycosylation enzymes in yeast are different from mammalian cells, the proteins produced by yeast are either unglycosylated or have non-mammalian glycosylation patterns. The produced proteins can be used as components in any of the compositions described herein.

[0291] Plasmids To express each protein, a plasmid was constructed that contained the following components: an inducible promoter (e.g., AOX1 promoter) or a constitutive promoter (GAP promoter or PGK promoter) for each protein to be expressed; a sequence encoding a signal peptide for each protein to be expressed, derived from the native bovine protein sequence or from a yeast protein sequence (alpha mating factor or OST1); a sequence encoding the milk protein to be expressed; a yeast transcription terminator sequence for each protein to be expressed (e.g., AOX1, AOD, or CYC1); a bacterial origin of replication derived from pUC19, allowing replication of the plasmid in E. coli; and a selection marker cassette (e.g., kanR or zeocinR), allowing selection in bacteria and yeast with antibiotics.

[0292] The different plasmids used to make the different proteins are listed in Table 10 below.

[0293] [Table 10] JPEG2025032348000013.jpg230156 JPEG2025032348000014.jpg117155

[0294] These plasmids were then transformed into wild-type P. pastoris for expression, and protein production was detected by SDS-PAGE, ELISA, and Western blot.

[0295] Alpha-lactalbumin Strain construction Expression of bovine alpha-lactalbumin (bvLAA) was driven by the methanol-inducible promoter P with the native LAA signal peptide (pJAG-nat-LAA), the full-length alpha mating factor signal peptide (pJAG-aMF-LAA), or the OST1 signal peptide (pJAG-OST-LAA).AOX1 Three plasmids were created, each of which was under the control of

[0296] Prior to transformation, 20 μg of each plasmid was linearized via digestion with the restriction enzyme Sac I. The digested plasmids were then concentrated by ethanol precipitation and resuspended in 10 μl of distilled water.

[0297] Competent Pichia pastoris cells were prepared as follows: A culture of P. pastoris was grown to log phase (OD600 approx. 1.0) in YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose). A 1.5 mL aliquot was taken by centrifugation and then resuspended in 1 mL of a 1:1 mixture of YPD + 20 mM HEPES (pH 8): 1 M lithium acetate. After adding 10 μL of 1 M dithiothreitol, the cells were incubated for 15 min at 30°C in a shaker at 300 rpm. The cells were pelleted by centrifugation and washed three times with 1 mL of 1 M ice-cold sorbitol. After the final wash, the cells were resuspended in 50 μL of 1 M sorbitol.

[0298] The cells were combined with the linearized plasmid DNA in a chilled 2 mm electroporation cuvette and subjected to a 1.5 kV pulse (25 μF, 200 Ω). The cells were transferred to a culture tube with 200 μL of cold 1:1 YPD:1 M sorbitol and allowed to recover for 2 hours at 30° C. (300 rpm). Finally, the cells were plated onto YPD agar plates containing Zeocin and grown at 30° C. for 2 days.

[0299] Protein expression Colonies were picked from the agar plates and grown in 750 μL of 1% BMD (0.2 M potassium phosphate buffer, 13.4 g / l yeast nitrogen base, 0.4 mg / ml biotin, 1.1% glucose) at 30° C. and 300 rpm. After 48 hours, 900 μL of culture was used to inoculate 750 μL of BMM2 (0.2 M potassium phosphate buffer, 13.4 g / l yeast nitrogen base, 0.4 mg / ml biotin, 1% methanol). After 24 hours, 150 μL of BMM10 (BMM10: 0.2 M potassium phosphate buffer, 13.4 g / l yeast nitrogen base, 0.4 mg / ml biotin, 5% methanol) and samples were taken for analysis one more day later.

[0300] analysis Protein expression was analyzed in culture samples that were centrifuged to remove cell clumps, and clarified supernatants were then assessed by SDS-PAGE, ELISA, and Western blot.

[0301] To visualize total protein via SDS-PAGE, cell-free supernatants were treated with SDS-PAGE sample buffer, boiled, and run on a 10% polyacrylamide gel. Gels were stained with SYPRO Ruby dye (Life Technologies). The resulting gels show that secretion of α-lactalbumin occurs using either OST1 or the native lactalbumin signal peptide (Figure 4).

[0302] To measure protein titers via ELISA, 25 μL of each sample was placed into a half-area 96-well microtiter plate and allowed to bind overnight at 4° C. After removing the samples, the binding surface was blocked by filling each well with 1% (w / v) bovine serum albumin (BSA) dissolved in Tris-buffered saline (50 mM Tris, pH 7.6, 150 mM NaCl) and incubating for 1 hour at room temperature. Samples were then incubated for 1.5 hours in primary antibody diluted in 1% BSA / TBS+0.1% (v / v) Tween-20. After three washes with TBS+Tween, samples were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) for an additional hour. After three final washes with TBS+Tween, a chromogenic substrate (TMB Single Solution, Life Technologies) was added and absorbance was measured at 650 nm. The resulting data show that α-lactalbumin was secreted using the native α-lactalbumin signal peptide or the OST1 signal peptide (Figure 5).

[0303] To analyze the samples via Western blot, one volume of sample was combined with an equal volume of SDS-PAGE sample buffer and run on a 10% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane, which was blocked via treatment with 1% BSA / TBS for 1 hour. After incubation with primary antibodies diluted in 1% BSA / TBS+Tween for 1.5 hours, the blot was washed three times with TBS+Tween. The blot was then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for an additional hour. After three final washes with TBS+Tween, a chromogenic substrate (1-Step Ultra TMB Blotting Solution, Thermo Fisher) was added. After staining was completed, the blot was washed with distilled water.

[0304] Beta-lactoglobulin Strain construction Expression of bovine beta-lactoglobulin (bvLGB) was driven by a methanol-inducible promoter (P AOX1 ) or two constitutive promoters (P GAP , or P in pLH45 PGK ) and three plasmids were assembled.

[0305] Prior to transformation, 20 μg of pLH37 was linearized via digestion with the restriction enzyme SacI. The same amounts of pLH44 and pLH45 were linearized with the enzyme ApaLI. The digested plasmids were then concentrated by ethanol precipitation and resuspended in 10 μl of distilled water.

[0306] Competent Pichia pastoris cells were prepared as follows: A culture of P. pastoris was grown to log phase (OD600 approx. 1.0) in YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose). A 1.5 mL aliquot was taken by centrifugation and then resuspended in 1 mL of a 1:1 mixture of YPD + 20 mM HEPES (pH 8): 1 M lithium acetate. After adding 10 μL of 1 M dithiothreitol, the cells were incubated for 15 min at 30°C in a shaker at 300 rpm. The cells were pelleted by centrifugation and washed three times with 1 mL of 1 M ice-cold sorbitol. After the final wash, the cells were resuspended in 50 μL of 1 M sorbitol.

[0307] The cells were combined with the linearized plasmid DNA in a chilled 2 mm electroporation cuvette and subjected to a 1.5 kV pulse (25 μF, 200 Ω). The cells were transferred to a culture tube with 200 μL of cold 1:1 YPD:1 M sorbitol and allowed to recover for 2 hours at 30° C. (300 rpm). Finally, the cells were plated onto YPD agar plates containing Zeocin and grown at 30° C. for 2 days.

[0308] Protein expression To assess expression in clones transformed with a plasmid containing a methanol-inducible promoter (pLH37), individual clones were grown in 750 μL of 1% BMD (0.2 M potassium phosphate buffer, 13.4 g / l yeast nitrogen base, 0.4 mg / ml biotin, 1.1% glucose) at 30° C. and 300 rpm. After 48 hours, 900 μL of culture was used to inoculate 750 μL of BMM2 (0.2 M potassium phosphate buffer, 13.4 g / l yeast nitrogen base, 0.4 mg / ml biotin, 1% methanol). After 24 hours, 150 μL of BMM10 (BMM10: 0.2 M potassium phosphate buffer, 13.4 g / l yeast nitrogen base, 0.4 mg / ml biotin, 5% methanol) and samples were taken for analysis one more day later.

[0309] To assess expression in clones transformed with plasmids supporting constitutive expression (pLH44 or pLH45), individual clones were grown overnight at 30° C. in PG medium (20 g / L peptone, 2% glycerol) with shaking at 300 rpm. Cultures were diluted 1:10 in minimal sulfate medium.

[0310] [Table 11]

[0311] After 48 hours, samples were taken for analysis.

[0312] analysis Protein expression was analyzed in culture samples that were centrifuged to remove cell clumps, and clarified supernatants were then assessed by ELISA and Western blot.

[0313] To measure protein titers via ELISA, 25 μL of each sample was placed in a half-area 96-well microtiter plate and allowed to bind overnight at 4° C. After removing the samples, the binding surface was blocked by filling each well with 1% (w / v) bovine serum albumin (BSA) dissolved in Tris-buffered saline (50 mM Tris, pH 7.6, 150 mM NaCl) and incubating for 1 hour at room temperature. Samples were then incubated for 1.5 hours in primary antibodies diluted in 1% BSA / TBS+0.1% (v / v) Tween-20. After washing three times with TBS+Tween, samples were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for an additional hour. After three final washes with TBS+Tween, a chromogenic substrate (TMB Single Solution, Life Technologies) was added and absorbance was measured at 650 nm. The resulting data indicate the secretion of β-lactoglobulin (Figure 6).

[0314] To analyze the samples via Western blot, one volume of sample was combined with an equal volume of SDS-PAGE sample buffer and run on a 10% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane, which was blocked via treatment with 1% BSA / TBS for 1 hour. After incubation with the primary antibody diluted in 1% BSA / TBS+Tween for 1.5 hours, the blot was washed three times with TBS+Tween. The blot was then incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) for an additional hour. After three final washes with TBS+Tween, a chromogenic substrate (1-Step Ultra TMB Blotting Solution, Thermo Fisher) was added. After staining was completed, the blot was washed with distilled water. The resulting Western blot shows that β-lactoglobulin was secreted from the recombinant yeast (Figure 7).

[0315] Bovine Casein Dual expression plasmids were constructed that support the expression of α-S1-casein along with β-casein in one plasmid and α-S2-casein along with kappa-casein in another plasmid. These pairings were chosen because the molar ratio of α-S1:α-S2:β:κ in fluid milk is approximately 5.5:1.5:4.0:1.5, and therefore it is desirable to have similar copy numbers of α-S1-casein and beta-casein, and similar copy numbers of α-S2-casein and kappa-casein.

[0316] Beta-casein and α-S2-casein were under the control of the PGAP constitutive promoter, while α-S1-casein and κ-casein were under the control of the PPGK constitutive promoter in their respective plasmids.

[0317] To direct the proteins into the secretory pathway, the proteins were expressed with their native signal peptides (pLH46 and pLH47), or with the OST1 signal peptide (pLH48 and pLH49). In addition, plasmids were used to express one protein with its native signal peptide and the other with the OST1 signal peptide: pLH0050:OST1-beta, native-alpha-S1 pLH0051: Natural-β, OST1-α-S1 pLH0054:OST1-α-S2, native-κ pLH0055: Natural-α-S2, OST1-κ I also made it.

[0318] To generate strains expressing all four casein proteins, yeast cells were first transformed with plasmids encoding beta-casein and α-S1-casein. Prior to transformation, 20 μg of each plasmid was linearized with the enzyme ApaLI. The digested plasmid was then concentrated by ethanol precipitation and resuspended in 10 μl of distilled water.

[0319] Competent Pichia pastoris cells were prepared as follows: A culture of P. pastoris was grown to log phase (OD600 approx. 1.0) in YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose). A 1.5 mL aliquot was taken by centrifugation and then resuspended in 1 mL of a 1:1 mixture of YPD + 20 mM HEPES (pH 8): 1 M lithium acetate. After adding 10 μL of 1 M dithiothreitol, the cells were incubated for 15 min at 30°C in a shaker at 300 rpm. The cells were pelleted by centrifugation and washed three times with 1 mL of 1 M ice-cold sorbitol. After the final wash, the cells were resuspended in 50 μL of 1 M sorbitol.

[0320] The cells were combined with the linearized plasmid DNA in a chilled 2 mm electroporation cuvette and subjected to a 1.5 kV pulse (25 μF, 200 Ω). The cells were transferred to a culture tube with 200 μL of cold 1:1 YPD:1 M sorbitol and allowed to recover for 2 hours at 30° C. (300 rpm). Finally, the cells were plated onto PG agar (20 g / L peptone, 2% (v / v) glycerol, 2% agar) plates containing Zeocin and grown for 2 days at 30° C.

[0321] Six clones from the beta + alpha S1 plates were then grown in culture and made competent for DNA uptake using the procedure described above, they were then transformed with the linearized alpha S2 + kappa plasmid and grown on PG plates containing G418 at 30°C for 2 days.

[0322] Expression To assess production of bovine casein proteins, five casein-expressing clones and a wild-type yeast negative control were grown overnight in PG medium (20 g / L peptone, 2% glycerol) at 30° C. with 300 rpm shaking. All five casein-expressing clones expressed alpha S2- and kappa-casein with their native casein signal peptides. Clones sLH115, 116, 117, and 118 expressed β-casein and α-S1-casein with their native signal peptides; clone sLH122 expressed beta-casein and α-S1-casein with the OST1 signal peptide. Cultures were diluted 1:10 in minimal sulfate medium.

[0323] [Table 12]

[0324] After 48 hours, samples were taken for analysis.

[0325] analysis Protein expression was analyzed in culture samples that were centrifuged to remove cell clumps, and clarified supernatants were then assessed by ELISA and Western blot.

[0326] To measure protein titers via ELISA, 25 μL of each sample was placed in a half-area 96-well microtiter plate and allowed to bind overnight at 4° C. After removing the samples, the binding surface was blocked by filling each well with 1% (w / v) bovine serum albumin (BSA) dissolved in Tris-buffered saline (50 mM Tris, pH 7.6, 150 mM NaCl) and incubating for 1 hour at room temperature. Samples were then incubated for 1.5 hours in primary antibodies diluted in 1% BSA / TBS+0.1% (v / v) Tween-20. After washing three times with TBS+Tween, samples were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for an additional hour. After three final washes with TBS+Tween, a chromogenic substrate (TMB Single Solution, Life Technologies) was added and absorbance was measured at 650 nm. The ELISA data show that the different yeast strains can secrete α-S1 casein and β-casein into the culture medium (Figure 8).

[0327] To analyze the samples via Western blot, one volume of sample was combined with an equal volume of SDS-PAGE sample buffer and run on a 10% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane, which was blocked via treatment with 1% BSA / TBS for 1 hour. After incubation with the primary antibody diluted in 1% BSA / TBS+Tween for 1.5 hours, the blot was washed three times with TBS+Tween. The blot was then incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) for an additional hour. After three final washes with TBS+Tween, a chromogenic substrate (1-Step Ultra TMB Blotting Solution, Thermo Fisher) was added. After staining was completed, the blot was washed with distilled water.

[0328] The data in this example demonstrate that the different expression vectors described herein can be used to generate transgenic yeast strains that secrete different milk proteins.

[0329] [Example 7] Method of Making the Composition The specific methods described below were used to generate the exemplary compositions described herein, and are outlined in FIG.

[0330] To prepare the dairy products, laboratory equipment such as mixers, stir plates, and ultrasonicators are utilized. For large scale production, standard fluid milk processing equipment shall be used.

[0331] As shown in Figure 9, there are three main components to the process of making this composition. These steps are: A. Preparation of Protein Solutions B. Preparation of Oil Mixtures C. Reconstitution of Milk Solids Includes.

[0332] In step A, powdered micellar casein protein and whey protein are combined and blended (step 1) and then mixed with demineralized (DI) water (step 2) to obtain protein solution 1. Typically, this contains 2.8% powdered micellar casein, 0.7% powdered whey protein, and 85.5% water in this solution. The mixing tank is covered to prevent water evaporation. This mixing is carried out by a mixer, a stirring plate, or an ultrasonicator for a sufficient time (about 30 minutes). This mixing time ensures that all the protein is dispersed in the water. The mixing speed is optimized as a medium speed, which provides enough force to disperse the protein and avoid air entrapment in the solution. The water content can be adjusted according to the use of other ingredients.

[0333] In step 3, separate solutions of CaCl2, KH2PO4, and NaCitrate in water are mineral sources utilized to prepare a mineral profile similar to natural cow's milk. Typically, the concentration of the CaCl2 solution is 0.1 g / mL, the concentration of the KH2PO4 is 0.27 g / mL, and the concentration of the NaCitrate solution is 0.21 g / ml NaCitrate. The water used to prepare the KH2PO4 along with the NaCitrate solution is typically hot water to ensure complete dissolution of the KH2PO4. While mixing protein solution 1, slowly add 0.015% CaCl (step 4). The volume of CaCl solution used is adjusted according to the weight percentage of CaCl required. Mixing is continued until the protein and Ca 2+The mixing is continued for about 30 minutes to allow complete interaction between the ions. Then, 0.27% KH2PO4 and 0.21% NaCitrate are divided into 5 portions, and each portion is added slowly to the mixture with an interval of 5 to 10 minutes (step 5). 0.085% CaCl2 is divided into 4 portions, and each portion is added slowly to the mixture with an interval of 5 to 10 minutes (step 6). The mixing is continued for at least 30 minutes, preferably 1 to 2 hours, to obtain protein solution 2.

[0334] In step B, low speed mixing is sufficient to achieve homogenous mixing of the different oil contents. The percentage of each component used below to prepare Oil Mixture 1 is based on the weight of the total Oil Mixture 1. First, 65% sunflower oil, 29% coconut oil, and 2% tributyrin are mixed together to form an oil base (step 7). The sunflower oil and coconut oil are deodorized to prevent undesirable odors. The combination of sunflower oil, coconut oil, and tributyrin can mimic the fatty acid profile similar to that of natural milk. The oil base content and its content can be adjusted according to different needs (different types of products). The scent mixture is prepared by mixing different scent components in sunflower oil (step 8). The compounds used to mimic the scent include but are not limited to ethyl butyrate, δ-decalactone, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, δ-undecalactone, acetoin, furfuryl alcohol, furfural, 2-methylfurfural, and 2-methylpyrazine. These contents can be adjusted according to different applications and preferences. 2.5% mono- and diglycerides, 0.6% free fatty acids, 0.5% phospholipids, and 0.4% scent mixture are added and mixed to prepare oil mixture 1 (step 9). Typically, the free fatty acids contain 0.15% butyric acid and 0.45% hexanoic acid. Soybean lecithin is used as the phospholipid source. Soy lecithin is readily available and inexpensive. A β-carotene solution is prepared at a concentration of 0.5 mg per gram in sunflower oil (step 10). 4% oil mixture 1 and 0.06% β-carotene solutions are mixed together to obtain oil mixture 2 (step 11). The use of β-carotene is adjusted to achieve different color levels in the milk. The use of oil mixture 1 can also be adjusted according to different dairy applications.

[0335] In step C, oil mixture 2 is slowly added to protein solution 2 and mixed thoroughly to prepare product mixture 1 (step 12). Mixing can be performed by a mixer or ultrasonic processor. Typically, oil mixture 2 and protein solution 2 are mixed under medium to high speed to ensure that the oil is uniformly dispersed in the aqueous solution. Then ultrasonic treatment is applied to break down the oil globules into smaller sizes, thereby increasing their stability in the solution. During mixing, it is necessary to avoid trapping air bubbles in the solution. Oil mixture 2 is added to the aqueous solution and stirred to completely disperse, using mixing for at least 20 minutes. 4% maltose solution is added to product mixture 1 and continued mixing for another 30 minutes to obtain product mixture 2 (step 13). Sweetness can be adjusted by sugar content according to different applications. The sugar source can also be adjusted as needed. Additional DI water may be required to bring the final total weight to 100%.

[0336] Intensive homogenization, pasteurization, and sterilization are not included in this step, but for scale-up production, these steps will need to be applied to prepare the product mixture in step C.

[0337] Equipment used Mixer: IKA-Labortechnik RW16 Basic, speed levels (4-6) Tip-type ultrasonic processor: Qsonica Model CL-188, Amplitude: 70% Water bath ultrasonic processor: Bransonic Model 1510R-MT

[0338] [Example 8] Exemplary Formulations Exemplary blend compositions having taste and texture profiles similar to whole milk, cream, high protein milk, fat free milk, and sugar free milk are presented below in Tables 11-15.

[0339] As would be appreciated by one of skill in the art, the compositions listed in Tables 11-15 are made by making the necessary modifications to the process described in Example 7.

[0340] [Table 13]

[0341] [Table 14]

[0342] [Table 15]

[0343] [Table 16]

[0344] [Table 17]

[0345] [Example 9] Exemplary Compositions An exemplary composition made by the methods described herein is shown in Figure 10. The composition in Figure 10 is similar in appearance (color), viscosity, foaming properties, flavor, and nutritional value to mammalian produced milk. The composition shown in Figure 10 includes mammalian derived proteins.

Claims

1. from about 0.3 g / L to about 1.1 g / L of κ-casein protein; from about 1.25 g / L to about 4.9 g / L of β-casein protein; one or more lipids at a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; One or more sweetening agents in a total final concentration of about 0.1% to about 6% by weight; and Ash content to a total final concentration of about 0.15% to about 1.5% by weight Including, Contains no animal-derived ingredients composition.

2. from about 0.3 g / L to about 1.1 g / L of κ-casein protein; from about 1.25 g / L to about 4.9 g / L of β-casein protein; one or more lipids at a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; One or more sweetening agents in a total final concentration of about 0.1% to about 6% by weight; and Ash content to a total final concentration of about 0.15% to about 1.5% by weight Including, Free of at least one component found in mammalian produced milk; and / or comprising at least one component not present in mammalian produced milk; Contains at least one component at a higher or lower concentration compared to the concentration of at least one component in mammalian produced milk. composition.

3. from about 0.3 g / L to about 1.1 g / L of κ-casein protein, said κ-casein protein being unglycosylated or having a non-mammalian glycosylation pattern; β-casein protein from about 1.25 g / L to about 4.9 g / L, said β-casein protein being unglycosylated or having a non-mammalian glycosylation pattern; one or more lipids at a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; One or more sweetening agents in a total final concentration of about 0.1% to about 6% by weight; and Ash content to a total final concentration of about 0.15% to about 1.5% by weight A composition comprising:

4. A composition comprising micelles comprising κ-casein protein and β-casein protein, wherein the micelles have a diameter of from about 50 nm to about 350 nm, and wherein the κ-casein protein and β-casein protein are non-glycosylated or have a non-mammalian glycosylation pattern.

5. The composition of claim 4, comprising micelles at a final concentration of about 2.0% to about 6% by weight.

6. 6. The composition of claim 4 or 5, wherein the ratio of β-casein protein to κ-casein protein in the micelles is from about 3.5:1 to about 5.5:

1.

7. 7. The composition of claim 6, wherein the ratio of β-casein protein to κ-casein protein in the micelles is from about 4:1 to about 5:

1.

8. one or more lipids at a total final concentration of about 0% to about 45% by weight; one or more flavor compounds in a total final concentration of about 0.01% to about 6% by weight; One or more sweetening agents in a total final concentration of about 0.1% to about 6% by weight; and Ash content to a total final concentration of about 0.15% to about 1.5% by weight The composition according to claims 4 to 7, further comprising:

9. 3. The composition of claim 2, comprising at least one component selected from the group consisting of calcium, phosphate, B complex vitamins, vitamin A, vitamin D, vitamin E, and vitamin K, at a high concentration compared to the concentration of the one or more components in mammalian produced milk.

10. 10. The composition of claim 2 or 9, which is free of at least one component found in produced mammalian milk selected from the group of lactose, bacteria, mycobacteria, allergens, viruses, prions, yeast, growth hormones, leukocytes, antibiotics, heavy metals, immunoglobulins, lactoferrin, lactoperoxidase, and lipase.

11. 11. The composition of claim 2, 9, or 10, comprising at least one component not present in produced mammalian milk selected from the group of artificial sweeteners, lipids of vegetable origin, β-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern, and κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern.

12. 12. The composition of any one of claims 1 to 11 comprising from about 0.27% to about 0.75% by weight of κ-casein protein and from about 1.23% to about 3.27% by weight of β-casein.

13. 13. The composition of any one of claims 1-3 and 8-12, wherein the total final concentration of the one or more lipids is from about 0% to about 4.5% by weight.

14. 14. The composition of any one of claims 1 to 3 and 8 to 13, wherein the one or more lipids are selected from the group consisting of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids.

15. sunflower oil to a final concentration of about 1% to about 28% by weight; coconut oil to a final concentration of about 0.5% to about 14% by weight; Tributyrin to a final concentration of about 0.05% to about 1.0% by weight; Mono- and diglycerides to a total final concentration of about 0.08% to about 1.2% by weight; Free fatty acids to a total final concentration of about 0.02% to about 0.28% by weight; and Phospholipids to a total final concentration of about 0.02% to about 0.3% by weight The composition of claim 14, comprising one or more of:

16. 16. The composition of claim 14 or 15, wherein the free fatty acids comprise at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid.

17. 17. The composition according to any one of claims 14 to 16, wherein the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid.

18. The composition according to any one of claims 14 to 17, wherein the mono- and diglycerides are mono- and diglycerides of vegetable origin or are mono- and diglycerides of bacterial origin.

19. 19. The composition of any one of claims 1 to 3 and 8 to 18, wherein the flavor compound comprises at least one flavor compound selected from the group consisting of δ-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, γ-undecalactone, and δ-undecalactone.

20. The composition of any one of claims 1 to 3 and 8 to 19, wherein one or more sweetening agents is a saccharide.

21. 21. The composition of claim 20, wherein the saccharide is selected from the group consisting of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose.

22. The composition of any one of claims 1 to 3 and 8 to 19, wherein the one or more sweetening agents is an artificial sweetener.

23. 23. The composition of claim 22, wherein the artificial sweetener is selected from the group consisting of stevia, aspartame, cyclamate, saccharin, sucralose, mogrosides, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000.

24. The composition of any one of claims 1 to 3 and 8 to 23, wherein the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride.

25. Ash content is CaCl 2 , K.H. 2 P.O. 4 , and sodium citrate 3 The composition according to any one of claims 1 to 3 and 8 to 24, comprising one or more of:

26. Ash content is CaCl 2 , K.H. 2 P.O. 4 , and sodium citrate 3 26. The composition of claim 25 comprising:

27. CaCl 2 has a final concentration of about 0.05 g / L to about 0.2 g / L; KH 2 P.O. 4 has a final concentration of about 0.2 g / L to about 0.4 g / L; Sodium Citrate 3 is about 0.1 g / L to about 0.3 g / L; 27. The composition of claim 26.

28. 28. A composition according to any one of claims 1 to 27, wherein the κ-casein protein is from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna or woolly mammoth.

29. 29. The composition of any one of claims 1 to 28, wherein the beta-casein protein is from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

30. α-lactalbumin protein to a final concentration of about 0.4% to about 2.5% by weight; and / or β-lactoglobulin protein at a final concentration of about 2.5% to about 4.5% by weight The composition of any one of claims 1 to 29, further comprising:

31. 31. The composition of claim 30, wherein the alpha-lactalbumin protein is from a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

32. 32. The composition of claim 30 or 31, wherein the β-lactoglobulin protein is a β-lactoglobulin protein from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

33. α-S1-casein protein to a final concentration of about 11% to about 16% by weight; α-S2-casein protein at a final concentration of about 2% to about 5% by weight The composition of any one of claims 1 to 32, further comprising:

34. the α-S1-casein protein is from a cow, a human, a sheep, a goat, a buffalo, a camel, a horse, a donkey, a lemur, a panda, a guinea pig, a squirrel, a bear, a macaque, a gorilla, a chimpanzee, a mountain goat, a monkey, an ape, a cat, a dog, a wallaby, a rat, a mouse, an elephant, a possum, a rabbit, a whale, a baboon, a gibbon, an orangutan, a mandrill, a pig, a wolf, a fox, a lion, a tiger, an echidna, or a woolly mammoth; and / or 34. The composition of claim 33, wherein the alpha-S2-casein protein is from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

35. 35. The composition of any one of claims 1 to 34, further comprising one or more of serum albumin, lactoferrin, and transferrin.

36. the serum albumin is from a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth; the lactoferrin is from a cow, a human, a sheep, a goat, a buffalo, a camel, a horse, a donkey, a lemur, a panda, a guinea pig, a squirrel, a bear, a macaque, a gorilla, a chimpanzee, a mountain goat, a monkey, an ape, a cat, a dog, a wallaby, a rat, a mouse, an elephant, a possum, a rabbit, a whale, a baboon, a gibbon, an orangutan, a mandrill, a pig, a wolf, a fox, a lion, a tiger, an echidna, or a woolly mammoth; and / or the transferrin is a transferrin protein from a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth; 36. The composition of claim 35.

37. The composition of any one of claims 1 to 35, further comprising one or more tone correcting agents.

38. 38. The composition of claim 37, wherein the one or more color correcting agents is beta-carotene or annatto.

39. The composition of any one of claims 1 to 37, wherein the pH of the composition is from about 6.2 to about 7.

2.

40. 41. The composition of claim 40, wherein the pH of the composition is from about 6.2 to about 6.

8.

41. Mammalian product milk or processed mammalian product milk; and One or both of κ-casein proteins that are unglycosylated or have a non-mammalian glycosylation pattern, and β-casein proteins that are unglycosylated or have a non-mammalian glycosylation pattern. A composition comprising:

42. 42. The composition of claim 41, wherein the final concentration of κ-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern in the composition is from 0.02% to about 3.0% by weight.

43. 42. The composition of claim 41, wherein the final concentration of beta-casein protein that is unglycosylated or has a non-mammalian glycosylation pattern in the composition is from 0.02% to about 3.0% by weight.

44. the final concentration of κ-casein protein that is unglycosylated and / or has a non-mammalian glycosylation pattern in the composition is from about 0.02% to about 0.6% by weight; the final concentration of unglycosylated and / or non-mammalian glycosylation pattern β-casein protein in the composition is from about 0.02% to about 2.5% by weight; 42. The composition of claim 41.

45. κ-casein protein to a final concentration of about 3.6% to about 5.4% by weight; β-casein protein to a final concentration of about 16.3% to about 24.5% by weight; a sweetener to a final concentration of about 35% to about 40% by weight; one or more lipids to a final concentration of about 25% to about 30% by weight; Ash to a final concentration of about 5% to about 7% by weight; and Water to a final concentration of about 2% to about 5% by weight Including, The κ-casein protein is non-glycosylated and / or has a non-mammalian glycosylation pattern, and / or the β-casein protein is non-glycosylated and / or has a non-mammalian glycosylation pattern. Powder composition.

46. promoter; A sequence encoding a signal sequence; A milk protein coding sequence; and Yeast termination sequence Including, The promoter is operably linked to a signal sequence, the signal sequence is operably linked to a sequence encoding a milk protein, and a termination sequence is operably linked to a sequence encoding a milk protein. Nucleic acid.

47. 47. The nucleic acid of claim 46, wherein the promoter is a constitutive promoter.

48. 48. The nucleic acid of claim 47, wherein the promoter is an inducible promoter.

49. 49. The nucleic acid of any one of claims 46 to 48, wherein the signal sequence is from the encoded milk protein or a different milk protein, or is a signal sequence from a yeast mating factor.

50. 50. The nucleic acid of any one of claims 46 to 49, wherein the encoded milk protein is selected from the group consisting of β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin, or transferrin.

51. 51. The nucleic acid of any one of claims 46 to 50, comprising a bacterial origin of replication.

52. 52. The nucleic acid of any one of claims 46 to 51, further comprising a selection marker.

53. The nucleic acid according to any one of claims 46 to 52, wherein the selection marker is an antibiotic resistance gene.

54. Additional promoter sequences; an additional sequence encoding a signal sequence; A sequence encoding an additional milk protein; and Additional yeast termination sequences, wherein the further promoter sequence is operably linked to a further sequence encoding a signal sequence, the sequence encoding the signal sequence is operably linked to a sequence encoding a further milk protein, and the sequence encoding the further milk protein is operably linked to a further yeast termination sequence. A nucleic acid according to any one of claims 46 to 53.

55. A host cell comprising a nucleic acid according to any one of claims 46 to 53.

56. 56. The host cell of claim 55 which is a yeast strain.

57. 57. The host cell of claim 56, wherein the yeast strain is Kluyveromyces sp., Pichia sp., Saccharomyces sp., Tetrahymena sp., Yarrowia sp., Hansenula sp., Blastobotrys sp., Candida sp., Zygosaccharomyces sp., and Debaryomyces sp.

58. 1. A method for making a recombinant milk protein that is unglycosylated or has a non-mammalian glycosylation pattern, comprising: Cultivating the host cell of any one of claims 55 to 57 in a culture medium under conditions sufficient to allow secretion of a milk protein that is unglycosylated or has a non-mammalian glycosylation pattern; and Recovering milk proteins that are unglycosylated or have a non-mammalian glycosylation pattern from the culture medium. The method includes:

59. 1. A method of making micelles comprising β-casein that is unglycosylated or has a non-mammalian glycosylation pattern and κ-casein that is unglycosylated or has a non-mammalian glycosylation pattern, comprising: Cultivating a host cell according to any one of claims 55 to 57 in a culture medium under conditions sufficient to allow release of micelles from the host cell, The method wherein the host cell comprises a nucleic acid comprising a sequence encoding β-casein and a sequence encoding κ-casein.

60. 1. A method for supplementing mammalian produced milk, comprising: Providing a mammalian product milk or a processed mammalian product milk; and β-casein proteins that are unglycosylated or have a non-mammalian glycosylation pattern; κ-casein proteins that are unglycosylated or have a non-mammalian glycosylation pattern; and Micelles containing β-casein proteins having a non-glycosylated or non-mammalian glycosylation pattern and κ-casein proteins having a non-glycosylated or non-mammalian glycosylation pattern mixing at least one of the following into the milk: The method includes:

61. 1. A method of making a composition, comprising: sonicating a liquid containing a protein mixture comprising β-casein protein and casein kappa protein or containing micelles comprising β-casein protein and kappa-casein protein; mixing the ash with the liquid; adding a mixture of one or more lipids, one or more flavor compounds, and one or more color correctors to the liquid and sonicating the liquid; and adding one or more sweetening agents to the liquid, thereby forming a composition; The method includes:

62. 62. The method of claim 61, wherein the β-casein protein is non-glycosylated or has a non-mammalian glycosylation pattern and / or the κ-casein protein is non-glycosylated or has a non-mammalian glycosylation pattern.

63. 63. The method of claim 61 or 62, wherein the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride.

64. The added ash is CaCl 2 , K.H. 2 P.O. 4 , and sodium citrate 3 The method of any one of claims 61 to 63, comprising one or more of the following:

65. The added ash is CaCl 2 , K.H. 2 P.O. 4 , and sodium citrate 3 65. The method of claim 64, comprising:

66. 66. The method of any one of claims 61-65, wherein the one or more lipids comprise at least one of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids.

67. 67. The method of claim 66, wherein the free fatty acids comprise at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid.

68. 68. The method of claims 66 and 67, wherein the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid.

69. 69. The method of any one of claims 66 to 68, wherein the mono- and diglycerides are plant-derived mono- and diglycerides or bacterial-derived mono- and diglycerides.

70. 70. The method of any one of claims 61 to 69, wherein the flavor compounds comprise at least one flavor compound selected from the group consisting of delta-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, gamma-undecalactone, and delta-undecalactone.

71. The method of any one of claims 61 to 70, wherein the one or more color correcting agents is β-carotene or annatto.

72. 72. The method of any one of claims 61 to 71, wherein the one or more sweetening agents is a saccharide.

73. 73. The method of claim 72, wherein the saccharide is selected from the group consisting of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose.

74. 72. The method of any one of claims 61 to 71, wherein the one or more sweetening agents is an artificial sweetener.

75. 75. The method of claim 74, wherein the artificial sweetener is selected from the group consisting of stevia, aspartame, cyclamate, saccharin, sucralose, mogrosides, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000.

76. 76. The method of any one of claims 61 to 75, wherein the pH of the liquid is between about 6.2 and 7.

4.

77. 77. The method of claim 76, wherein the pH of the liquid is between about 6.4 and about 6.

8.

78. the β-casein protein is from a cow, a human, a sheep, a goat, a buffalo, a camel, a horse, a donkey, a lemur, a panda, a guinea pig, a squirrel, a bear, a macaque, a gorilla, a chimpanzee, a mountain goat, a monkey, an ape, a cat, a dog, a wallaby, a rat, a mouse, an elephant, a possum, a rabbit, a whale, a baboon, a gibbon, an orangutan, a mandrill, a pig, a wolf, a fox, a lion, a tiger, an echidna, or a woolly mammoth; and / or the kappa-casein protein is from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth; 78. The method according to any one of claims 61 to 77.

79. 79. The method of any one of claims 61 to 78, wherein the protein mixture further comprises one or more proteins selected from the group of alpha-lactalbumin, beta-lactoglobulin, alpha-S1-casein, alpha-S2-casein, lactoferrin, transferrin, and serum albumin.

80. A composition made by the method of any one of claims 61 to 79.

81. 1. A method for making butter, cheese, caseinate, or yogurt, comprising the steps of: Providing a composition according to any one of claims 1 to 44 and 80; and making butter, cheese, caseinate or yogurt using the composition of any one of claims 1 to 44 and 80 as a starting material. The method includes:

82. (a) a mixture of one or more milk proteins, one or more fats, and one or more flavor compounds; and (b) a mixture of ash and at least one sweetening agent; Kit including:

83. 82. The kit of claim 81, wherein the one or more milk proteins are selected from the group consisting of β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, α-S1-casein, α-S2-casein, lactoferrin, transferrin, and serum albumin.

84. 84. The kit of claim 82 or 83, wherein the one or more milk proteins are milk proteins from cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth.

85. 85. The kit of any one of claims 82 to 84, wherein the one or more fats are selected from the group consisting of sunflower oil, coconut oil, tributyrin, mono- and diglycerides, free fatty acids, and phospholipids.

86. 86. The kit of claim 85, wherein the free fatty acid comprises at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid.

87. 87. The kit of claims 85 and 86, wherein the phospholipid is soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, or rapeseed lecithin phospholipid.

88. 88. The kit according to any one of claims 85 to 87, wherein the mono- and diglycerides are plant-derived mono- and diglycerides or bacterial-derived mono- and diglycerides.

89. 89. The kit of any one of claims 82 to 88, wherein the flavor compound comprises at least one flavor compound selected from the group consisting of delta-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, gamma-undecalactone, and delta-undecalactone.

90. The kit of any one of claims 82 to 89, wherein the mixture in (a) further comprises one or more color correcting agents.

91. 91. The kit of claim 90, wherein the one or more color correcting agents is beta-carotene or annatto.

92. 92. The kit of any one of claims 82 to 91, wherein one or more sweetening agents is a saccharide.

93. 93. The kit of claim 92, wherein the saccharide is selected from the group consisting of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose.

94. The kit of any one of claims 82 to 91, wherein the one or more sweetening agents are artificial sweeteners.

95. 95. The kit of claim 94, wherein the artificial sweetener is selected from the group consisting of stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lactitol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, and P-4000.

96. The kit of any one of claims 82 to 95, wherein the ash comprises one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride.

97. Ash content is CaCl 2 , K.H. 2 P.O. 4 , and sodium citrate 3 97. The kit of claim 96, comprising one or more of:

98. Ash content is CaCl 2 , K.H. 2 P.O. 4 , and sodium citrate 3 98. The kit of claim 97, comprising:

99. A kit according to any one of claims 82 to 99, further comprising instructions for making the composition according to any one of claims 1 to 44.

100. A kit comprising at least one nucleic acid according to any one of claims 46 to 54.

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