Artificial protein composition

Abstract

Micellar solutions comprising isolated non-human beta-casein and isolated non-human kappa-casein and substantially free of alpha S-casein artificial casein micelles, including methods of making the same, methods of coagulating the same, and compositions comprising the same; a method of preparing a curd composition from kappa-casein (whether phosphorylated, dephosphorylated or non-phosphorylated) with or without beta-casein (whether phosphorylated, dephosphorylated or non-phosphorylated) for use in the manufacture of downstream derivative products, such as cheese having functional characteristics comparable to equivalent products produced from milk; casein and protein emulsions of non-animal origin, curd compositions and cheese products and methods of producing the same; and a method of treating non-micellar casein to form a Parefirata style cheese product, such as a mozzarella style cheese product, comprising treating dephosphorylated casein or caseinate with a transglutaminase, in combination with oil or fat to form an emulsion useful for forming a cheese product, and treating non-micellar casein to form a Parefirata style cheese product, such as a mozzarella style cheese product. The cheese products may be processed using an acid-induced or chymosin-induced coagulation, such as acid-induced or chymosin-induced coagulation, and using standard cheese processing steps to produce different styles of cheese products.

Description

Technical Field

[0001] The technology described herein belongs to the field of protein-based food products and dairy substitutes. More specifically, the technology relates to artificial protein compositions comprising protein components derived from milk or protein components identical to those derived from milk. Provided herein are methods for producing emulsions, micelles, coagulants, and curd compositions from phosphorylated, dephosphorylated, or non-phosphorylated casein, as well as food products derived therefrom. Background Art

[0002] The following discussion of the background art is intended only to facilitate an understanding of the present invention and is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0003] As of July 5, 2022, the global dairy market size was approximately US$830 billion and is expected to grow to approximately US$1.13 trillion by 2026. Cow's milk accounts for the largest share of the global dairy market, while plant-based dairy alternatives, lactose-intolerant milk, low-carbohydrate high-protein dairy products, and other improved dairy products, such as milk without A1 β-casein (A2 milk), are also becoming increasingly popular in the increasingly diet-conscious consumer market.

[0004] Milk of mammalian origin is a highly complex liquid composition that contains, in addition to water, thousands of different compounds from lipids, triglycerides, carbohydrates, sugars, peptides, inorganic salts and other molecular entities. Although many consider milk of mammalian origin, including cow's milk, to be an ideal source of nutrition, a variety of alternatives to milk of mammalian origin are now successfully marketed, including plant- or nut-based milks, such as soy milk, almond milk or coconut milk, and are being accepted by consumers for reasons related to the allergenicity of milk of mammalian origin, lactose intolerance of certain components, personal preference or perceptions of adverse environmental impacts caused by the dairy industry.

[0005] For example, most mammalian milk comes from ruminants, including cows, buffalo, yaks, goats and sheep, as well as pseudo-ruminants such as camels, alpacas and llamas. According to a recent UN assessment, cattle farming and ruminant livestock farming generally produce more global warming greenhouse gases, measured in carbon dioxide (CO2) equivalents, than transportation. It is estimated that ruminants account for 10% of Australia's total greenhouse gas emissions. Ruminants produce methane (CH4) as a by-product of digestion through anaerobic microbial fermentation in the rumen and, to a lesser extent, the large intestine. This process is called methanogenesis.

[0006] Methane is an effective absorber of solar infrared radiation and has a global warming potential 25 times that of CO2. The rumen microbiome consists of bacteria, protozoa, fungi, and bacteriophages, all of which work together to digest ingested organic matter and produce CO2, H2, volatile fatty acids, and formate. These end products are utilized by methanogenic archaea in the rumen, which produce CH4. Although the production of CH4 reduces the partial pressure of H2, this can also cause problems because it also limits the amount of energy and carbon available for the synthesis of volatile fatty acids, which are critical for ruminant nutrition and may additionally limit rumen fermentation. Most of the CH4 produced by ruminants is exhaled or excreted through the mouth, resulting in up to 12% of the total caloric intake in the ruminant diet being wasted. In addition, producing a cup of dairy milk from a cow consumes up to 9 times more land and significantly more water than any plant-based milk substitute.

[0007] Attempts to address these environmental concerns with plant-based milks (including soy, almond, or coconut milk) fall short in terms of flavor and practicality. Furthermore, much of the industrial and cultural value of dairy milk derives from its use in derivative products such as cheese, yogurt, cream, or butter. While plant-based dairy alternatives address some environmental and health concerns (and offer sufficient flavor for a small consumer base), they almost always fail to produce such derivative products when produced using the same processes as dairy milk.

[0008] Therefore, there is a need for an alternative dairy product or composition having desirable flavor and performance characteristics, such as a composition that replicates the flavor of dairy products, minimizes foodborne pathogens, and reduces environmental impact during production, while retaining its ability to be used in dairy milk-derived products or downstream applications and providing a nutritional profile similar or comparable to that of milk of mammalian origin. There is also a need for improved processes for producing derived products (e.g., curd compositions and cheeses) from artificial protein micellar solutions that mimic the behavior of dairy milk or milk of mammalian origin.

[0009] The protein content of cow's milk required for most derivative products such as cheese is mainly composed of four different caseins; α s1 -Casein, α s2-casein, beta-casein and kappa-casein. Cheese is the third most unsustainable animal product in the world (in terms of greenhouse gas emissions per kilogram of product), but the plant-based alternatives that have been launched on the market in the past decade have not reduced the demand for dairy cheese. On the contrary, the consumption of mozzarella cheese (mozzarellacheese) in the United States and other developing countries has increased year by year. Due to the lack of casein, current cheese substitutes are inferior to dairy cheese in functionality (including melting properties and browning properties when cooked or grilled), texture, nutrition and taste. At the same time, the most common cause of human allergy to dairy products is the presence of α-casein in dairy milk. s1 -Casein.

[0010] In milk, casein exists in colloidal particles called "casein micelles." The term "casein micelles" describes the colloidal calcium phosphate / calcium caseinate particles in milk. Casein micelles are assemblies of four different caseins: α s1 -Casein, α s2 The molar ratio of casein is approximately 4:1:4:1. In casein micelles, kappa-casein is primarily localized to the surface, forming a "coat" or outer layer within the micelle, while the remaining caseins reside in the more hydrophobic core. Thus, kappa-casein is responsible for maintaining the other caseins in solution. Colloidal calcium phosphate stabilizes the casein micelle structure.

[0011] The production process of cheese is as follows: a suspension containing casein micelles ((containing α s1 -Casein, α s2 In the present invention, the present invention relates to the production of milk products of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention. The present invention relates to a kind of milk product of the present invention.

[0012] Milk-derived caseinate is produced by precipitating micellar casein at the isoelectric point of micellar casein, and then neutralizing to dissolve single casein again. However, re-dissolving does not cause the reformation of casein micelles, and casein remains in the solution as caseinate. This non-micellar casein can form a uniform solid texture by mixing and cooking raw materials, thereby making processed cheese and cheese analogues." cheese analogue (analogue cheese) " is a kind of economical and efficient cheese-like product, wherein milk protein (milk protein) and / or milk fat are substituted by non-milk natural protein and fat such as caseinate and / or edible vegetable oil (vegetable oil). Usually, " cheese analogue " is produced by mixing the various components comprising non-milk fat or protein. In caseinate solution, adding rennet can not cause the formation of gel (although κ-casein is still hydrolyzed, there is no micelle to gather together).

[0013] With the growing awareness of dairy alternatives, a certain proportion of non-micellar casein in mozzarella cheese can be replaced with plant proteins without affecting its protein content. However, it is challenging to achieve the desired emulsification, stretchability, and meltability of cheese containing plant proteins. This leads to low consumer acceptance and preference.

[0014] There is a need to provide a composition comprising non-micellar casein and vegetable protein that is suitable for producing a product having organoleptic properties similar to cheese products such as mozzarella.

[0015] Chymosin, also known as rennin, is a proteolytic enzyme that hydrolyzes κ-casein between amino acids 105 and 106, thereby removing the hydrophilic C-terminal fragment ("glycomacropeptide") from the κ-casein. When rennin is added to casein micelles, the κ-casein is still hydrolyzed, and the truncated κ-casein formed after removal of the glycomacropeptide (called para-κ-casein) remains associated with the casein micelles. However, once cleaved by rennin, the residual stability of para-κ-casein is insufficient to fully stabilize the micelles. Due to κ-casein hydrolysis, the micelles aggregate into para-casein micelles, ultimately fully coagulating to form a gel. In milk, approximately 85% of the κ-casein must be hydrolyzed for coagulation to occur.

[0016] Caseins can be produced recombinantly by precise fermentation. In addition to the amino acid sequence, chemical modifications of proteins after their translation, so-called post-translational modifications, have an important influence on their final structure and function. In the case of α-casein and β-casein, phosphorylation is crucial for their coagulation and gelling properties, which are key to cheese production. However, in some groups of microorganisms, especially bacteria, phosphorylation is less common and has different properties. Therefore, caseins produced recombinantly by bacteria lack phosphate groups and cannot be synthesized by the typical CaCl2 reaction. 2+ Bridges build and stabilize the gel network. Previous experiments and scientific papers have shown that non-phosphorylated caseins only build fragile, brittle gels with very low cohesiveness. Therefore, their suitability for cheese production is very low.

[0017] It is known in the art that dephosphorylated bovine β-casein is less prone to aggregation (see, McCarthy et al., Food Chemistry, vol 138: 1304-1311 (2013); Ohmiya et al., Agricultural and Biological Chemistry, Volume 47: 535–542 (1983)), and that partially dephosphorylated casein in casein micelles adversely affects the coagulation ability of casein (see, Pearse et al., Journal of Dairy Research, Vol 53: 381-390 (1986)). The ability of rennet to induce coagulation of dephosphorylated casein has also been observed (see Yamauchi et al., Agricultural and Biological Chemistry 42: 1031-1035 (1978). Van Hekken et al, noted (J Dairy Science 77: 907-916 (1994)). Dephosphorylation affects the way casein associates with itself and other proteins to form micelles. Dephosphorylation removes most of the Ca2+ binding sites, leaving only weaker binding sites for glutamic acid and aspartic acid residues. Thus, the loss of phosphate groups results in abnormal micelles that are less stable in the presence of calcium ions.

[0018] A common challenge facing many dairy alternative manufacturers is how to scale up quickly and cost-effectively. Manufacturing recombinant proteins can be costly and time-consuming. This is partly because downstream costs for protein purification can account for up to 80% of the total protein production process cost, and protein yields can drop by up to 70%, depending on the purity of the product.

[0019] Because recombinant casein is expensive and difficult to develop and produce, it would be desirable to produce artificial casein micelles that have structural and functional properties similar to those observed in dairy milk (including in terms of mineral content and micelle size), but without requiring all four caseins present in dairy milk (α s1 -Casein, α s2 -casein, β-casein and κ-casein), thereby greatly simplifying the production of such artificial casein micelles, especially when the casein used is derived from non-dairy sources (e.g., by recombinant microorganisms). It is also desirable to produce a curd composition that has structural and functional properties similar to those observed in such curd compositions and downstream derivative products derived from whole milk, including texture, firmness, elasticity, melting behavior in downstream derivative products (such as cheese) and desirable browning behavior when cooked or grilled, but without requiring all four caseins present in milk (α-casein, β-casein and κ-casein). s1 -Casein, α s2 -casein, β-casein and κ-casein), thereby greatly simplifying the production of such artificial curd compositions and downstream derived products, especially when the casein used is derived from non-dairy sources (e.g. by recombinant microorganisms).

[0020] It is against this background that the present invention has been developed. Summary of the Invention

[0021] By developing a new method for preparing curd compositions, the present inventors have produced artificial curd products from only κ-casein (whether phosphorylated or not) or a combination of κ-casein (whether phosphorylated or not) and β-casein (whether phosphorylated or not), without the need for α-casein. s1 -Casein and α s2 - casein, and whose properties are very similar to those of natural curd products of bovine origin in terms of texture, firmness, elasticity, melting behavior in downstream derived products (e.g. cheese) and desirable browning behavior when cooked or grilled, similar to those observed in such curd compositions and downstream derived products derived from whole milk.

[0022] The components of natural bovine milk used to form conventional curd products include κ-casein, β-casein, α-casein, s1 -Casein and α s2 -caseins, each of which exists in different degrees of phosphorylation. However, from the equivalent phosphorylation of κ-casein, β-casein, α s1 -Casein and α s2Producing alternative curd products from non-mammalian sources of κ-casein is a daunting processing challenge, characterized by high costs and difficulty. On the other hand, precision fermentation processes enable the cost-effective production of non-phosphorylated, mono-phosphorylated, or di-phosphorylated forms of κ-casein. Therefore, the present invention provides a cost-effective approach for producing non-animal-derived curd products and their downstream derivatives.

[0023] These artificial curd products are coagulated by acid and / or microbial agents and / or fermentation and / or the action of rennet and / or enzymes and / or via hydrolysis or decomposition of glucono-delta-lactone to produce curds having the same cheese texture as those produced from cow's milk, allowing to fully (or partially) replace the functionality of bovine-derived curd products and their downstream derivatives such as cheese.

[0024] Furthermore, by developing a method to (re)assemble non-micellar caseins into casein micelles, the present inventors produced artificial casein micelles from only β-casein and κ-casein without the need for α-casein. s1 -Casein and α s2 -casein and has properties very similar to those of natural bovine casein micelles in terms of micelle size and mineral content. These artificial micelles can also be coagulated by the action of rennet to produce a cheese texture identical to those produced from cow's milk, allowing a complete replacement of the functionality of bovine casein micelles.

[0025] In a first aspect, the present disclosure provides a micellar solution comprising a plurality of artificial casein micelles, wherein the artificial casein micelles comprise isolated non-human β-casein and isolated non-human κ-casein, and wherein the artificial casein micelles are substantially free of α-casein. s -Casein.

[0026] In some embodiments, the Z-average diameter of the artificial casein micelles is greater than 30 nm.

[0027] In a preferred embodiment, the Z-average diameter of the artificial casein micelles falls within the range of 40 to 500 nm.

[0028] In some embodiments, the micellar solutions described herein comprise less than 7 wt% α-casein, based on the total casein present in the micellar solution. s -Casein.

[0029] In some embodiments, the micellar solutions described herein comprise a wt% ratio of non-human β-casein to non-human κ-casein that falls within the range of 10:90 to 90:10.

[0030] In some embodiments, the micellar solutions described herein comprise a total casein concentration falling within the range of 10 g / L to 95 g / L.

[0031] In some embodiments, the micellar solutions described herein comprise a total non-human β-casein concentration falling within the range of 1 g / L to 90 g / L.

[0032] In some embodiments, the micellar solutions described herein comprise a total non-human kappa-casein concentration falling within the range of 1 g / L to 90 g / L.

[0033] In some embodiments, the micellar solutions described herein comprise α- s1 -Casein concentration.

[0034] In some embodiments, the micellar solutions described herein comprise α s2 -Casein concentration.

[0035] In some embodiments, the hydration of the artificial casein micelles in the micellar solutions of the present invention falls within the range of 1 to 8 (g water / g micellar protein).

[0036] In some embodiments, the percentage of non-micellar casein to total casein in the micellar solutions of the present invention falls within the range of 5 to 20%.

[0037] In some embodiments, the micellar calcium content of the micellar solution of the present invention is at least 70% of the total calcium in the micellar solution.

[0038] In some embodiments, the content of micellar magnesium in the micellar solution of the present invention is at least 30% of the total magnesium in the micellar solution. In a preferred embodiment, the content of micellar magnesium in the micellar solution of the present invention is at least 35% of the total magnesium in the micellar solution.

[0039] In some embodiments, the content of micellar inorganic phosphate in the micellar solution of the present invention is at least 50% of the total inorganic phosphate in the micellar solution.

[0040] In some embodiments, the micellar citrate content in the micellar solution of the present invention is at least 5% of the total citrate in the micellar solution.

[0041] In one embodiment, the present disclosure provides a curd composition comprising the micellar solution of the present invention in a coagulated form.

[0042] In some embodiments, the curd composition further comprises a renneting agent.

[0043] In a preferred embodiment, the curd composition has a maximum G' (storage modulus) falling within the range of 5 to 200 Pa, preferably after 1 hour of incubation with rennet.

[0044] In other embodiments, the present disclosure provides edible compositions comprising the micellar solution of the present invention or the curd composition of the present invention.

[0045] In some embodiments, the edible composition does not contain any protein from animal sources.

[0046] In one embodiment, the present disclosure provides a method for producing an edible composition comprising: mixing isolated non-human β-casein, isolated non-human κ-casein, and at least one salt under conditions where the β-casein and κ-casein form a micellar solution, wherein the micellar solution is substantially free of α-casein. s - casein; and subjecting the micellar solution to a first condition to form a coagulum.

[0047] In some embodiments of the method for producing an edible composition, the first condition is adding acid or acidifying the micellar solution with a microorganism.

[0048] In some embodiments of the method for producing an edible composition, the method further comprises subjecting the coagulum to a coagulant to form a coagulated curd.

[0049] In some embodiments of the method for producing an edible composition, the method further comprises aging and / or ripening the curdled curd to form a cheese composition.

[0050] In some embodiments of the method for producing an edible composition, the edible composition does not contain any protein of animal origin.

[0051] In a second aspect, the present disclosure provides a method for preparing a curd composition, wherein the method comprises the following steps: a) preparing an emulsion comprising: (i) phosphorylated κ-casein; or (ii) dephosphorylated or non-phosphorylated κ-casein; or (iii) phosphorylated κ-casein and dephosphorylated or non-phosphorylated κ-casein; or (iv) phosphorylated kappa-casein, dephosphorylated or non-phosphorylated kappa-casein, and dephosphorylated or non-phosphorylated beta-casein; or (v) dephosphorylated or non-phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; or (vi) phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; and (vii) optionally, a lipid source; b) adding a calcium salt to the emulsion; and c) causing the emulsion to coagulate.

[0052] In some embodiments, the coagulation step c) of the method comprises adding acid; and / or adding rennet; and / or adding enzymes; and / or adding chymosin to provide a coagulated emulsion.

[0053] In some embodiments, the method further comprises the additional steps of heating the emulsion prior to step b); and / or heating the emulsion prior to step c); maintaining the emulsion at a suitable temperature for a suitable time; preferably, wherein the temperature falls within the range of 45°C to 70°C; preferably, wherein the time period is within the range of 1 minute to 180 minutes. The most preferred heat treatment time period is 30 minutes, and the most preferred heat treatment temperature is 63°C.

[0054] In some embodiments, the method further comprises the additional step of treating the emulsion with transglutaminase prior to step b); and / or treating the emulsion with transglutaminase prior to step c); preferably, the additional step of treating the emulsion with transglutaminase comprises adding transglutaminase to the emulsion and incubating at a suitable temperature and for a suitable period of time; preferably, the temperature falls within the range of 45°C to 60°C; preferably, the period of time falls within the range of 1 minute to 180 minutes. The most preferred incubation period is 30 minutes, and the most preferred incubation temperature is 50°C.

[0055] The heat treatment step and the transglutaminase incubation step are independent optional steps that can be performed independently of each other. However, in some embodiments, the heat treatment step can be advantageously performed after the transglutaminase incubation step, so that the heat treatment step performs the dual function of enhancing the functionality of the cheese (particularly stretchability) and inactivating the transglutaminase.

[0056] In some embodiments of the method, the emulsion comprises a total protein concentration falling within the range of 2 wt.% to 20 wt.%; preferably, wherein the emulsion comprises a total protein concentration falling within the range of 3 wt.% to 10 wt.%; most preferably, wherein the emulsion comprises a total protein concentration falling within the range of 4 wt.% to 8 wt.%.

[0057] In some embodiments of the method, the emulsion comprises a fat to protein ratio falling within the range of fat free to 2:0.5; preferably, wherein the emulsion comprises a fat to protein ratio falling within the range of 1:1.5 to 1.5:1; most preferably, wherein the emulsion comprises a fat to protein ratio of about 1:1.

[0058] In some embodiments of the method, the calcium salt added in step b) is added so as to bring the concentration of calcium in the emulsion to a concentration falling within the range of 2 mM to 20 mM; preferably, wherein the calcium salt is added so as to bring the concentration of calcium in the emulsion to a concentration falling within the range of 5 mM to 15 mM; most preferably, wherein the calcium salt is added so as to bring the concentration of calcium in the emulsion to a concentration falling within the range of 8 mM to 12 mM.

[0059] In some embodiments of the method, the calcium salt added in step b) is selected from the group consisting of calcium chloride, calcium hydroxide, calcium carbonate, calcium citrate, calcium phosphate, calcium stearate, calcium malate, calcium glycerophosphate, calcium lactate and calcium gluconate, or a mixture thereof.

[0060] In some embodiments of the method, the lipid source added in step a)(vii) is selected from the group consisting of non-animal fats or oils, plant fats or oils, microbial fats or oils, fungal fats or oils, recombinantly produced fats or oils, or a mixture of any of the foregoing lipid sources; preferably, wherein the lipid source is an edible vegetable oil selected from the group consisting of peanut oil, soybean oil, sunflower oil, safflower oil, rapeseed oil, corn oil, avocado oil, almond oil, olive oil, cottonseed oil, coconut oil, sesame oil, chia (Salvia Hispanica L.) seed oil, wheat germ oil, grapeseed oil, rice bran oil, linseed oil, mustard oil, palm oil, castor oil, hydrogenated castor oil, hemp seed oil, and any mixture thereof.

[0061] In some embodiments of the method, step c) of coagulating the emulsion comprises acidifying the emulsion by adding acid, or by treating with a microbial acidifier, or by a fermentation process, or by hydrolysis or cleavage of glucono-delta-lactone.

[0062] In some embodiments of the method, step c) of coagulating the emulsion comprises acidifying the emulsion to a pH falling within the range of pH 4.2 to pH 6.5; preferably, wherein coagulating the emulsion comprises acidifying the emulsion to a pH falling within the range of pH 4.8 to pH 5.8; most preferably, wherein acidifying the emulsion comprises bringing the pH of the emulsion to about 5.2.

[0063] In some embodiments, the method further comprises the following steps: d) incubating the coagulated emulsion produced in step c) at a suitable temperature for a period of time to produce a coagulum comprising a crude curd and a liquid phase; preferably, wherein the period of time falls within the range of 10 min to 180 min; most preferably, wherein the period of time falls within the range of 20 min to 60 min; preferably, wherein the temperature falls within the range of 18° C. to 50° C.; most preferably, wherein the temperature falls within the range of 25° C. to 40° C.

[0064] In some embodiments, the method further comprises the following steps: e) removing the crude curd of the coagulant produced in step d) from the liquid phase of the coagulant produced in step d) to provide a curd composition.

[0065] In some embodiments, the method further comprises the following steps: f) texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey, at elevated temperature to provide a textured curd composition.

[0066] In some embodiments of the method, step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at an elevated temperature comprises an elevated temperature falling within the range of 60°C to 95°C; preferably an elevated temperature falling within the range of 70°C to 90°C; most preferably an elevated temperature falling within the range of 75°C to 85°C.

[0067] In some embodiments of the method, step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises texturizing at a ratio of curd composition to water falling within the range of 0.5:3 to 3:0.5; preferably a ratio of curd composition to water falling within the range of 1:2 to 2:1; most preferably a ratio of curd composition to water of 1:2.

[0068] In some embodiments of the method, step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises texturizing by kneading and / or stretching and / or folding the curd composition, and optionally forming the curd composition into balls.

[0069] In some embodiments, the method further comprises the following steps: g) Cooling the textured curd composition; preferably in brine or water.

[0070] In some embodiments, the textured curd composition provided by step f) is a cheese product of non-animal origin; preferably a mozzarella type cheese product of non-animal origin.

[0071] In some embodiments, the present disclosure provides a food product produced by the methods of the present invention, comprising casein.

[0072] In some embodiments, the food product is a cheese product of non-animal origin.

[0073] In a preferred embodiment, the food product is a mozzarella-type cheese product of non-animal origin.

[0074] In a third aspect, the present invention provides an emulsified composition comprising one or more lipids and a protein component, wherein the protein component has a protein content of at least 80% (by weight) protein, wherein about 25-40% (by weight) of the protein content comprises zein protein, the remainder being micellar and / or non-micellar casein.

[0075] The present invention also provides a method of forming an emulsified composition according to the present invention, wherein the method comprises the following steps: i) preparing a mixture of non-micellar casein and / or zein proteins; ii) adding one or more lipids; iii) emulsifying the mixture to obtain an emulsified composition.

[0076] In the method of the present invention, the zein component may first be dissolved in an alkaline aqueous solution. Thus, the present invention may provide a method of forming an emulsified composition according to the present invention, wherein the method comprises the following steps: i) preparing a zein protein solution at an alkaline pH; ii) adding non-micellar casein; ii) adding one or more lipids; iii) emulsifying the mixture to obtain an emulsified composition.

[0077] In an alternative method of forming the emulsified composition of the present invention, the method comprises the steps of: i) preparing an aqueous solution of non-micellar casein; ii) adding one or more lipids; iii) emulsifying the mixture and zein protein to obtain an emulsified composition.

[0078] The present invention also provides a method for forming a fiber or pasta filata cheese product, wherein the method comprises forming an emulsified composition according to the first aspect of the present invention. Specifically, by providing an emulsified composition according to the first aspect of the present invention and inducing gel formation by acidifying the emulsion and / or by adding a calcium salt such as CaCl2.

[0079] Optionally, the addition of a calcium salt (CaCl2) can be carried out in the presence of a base to increase the pH (e.g., to pH 6 or higher). Typically, when the pH is alkaline, the calcium salt is added to induce gel formation. Optionally, temperature variation can also be used to promote gel formation. The coagulated product formed by gel formation can be processed using standard cheese processing steps to produce a pasta filata-style (e.g., mozzarella-style) cheese product.

[0080] The pasta filata style cheese product obtained forms another embodiment of the invention.

[0081] In a fourth aspect, the invention provides a method for forming a cheese product, wherein the method comprises the step of treating dephosphorylated casein or caseinate with transglutaminase. The treated casein or caseinate can then be combined with fat to form an emulsion and processed into a cheese product, for example by adding rennet to the emulsion or by acidification (for example to a pH of 4 to 6). Acidification can be performed by directly adding acid or by adding a microbial (starter) culture. The method can be performed at ambient temperature. Alternatively, curd can be produced by adding acid or by acidifying the emulsion with a starter culture.

[0082] In one embodiment, the present invention provides a cheese product comprising transglutaminase-treated dephosphorylated casein. Optionally, the cheese product can be a pasta filata-style cheese product. Optionally, the cheese product can be a mozzarella-style cheese product. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Other features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention. It should not be construed as limiting the broad overview, disclosure, or description of the invention as described above. The description will be made with reference to the accompanying drawings, in which: Figure 1.1 Schematic diagram of the method for forming artificial casein micelles of the present invention.

[0084] Figure 2.1 3 is a graph showing the relationship between the Z-average diameter (intensity-weighted average hydrodynamic size) of the artificial casein micelles of the prepared micelle solution sample containing the artificial casein micelles and the total β-casein content (%) in the sample.

[0085] Figure 3.1Figure 1 is a series of scanning electron microscopy (SEM) images of artificial casein micelles (ACMs) of the present invention, having different ratios of β-casein to κ-casein (b:k): (1) ACM b:k 70:30, (2) ACM b:k 75:25, (3) ACM b:k 80:20, (4) ACM b:k 85:15. Scale bar = 0.5 μm.

[0086] Figure 4.1 This is a scanning electron microscope (SEM) image of skim milk. Scale bar = 0.5 μm.

[0087] Figure 5.1 The graph is a function of the micellar casein or precipitable casein (casein fraction precipitated during ultracentrifugation) of the prepared ACM micelle solution sample containing artificial casein micelles and the total β-casein content (%) in the sample.

[0088] Figure 6.1 Graph showing the relationship between the ACM micelle hydration of the prepared micelle solution sample containing artificial casein micelles and the total β-casein content (%) in the sample.

[0089] Figure 7.1 The maximum G' (storage modulus; a measure of firmness) of curd produced when the ACM micelle solution sample containing artificial casein micelles was coagulated at pH 6.3 with the addition of 0.10% CaCl2 and rennet-induced coagulation was plotted as a function of the total β-casein content (%) in the sample.

[0090] Figure 1.2 is a series of photographs of curd compositions made from a) phosphorylated κ-casein alone; and b) dephosphorylated κ-casein alone according to the method of the present invention at the following stages: 1. coarse curd separation; 2. texturization; and 3. testing meltability after heating the textured compositions.

[0091] Figure 2.2 is a series of photographs of curd compositions made according to the method of the present invention from a combination of dephosphorylated β-casein and phosphorylated κ-casein according to Schemes 1 to 5 [a)-e)] described in Example 3.2 at the following stages: 1. coarse curd separation; 2. texturization; and 3. testing meltability after heating the textured composition.

[0092] Figure 3.2is a series of photographs of curd compositions made according to the method of the present invention from a combination of dephosphorylated β-casein and phosphorylated κ-casein [a) & b)]; and dephosphorylated κ-casein [c) & d)]; according to Schemes I to IV described in Example 4.2 at the following stages: 1. coarse curd separation; and 2. texturization.

[0093] Figure 1.3 is a flow chart of a method of the present invention for forming mozzarella cheese.

[0094] Figure 2.3 Shown are a) curd after whey drainage; b) curd during kneading and stretching; c) mozzarella cheese after refrigeration; and d) cheese texture.

[0095] Figure 1.4 A photograph of a dephosphorylated sodium caseinate acid gel not treated with transglutaminase is shown.

[0096] Figure 2.4 A photograph of a dephosphorylated sodium caseinate acid gel is shown. Figure 2.2 A shows the uncut gel. Figure 2.2 B shows excised gels, each treated with transglutaminase: a) 2 U enzyme / g protein; b) 3 U enzyme / g protein; and c) 4 U enzyme / g protein.

[0097] Figure 3.4 is a bar graph showing gel firmness with increasing transglutaminase concentration during treatment of dephosphorylated casein / caseinate.

[0098] Figure 4.4 is a bar graph showing curd yield with increasing transglutaminase concentration during treatment with dephosphorylated casein / caseinate.

[0099] Figure 5.4 Photographs of curds of dephosphorylated casein / caseinate during whey drainage are shown: a) without transglutaminase treatment, pH 5.2; b) without transglutaminase treatment, pH 5.6; and c) after transglutaminase (4 U / g protein) treatment, pH 5.6.

[0100] Figure 6.4 A photograph of a mozzarella-like cheese product formed in accordance with the present invention is shown.

[0101] Figure 7.4 is a schematic diagram showing the method of the present invention.

[0102] definition

[0103] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0104] Unless expressly stated otherwise, the term "%" in this specification should be understood to mean weight % or wt.%.

[0105] As used herein, the term "micelle" and its grammatical variants are understood to mean a generally (or approximately) spherical supramolecular structure present as a dispersion within a composition or solution. A micelle can have a surface consisting, for example, of a charged outer layer. A micelle can encapsulate one or more biomolecules. For example, a micelle can encapsulate two or more proteins (e.g., β-casein and κ-casein). A micelle can have a diameter between about 10 nm and about 500 nm. Other aspects and features of micelles are known in the art.

[0106] As used herein, the phrase "substantially free of s -casein" when applied to the micellar solution of the present invention should be understood to mean that although some α s - Caseins may be present as minor impurities in the micellar solution, but they will represent a very minor component of the micellar solution, comprising at most 0.3% of the total micellar solution, preferably at most 0.2% of the total micellar solution, and most preferably not more than 0.18% of the total micellar solution. The phrase "substantially free of α-casein" with respect to the total casein present in the micellar solution of the present invention is s -casein" should be understood to mean that although some α s -Casein may be present as a small amount of impurity in the total casein in micellar solution, but α s -casein will be less than 10 wt% of the total casein present in the micellar solution, preferably less than 8 wt% of the total casein present in the micellar solution, and most preferably no more than 7 wt% of the total casein present in the micellar solution. It will be appreciated by those skilled in the art that when the protocols described herein are applied to assemble artificial casein micelles comprising recombinantly produced β-casein and κ-casein, the micellar solution of the present invention should be completely free of α-casein. s - caseins (ie, they will contain 0% α s - casein, and they will contain 0 wt% α-casein relative to the total casein present in the micellar solution. s -casein).

[0107] As used herein, the terms "about" or "approximately" and grammatical variations thereof refer to within an acceptable error range 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, or on limitations of the measurement system. It should be understood that all ranges and amounts described below are approximate and are not intended to limit the invention. Where ranges and amounts are used, these may be approximate to include statistical ranges or measurement errors or variations. In some embodiments, for example, a measured value may be plus or minus 10%.

[0108] As used herein, the term "isolated non-human β-casein" should be understood to refer to any β-casein isolated from any non-human source, including β-casein isolates from any non-human mammalian species, as well as any synthetically or recombinantly produced β-casein, and including variants having at least 80% sequence homology to any mammalian β-casein sequence, and including such variants having at least 80% sequence homology to any mammalian β-casein sequence with or without post-translational modifications, such as glycosylation and / or phosphorylation. Some embodiments of the artificial micelles of the present invention include synthetically or recombinantly produced β-casein variants having sequence homology to any mammalian β-casein sequence, wherein the sequence homology to any mammalian β-casein sequence is selected from the group of sequence homologies consisting of: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence homology to any mammalian β-casein sequence, with or without post-translational modifications, such as glycosylation and / or phosphorylation.

[0109] As used herein, unless the context indicates otherwise, the term "κ-casein" should be understood to refer to any κ-casein isolated from any source, including κ-casein isolates from any mammalian species, as well as any synthetically or recombinantly produced κ-casein, and includes variants having at least 80% sequence homology to any mammalian κ-casein sequence, and includes such variants having at least 80% sequence homology to any mammalian κ-casein sequence with or without post-translational modifications, such as glycosylation and / or phosphorylation. Some embodiments of the curd composition of the present invention comprise a synthetically or recombinantly produced κ-casein variant having sequence homology to any mammalian κ-casein sequence, wherein the sequence homology to any mammalian κ-casein sequence is selected from the group of sequence homologies consisting of: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence homology to any mammalian κ-casein sequence, with or without post-translational modifications, such as glycosylation and / or phosphorylation.

[0110] As used herein, the term "isolated non-human κ-casein" should be understood to refer to any κ-casein isolated from any non-human source, including κ-casein isolates from any non-human mammalian species, as well as any synthetically or recombinantly produced κ-casein, and including variants having at least 80% sequence homology to any mammalian κ-casein sequence, and including such variants having at least 80% sequence homology to any mammalian κ-casein sequence with or without post-translational modifications, such as glycosylation and / or phosphorylation. Some embodiments of the artificial micelles of the present invention include synthetically or recombinantly produced κ-casein variants having sequence homology to any mammalian κ-casein sequence, wherein the sequence homology to any mammalian κ-casein sequence is selected from the group of sequence homologies consisting of: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence homology to any mammalian κ-casein sequence, with or without post-translational modifications, such as glycosylation and / or phosphorylation.

[0111] As used herein, unless the context indicates otherwise, the term "β-casein" should be understood to refer to any β-casein isolated from any source, including β-casein isolates from any mammalian species, as well as any synthetically or recombinantly produced β-casein, and includes variants having at least 80% sequence homology to any mammalian β-casein sequence, and includes such variants having at least 80% sequence homology to any mammalian β-casein sequence with or without post-translational modifications, such as glycosylation and / or phosphorylation. Some embodiments of the curd composition of the present invention include a synthetically or recombinantly produced β-casein variant having sequence homology to any mammalian β-casein sequence, wherein the sequence homology to any mammalian β-casein sequence is selected from the group of sequence homologies consisting of: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence homology to any mammalian β-casein sequence, with or without post-translational modifications, such as glycosylation and / or phosphorylation.

[0112] As used herein, the term "phosphorylated" as applied to the kappa-casein and beta-casein components used in the methods of the present invention will be understood to mean that the component to which the term "phosphorylated" is applied is at least partially phosphorylated, that is, phosphorylated kappa-casein will have at least one phosphate group attached thereto, and may have multiple phosphate groups attached thereto, and phosphorylated beta-casein will have at least one phosphate group attached thereto, and may have multiple phosphate groups attached thereto. In other words, the term "phosphorylated" will be understood to include components that are partially phosphorylated. Phosphorylated as well as fully phosphorylated. Thus, the term "phosphorylated κ-casein" should be understood to include native κ-casein (i.e., κ-casein isolated from mammalian milk without any dephosphorylation step), as well as partially dephosphorylated κ-casein, and recombinantly produced κ-casein that has been partially or fully phosphorylated. Similarly, the term "phosphorylated β-casein" should be understood to include native β-casein (i.e., β-casein isolated from mammalian milk without any dephosphorylation step), as well as partially dephosphorylated β-casein, and recombinantly produced β-casein that has been partially or fully phosphorylated.

[0113] As used herein, the term "non-phosphorylated" when applied to the κ-casein and β-casein components used in the methods of the present invention will be understood to mean that the components to which the term "non-phosphorylated" is applied do not have any phosphate groups attached thereto, that is, non-phosphorylated κ-casein will not have any phosphate groups attached thereto, and non-phosphorylated β-casein will not have any phosphate groups attached thereto. In other words, the term "non-phosphorylated" will be understood to include recombinant κ-casein and recombinant β-casein that have not undergone any phosphorylation, as well as mammalian-derived κ-casein and mammalian-derived β-casein that have been completely dephosphorylated.

[0114] As used herein, the term "dephosphorylated" as applied to the κ-casein and β-casein components used in the methods of the present invention will be understood to mean that the components to which the term "dephosphorylated" applies are at least partially dephosphorylated, that is, the dephosphorylated κ-casein may have no phosphate groups attached thereto, may have at least one phosphate group attached thereto, or may have multiple phosphate groups attached thereto, and the dephosphorylated β-casein may have no phosphate groups attached thereto, may have at least one phosphate group attached thereto, or may have multiple phosphate groups attached thereto. In other words, the term "dephosphorylated" will be understood to include partial dephosphorylation as well as complete dephosphorylation.

[0115] As used herein, the term "animal-derived protein" should be understood to refer to any protein from any animal source. It should also be understood that the term "animal-derived protein" does not include any protein that is synthetically or recombinantly produced.

[0116] As used herein, the term "non-animal origin" when applied to curd and downstream products derived from curd (e.g., cheese products) and any individual ingredients thereof, will be understood to mean that the ingredient or product or composition to which the term "non-animal origin" is applied will not contain any material from an animal, whether the animal is a mammal, a bird, a reptile, a fish, an insect or another type of animal. However, as used herein, the term "non-animal origin" does include ingredients or products or compositions derived from microbial species. That is, vegetable fats or oils are lipids of non-animal origin, and the β-casein of recombinant origin is β-casein of non-animal origin. Plant protein is protein of non-animal origin, etc. In other words, the term "non-animal origin" includes any ingredient or composition or product derived from plant origin, or synthetic origin, or microbial origin, or fungal origin, or recombinant origin.

[0117] As used herein, the term "fat-free" when applied to the emulsions of the present method is understood to mean that although some fat may be present as a minor component of the emulsion, they will represent a minor component of the emulsion, at most 3% of the emulsion, preferably at most 2% of the emulsion, and most preferably not more than 1% of the emulsion.

[0118] Other definitions of selected terms used herein can be found in the detailed description of the invention and are applicable to the entire text. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. DETAILED DESCRIPTION

[0119] Artificial micelles

[0120] The present disclosure provides an unprecedented approach that allows for the preparation of a micellar solution comprising a plurality of artificial casein micelles, wherein the artificial casein micelles comprise isolated non-human β-casein and isolated non-human κ-casein, and wherein the artificial casein micelles are substantially free of α-casein. s -Casein.

[0121] Advantageously, the artificial micellar solutions of the present invention have suitable micelle size and micellar mineral salt content for use in downstream product manufacturing, such as cheese making.

[0122] This protocol can be directly applied to prepare artificial casein micelles from recombinantly produced β-casein and κ-casein, which are essentially completely free of α s To provide preliminary proof of principle, artificial casein micelles were prepared from bovine β-casein (about 98% pure, containing a small amount of α-casein) and bovine κ-casein (70% pure, also containing a small amount) in a ratio of 75:25. Figure 1.1 The process flow is explained.

[0123] The glass beaker shown in the figure ( Figure 1.1 ) represents a jacketed titration vessel connected to a water bath at the preferred temperature (37° C.). Three separate salt solutions (solution I: CaCl and MgCl; solution II: KH and Na; solution III: trisodium C6H5O7) of the preferred salts at the preferred concentrations (solution I: 325 mM CaCl and 61.2 mM MgCl, adjusted to pH 6.70 with 0.1 M HCl; solution II: 155 mM KH2PO4 and 155 mM Na2HPO4; solution III: 14.5 mM trisodium citrate) are titrated into the titration vessel at the preferred rate (10 mL / h) simultaneously with the isolated non-human β-casein and isolated non-human κ-casein (75:25, 64.1 g L-1) at the preferred ratio and preferred concentration. -1) was metered into the titration vessel at a preferred rate (60 mL / h). After a preferred period of time (1 hour) of pumping the solution, the micellar solution was allowed to equilibrate at a preferred pH (pH 6.7) and a preferred temperature (37° C.) for a preferred period of time (30 minutes).

[0124] Those skilled in the art will appreciate that although the above-described preferred conditions are used to provide proof of principle for the present scheme, each of these preferred conditions may be modified without departing from the general principles of application provided by the disclosure of the core inventive concepts set forth herein.

[0125] For example, the water bath temperature can be varied within any suitable range above or below the preferred temperature of 37° C. For example, the water bath temperature can be set at any temperature selected from the group consisting of 15±0.5° C., 16±0.5° C., 17±0.5° C., 18±0.5° C., 19±0.5° C., 20±0.5° C., 21±0.5° C., 22±0.5° C., 23±0.5° C., 24±0.5° C., 25±0.5° C., 26±0.5° C., 27±0.5° C., 28±0.5° C., 29±0.5° C., 30±0.5° C., 31±0.5° C., 32±0.5° C., 33±0.5° C., 34±0.5° C., 35±0.5° C., 36±0.5° C., 37±0.5° C., 38±0.5° C., 39±0.5° C., 40±0.5° C., 41±0.5° C., 42±0.5° C., 43±0.5° C., 44±0.5° C., 45±0.5° C., 46±0.5° C., 47±0.5° C., 48±0.5° C., 49±0.5° C., 50±0.5° C., 51±0.5° C., 52±0.5° C., 53±0.5° C., 54±0.5° C. 31±0.5℃, 32±0.5℃, 33±0.5℃, 34±0.5℃, 35±0.5℃, 36±0.5℃, 37±0.5℃, 38±0.5℃, 39±0.5℃, 40±0.5℃, 41±0.5℃, 42±0.5℃, 43±0.5℃, 44±0.5℃, 45±0.5℃, 46±0.5℃, 47±0.5℃, 48±0.5℃, 49±0.5℃ and 50±0.5℃.

[0126] Furthermore, the preferred salt solutions ((Solution I: CaCl2 and MgCl2; Solution II: KH2PO4 and Na2HPO4; Solution III: trisodium C6H5O7) can be varied by using alternative salts to provide the desired Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- Ion source. For example, other salts such as, but not limited to, calcium acetate, calcium carbonate, calcium citrate, calcium gluconate, calcium sulfate, calcium phosphate, calcium nitrate, magnesium acetate, magnesium carbonate, magnesium citrate, magnesium gluconate, magnesium sulfate, magnesium phosphate, magnesium nitrate, potassium acetate, potassium carbonate, potassium citrate, potassium gluconate, potassium sulfate, dipotassium hydrogen phosphate, tripotassium phosphate, potassium nitrate, sodium acetate, sodium carbonate, monosodium citrate, disodium citrate, sodium gluconate, sodium sulfate, monosodium phosphate, trisodium phosphate, and sodium nitrate can be used as the Ca source. 2+ Mg 2+ , K+ 、Na + PO4 3- and C6H5O7 3- Alternative source of ions.

[0127] In addition, although Figure 1.1 In the embodiment shown, four separate solutions are used (three salt solutions and one casein solution), but any number of solutions may be used and titrated into the titration vessel to provide the artificial micelles of the present invention. 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- ions, and each of β-casein and κ-casein, and additionally for any acid, base or buffer solution, separate solutions are prepared and titrated into the titration vessel. Without departing from the invention as described herein, the method of the present invention may be performed using 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 separate solutions. For example, solution I may be combined with solution II before titration into the titration vessel, and solution III may be combined with the β-casein and κ-casein solutions before titration into the titration vessel, so that only two solutions need to be titrated into the titration vessel. Alternatively, a titration may be performed for Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- Separate solutions are prepared for each of the ions, β-casein, and κ-casein and titrated into the titration vessel, so eight solutions need to be titrated into the titration vessel. In addition, if additional buffer solutions or pH adjustment solutions are provided, nine solutions will be titrated into the titration vessel.

[0128] In addition, the preferred concentration of Ca in the salt solution is 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- Ion concentrations can vary. For example, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3-The concentration of the ions can be provided in different concentrations independently of each other, and the concentration falls within the range of 0.01 mM to 1.0 M, depending on the process scale, titration rate and the desired properties of the resulting micellar solution. Preferably, but not limited to, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3-The concentration of the ions may be provided at a concentration each independently selected from the group consisting of 1 ± 0.5 mM, 2 ± 1 mM, 4 ± 1 mM, 6 ± 1 mM, 8 ± 1 mM, 10 ± 1 mM, 12 ± 1 mM, 14 ± 1 mM, 16 ± 1 mM, 18 ± 1 mM, 20 ± 1 mM, 22 ± 1 mM, 24 ± 1 mM, 26 ± 1 mM, 28 ± 1 mM, 30 ± 1 mM, 32 ± 1 mM, 34 ± 1 mM, 36 ± 1 mM, 38 ± 1 mM, 40 ± 1 mM, 42 ± 1 mM, 44 ± 1 mM, 46 ± 1 mM, 48 ± 1 mM, 50 ± 1 mM, 52 ± 1 mM, 54 ± 1 mM, 56 ± 1 mM, 58 ± 1 mM, 60 ± 1 mM, 62 ± 1 mM, 64 ± 1 mM, 4±1mM, 66±1mM, 68±1mM, 70±1mM, 72±1mM, 74±1mM, 76±1mM, 78±1mM, 80±1mM, 82±1mM, 84±1mM, 86±1mM, 88±1mM, 90±1mM, 92±1mM, 94±1mM, 96±1mM, 98±1mM, 100±1mM, 102±1mM, 104±1mM, 106±1mM, 108±1mM, 110±1mM, 112±1mM, 114±1mM, 116±1mM, 118±1mM, 120±1mM, 122±1mM, 124±1mM, 126±1mM, 128±1mM, 130±1 mM, 132±1mM, 134±1mM, 136±1mM, 138±1mM, 140±1mM, 142±1mM, 144±1mM, 146 ±1mM, 148±1mM, 150±1mM, 152±1mM, 154±1mM, 156±1mM, 158±1mM, 160±1mM, 1 62±1mM, 164±1mM, 166±1mM, 168±1mM, 170±1mM, 172±1mM, 174±1mM, 176±1mM, 178±1mM, 180±1mM, 182±1mM, 184±1mM, 186±1mM, 188±1mM, 190±1mM, 192±1mM M, 194±1mM, 196±1mM, 198±1mM, 200±1mM, 202±1mM, 204±1mM, 206±1mM, 208± 1mM, 210±1mM, 212±1mM, 214±1mM, 216±1mM, 218±1mM, 220±1mM, 222±1mM, 224 ±1mM, 226±1mM, 228±1mM, 230±1mM, 232±1mM, 234±1mM, 236±1mM, 238±1mM, 240±1mM, 242±1mM, 244±1mM, 246±1mM, 248±1mM, 250±1mM, 252±1mM, 254±1mM,256±1mM, 258±1mM, 260±1mM, 262±1mM, 264±1mM, 266±1mM, 268±1mM, 270±1mM, 272±1mM, 274±1mM, 276±1mM, 278±1mM, 280±1mM, 282±1mM, 284±1mM, 286 ±1mM, 288±1mM, 290±1mM, 292±1mM, 294±1mM, 296±1mM, 298±1mM, 300±1mM, 302±1mM, 304±1mM, 306±1mM, 308±1mM, 310±1mM, 312±1mM, 314±1mM, 316±1mM, 318±1mM, 320±1mM, 322±1mM, 324±1mM, 326±1mM, 328±1mM, 330±1mM, 332±1mM, 334±1mM, 336±1mM, 338±1mM, 340±1mM, 342±1mM, 344±1mM, 346±1mM, 348 48±1mM, 350±1mM, 352±1mM, 354±1mM, 356±1mM, 358±1mM, 360±1mM, 362±1mM, 364±1mM, 366±1mM, 368±1mM, 370±1mM, 372±1mM, 374±1mM, 376±1mM, 378± 1mM, 380±1mM, 382±1mM, 384±1mM, 386±1mM, 388±1mM, 390±1mM, 392±1mM, 3 94±1mM, 396±1mM, 398±1mM, 400±1mM, 402±1mM, 404±1mM, 406±1mM, 408±1mM , 410±1mM, 412±1mM, 414±1mM, 416±1mM, 418±1mM, 420±1mM, 422±1mM, 424±1mM, 426±1mM, 428±1mM, 430±1mM, 432±1mM, 434±1mM, 436±1mM, 438±1mM, 44 0±1mM, 442±1mM, 444±1mM, 446±1mM, 448±1mM, 450±1mM, 452±1mM, 454±1mM, 456±1mM, 458±1mM, 460±1mM, 462±1mM, 464±1mM, 466±1mM, 468±1mM, 470±1 mM, 472±1mM, 474±1mM, 476±1mM, 478±1mM, 480±1mM, 482±1mM, 484±1mM, 486±1mM, 488±1mM, 490±1mM, 492±1mM, 494±1mM, 496±1mM, 498±1mM and 500±1mM.

[0129] Similarly, in the solution of isolated non-human β-casein and isolated non-human κ-casein at preferred concentrations, the concentrations of isolated non-human β-casein and isolated non-human κ-casein can vary. For example, the concentrations of isolated non-human β-casein and isolated non-human κ-casein can be provided independently at different concentrations, and the concentrations fall within the range of 1 g / L to 500 g / L, depending on the process scale, titration rate, and the desired properties in the resulting micellar solution. Preferably, but not limiting, the concentrations of isolated non-human β-casein and isolated non-human κ-casein can be provided independently at a concentration selected from the group consisting of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 ± 5 g / L, 20 ± 5 g / L, 30 ± 5 g / L, 40 ± 5 g / L, 50 ± 5 g / L, 60 ± 5 g / L, 70 ± 5 g / L, 80 ± 5 g / L, 90 ± 5 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L, 100 ± 100 g / L 0±5g / L, 60±5g / L, 70±5g / L, 80±5g / L, 90±5g / L, 100±5g / L, 110±5g / L, 120±5g / L, 13 0±5g / L, 140±5g / L, 150±5g / L, 160±5g / L, 170±5g / L, 180±5g / L, 190±5g / L, 200±5g / L, 210±5g / L, 220±5g / L, 230±5g / L, 240±5g / L, 250±5g / L, 260±5g / L, 270±5g / L, 280 ±5g / L, 290±5g / L, 300±5g / L, 310±5g / L, 320±5g / L, 330±5g / L, 340±5g / L, 350±5g / L , 360±5g / L, 370±5g / L, 380±5g / L, 390±5g / L, 400±5g / L, 410±5g / L, 420±5g / L, 430± 5g / L, 440±5g / L, 450±5g / L, 460±5g / L, 470±5g / L, 480±5g / L, 490±5g / L and 500±5g / L.

[0130] In addition, when solutions of isolated non-human β-casein and isolated non-human κ-casein are combined in a single solution at a preferred ratio, the ratio of their isolated non-human β-casein and isolated non-human κ-casein can be varied. For example, isolated non-human β-casein and isolated non-human κ-casein can be provided in different β-casein:κ-casein ratios, and the ratios fall within the range of 5:95 to 95:5, depending on the process scale, titration rate, and the desired properties in the resulting micellar solution. Preferably, but not limiting, the isolated non-human β-casein and the isolated non-human κ-casein can be provided in a ratio (β-casein:κ-casein) selected from the group consisting of 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, and 95: 5. Alternatively, the isolated non-human β-casein and the isolated non-human κ-casein can be provided in separate solutions, and their respective concentrations and titration rates into the titration vessel can be appropriately controlled to achieve the aforementioned ratio of β-casein:κ-casein in the final micellar solution product produced by the method of the present invention.

[0131] Furthermore, by adding a suitable acid or base before titration into the titration vessel, the Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- The pH of each of the ionic solution and the isolated non-human β-casein and isolated non-human κ-casein solutions is adjusted to be above or below the preferred pH of 6.70. Suitable acids include HCl, acetic acid, citric acid, malic acid, tartaric acid, folic acid, fumaric acid, ascorbic acid, phosphoric acid or any other organic or inorganic acid that is "generally recognized as safe" (GRAS), which is consistent with the U.S. Food and Drug Administration (FDA) regulations that a chemical or substance added to food is recognized by experts as safe under the conditions of its intended use. Suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, ammonium bicarbonate, calcium carbonate, potassium carbonate, potassium bicarbonate or any other organic or inorganic base that is GRAS. Ca 2+ Mg 2 + , K + 、Na + PO4 3- and C6H5O7 3-The pH of each of the solution of Ca ions and the solution of isolated non-human β-casein and isolated non-human κ-casein can vary within the range of pH 4 to pH 10, depending on the process scale, titration rate, and desired properties of the resulting micellar solution. Preferably, but not limited to, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- The solution of ions and the solution of isolated non-human β-casein and isolated non-human κ-casein can each independently be provided at a pH selected from the group consisting of pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.1, pH9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9 and pH 10.

[0132] In addition, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- Each of the solutions of Ca ions and the solutions of isolated non-human β-casein and isolated non-human κ-casein can be independently titrated into the titration vessel at different rates above and below the preferred rate (10 mL / H). For example, but not by way of limitation, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3-Each of the solutions of Ca ions and the solutions of isolated non-human β-casein and isolated non-human κ-casein can be independently titrated into the titration vessel at a rate falling within the range of 1.0 mL / h to 1000 L / h, depending on the process scale, titration rate, and desired properties of the resulting micellar solution. Preferably, but not limiting, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3-Each of the solutions of isolated non-human β-casein and isolated non-human κ-casein can be independently titrated into the titration vessel at a rate selected from the group consisting of: 1 mL / h, 2 mL / h, 3 mL / h, 4 mL / h, 5 mL / h, 6 mL / h, 7 mL / h, 8 mL / h, 9 mL / h, 10 ± 5 mL / h, 20 ± 5 mL / h, 30 ± 5 mL / h, 40 ± 5 mL / h, 50 ± 5 mL / h, 60 ± 5 mL / h, 70 ± 5 mL / h, 80 ± 5 mL / h, 90 ± 5 mL / h, 100 ± 5 mL / h, 110 ± 5 mL / h, 120 ± 5 mL / h, 130 ± 5 mL / h, 140 ± 5 mL / h, 150 ± 5 mL / h, 160 ± 5 mL / h, 170 ± 5 mL / h, 180 ± 5 mL / h, 190 ± 5 mL / h, 200 ± 5 mL / h, 210 ± 5 mL / h, 220 ± 5 mL / h, 230 ± 5 mL / h, 240 ± 5 mL / h, 250 ± 5 mL / h, 260 ± 5 mL / h, 270 ± 5 mL / h, 280 ± 5 mL / h, 290 ± 5 mL / h, 300 ± 5 mL / h, 310 ± 5 mL / h, 320 ± 3 0±5mL / h, 160±5mL / h, 170±5mL / h, 180±5mL / h, 190±5mL / h, 200±5mL / h, 210±5mL / h, 220±5mL / h, 230±5mL / h, 240±5mL / h, 250±5mL / h, 260±5mL / h, 270±5m 4 00±5mL / h, 410±5mL / h, 420±5mL / h, 430±5mL / h, 440±5mL / h, 450±5mL / h, 460±5mL / h, 470±5mL / h, 480±5mL / h, 490±5mL / h, 500±5mL / h, 510±5mL / h, 520±5 mL / h, 530±5mL / h, 540±5mL / h, 550±5mL / h, 560±5mL / h, 570±5mL / h, 580±5mL / h, 590±5mL / h, 600±5mL / h, 610±5mL / h, 620±5mL / h, 630±5mL / h, 640±5mL / h, 650±5mL / h, 660±5mL / h, 670±5mL / h, 680±5mL / h, 690±5mL / h, 700±5mL / h, 710±5mL / h, 720±5mL / h, 730±5mL / h, 740±5mL / h, 750±5mL / h, 760±5mL / h, 770±5 mL / h, 780±5mL / h, 790±5mL / h, 800±5mL / h, 810±5mL / h, 820±5mL / h, 830±5mL / h, 840±5mL / h, 850±5mL / h, 860±5mL / h, 870±5mL / h, 880±5mL / h, 890±5mL / h,900±5mL / h、910±5mL / h、920±5mL / h、930±5mL / h、940±5mL / h、950±5mL / h、960±5mL / h、970±5mL / h、980±5mL / h、990±5mL / h、1000±5mL / h、2L / h、3L / h、4L / h、5L / h、6L / h、7L / h、8L / h、9L / h、0±5L / h、20±5L / h、30±5L / h、40±5L / h、50±5L / h、60±5L / h、70±5L / h、80±5L / h、90±5L / h、100±5L / h、110±5L / h、120±5L / h、130±5L / h、140±5L / h、150±5L / h、160±5L / h、170±5L / h、180±5L / h、190±5L / h、200±5L / h、210±5L / h、220±5L / h、230±5L / h、240±5L / h、250±5L / h、260±5L / h、270±5L / h、280±5L / h、290±5L / h、300±5L / h、310±5L / h、320±5L / h、330±5L / h、340±5L / h、350±5L / h、360±5L / h、370±5L / h、380±5L / h、390±5L / h、400±5L / h、410±5L / h、420±5L / h、430±5L / h、440±5L / h、450±5L / h、460±5L / h、470±5L / h、480±5L / h、490±5L / h、500±5L / h、510±5L / h、520±5L / h、530±5L / h、540±5L / h、550±5L / h、560±5L / h、570±5L / h、580±5L / h、590±5L / h、600±5L / h、610±5L / h、620±5L / h、630±5L / h、640±5L / h、650±5L / h、660±5L / h、670±5L / h、680±5L / h、690±5L / h、700±5L / h、710±5L / h、720±5L / h、730±5L / h、740±5L / h、750±5L / h、760±5L / h、770±5L / h、780±5L / h、790±5L / h、800±5L / h、810±5L / h、820±5L / h、830±5L / h、840±5L / h、850±5L / h、860±5L / h、870±5L / h、880±5L / h、890±5L / h、900±5L / h、910±5L / h、920±5L / h、930±5L / h、940±5L / h、950±5L / h、960±5L / h, 970±5L / h, 980±5L / h, 990±5L / h and 1000±5L / h.

[0133] Furthermore, the preferred time period (1 hour) for pumping the solution may be varied without departing from the methods of certain embodiments of the invention described herein. For example, but not limitation, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- The time period for each of the solutions of isolated non-human β-casein and isolated non-human κ-casein to be independently pumped into the titration vessel ranges from 0.1 h to 96 h, depending on the process scale, titration rate, and desired properties of the resulting micellar solution. Preferably, but not limited to, Ca 2+ Mg 2+ , K + 、Na + PO4 3- and C6H5O7 3- The time period over which each of the solution of ions and the solution of isolated non-human β-casein and isolated non-human κ-casein can be pumped into the titration vessel is selected from the group consisting of 0.1 h, 0.2 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 2 ± 1 h, 4 ± 1 h, 6 ± 1 h, 8 ± 1 h, 10 ± 1 h, 12 ± 1 h, 14 ± 1 h, 16 ± 1 h, 18 ± 1 h, 20 ± 1 h, 22 ± 1 h, 24 ± 1 h, 26 ± 1 h, 28 ± 1 h, 30 ± 1 h, 32 ± 1 h, ±1h, 34±1h, 36±1h, 38±1h, 40±1h, 42±1h, 44±1h, 46±1h, 48±1h, 50±1h, 52±1h, 54±1h, 56±1h, 58±1h, 60±1h, 62±1h, 64±1h, 66±1h, 68±1h, 70±1h, 72±1h, 74±1h, 76±1h, 78±1h, 80±1h, 82±1h, 84±1h, 86±1h, 88±1h, 90±1h, 92±1h, 94±1h and 96±1h.

[0134] In addition, the time period for the micellar solution to reach equilibrium can vary above and below the preferred time period (30 minutes). For example, but not limiting, the time period for the micellar solution to reach equilibrium can be selected from a time falling within the range of 1 min to 300 min, depending on the process scale, titration rate, and the desired properties of the resulting micellar solution. Preferably, but not limiting, the time period for the micellar solution to reach equilibrium can be selected from the group comprising the following: 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min ,52min,53min,54min,55min,56min,57min,58min,59min,60min,61min,62min,63min,64min,65min,66min,67min,68min,69min,70 min, 71min, 72min, 73min, 74min, 75min, 76min, 77min, 78min, 79min, 80min, 81min, 82min, 83min, 84min, 85min, 86min, 87min, 88min , 89min, 90min, 91min, 92min, 93min, 94min, 95min, 96min, 97min, 98min, 99min, 100min, 101min, 102min, 103min, 104min, 105min, 10 6min, 107min, 108min, 109min, 110min, 111min, 112min, 113min, 114min, 115min, 116min, 117min, 118min, 119min, 120min, 121min, 1 22min, 123min, 124min, 125min, 126min, 127min, 128min, 129min, 130min, 131min, 132min, 133min, 134min, 135min, 136min, 137min,138min、139min、140min、141min、142min、143min、144min、145min、146min、147min、148min、149min、150min、151min、152min、153min、154min、155min、156min、157min、158min、159min、160min、161min、162min、163min、164min、165min、166min、167min、168min、169min、170min、171min、172min、173min、174min、175min、176min、177min、178min、179min、180min、181min、182min、183min、184min、185min、186min、187min、188min、189min、190min、191min、192min、193min、194min、195min、196min、197min、198min、199min、200min、201min、202min、203min、204min、205min、206min、207min、208min、209min、210min、211min、212min、213min、214min、215min、216min、217min、218min、219min、220min、221min、222min、223min、224min、225min、226min、227min、228min、229min、230min、231min、232min、233min、234min、235min、236min、237min、238min、239min、240min、241min、242min、243min、244min、245min、246min、247min、248min、249min、250min、251min、252min、253min、254min、255min、256min、257min、258min、259min、260min、261min、262min、263min、264min、265min、266min、267min、268min、269min、270min、271min、272min、273min、274min、275min、276min、277min、278min、279min、280min、281min, 282min, 283min, 284min, 285min, 286min, 287min, 288min, 289min, 290min, 291min, 292min, 293min, 294min, 295min, 296min, 297min, 298min, 299min and 300min.

[0135] In addition, the pH at which the micellar solution reaches equilibrium can vary above and below the preferred pH of 6.7. For example, but not by way of limitation, the pH at which the micellar solution reaches equilibrium can be selected from a pH falling within the range of pH 4 to pH 10, depending on the process scale, titration rate, and desired properties of the resulting micellar solution. Preferably, but not limiting, the pH at which the micellar solution reaches equilibrium can be selected from the group consisting of pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7 7.7, pH 7.8, pH 7.9, pH8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9 and pH 10.

[0136] In addition, the temperature at which the micellar solution is in equilibrium can be varied within any suitable range above or below the preferred temperature of 37°C. For example, the temperature at which the micellar solution is in equilibrium can be set at any temperature selected from the group consisting of 15±0.5°C, 16±0.5°C, 17±0.5°C, 18±0.5°C, 19±0.5°C, 20±0.5°C, 21±0.5°C, 22±0.5°C, 23±0.5°C, 24±0.5°C, 25±0.5°C, 26±0.5°C, 27±0.5°C, 28± 0.5℃, 29±0.5℃, 30±0.5℃, 31±0.5℃, 32±0.5℃, 33±0.5℃, 34±0.5℃, 35±0.5℃, 36±0.5℃, 37±0.5℃, 38±0.5℃, 39±0.5℃, 40±0.5℃, 41±0.5℃, 42±0.5℃, 43±0.5℃, 44±0.5℃, 45±0.5℃, 46±0.5℃, 47±0.5℃, 48±0.5℃, 49±0.5℃ and 50±0.5℃.

[0137] Those skilled in the art will appreciate that infringement of the claims of this specification can be detected in a variety of ways. Without limitation, one method by which infringement of the claims of this specification can be detected is by performing high performance liquid chromatography (HPLC) analysis of the protein content in the potentially infringing product or micelle, where no or only minimal amounts of α s - Casein may indicate the presence of an infringement. Alternatively, but not limiting, infringement may be determined by microscopic methods such as scanning electron microscopy (SEM).

[0138] The embodiments described herein provide a micellar solution comprising a plurality of artificial casein micelles, wherein the artificial casein micelles comprise isolated non-human β-casein and isolated non-human κ-casein, and wherein the artificial casein micelles are substantially free of α-casein. s -Casein.

[0139] In some embodiments, the Z-average diameter of the artificial casein micelles is greater than 30nm. The Z-average diameter of the artificial casein micelles of the present invention can be determined, for example, but not limited to, by dynamic light scattering measurements or by scanning electron microscopy (SEM) as performed in the examples herein. In a non-limiting manner, the Z-average diameter of the artificial casein micelles of the present invention can be greater than: 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm, 75nm, 76nm, 77nm, 78nm, 79nm, 80nm, 9nm, 70nm, 71nm, 72nm, 73nm, 74nm, 75nm, 76nm, 77nm, 78nm, 79nm, 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91n m, 92nm, 93nm, 94nm, 95nm, 96nm, 97nm, 98nm, 99nm, 100nm, 101nm, 102nm, 103nm, 104nm, 105nm, 106nm, 107nm, 108nm, 109nm, 110nm, 111nm , 112nm, 113nm, 114nm, 115nm, 116nm, 117nm, 118nm, 119nm, 120nm, 121nm, 122nm, 123nm, 124nm, 125nm, 126nm, 127nm, 128nm, 129nm, 130 nm, 131nm, 132nm, 133nm, 134nm, 135nm, 136nm, 137nm, 138nm, 139nm, 140nm, 141nm, 142nm, 143nm, 144nm, 145nm, 146nm, 147nm, 148nm, 14 9nm, 150nm, 151nm, 152nm, 153nm, 154nm, 155nm, 156nm, 157nm, 158nm, 159nm, 160nm, 161nm, 162nm, 163nm, 164nm, 165nm, 166nm, 167nm, 1 68nm, 169nm, 170nm, 171nm, 172nm, 173nm, 174nm, 175nm, 176nm, 177nm, 178nm, 179nm, 180nm, 181nm, 182nm, 183nm, 184nm, 185nm, 186nm,187nm, 188nm, 189nm, 190nm, 191nm, 192nm, 193nm, 194nm, 195nm, 196nm, 197nm, 198nm, 199nm, 200nm, 2 01nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215 nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm, 226nm, 227nm, 228nm, 229n m, 230nm, 231nm, 232nm, 233nm, 234nm, 235nm, 236nm, 237nm, 238nm, 239nm, 240nm, 241nm, 242nm, 243nm, 244nm, 245nm, 246nm, 247nm, 248nm, 249nm, 250nm, 251nm, 252nm, 253nm, 254nm, 255nm, 256nm, 257nm, 2 58nm, 259nm, 260nm, 261nm, 262nm, 263nm, 264nm, 265nm, 266nm, 267nm, 268nm, 269nm, 270nm, 271nm, 272 nm, 273nm, 274nm, 275nm, 276nm, 277nm, 278nm, 279nm, 280nm, 281nm, 282nm, 283nm, 284nm, 285nm, 286n m, 287nm, 288nm, 289nm, 290nm, 291nm, 292nm, 293nm, 294nm, 295nm, 296nm, 297nm, 298nm, 299nm or 300nm. ,

[0140] In a preferred embodiment, the Z-average diameter of the artificial casein micelles falls within the range of 40 to 500 nm. Without limitation, the Z-average diameter of the artificial casein micelles of particularly preferred embodiments may fall within any range selected from the group comprising the following ranges: 40 to 500 nm, 45 to 500 nm, 50 to 500 nm, 55 to 500 nm, 60 to 500 nm, 65 to 500 nm, 70 to 500 nm, 75 to 500 nm, 80 to 500 nm, 85 to 500 nm, 90 to 500 nm, 95 to 500 nm, 100 to 500 nm, 105 to 500 nm, 110 to 500 nm, 115 to 500 nm, 120 to 500 nm, 125 to 500 nm, 130 to 500 nm, 135 to 500 nm, 140 to 500 nm, 145 to 500 nm, 150 to 500 nm, 155 to 500 nm, 160 to 500 nm, 165 to 500 nm, 170 to 500 nm, 175 to 500 nm, 180 to 500 nm, 185 to 500 nm, 190 to 500 nm, 200 to 500 nm, 210 to 500 nm, 220 to 500 nm, 225 to 500 nm, 230 to 500 nm, 235 to 500 nm, 240 to 500 nm, 250 to 500 nm, 260 to 500 nm, 270 to 500 nm, 2 500 nm, 210 to 500 nm, 215 to 500 nm, 220 to 500 nm, 225 to 500 nm, 230 to 500 nm, 235 to 500 nm, 240 to 500 nm, 245 to 500 nm, 250 to 500 nm, 255 to 500 nm, 500 nm, 340 to 500 nm, 345 to 500 nm, 350 to 500 nm, 355 to 500 nm, 360 to 500 nm, 365 to 500 nm, 370 to 500 nm, 375 to 500 nm, 500 nm, 460 to 500 nm, 465 to 500 nm, 470 to 500 nm, 475 to 500 nm, 480 to 500 nm, 485 to 500 nm, 490 to 500 nm, and 495 to 500 nm.

[0141] In some embodiments, the micellar solutions described herein comprise less than 7 wt% α-casein, based on the total casein present in the micellar solution. s -Casein. Preferably, the micellar solution described herein comprises a certain amount of α s - casein, as a percentage of the total casein present in the micellar solution, selected from the group consisting of: 7 wt%, 6.9 wt%, 6.8 wt%, 6.7 wt%, 6.6 wt%, 6.5 wt%, 6.4 wt%, 6.3 wt%, 6.2 wt%, 6.1 wt%, 6 wt%, 5.9 wt%, 5.8 wt%, 5.7 wt%, 5.6 wt%, 5.5 wt%, 5.4 wt%, 5.3 wt%, 5.2 wt%, 5.1 wt%, 5 wt%, 4.9 wt%, 4.8 wt%, 4.7 wt%, 4.6 wt%, 4.5 wt%, 4.4 wt%, 4.3 wt%, 4.2 wt%, 4.1 wt%, 4 wt%, 3.9 wt%, 3.8 wt%, 3.7wt%, 3.6wt%, 3.5wt%, 3.4wt%, 3.3wt%, 3.2wt%, 3.1wt%, 3wt%, 2.9wt%, 2.8wt%, 2.7wt%, 2.6wt%, 2.5wt%, 2.4wt%, 2.3wt%, 2.2wt%, 2.1wt%, 2wt%, 1.9wt%, 1.8wt%, 1.7wt%, 1.6wt%, 1.5wt%, 1.4wt%, 1.3wt%, 1.2wt%, 1.1wt%, 1wt%, 0.9wt%, 0.8wt%, 0.7wt%, 0.6wt%, 0.5wt%, 0.4wt%, 0.3wt%, 0.2wt%, 0.1wt% and 0wt%.

[0142] In some embodiments, the micellar solutions described herein comprise a wt% ratio of non-human β-casein to non-human κ-casein falling within the range of 10:90 to 90: 10. Without limitation, the wt% ratio of non-human β-casein to non-human κ-casein (b:k) can be selected from the group consisting of 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, and 95:5.

[0143] In some embodiments, the micellar solutions described herein comprise a total casein concentration ranging from 10 g / L to 95 g / L. Without limitation, the total casein concentration in the micellar solutions of the present invention can be selected from the group consisting of 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L, 33g / L, 34g / L, 35g / L, 36g / L, 37g / L, 38g / L , 39g / L, 40g / L, 41g / L, 42g / L, 43g / L, 44g / L, 45g / L, 46g / L, 47g / L, 48g / L, 49g / L, 50 g / L, 51g / L, 52g / L, 53g / L, 54g / L, 55g / L, 56g / L, 57g / L, 58g / L, 59g / L, 60g / L, 61g / L, 62g / L, 63g / L, 64g / L, 65g / L, 66g / L, 67g / L, 68g / L, 69g / L, 70g / L, 71g / L, 72g / L, 7 3g / L, 74g / L, 75g / L, 76g / L, 77g / L, 78g / L, 79g / L, 80g / L, 81g / L, 82g / L, 83g / L, 84g / L, 85g / L, 86g / L, 87g / L, 88g / L, 89g / L, 90g / L, 91g / L, 92g / L, 93g / L, 94g / L and 95g / L.

[0144] In some embodiments, the micellar solutions described herein comprise a total non-human β-casein concentration falling within the range of 1 g / L to 90 g / L. Without limitation, the total non-human β-casein concentration in the micellar solutions of the present invention can be selected from the group consisting of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L 0g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L, 33g / L, 34g / L, 35g / L, 36g / L, 37g / L, 38g / L, 39g / L, 40g / L, 41g / L, 42g / L, 43g / L , 44g / L, 45g / L, 46g / L, 47g / L, 48g / L, 49g / L, 50g / L, 51g / L, 52g / L, 53g / L, 54g / L, 55g / L, 56g / L, 57g / L, 58g / L, 59g / L, 60g / L, 61g / L, 62g / L, 63g / L, 64g / L, 65g / L, 66g / L, 67 g / L, 68g / L, 69g / L, 70g / L, 71g / L, 72g / L, 73g / L, 74g / L, 75g / L, 76g / L, 77g / L, 78g / L, 7 9g / L, 80g / L, 81g / L, 82g / L, 83g / L, 84g / L, 85g / L, 86g / L, 87g / L, 88g / L, 89g / L and 90g / L.

[0145] In some embodiments, the micellar solutions described herein comprise a total non-human κ-casein concentration falling within the range of 1 g / L to 90 g / L. Without limitation, the total non-human κ-casein concentration in the micellar solutions of the present invention can be selected from the group consisting of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, 31g / L , 32g / L, 33g / L, 34g / L, 35g / L, 36g / L, 37g / L, 38g / L, 39g / L, 40g / L, 41g / L, 42g / L, 43g / L, 44g / L, 45g / L, 46g / L, 47g / L, 48g / L, 49g / L, 50g / L, 51g / L, 52g / L, 53g / L, 54g / L, 55g / L, 56g / L, 57g / L, 58g / L, 59g / L, 60g / L, 61g / L, 62g / L, 63g / L, 64g / L, 65g / L, 66g / L, 67 g / L, 68g / L, 69g / L, 70g / L, 71g / L, 72g / L, 73g / L, 74g / L, 75g / L, 76g / L, 77g / L, 78g / L, 7 9g / L, 80g / L, 81g / L, 82g / L, 83g / L, 84g / L, 85g / L, 86g / L, 87g / L, 88g / L, 89g / L and 90g / L.

[0146] In some embodiments, the micellar solutions described herein comprise α- s1 -Casein concentration. Without limitation, the α s1 - The casein concentration may fall within any range selected from the group consisting of 0 g / L to 2 g / L, 0 g / L to 1.9 g / L, 0 g / L to 1.8 g / L, 0 g / L to 1.7 g / L, 0 g / L to 1.6 g / L, 0 g / L to 1.5 g / L, 0 g / L to 1.4 g / L, 0 g / L to 1.3 g / L, 0 g / L to 1.2 g / L , 0g / L to 1.1g / L, 0g / L to 1g / L, 0g / L to 0.9g / L, 0g / L to 0.8g / L, 0g / L to 0.7g / L, 0g / L to 0.6g / L, 0g / L to 0.5g / L, 0g / L to 0.4g / L, 0g / L to 0.3g / L, 0g / L to 0.2g / L, and 0g / L to 0.1g / L.

[0147] In some embodiments, the micellar solutions described herein comprise α s2 -Casein concentration. Without limitation, the α s2 - The casein concentration can fall within any range selected from the group of ranges comprising 0 g / L to 1 g / L, 0 g / L to 0.9 g / L, 0 g / L to 0.8 g / L, 0 g / L to 0.7 g / L, 0 g / L to 0.6 g / L, 0 g / L to 0.5 g / L, 0 g / L to 0.4 g / L, 0 g / L to 0.3 g / L, 0 g / L to 0.2 g / L, and 0 g / L to 0.1 g / L.

[0148] In some embodiments, the hydration of the artificial casein micelles in the micellar solution of the present invention falls within the range of 1 to 8 (g water / g micellar protein). Without limitation, the hydration of the artificial casein micelles in the micellar solution of the present invention can be selected from the group consisting of: 1 (g water / g micellar protein), 1.1 (g water / g micellar protein), 1.2 (g water / g micellar protein), 1.3 (g water / g micellar protein), 1.4 (g water / g micellar protein), 1.5 (g water / g micellar protein), 1.6 (g water / g micellar protein), 1.7 (g water / g micellar protein), 1.8 (g water / g micellar protein), 1.9 (g water / g micellar protein), 2 (g water / g micellar protein), 2.1 (g water / g micellar protein), 2.2 (g water / g micellar protein), 2.3 (g water / g micellar protein), 2.4 (g water / g micellar protein), (g water / g micellar protein), 2.5 (g water / g micellar protein), 2.6 (g water / g micellar protein), 2.7 (g water / g micellar protein), 2.8 (g water / g micellar protein), 2.9 (g water / g micellar protein), 3 (g water / g micellar protein), 3.1 (g water / g micellar protein), 3.2 (g water / g micellar protein), 3.3 (g water / g micellar protein), 3.4 (g water / g micellar protein), 3.5 (g water / g micellar protein), 3.6 (g water / g micellar protein), 3.7 (g water / g micellar protein), 3.8 (g water / g micellar protein), 3.9 (g water / g micellar protein), 4 (g water / g micellar protein), 4.1 (g water / g micellar protein ), 4.2 (g water / g micellar protein), 4.3 (g water / g micellar protein), 4.4 (g water / g micellar protein), 4.5 (g water / g micellar protein), 4.6 (g water / g micellar protein), 4.7 (g water / g micellar protein), 4.8 (g water / g micellar protein), 4.9 (g water / g micellar protein), 5 (g water / g micellar protein), 5.1 (g water / g micellar protein), 5.2 (g water / g micellar protein), 5.3 (g water / g micellar protein), 5.4 (g water / g micellar protein), 5.5 (g water / g micellar protein), 5.6 (g water / g micellar protein), 5.7 (g water / g micellar protein), 5.8 (g water / g micellar protein) , 5.9 (g water / g micellar protein), 6 (g water / g micellar protein), 6.1 (g water / g micellar protein), 6.2 (g water / g micellar protein), 6.3 (g water / g micellar protein), 6.4 (g water / g micellar protein), 6.5 (g water / g micellar protein), 6.6 (g water / g micellar protein), 6.7 (g water / g micellar protein), 6.8 (g water / g micellar protein), 6.9 (g water / g micellar protein), 7 (g water / g micellar protein), 7.1 (g water / g micellar protein), 7.2 (g water / g micellar protein), 7.3 (g water / g micellar protein), 7.4 (g water / g micellar protein), 7.5 (g water / g micellar protein), 7.6 (g water / g micellar protein), 7.7 (g water / g micellar protein), 7.8 (g water / g micellar protein), 7.9 (g water / g micellar protein) and 8 (g water / g micellar protein).

[0149] In some embodiments, the percentage of non-micellar casein to total casein in the micellar solution of the present invention falls within the range of 5 to 20%. Without limitation, the percentage of non-micellar casein to total casein in the micellar solution of the present invention can be selected from the group consisting of: 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%. %, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 1 2.6%, 12.7%, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%, 15.9%, 16%, 16.1%, 16.2%, 16. 3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18%, 18.1%, 18.2%, 18.3%, 18.4%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19%, 19.1%, 19.2%, 19.3%, 19.4%, 19.5%, 19.6%, 19.7%, 19.8%, 19.9% ​​and 20%.

[0150] In some embodiments, the micellar calcium content of the micellar solution of the present invention is at least 70% of the total calcium in the micellar solution. Without limitation, the micellar calcium content of the micellar solution of the present invention can be greater than any percentage selected from the group consisting of: 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the total calcium in the micellar solution.

[0151] In some embodiments, the micellar magnesium content of the micellar solution of the present invention is at least 30%, preferably at least 35%, of the total magnesium in the micellar solution. Without limitation, the micellar magnesium content of the micellar solution of the present invention can be greater than any percentage selected from the group consisting of: 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.

[0152] In some embodiments, the micellar inorganic phosphate content of the micellar solution of the present invention is at least 50% of the total inorganic phosphate in the micellar solution. Without limitation, the micellar inorganic phosphate content of the micellar solutions of the present invention can be greater than any percentage selected from the group consisting of: 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the total inorganic phosphate in the micellar solution.

[0153] In some embodiments, the micellar citrate content of the micellar solution of the present invention is at least 5% of the total citrate in the micellar solution. Without limitation, the micellar citrate content of the micellar solution of the present invention can be greater than any percentage selected from the group consisting of: 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 111%, 11 %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.

[0154] In one embodiment, the present disclosure provides a curd composition comprising the micellar solution of the present invention in a coagulated form.

[0155] The curd composition can be a useful precursor for the manufacture of downstream products such as yogurt or cheese. The curd composition can be coagulated by the action of an acid or a coagulant. Suitable acids for coagulation include, but are not limited to, citric acid and lactic acid.

[0156] In some embodiments, a yogurt composition can be formed using the methods described herein. Yogurt can be formed using the micellar solution described herein. The method can include heating and then cooling the micellar solution, and acidifying the micellar solution with an acid or a microorganism. The microorganism can include one or more of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, lactobacilli, or bifidobacteria.

[0157] In some embodiments, after acidification, a coagulant can be added to form a coagulated curd (coagulated curd matrix), which can then be used to prepare cheese. The micelles in the micellar solution (such as milk and micellar solution as described herein) are stable and repel each other in a colloidal suspension. In the presence of a coagulant or milk-clotting enzyme, when acidified, the micelles are unstable and attract each other, thereby coagulating. In the presence of a coagulant or chymosin, a cross-linked coagulated curd matrix is ​​formed.

[0158] In some embodiments, the curd composition further comprises a renneting agent. Suitable renneting agents for practicing the present invention include, but are not limited to, proteases, chymosins, pepsins, lipases, animal-derived rennets, plant-derived rennets (including extracts from Galium spp., dried caper leaves, nettles, thistles, mallows, Withania coagulans, groundivy, Cynara, soybeans), calf rennet, kid rennet, fungal-derived rennet, microbial-derived rennet (e.g., extracts of Rhizomucor miehei), and recombinantly produced rennet.

[0159] In a preferred embodiment, the curd composition has a maximum G' (storage modulus) falling within the range of 5 to 200 Pa, preferably after incubation with rennet for 1 hour. Without limitation, the curd composition of the present invention has a maximum G' (storage modulus) falling within the range of 5 to 200 Pa after incubation with rennet for 0.1 h, 0.2 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3 h, 3.1 h, 3.2 h , 3.3h, 3.4h, 3.5h, 3.6h, 3.7h, 3.8h, 3.9h, 4h, 4.1h, 4.2h, 4.3h, 4.4h, 4.5h, 4.6h, 4.7h, 4.8h, 4.9h, 5h, 5.1h, 5.2h, 5.3h, 5.4h, 5.5h, 5.6h, 5.7h, 5.8h, 5.9h and 6h. The maximum G' after any time period may fall within any range selected from the group consisting of: 5 to 200 Pa, 10 to 2 00Pa, 15 to 200Pa, 20 to 200Pa, 25 to 200Pa, 30 to 200Pa, 35 to 200Pa, 40 to 200Pa, 45 to 200Pa, 50 to 200Pa, 55 to 200Pa, 60 to 200Pa, 65 to 200Pa, 70 to 200Pa, 75 to 200Pa, 80 to 200Pa, 85 to 200Pa, 90 to 200Pa, 95 to 200Pa, 100 to 200Pa, 105 to 200Pa, 1 10 to 200Pa, 115 to 200Pa, 120 to 200Pa, 125 to 200Pa, 130 to 200Pa, 135 to 200Pa, 140 to 200Pa, 145 to 200Pa, 150 to 200Pa, 155 to 200Pa, 160 to 200Pa, 165 to 200Pa, 170 to 200Pa, 175 to 200Pa, 180 to 200Pa, 185 to 200Pa, 190 to 200Pa and 195 to 200Pa.

[0160] In other embodiments, the present disclosure provides edible compositions comprising the micellar solution of the present invention or the curd composition of the present invention.Such edible compositions include, but are not limited to, yogurt, cheese, and milk substitutes.

[0161] In some embodiments, the edible composition does not contain any protein from animal sources.

[0162] In one embodiment, the present disclosure provides a method for producing an edible composition comprising: mixing isolated non-human β-casein, isolated non-human κ-casein, and at least one salt under conditions where the β-casein and κ-casein form a micellar solution, wherein the micellar solution is substantially free of α-casein. s - casein; and subjecting the micellar solution to a first condition to form a coagulum.

[0163] In some embodiments of the method for producing an edible composition, the first condition is adding acid or acidifying the micellar solution with a microorganism.

[0164] In some embodiments of the method for producing an edible composition, the method further comprises subjecting the coagulum to a coagulant to form a coagulated curd.

[0165] In some embodiments of the method for producing edible compositions, the method further comprises aging and / or ripening the curd of the curdization to form a cheese composition.The curd of curdization can be further processed to produce cheese or cheese-like product. In some cases, for example, mozzarella cheese product, the curd of curdization can be heated and stretched. In other embodiments, the curd of curdization is aged, for example, for Brie, Camembert, Feta, Halloumi, Gouda, Edam, Cheddar, Manchego, Swiss, Colby, Muenster, blue cheese or Parmesan cheese or cheese-like product.

[0166] In some embodiments, the micellar solution or the curdled curd can be treated with hot water to form cheese, for example, mozzarella-type cheese. The hot water treatment can be carried out at a temperature of about 50°C to about 90°C. The hot water treatment can be carried out at a temperature of at least 55°C. The hot water treatment can be carried out at a temperature of up to 75°C. The hot water treatment can be carried out at a temperature of 50°C to 55°C, 55°C to 60°C, 55°C to 65°C, 55°C to 70°C, 55°C to 75°C, 60°C to 65°C, 60°C to 70°C, 60°C to 75°C, 65°C to 70°C, 65°C to 75°C, 70°C to 75°C, 75°C to 80°C, 80°C to 85°C, or 85°C to 90°C. The hot water treatment can be carried out at a temperature of about 50°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, or about 90°C. The hydrothermal treatment may be carried out at a temperature of at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., or 85° C. The hydrothermal treatment may be carried out at a temperature of up to 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or 90° C. In some cases, after the hydrothermal treatment, the product is stretched into cheese.

[0167] In some embodiments of the method for producing an edible composition, the edible composition does not contain any protein from animal sources.

[0168] For example, when casein from a recombinant source is used, the cheese composition formed using the methods described herein may optionally not include any animal-derived components. The cheese composition formed using the methods described herein may optionally not include any animal-derived dairy-based components, such as animal-derived dairy proteins. The cheese composition formed using the methods described herein may optionally not include any whey proteins. The cheese composition formed using the methods described herein may optionally not include any α s - casein. The cheese compositions described herein can be pasta filata-like cheeses, such as mozzarella. Soft cheeses such as paneer, cream cheese, or cottage cheese can also be formed using the methods described herein. Other types of cheese, such as aged and ripened cheeses, can also be formed using the methods described herein, such as brie, camembert, feta, halloumi, gouda, edam, cheddar, manchego, Swiss-type cheese, colby, muenster, blue cheese, and parmesan.

[0169] The texture of cheese made by the methods described herein can be comparable to the texture of similar types of cheese made using dairy-derived protein from animal sources (e.g., cheese made from animal milk). The texture of cheese can be tested using a trained panel of human subjects or a machine such as a texture analyzer.

[0170] The taste of cheese made by the methods described herein is comparable to similar types of cheese made using animal-derived dairy proteins.The taste of cheese can be tested using a trained panel of human subjects.

[0171] The cheese compositions described herein can have browning capabilities comparable to similar types of cheese made using animal-derived dairy proteins. The cheese compositions described herein can have melting capabilities comparable to similar types of cheese made using animal-derived dairy proteins.

[0172] The texture of the yogurt prepared by the method described herein can be comparable to the texture of similar types of yogurt made using animal-derived milk proteins (e.g., yogurt made from animal milk). The texture of the yogurt can be tested using a trained group of human subjects or a machine such as a texture analyzer.

[0173] The taste of yogurt prepared by the method described herein is comparable to similar types of yogurt made using animal-derived milk-derived proteins.The taste of yogurt can be tested using a trained panel of human subjects.

[0174] Emulsion and curd compositions

[0175] The present disclosure provides an unprecedented method that allows for the preparation of a curd composition comprising only κ-casein or optionally a combination of κ-casein and β-casein, and wherein the curd composition is substantially free of α-casein. s -Casein.

[0176] Advantageously, the curd composition of the present invention has suitable chemical, physical and functional properties for use in downstream derivative product manufacturing such as cheese making.

[0177] The method is directly applicable to the preparation of curd compositions and downstream derived products from recombinantly produced kappa-casein and optionally beta-casein, which are essentially completely free of alpha-casein. s -Casein.

[0178] Therefore, the present disclosure provides a method for preparing a curd composition, wherein the method comprises the following steps: a) preparing an emulsion comprising: (i) phosphorylated κ-casein; or (ii) dephosphorylated or non-phosphorylated κ-casein; or (iii) phosphorylated κ-casein and dephosphorylated or non-phosphorylated κ-casein; or (iv) phosphorylated kappa-casein, dephosphorylated or non-phosphorylated kappa-casein, and dephosphorylated or non-phosphorylated beta-casein; or (v) dephosphorylated or non-phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; or (vi) phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; and (vii) a lipid source; b) adding a calcium salt to the emulsion; and c) causing the emulsion to coagulate.

[0179] To provide preliminary proof of principle, purified samples of bovine κ-casein and β-casein were obtained to investigate the methods of the present invention. Portions of these purified bovine κ-casein and β-casein samples were also dephosphorylated to further obtain dephosphorylated κ-casein and dephosphorylated β-casein for further investigation of the methods of the present invention.

[0180] Since these κ-casein, β-casein, dephosphorylated κ-casein and dephosphorylated β-casein starting materials are of bovine origin, there will be a small amount of α s -Casein. Thus, the process of the present invention comprises an emulsion comprising, consisting essentially of, or consisting of the specified kappa-casein and optionally beta-casein starting materials.

[0181] Step a) of the method of the present invention comprises preparing an emulsion comprising; (i) phosphorylated κ-casein; or (ii) dephosphorylated or non-phosphorylated κ-casein; or (iii) phosphorylated κ-casein and dephosphorylated or non-phosphorylated κ-casein; or (iv) phosphorylated kappa-casein, dephosphorylated or non-phosphorylated kappa-casein, and dephosphorylated or non-phosphorylated beta-casein; or (v) dephosphorylated or non-phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; or (vi) phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; and (vii) Lipid source.

[0182] Thus, in some embodiments of the present invention, step a) of the method of the present invention comprises preparing an emulsion consisting essentially of; (i) phosphorylated κ-casein; or (ii) dephosphorylated or non-phosphorylated κ-casein; or (iii) phosphorylated κ-casein and dephosphorylated or non-phosphorylated κ-casein; or (iv) phosphorylated kappa-casein, dephosphorylated or non-phosphorylated kappa-casein, and dephosphorylated or non-phosphorylated beta-casein; or (v) dephosphorylated or non-phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; or (vi) phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; and (vii) Lipid source.

[0183] Furthermore, in some embodiments of the present invention, step a) of the method of the present invention comprises preparing an emulsion consisting of; (i) phosphorylated κ-casein; or (ii) dephosphorylated or non-phosphorylated κ-casein; or (iii) phosphorylated κ-casein and dephosphorylated or non-phosphorylated κ-casein; or (iv) phosphorylated kappa-casein, dephosphorylated or non-phosphorylated kappa-casein, and dephosphorylated or non-phosphorylated beta-casein; or (v) dephosphorylated or non-phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; or (vi) phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; and (vii) Lipid source.

[0184] The lipid source for the preparation of the emulsion precursor of the inventive method can be any lipid source applicable to food, such as any lipid source " generally recognized as safe " (GRAS) according to the regulation of U.S. Food and Drug Administration (FDA), namely the chemicals or substances added in food are considered to be safe by experts under the condition of its intended use. If not subject to the impact of dietary restrictions such as vegetarianism (vegetarianism) or veganism (veganism), lipid source can be an animal-derived lipid source, such as fish oil, krill oil or milk fat. For example, when the product of the inventive method is intended to be consumed by vegans (vegan), the selection of lipid source should be limited to non-animal-derived lipid sources, such as non-animal fat or oil, vegetable fat or oil, microbial fat or oil, fungal fat or oil, the fat of recombinant production or oil or the mixture of any above-mentioned lipid sources. Preferably, lipid source is edible plant oil. For example, without limitation, the lipid source can be an edible vegetable oil selected from the group consisting of peanut oil, soybean oil, sunflower oil, safflower oil, rapeseed oil, corn oil, avocado oil, almond oil, olive oil, cottonseed oil, coconut oil, sesame oil, chia (Salvia Hispanica L.) seed oil, wheat germ oil, grapeseed oil, rice bran oil, linseed oil, mustard oil, palm oil, castor oil, hydrogenated castor oil, hemp seed oil, and any mixture of any of the foregoing edible vegetable oils.

[0185] The emulsion precursor used in the method of the present invention comprises a total protein concentration falling within the range of 2 wt.% to 20 wt.%; preferably, wherein the emulsion comprises a total protein concentration falling within the range of 3 wt.% to 10 wt.%; most preferably, wherein the emulsion comprises a total protein concentration falling within the range of 4 wt.% to 8 wt.%. Without departing from the scope of the present method, the emulsion precursor used in the present method may comprise a total protein concentration selected from the group consisting of: 1±0.5 wt.%, 2±0.5 wt.%, 3±0.5 wt.%, 4±0.5 wt.%, 5±0.5 wt.%, 6±0.5 wt.%, 7±0.5 wt.%, 8±0.5 wt.%, 9±0.5 wt.%, 10±0.5 wt.%, 11±0.5 wt.%, 12±0.5 wt.%, 13±0.5 wt.%, 14±0.5 wt.%, 15±0.5 wt.%, 16±0.5 wt.%, 17±0.5 wt.%, 18±0.5 wt.%, 19±0.5 wt.% and 20±0.5 wt.%.

[0186] The emulsion precursor used in the method of the present invention comprises a fat to protein ratio falling within the range of fat-free to 2:0.5; preferably, wherein the emulsion comprises a fat to protein ratio falling within the range of 1:1.5 to 1.5:1; most preferably, wherein the emulsion comprises a fat to protein ratio of about 1:1. Without departing from the scope of the present method, the emulsion precursor used in the present method may comprise a fat to protein ratio selected from the group consisting of: 0:∞, 0.1:0.5, 0.2:0.5, 0.3:0.5, 0.4:0.5, 0.5:0.5, 0.6:0.5, 0.7:0.5, 0.8:0.5, 0.9:0.5, 1:0.5, 1.1:0.5, 1.2:0.5, 1.3:0.5, 1.4:0.5, 1.5:0.5, 1.6:0.5, 1.7:0.5, 1.8:0.5, 1.9:0.5, 2: :0.5, 0.1:0.4, 0.2:0.4, 0.3:0.4, 0.4:0.4, 0.5:0.4, 0.6:0.4, 0.7:0.4, 0.8:0.4, 0.9:0.4, 1:0.4, 1.1:0.4, 1.2:0.4, 1.3:0.4, 1.4:0.4, 1.5:0.4, 1.6:0.4, 1.7:0.4, 1.8:0.4, 1.9:0.4, 2:0.4, 0.1:0.3, 0.2:0.3, 0.3:0.3, 0.4:0.3, 0.5:0.3, 0.6:0.3, 0 .7:0.3、0.8:0.3、0.9:0.3、1:0.3、1.1:0.3、1.2:0.3、1.3:0.3、1.4:0.3、1.5:0.3、1.6:0.3、1.7:0.3、1.8:0.3、1.9:0.3、2:0.3、0.1:0.2、0.2:0.2、0.3:0.2、0.4:0.2、0.5:0.2、0.6:0.2、0.7:0.2、0.8:0.2、0.9:0.2、1:0.2、1.1:0.2、1.2:0.2、1.3:0.2、 : 0.1, 1.4: 0.2, 1.5: 0.2, 1.6: 0.2, 1.7: 0.2, 1.8: 0.2, 1.9: 0.2, 2: 0.2, 0.1: 0.1, 0.2: 0.1, 0.3: 0.1, 0.4: 0.1, 0.5: 0.1, 0.6: 0.1, 0.7: 0.1, 0.8: 0.1, 0.9: 0.1, 1: 0.1, 1.1: 0.1, 1.2: 0.1, 1.3: 0.1, 1.4: 0.1, 1.5: 0.1, 1.6: 0.1, 1.7: 0.1, 1.8: 0.1, 1.9: 0.1 and 2: 0.1.

[0187] Step b) of the method of the present invention comprises adding a calcium salt to the emulsion. Without wishing to be bound by theory, the inventors believe that the addition of calcium ions to a solution comprising kappa-casein induces the formation of micelles, thereby producing a micellar solution of casein suitable for treatment according to the present invention to induce coagulation.

[0188] Although calcium chloride is the preferred salt for carrying out step b) of the process of the present invention, those skilled in the art will appreciate that any suitable calcium chloride may be used. 2+ Alternative sources of ions may be used without departing from the scope of the present invention. For example, but not limited to, the calcium salt can be any calcium salt selected from the group consisting of calcium chloride, calcium hydroxide, calcium carbonate, calcium acetate, calcium sulfate, calcium nitrate, calcium citrate, calcium phosphate, calcium stearate, calcium malate, calcium glycerophosphate, calcium lactate, and calcium gluconate, or a mixture of any of the foregoing calcium salts.

[0189] In some embodiments, a calcium salt is added in step b) of the method of the present invention in an amount sufficient to bring the concentration of calcium in the emulsion to a concentration falling within the range of 2 mM to 20 mM; preferably, wherein the calcium salt is added to bring the concentration of calcium in the emulsion to a concentration falling within the range of 5 mM to 15 mM; most preferably, wherein the calcium salt is added to bring the concentration of calcium in the emulsion to a concentration falling within the range of 8 mM to 12 mM. Without departing from the scope of the present method, calcium salts may be added to bring the calcium concentration in the emulsion to any concentration selected from the group consisting of 2±0.5 mM, 3±0.5 mM, 4±0.5 mM, 5±0.5 mM, 6±0.5 mM, 7±0.5 mM, 8±0.5 mM, 9±0.5 mM, 10±0.5 mM, 11±0.5 mM, 12±0.5 mM, 13±0.5 mM, 14±0.5 mM, 15±0.5 mM, 16±0.5 mM, 17±0.5 mM, 18±0.5 mM, 19±0.5 mM and 20±0.5 mM.

[0190] Step c) of the process of the invention comprises coagulating the emulsion.

[0191] Those skilled in the art will appreciate that the key step in producing any curd composition from micellar protein solution is the coagulation step. Coagulation can be promoted to achieve necessary coagulation by several different ways, including coagulation by acidification at any stage before step c), and / or by coagulation with enzyme treatment, and / or by coagulation with rennet treatment, and / or by coagulation of microbial fermentation, and / or by auxiliary coagulation of heat treatment. Each of these possible methods of inducing coagulation can be combined with other methods, and within certain practical limitations, they can be carried out in parallel, or carried out successively in any order. For example, according to the temperature adopted, heat treatment may not be suitable for carrying out in parallel with enzyme treatment or microbial fermentation, because the temperature of heat treatment may denature the enzyme or kill the microorganism, so heat treatment can be carried out before or after enzyme treatment, or before or after microbial fermentation, to avoid possibly denaturing the enzyme and / or killing the microorganism.

[0192] When coagulation is assisted or promoted by heat treatment, those skilled in the art will understand that the method of the present invention may further comprise the additional steps of heating the emulsion prior to step b) (addition of the calcium salt); and / or heating the emulsion prior to step c) (coagulating the emulsion); maintaining the emulsion at a suitable temperature for a suitable time; preferably, wherein the temperature falls within the range of 45°C to 70°C; preferably, wherein the time period falls within the range of 1 min to 180 min. Without departing from the scope of the present method, a suitable temperature may be any temperature selected from the group consisting of: 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C and 70°C; and a suitable time period may be any time period selected from the group consisting of: 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min ,27min,28min,29min,30min,31min,32min,33min,34min,35min,36min,37min,38min,39min,40min,41min,42min,43min,44m in, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min, 60min, 61min, 6 2min, 63min, 64min, 65min, 66min, 67min, 68min, 69min, 70min, 71min, 72min, 73min, 74min, 75min, 76min, 77min, 78min, 79min , 80min, 81min, 82min, 83min, 84min, 85min, 86min, 87min, 88min, 89min, 90min, 91min, 92min, 93min, 94min, 95min, 96min, 97m in, 98min, 99min, 100min, 101min, 102min, 103min, 104min, 105min, 106min, 107min, 108min, 109min, 110min, 111min, 112min,113min, 114min, 115min, 116min, 117min, 118min, 119min, 120min, 121min, 122min, 123min, 124min, 125min, 126min, 127min, 128min, 129min, 130min, 131min, 132min, 133min, 134min, 135min, 136min, 137min, 138min, 139min, 140min, 141min, 142min, 143min, 144min, 145min, 146min, 147min, 148min, 149min, 150min, 151min, 152min, 153min, 154min, 155min, 156min, 157min, 158min, 159min, 160min, 161min, 162min, 163min, 164min, 165min, 166min, 167min, 168min, 169min, 170min, 171min, 172min, 173min, 174min, 175min, 176min, 177min, 178min, 179min and 180min.

[0193] When coagulation is promoted by acidification, it will be understood by those skilled in the art that sufficient acidification can be suitably achieved by adding acid, or by treating with a microbial acidifier, or by a fermentation process, or by hydrolysis or dissociation of glucono-δ-lactone. In certain embodiments, sufficient acidification can be suitably achieved by adding a calcium salt in step b) of the method of the present invention. For example, when calcium phosphate or calcium sulfate is added, or when the casein in the emulsion is fully phosphorylated and calcium chloride is added, the pH value may be fully reduced to a suitable acidic level. In other embodiments, sufficient acidification can be suitably achieved by directly adding an acid. Suitable acids include HCl, acetic acid, citric acid, malic acid, tartaric acid, folic acid, fumaric acid, ascorbic acid, phosphoric acid, salicylic acid, lactic acid, calcium phosphate or any other organic or inorganic acid that is "generally recognized as safe" (GRAS), which is in accordance with the regulations of the U.S. Food and Drug Administration (FDA), meaning that the chemical or substance added to food is considered safe by experts under the conditions of its intended use. Suitable microbial acidifiers that can be used for acidification by fermentation include, but are not limited to, enterobacteria, acetic acid bacteria, lactic acid bacteria, Saccharomyces, and yeast.

[0194] In some embodiments, step c) of coagulating the emulsion comprises acidifying the emulsion to a pH falling within the range of pH 4.2 to pH 6.5; preferably, wherein coagulating the emulsion comprises acidifying the emulsion to a pH falling within the range of pH 4.8 to pH 5.8; most preferably, wherein acidifying the emulsion comprises bringing the emulsion to a pH of about 5.2. Without departing from the scope of the process of the present invention, the emulsion may be brought in step c) to any pH selected from the group consisting of pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6, pH 6.1, pH 6.2, pH 6.3, pH 6.4 and pH 6.5.

[0195] When coagulation is promoted by enzymatic treatment, suitable enzymes include, but are not limited to, aspartic (acid) proteinase, aspartic protease, aspartic endopeptidase, pepsin, lysyl oxidase, chymosin and / or transglutaminase. Suitable chymosin includes, but is not limited to, recombinantly produced chymosin and fermentation-produced chymosin (FPC). Suitable transglutaminases include, but are not limited to, recombinantly produced transglutaminases and fermentation-produced transglutaminases, keratinocyte transglutaminase (TGM1), tissue transglutaminase (TGM2), epidermal transglutaminase (TGM3), prostate transglutaminase (TGM4), TGM X (TGM5), TGM Y (TGM6), TGM Z (TGM7), protein 4.2 (EPB42), factor XIII (F13A1), or a transglutaminase from Streptoverticilium mobaraense.

[0196] When coagulation is assisted or promoted by treatment with transglutaminase, those skilled in the art will understand that the method of the present invention may also include an additional step of treating the emulsion with transglutaminase before step b) (addition of calcium salt); and / or treating the emulsion with transglutaminase before step c) (coagulation of the emulsion); preferably, the additional step of treating the emulsion with transglutaminase comprises adding transglutaminase to the emulsion and incubating at a suitable temperature for a suitable period of time; preferably, wherein the temperature falls within the range of 45°C to 60°C; preferably, wherein the time period falls within the range of 1 min to 180 min. Without departing from the scope of the present invention, the suitable temperature may be any temperature selected from the group consisting of: 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C and 60°C; and the suitable time period may be any time period selected from the group consisting of: 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29mi n, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 4 6min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62m in, 63min, 64min, 65min, 66min, 67min, 68min, 69min, 70min, 71min, 72min, 73min, 74min, 75min, 76min, 77min, 78min ,79min,80min,81min,82min,83min,84min,85min,86min,87min,88min,89min,90min,91min,92min,93min,94min,9 5min, 96min, 97min, 98min, 99min, 100min, 101min, 102min, 103min, 104min, 105min, 106min, 107min, 108min, 109min,110min, 111min, 112min, 113min, 114min, 115min, 116min, 117min, 118min, 119min, 120min, 121min, 122min, 123min, 124min, 125min, 126min, 127mi n, 128min, 129min, 130min, 131min, 132min, 133min, 134min, 135min, 136min, 137min, 138min, 139min, 140min, 141min, 142min, 143min, 144min, 145 min, 146min, 147min, 148min, 149min, 150min, 151min, 152min, 153min, 154min, 155min, 156min, 157min, 158min, 159min, 160min, 161min, 162min, 1 63min, 164min, 165min, 166min, 167min, 168min, 169min, 170min, 171min, 172min, 173min, 174min, 175min, 176min, 177min, 178min, 179min and 180min. ,

[0197] A person skilled in the art will appreciate that the additional steps of heat treatment and transglutaminase incubation are unrelated optional steps that can be performed independently of each other, either before or after the addition of the calcium salt in step b) of the process of the invention, and that both additional steps are intended to promote favorable coagulation (in step c) and improve the functional and physical properties of the resulting curd product. However, in some embodiments, the heat treatment step can advantageously be performed after the transglutaminase incubation step, so that the heat treatment step performs the dual function of enhancing the functionality of the cheese (particularly stretchability) and also inactivating the transglutaminase prior to final coagulation via the addition of acid, enzymes, microorganisms or rennet.

[0198] When coagulation is promoted by treatment with rennet, suitable coagulants include, but are not limited to, proteases, chymosin, pepsin, lipase, rennet of animal origin, rennet of plant origin (including extracts from Galium spp., dried caper leaves, nettle, thistle, mallow, Withania coagulans, ground ivy, Cynara, soy), calf rennet, kid rennet, rennet of fungal origin, rennet of microbial origin (e.g., extracts of Rhizomucor miehei), and recombinantly produced rennet.

[0199] When coagulation is promoted by microbial fermentation, suitable microorganisms include acidifying fungi, bacteria or yeasts, or species of fungi, bacteria or yeasts that produce enzymes including, but not limited to, aspartic (acid) proteinase, aspartic protease, aspartic endopeptidase, pepsin, lysyl oxidase, chymosin and / or transglutaminase. For example, without limitation, suitable microorganisms can be selected from the group consisting of enterobacteria, acetic acid bacteria, lactic acid bacteria, Saccharomyces, yeast, Rhizomucor miehei and Streptoverticilium mobaraense.

[0200] In some embodiments, upon completion of step c), optimal coagulation is observed after incubation for a period of time at a suitable temperature. Thus, in some embodiments, the method of the present invention further comprises the following steps:

[0201] d) incubating the coagulated emulsion produced in step c) at a suitable temperature for a period of time to produce a coagulum comprising a crude curd and a liquid phase; preferably, wherein the period of time falls within the range of 10 min to 180 min; most preferably, wherein the period of time falls within the range of 20 min to 60 min; preferably, wherein the temperature falls within the range of 18° C. to 50° C.; most preferably, wherein the temperature falls within the range of 25° C. to 40° C.

[0202] Without departing from the scope of the present method, a suitable temperature for incubating step d) may be any temperature selected from the group consisting of: 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C and 50°C; and a suitable time period for incubating step d) may be any time period selected from the group consisting of: 10 min, 11 min, 12 min, 13 min, 14 min, 15 min n, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36m in, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57 min, 58min, 59min, 60min, 61min, 62min, 63min, 64min, 65min, 66min, 67min, 68min, 69min, 70min, 71min, 72min, 73min, 74min, 75min, 76min, 77min, 7 8min, 79min, 80min, 81min, 82min, 83min, 84min, 85min, 86min, 87min, 88min, 89min, 90min, 91min, 92min, 93min, 94min, 95min, 96min, 97min, 98min, 99min, 100min, 101min, 102min, 103min, 104min, 105min, 106min, 107min, 108min, 109min, 110min, 111min, 112min, 113min, 114min, 115min, 116min, 117min, 118min, 119min, 120min, 121min, 122min, 123min, 124min, 125min, 126min, 127min, 128min, 129min, 130min, 131min, 132min, 133min, 134min,135min, 136min, 137min, 138min, 139min, 140min, 141min, 142min, 143min, 144min, 145min, 146min, 147min, 148min, 149min, 150min, 151min, 152min, 153min, 154min, 155min, 156min, 157min, 158min, 159min, 160min, 161min, 162min, 163min, 164min, 165min, 166min, 167min, 168min, 169min, 170min, 171min, 172min, 173min, 174min, 175min, 176min, 177min, 178min, 179min and 180min.

[0203] The method of the present invention provides a curd composition in step e); e) removing the crude curd of the coagulant produced in step d) (incubating) from the liquid phase of the coagulant produced in step d) to provide a curd composition.

[0204] The curd composition produced by the method of the present invention can be further processed to provide advantageous functional and physical properties, depending on the downstream derivative products to be produced using the curd composition. Therefore, in some embodiments, the method of the present invention further comprises the following steps: f) texturizing the curd composition produced in step e) (separation of the curd composition) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature to provide a textured curd composition.

[0205] In some embodiments, step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises an elevated temperature falling within the range of 60°C to 95°C; preferably an elevated temperature falling within the range of 70°C to 90°C; most preferably an elevated temperature falling within the range of 75°C to 85°C. Without departing from the scope of the process of the present invention, the elevated temperature for texturizing step f) may be any temperature selected from the group consisting of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C and 95°C.

[0206] In some embodiments, step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises texturizing at a ratio of curd composition to water falling within the range of 0.5:3 to 3:0.5; preferably a ratio of curd ingredients to water falling within the range of 1:2 to 2:1; most preferably a ratio of curd ingredients to water of 1:2. Without departing from the scope of the process of the present invention, the ratio of curd composition to water for texturizing the curd composition in step f) may be any ratio selected from the group consisting of: 0.5:3, 0.6:3, 0.7:3, 0.8:3, 0.9:3, 1:3, 1.1:3, 1.2:3, 1.3:3, 1.4:3, 1.5:3, 1.6:3, 1.7:3, 1.8:3, 1.9:3, 2:3, 2.1:3, 2.2:3, 2.3:3, 2.4:3, 2 .5:3, 2.6:3, 2.7:3, 2.8:3, 2.9:3, 3:3, 3:2.9, 3:2.8, 3:2.7, 3:2.6, 3:2.5, 3:2.4, 3:2.3, 3:2.2, 3:2.1, 3:2, 3:1.9, 3:1.8, 3:1.7, 3:1.6, 3:1.5, 3:1.4, 3:1.3, 3:1.2, 3:1.1, 3:1, 3:0.9, 3:0.8, 3:0.7, 3:0.6, and 3:0.5.

[0207] In some embodiments, step f) of texturizing the curd composition produced in step e) further comprises texturizing by kneading and / or stretching and / or folding the curd composition, and optionally forming the curd composition into a ball.

[0208] In some embodiments, the method of the present invention further comprises the following steps: g) Cooling the textured curd composition; preferably in brine or water.

[0209] By developing a method to (re)assemble non-micellar caseins into casein micelles and then coagulate them, the present inventors have produced artificial casein micelles from only κ-casein, and only κ-casein and β-casein, without the need for α-casein. s1 -Casein and α s2 -casein, and has coagulation properties very similar to those of natural bovine casein micelles in terms of micelle size and mineral content. These artificial micelles can also be coagulated by the action of heat, or rennet, or enzymes, or acids, or microorganisms, or a combination thereof, to produce cheese textures identical to those produced from cow's milk, allowing for a complete replacement of the functionality of bovine casein micelles.

[0210] The curdled milk composition produced by the method of the present invention can be a useful precursor for making downstream products such as yogurt or cheese. Therefore, in other embodiments, the disclosure provides edible compositions or food products comprising the curdled milk composition of the present invention. Such edible compositions or food products include but are not limited to yogurt and cheese.

[0211] In some embodiments, the present invention provides a food product produced by the method of the present invention, comprising casein. In preferred embodiments, the food product is a cheese product of non-animal origin. In particularly preferred embodiments, the food product is a mozzarella-type cheese product or a pasta filata-type cheese product of non-animal origin.

[0212] In some embodiments, a yogurt composition food product can be formed using the methods described herein. Yogurt can be formed using a curd composition produced using the methods described herein. The method can include incubating the curd composition with a microorganism. The microorganism can include one or more of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus, or Bifidobacterium.

[0213] The texture of the yogurt prepared by the method described herein can be comparable to the texture of similar types of yogurt prepared using animal-derived or dairy-derived proteins (e.g., yogurt prepared from animal milk). The texture of the yogurt can be tested using a trained group of human subjects or a machine such as a texture analyzer.

[0214] The taste of yogurt prepared by the method described herein is comparable to similar types of yogurt prepared using animal-derived milk-derived proteins.The taste of yogurt can be tested using a trained panel of human subjects.

[0215] In some embodiments of the method for producing food products, said method also comprises aging and / or ripening the curdled milk composition to form cheese composition.Curdled milk composition can be further processed to produce cheese or cheese-like product.In some cases, for example mozzarella cheese product, curdled milk can be heated and stretched or kneaded with texturization.In these and other embodiments, curdled milk composition or texturized curdled milk composition can be aged, for example, for Brie, Camembert, Feta, Halloumi, Gouda, Edam, Cheddar, Manchego, Swiss-type cheese, Colby, Muenster, blue cheese or Parmesan type cheese or cheese-like product.

[0216] The cheese food product described herein can be a pasta filata-like cheese, such as mozzarella cheese. Soft cheeses such as paneer cheese, cream cheese or cottage cheese can also be formed using the methods described herein.

[0217] The inventors have found that the method of the present invention is particularly suitable for producing pasta filata-type cheeses, as well as stretched-curd, pulled-curd and plastic-curd cheeses, including akkawi, braided cheese, cacio figurato, caciocavallo, galbanino, halloumi, kashkaval, mozzarella, bocconcini, buffalo mozzarella, Oaxaca cheese, oscypek, Pallone digravina, palmito cheese, provolone, ragusano, queso de mano, scamorza, stracciata, stracciatella di bufala, string cheese, chechil, Sulguni and Vastedda della Valledel Belice.

[0218] In some embodiments, the textured curd composition provided by step f) of the method of the present invention is a cheese product of non-animal origin; preferably a mozzarella-type cheese product of non-animal origin.

[0219] The texture of cheese made by the methods described herein can be comparable to the texture of similar types of cheese made using animal-derived or dairy-derived proteins, such as cheese made from animal milk. The texture of cheese can be tested using a trained panel of human subjects or a machine such as a texture analyzer.

[0220] The taste of cheese produced by the methods described herein is comparable to similar types of cheese produced using dairy proteins from animal sources.The taste of cheese can be tested using a trained panel of human subjects.

[0221] The cheese compositions described herein can have browning capabilities comparable to similar types of cheeses made using animal-derived milk proteins. The cheese compositions described herein can have melting capabilities comparable to similar types of cheeses made using animal-derived milk proteins.

[0222] Emulsion and curd compositions containing non-animal derived protein

[0223] The present invention provides an emulsified composition comprising one or more lipids and a protein component, wherein the protein component has a protein content of at least 80% protein by weight, wherein about 25-40% by weight of the protein content comprises zein and the remainder is non-micellar casein.

[0224] Optionally, the protein component starting material may be in powder form.

[0225] Optionally, the protein component starting material may also include non-protein components, but will typically comprise at least 80% protein (by weight), such as about 85% protein, such as about 90% protein.

[0226] The protein component includes non-micellar casein, such as β-casein or sodium caseinate, and zein as the major protein. That is, the protein component may include about 40% (by weight) caseinate / β-casein and about 40% (by weight) zein in the protein fraction (i.e., the total protein content of the protein component). Optionally, the protein component is primarily β-casein.

[0227] In the method of the present invention, starting material, such as β-casein is in free form, i.e., not in the form of casein micelles. β-casein may be induced to form a dimer or micelle-like assembly consisting only of β-casein. β-casein can be produced from milk, such as cow's milk, by cold microfiltration. Alternatively, other milk sources can be used. β-casein can also be obtained by cold separation using a sedimentation centrifuge or separator (or any type of centrifuge). For this method, it is necessary to cool the protein solution, suspension, dispersion or emulsion to a temperature below 10°C, then acidify to precipitate α-casein and κ-casein, which can then be separated by centrifugation into α-casein and κ-casein. Alternatively, β-casein (or any casein provided herein) can be produced by recombinant expression using techniques known in the art. When using cold microfiltration of milk to provide β-casein, the resulting powder may include non-protein components, but will typically contain at least 80% protein by weight, such as more than 82% protein, such as about 85% protein. The protein component will comprise beta-casein and zein as major proteins, i.e. the protein component will comprise at least 80% (by weight) protein content, of which approximately 25-40% (by weight) of the protein content consists of zein and the remainder is beta-casein.

[0228] The β-casein may conveniently be derived from bovine milk. Other suitable sources include milk from other ungulates, such as sheep's milk, goat's milk, horse's milk, camel's milk or buffalo's milk.

[0229] The beta-casein of the present invention mentioned herein or any other independent casein protein also can be produced by expressing its gene in a suitable recombinant construct using a suitable host expression system.Term "recombination" is known to those of ordinary skill in the art.When referring to nucleic acid (such as gene), the term "recombination" can be used to describe the nucleic acid removed from its natural background, when found in nature, not with the nucleic acid associated with all or part of the adjacent or adjacent nucleic acid, operably connected to the nucleic acid of its nucleic acid not connected in nature, or nucleic acid not present in nature.Term "recombination" can be used to describe the DNA isolate of the clone, or the nucleic acid including chemically synthesized nucleotide analogs.When "recombination" is used to describe protein, such as recombinant beta-casein or recombinant kappa-casein, it can refer to, for example, compared with the kind or type of the cell of natural production protein, such as by using "recombination" nucleic acid, the protein produced in different kinds or types of cells.

[0230] The inventors conducted three different zein hydration methods: hydration at alkaline pH, enzymatic hydrolysis of zein, and incorporation of zein into an emulsion using high shear. All three different hydration methods resulted in stretchable and meltable cheese in the zein-caseinate system.

[0231] Therefore, the present invention also provides a method of forming an emulsified composition according to the present invention, wherein the method comprises the following steps: i) preparing a mixture of non-micellar casein and zein protein; ii) adding one or more lipids; iii) homogenizing the mixture to obtain an emulsified composition.

[0232] In the method of the present invention, the zein component can first be dissolved in an alkaline aqueous solution. The pH of the aqueous solution can be in the range of pH 11.3 to 12.7, for example pH 12.5. Alternatively, the pH range can be at least pH 12.0.

[0233] The dissolution of zein to form an aqueous solution can be carried out at a temperature of 5-50° C. and ambient pressure. For convenience, room temperature (20-25° C.) is generally used.

[0234] Therefore, the present invention preferably provides a method for forming an emulsified composition according to the present invention, wherein the method comprises the following steps: i) preparing an aqueous solution of zein protein at an alkaline pH; ii) adding non-micellar casein; ii) adding one or more lipids and optionally sugars; iii) homogenizing the mixture to obtain an emulsified composition The mixture of non-micellar casein and zein protein in step (i) of the process of the present invention can be prepared as follows: (1) i) preparing a mixture of an aqueous solution of zein and non-micellar casein at an alkaline pH; ii) Lowering the pH of the solution to neutral (about pH 7.0).

[0235] Dissolving steps (i) and (ii) may be performed at room temperature and ambient pressure.

[0236] The mixture of non-micellar casein and zein in step (i) of the process of the present invention may alternatively be prepared as follows: i) preparing an aqueous solution of zein protein at an alkaline pH; ii) enzymatic hydrolysis of zein protein at pH 9.0; iii) Non-micellar casein was added to the zein protein solution at pH 7.0.

[0237] The enzymatic hydrolysis step can be carried out at a temperature of about 50°C and ambient pressure. Enzymatic hydrolysis can be carried out with different enzymes, and the process conditions depend on the enzyme. For example, Alcalase (2.4L FG) can be used at pH 8.0-9.0 and 50°C-60°C.

[0238] The mixture of non-micellar casein and zein protein in step (i) of the process of the present invention can be prepared by dissolving the zein protein in the alkaline aqueous solution described above and then adding the non-micellar casein component. The pH is then lowered to neutral, typically to about pH 7.0, by acidification.

[0239] In an alternative method of forming the emulsified composition of the present invention, the method comprises the steps of: i) preparing an aqueous solution of non-micellar casein; ii) adding one or more lipids; iii) emulsifying the mixture and the zein protein to obtain an emulsified composition.

[0240] The emulsification step is carried out by a high shear method sufficient to incorporate the zein into the emulsion. This mechanical energy can be introduced by a "normal" high-speed mixer using a rotor-stator system or a high-pressure valve or ultrasonic treatment (ultrasound). For high shear, the range can be 3000-25000 rpm (about 50-420 rev / sec). An Ultraturrax, a rotor-stator dispersion device, also known as a high shear mixer, can be used.

[0241] The method may comprise a step (iv) comprising homogenising the mixture obtained from step (iii). Thus, the claimed method may comprise the steps of: i) preparing an aqueous solution of non-micellar casein; ii) adding one or more lipids and optionally sugars; iii) incorporating the zein into the emulsion by high shear emulsifying the mixture and the zein protein to obtain an emulsified composition; iv) homogenizing the mixture obtained from step (iii).

[0242] (i) The hydration of non-micellar casein is usually carried out at neutral pH and room temperature.

[0243] In the method of the present invention, the protein components, typically β-casein and zein, can first be dissolved in an aqueous solution. Solutions containing up to 10% (by weight), typically up to 2% by weight, of the protein components can be prepared. Optionally, a salt capable of forming a soluble calcium salt, such as sodium citrate, can also be included to facilitate the dissolution of the protein components. For example, a 10% solution of the protein components (β-casein and zein) can be prepared by adding 10 g of β-casein / zein to 90 g of water, optionally adding 0.15 g of trisodium citrate. For example, a concentration of 0.05 to 0.055 mmol citrate / g of β-casein / zein can be used. The dissolution step can be carried out at low temperatures (e.g., at about 5°C), or can be carried out at higher temperatures, such as at temperatures up to about 50°C. However, at higher protein concentrations, β-casein will aggregate at temperatures above 50°C.

[0244] Optionally, the dissolving step is performed at ambient pressure.

[0245] Dissolving β-casein to form an aqueous solution can be carried out at a temperature of 5-50°C and ambient pressure. For convenience, room temperature (20-25°C) can optionally be used. However, lower temperatures (less than 10°C) may be advantageous for solubilizing higher protein concentrations.

[0246] The method of the present invention requires adding fat and optionally sugar to the β-casein / zein solution, followed by an emulsification step. Optionally, the fat can be an oil at the processing temperature. Edible vegetable oils and butter are suitable fats. Suitable edible vegetable oils include rapeseed oil and sunflower oil, but other edible vegetable oils or nut oils can also be used. The fat can be a fusion of these oils and / or a mixture of oil and butter. The fat can be added in a ratio of 3:1 to 1:3 relative to the weight of the protein, for example, a fat to protein ratio of 1:1. After combination, the mixture thus formed can be emulsified by any suitable means, for example, by simple dispersion, or high-speed stirring and / or by homogenization. Optionally, the emulsification step occurs at a temperature of 5°C to 60°C, for example, a temperature of 30 to 60°C, for example, a temperature of 45 to 55°C. A temperature of approximately 50°C may be suitable. Alternatively, a lower temperature of 5°C to 25°C can be used. Optionally, the emulsification step is performed at a pressure of at least 2,000 kPa (20 bar), for example at a pressure of 5,000 kPa (50 bar) to 45,000 kPa (450 bar).

[0247] The emulsification process may be carried out in a two-stage process using a first higher pressure (e.g. 20,000 kPa (200 bar) followed by a second lower pressure (e.g. 5,000 kPa (50 bar). The temperature may be from 5°C to 25°C, e.g. 20°C.

[0248] In another aspect, the present invention provides a method of forming a fibrous or "pasta filata" style cheese product, wherein the method comprises preparing an emulsified composition of the present invention and forming a treated emulsion by the following steps: i) acidified emulsion, and / or ii) Adding a calcium salt, such as CaCl2.

[0249] When the treated emulsion is formed using the addition of a calcium salt such as CaCl2, the emulsion is optionally also treated with a base to adjust the pH to at least 6, such as at least pH 7, such as pH 7.5 or higher.

[0250] Optionally, during formation of the treated emulsion, the temperature of the emulsion may also be changed (reduced) to promote gel formation. Optionally, the temperature will be reduced to 10°C or less.

[0251] In the treated emulsion, gel formation may occur, which may result in a coagulated product. The coagulated product may then be subjected to traditional pasta filata cheese preparation steps.

[0252] The emulsion can be used to form a fiber-rich cheese product, such as a mozzarella-style cheese product, by forming a gel. Gel formation can be induced by acidification and / or addition of a calcium salt such as CaCl2. Alternatively, when the protein source is caseinate / zein, coagulation can be induced by rennet.

[0253] Optionally, gel formation is induced by adding a calcium salt (CaCl2) and alkalizing. Any food grade reagent can be used to increase the pH as needed. Examples include sodium hydroxide, potassium hydroxide, etc.

[0254] One option for inducing gel formation is by acidification. Typically, this step results in a pH of 4.0 to 8.0, for example a pH of 5.0 to 6.0, for example a pH of 5.0 to 5.5. Optionally, the pH of the emulsion is about 7.3, and may be between 6.5 and 7.5, depending on the protein, citrate and salt concentrations. Optionally, the emulsion is cooled to a temperature of, for example, 0-20°C, for example 5-10°C, before acidification. The cooling step may be carried out over a period of several minutes, for example, about 10 minutes. The exact time taken to cool the emulsified mixture is not particularly critical.

[0255] Any suitable food grade acid can be used to reach the desired pH, such as hydrogen chloride, lactic acid, citric acid, acetic acid, D-(+)-glucono-δ-lactone ("GDL") or any combination thereof. Optionally, a starter culture can be used to produce the pH reduction by fermentation. Thermophilic and / or mesophilic lactic acid bacteria suitable for producing yogurt or cheese are well known in the art and can be used, such as Lactococcus lactis or Streptococcus salivarius. When fermentation is used to reduce the pH, sugar can be added (e.g., up to 2-5% by weight relative to the mixture) and a temperature of ambient temperature to about 37° C. is used for the fermentation step. When an acid is added to reduce the pH, the preferred acid used is citric acid and / or lactic acid. A pH below 5 can reduce the moisture content of the cheese product.

[0256] A suitable calcium salt is calcium chloride, but any soluble calcium salt suitable for food production can be used. Calcium ions will be bound by proteins, so the "free calcium" mentioned refers to the availability of calcium ions to the composition, which will depend on the solubility of the selected calcium salt and the ratio of calcium to protein. Preferably, the calcium salt is calcium chloride, calcium hydroxide, calcium carbonate, calcium citrate, calcium phosphate, calcium stearate, calcium malate, calcium glycerophosphate, calcium lactate, calcium gluconate, or a mixture thereof. In a particularly preferred embodiment of the present invention, the calcium salt is calcium chloride.

[0257] A further option is to use acidification and the addition of a calcium salt to induce gel formation. Optionally, acidification of the emulsion occurs first, and then the calcium salt is added to the emulsion. Alternatively, the calcium salt is first added to the emulsion, and then the emulsion is acidified.

[0258] Once the gel is formed, the usual processing steps for traditional mozzarella-style cheese can be followed. Typically, these steps include cutting the curds, repeated periods of stirring the cut curds, allowing the whey to be removed by draining, plasticizing and shaping the cheese, cooling the cheese in solution at a temperature below 15°C, and then storing it in a storage solution at a refrigerated temperature (e.g., 5°C).

[0259] In more detail, after inducing coagulation due to the addition of calcium salts / lowering of pH, the coagulated product (curd) can be incubated as described above and optionally cut and repeatedly stirred in the latter half of the incubation period. Thereafter, whey can be drained from the curd in a manner similar to the whey draining step in forming traditional mozzarella-style cheese. Generally, the curd can be placed in a sieve or cloth and the whey drained for a period of 5 to 60 minutes, for example, 5 to 45 minutes. Once the whey has been drained from the curd, the curd can be plasticized or textured and formed at a temperature of about 60 to 80°C for a period of up to 5 minutes, for example, 1 to 3 minutes. Temperatures above 80°C produce viscous curds, while temperatures below 60°C limit the ability of the curd to stretch. The curd can be textured in a liquid containing water (e.g., water or whey). Conveniently, a curd:water ratio of 1:1 to 1:10 (by weight) can be used. The stretching liquid may optionally include up to 5% (by weight) of a calcium salt (e.g., calcium chloride) or a sodium salt (e.g., sodium chloride). The cheese may then be cooled to a temperature below 15° C. and stored in a refrigerator (e.g., at a temperature of approximately 5° C.). Optionally, the cheese may be stored in a storage solution, which is typically an aqueous solution. For example, water may be used to form the storage solution. Suitable storage solutions include, for example, 5-25% (by weight) sodium chloride and / or 0.1-1% or more calcium chloride and / or 0.2% (by weight) lactic acid.

[0260] Figure 2.3 Shown are cheeses according to the invention at different stages of their processing and after melting (d).

[0261] As mentioned above, in addition to the addition of a calcium salt, the pH of the emulsion may optionally be increased (basified).

[0262] Excessive kneading of the product should be avoided during the shaping step as this will reduce the moisture content of the final product.

[0263] The process can be carried out at standard atmospheric pressure, thus avoiding any expensive pressurization steps.

[0264] Transglutaminase treatment

[0265] The present invention provides a method of forming a cheese product, wherein the method comprises the step of treating dephosphorylated casein or caseinate with a transglutaminase.

[0266] The resulting cheese product may be a pasta filata style cheese product.

[0267] The cheese product formed may be a cheese product, for example, a high moisture cheese product.

[0268] "Pastafilata-style" cheese products refer to cheeses that have been stretched or elongated into curds, typically after the curds have been immersed in hot water. Stretching or elongating the curds creates a fibrous structure in the cheese.

[0269] As used herein, the term "high-moisture cheese product" refers to any cheese food product with a moisture content exceeding 60% by weight. Moisture content can be determined using an infrared dryer (e.g., MA 30, Sartorius). For example, 3.00 ± 0.05 g of grated pasta filata-style cheese can be weighed in an aluminum pan and heated using infrared light to a temperature of 105°C until a constant weight is reached. The moisture loss can then be determined.

[0270] In the method for the present invention, the starting material of casein or caseinate for forming emulsion is dephosphorylated, i.e., the casein starting material lacks the phosphorylation of the usual level present in casein produced by mammals. Casein is usually in free form, i.e., not in the form of micelle-like assemblies. Casein can be in the form of a salt (i.e., caseinate), or casein can be in solution as free casein. Examples of suitable caseins are caseinate and single casein fractions. Usually, dephosphorylated casein or caseinate is produced using genetic engineering recombination. Alternatively, an enzyme can be used to remove the phosphate group, such as using alkaline phosphatase, to produce dephosphorylated casein or caseinate. Optionally, compared with natural casein protein, dephosphorylated casein or caseinate starting material has no more than 50%, for example, no more than 40%, for example, no more than 30%, for example, no more than 20%, for example, no more than 10%, for example, no more than 5%, for example, no more than 3% phosphoryl groups.

[0271] The dephosphorylated or non-phosphorylated casein can be a caseinate, such as sodium caseinate, potassium caseinate, calcium caseinate, or a mixture of any of these. Optionally, the caseinate is sodium caseinate. The dephosphorylated or non-phosphorylated casein or caseinate can be a mixture of caseins or caseinates, such as a combination of four different casein proteins: αs1-casein, αs2-casein, β-casein, and κ-casein, optionally present in a naturally occurring molar ratio of about 4:1:4:1.

[0272] As mentioned above, dephosphorylated or non-phosphorylated casein can be used in the form of salt (caseinate) or as a single casein in solution. Dephosphorylated casein will not be in the form of micelle-like assembly. Examples of suitable caseins are caseinate and single casein fractions. Typically, casein will comprise a mixture of more than one casein protein type. For example, casein can comprise two or more of αs1-casein, αs2-casein, β-casein and κ-casein, optionally, casein can comprise three or more of αs1-casein, αs2-casein, β-casein and κ-casein, and casein can comprise casein, αs2-casein, β-casein and κ-casein.

[0273] While recombinantly produced or non-phosphorylated casein or caseinate is envisioned as the primary source of starting material, dephosphorylated or non-phosphorylated casein or caseinate may alternatively be derived from mammalian milk. Suitable sources include milk from ungulates, such as cow's milk, sheep's milk, goat's milk, horse's milk, camel's milk, or buffalo's milk. The casein may optionally be present as sodium caseinate. The casein may be sodium kappa-caseinate.

[0274] Optionally, no milk proteins other than dephosphorylated or non-phosphorylated casein (or caseinate) are used in the process.

[0275] The dephosphorylated or non-phosphorylated casein / caseinate solution used as starting material can have a protein content of 1 to 300 g / L, such as 10 to 200 g / L, such as 50 to 60 g / L. A suitable casein / caseinate solution can have 5% (by weight) dephosphorylated casein / caseinate.

[0276] Ideally, the dephosphorylated or non-phosphorylated casein or caseinate solution starting material is formed using conditions which ensure complete hydration of the casein or caseinate.For example, the solution may be heated to a temperature of 50°C with stirring for about 30 minutes to one hour.

[0277] Transglutaminase is added to the casein / caseinate solution to induce protein polymerization. The temperature used for incubation is generally below 37°C to avoid denaturation of the enzyme, which would reduce activity. However, higher temperatures can cause partial unfolding of the substrate protein and increase the substrate sites available to the transglutaminase. Therefore, temperatures above 37°C are useful, for example temperatures of 40-50°C. One skilled in the art will be able to determine appropriate incubation times and temperatures. An example is an incubation period of up to 60 minutes, for example 20 to 40 minutes, at a suitable incubation temperature, for example a temperature above 30°C, or even higher temperatures such as 50°C.

[0278] The transglutaminase may conveniently be added in a concentration of at least 0.5 U / g protein. For example, a concentration of 1 U / g protein, for example 2 U / g protein, for example 3 U / g protein, for example 4 U / g protein, for example 5 U / g protein may be used. Where it is desired to shorten the incubation time, a higher concentration of enzyme may be suitable. The duration of the incubation may be several hours (e.g. overnight). In addition, the temperature used for the incubation period will affect the duration of the incubation and the amount of enzyme required. For example, an overnight incubation at 4°C may be used. For example, an incubation at 50°C for 60 minutes, for example using a transglutaminase concentration of at least 0.5 U / g protein, may alternatively be used. What is required is to achieve a suitable level of polymerization of the protein starting material, and this can be readily determined by one skilled in the art.

[0279] Once the polymerization step has been performed, the transglutaminase is denatured, for example, by heating. An exemplary method of denaturing the enzyme is to heat the polymerization mixture to 80°C or higher for several minutes.

[0280] The polymerized protein mixture is then combined with an oil or fat (e.g., an edible vegetable oil or fat) and processed into an emulsion, for example, using high speed mixing. Optionally, the emulsification process is carried out at ambient pressure in combination with high shear mixing. Optionally, the emulsification step is carried out at elevated pressure, i.e., above ambient pressure. Optionally, the emulsification step is carried out at a temperature between ambient temperature and 60°C.

[0281] The method of the present invention requires adding fat to the polymerized casein solution and then performing an emulsification step. Optionally, the fat can be an oil at the processing temperature. Edible vegetable oils and butter are suitable fats. Suitable edible vegetable oils include rapeseed oil, but other edible vegetable oils or nut oils can also be used. The fat can be a fusion of these oils and / or can be a mixture of oil and butter. A suitable amount of fat will provide a protein:fat ratio of 3:1 to 1:3. The preferred ratio is 2:1 to 1:2. After combination, the mixture thus formed can be emulsified by any suitable means, for example by high-speed stirring and / or by homogenization. Optionally, the emulsification step occurs at a temperature of ambient temperature to 60°C, for example a temperature of 30 to 60°C, for example a temperature of 45 to 55°C. A temperature of about 50°C may be suitable. Alternatively, a lower temperature of 5°C to 25°C can be used. Optionally, the emulsifying step is performed at a pressure of at least 5,000 kPa (50 bar), for example at a pressure of 5,000 kPa (50 bar) to 45,000 kPa (450 bar).

[0282] Optionally, the emulsification process is carried out at ambient pressure in combination with high shear mixing. Optionally, the emulsification step is carried out at elevated pressure, i.e., above ambient pressure. For example, the pressure used can be greater than 1,000 kPa, such as greater than 2,000 kPa. Optionally, at least a portion of the emulsification process is carried out at a pressure of 3,000-7,000 kPa, such as 4,000-6,000 kPa. Optionally, the pressure is about 5,000 kPa. Optionally, at least a portion of the emulsification process is carried out at a temperature between ambient temperature and 60°C.

[0283] Optionally, the emulsification process can be carried out in a two-stage process, wherein the first stage is carried out using a first higher pressure of greater than 10,000 kPa (e.g., 20,000 kPa (200 bar)), followed by a second stage carried out at a lower pressure of about 3,000 to 7,000 kPa (e.g., 5,000 kPa (50 bar)). The temperature can be 5°C to 25°C, e.g., 20°C.

[0284] By using rennet or acid to produce a gel, the emulsion can be used to form a cheese product, such as a fibrous cheese product, such as a mozzarella-style cheese product. An alternative to acid is to use D-(+)-glucono-δ-lactone or a bacterial culture that produces acid by fermentation. Optionally, rennet can be added in the form of rennet. Optionally, the temperature for adding rennet can be a temperature from ambient temperature to about 40°C, such as a temperature from 25°C to 40°C, such as a temperature from 30°C to 40°C, such as a temperature from 34°C to 37°C. A temperature of about 35°C can be conveniently used. Typically, rennet is added together under stirring to ensure that the mixture is homogeneous. Optionally, at least 2 IMCU (international clotting units) of rennet are added to every 100 mL of emulsion, such as 2 to 10 IMCU of rennet are added to every 100 mL of emulsion. As an example, 5 IMCU of rennet can be used for every 100 mL of emulsion.

[0285] After adding rennet, the mixture may be incubated for 5 to 30 minutes, for example 10 to 20 minutes, typically about 15 minutes to allow gel formation. As described above, the temperature of the mixture during incubation may be 25°C to 40°C, for example 30°C to 40°C, for example 34°C to 37°C. A temperature of about 35°C may be conveniently used during incubation.

[0286] Once the gel is formed, the usual processing steps for traditional pasta filata style cheese can follow. Typically, these steps include cutting the curd, repeated periods of stirring the cut curd, allowing the whey to be removed by draining, plasticizing and cheese shaping, cooling the cheese in solution at a temperature below 15°C, and then storing it in a storage solution at a refrigerated temperature (e.g., 5°C).

[0287] In more detail, after coagulation is induced due to the addition of rennet, the coagulated product (curd) can be incubated as described above, cut and repeatedly stirred during the second half of the incubation period. Thereafter, the whey can be drained from the curd in a manner similar to the whey draining step in forming traditional mozzarella-style cheese. Typically, the curd can be placed in a sieve and the whey drained for a period of 10 to 20 minutes. Once the whey has been drained from the curd, the curd can be plasticized and shaped at a temperature of about 60 to 80° C. for a period of up to 5 minutes, for example. For example, 1 to 3 minutes. The curd can be textured in a liquid comprising water (e.g., water or whey). Conveniently, a curd:water ratio of 1:1 to 1:2 (by weight) can be used. Thereafter, the cheese can be cooled to a temperature below 15°C and stored in a refrigerator (e.g., at a temperature of about 5°C). Optionally, the cheese can be stored in a storage solution, which is typically an aqueous solution. For example, water can be used to form the storage solution. Suitable storage solutions include, for example, 5-25% (by weight) sodium chloride and / or 0.2% (by weight) lactic acid.

[0288] Optionally, the method of the present invention comprises mixing non-phosphorylated casein or caseinate with transglutaminase to form a polymerized protein mixture. Fat is then added to the polymerized protein mixture to form an emulsion. The emulsion can be induced to form a gel by adding rennet or by acidification. The gel can then be processed by at least one of the following processing steps: a) incubating the mixture, optionally performing conventional cleavage; and / or b) whey drainage; and / or c) plasticizing and shaping to produce cheese products; and / or d) cooling the cheese product.

[0289] Excessive kneading of the product should be avoided during the shaping step as this will reduce the moisture content of the final product.

[0290] The method of the present invention may further comprise one or more of the following additional steps: i) Cooling step after emulsification and before addition of rennet.After emulsification, the emulsion may be cooled to below ambient temperature, such as 10°C or lower, such as 4 to 8°C, such as about 5°C.

[0291] ii) After emulsification (and optionally before acidification or addition of rennet), a salt, such as a calcium salt, is added. Typically, the calcium salt is water-soluble and may conveniently be calcium chloride, for example. Optionally, sodium chloride may be added in addition or in addition, for example in an amount of 1-2% by weight. Optionally, a polyphosphate, such as sodium polyphosphate, may be added in an amount of, for example, 0.3% by weight, in addition or in addition.

[0292] iii) an acidification step following emulsification (and optionally after or simultaneously with the calcium salt addition step, if present). Typically, a pH of 5.0 to 6.0 is obtained, such as a pH of 5.2 to 5.8, such as about 5.6.

[0293] Thus, optional step i) is a cooling step immediately after emulsification.After emulsification, the emulsion may be cooled to below ambient temperature, for example 5 to 15°C for a few minutes, for example up to 10 minutes.

[0294] In optional step iii), the pH of the emulsion is reduced. Optionally, the pH of the emulsification is reduced to a pH of 5.0 to 6.0, for example, to a pH of 5.2 to 5.8. An acidification step (if present) will occur after the emulsification and cooling step (if present). Optionally, the acidification step can occur at a temperature below ambient temperature, for example, 5 to 15°C. Any suitable food-grade acid can be used to achieve the desired pH, such as hydrogen chloride, lactic acid, citric acid, acetic acid, D-(+)-glucono-δ-lactone, or any combination thereof.

[0295] Optionally, a starter culture can be used to produce a pH reduction by fermentation. Thermophilic and / or mesophilic lactic acid bacteria suitable for producing yogurt or cheese are well known in the art and can be used, such as Lactococcus lactis or Streptococcus salivarius. Optionally, sugar or other nutrients for bacteria can be added, for example, up to 2% of sugar by weight. When using bacteria to cause acidification, the temperature of the emulsion is adjusted to a suitable temperature to promote fermentation, for example, adjusted to approximately 37°C to 40°C. Preferably, the acid added is citric acid and / or lactic acid. A pH lower than 5 can reduce the moisture content of the cheese product. Optionally, the acidification step can be carried out at a temperature of 5 to 15°C, for example, 8 to 12°C. Optionally, the acidification step can occur simultaneously with cooling.

[0296] In the optional step ii), a salt may be added. Suitable salts may be calcium salts, sodium chloride and / or polyphosphates. A suitable calcium salt is calcium chloride, but any soluble calcium salt suitable for food production may be used. Calcium ions will be bound by proteins, so the "free calcium" referred to herein refers to the availability of calcium ions to the composition, which will depend on the solubility of the selected calcium salt. Preferably, the calcium salt is calcium chloride, calcium hydroxide, calcium carbonate, calcium citrate, calcium phosphate, calcium stearate, calcium malate, calcium glycerophosphate, calcium lactate, calcium gluconate or a mixture thereof. In a particularly preferred embodiment of the present invention, the calcium salt is calcium chloride.

[0297] Optionally, a calcium salt is added to the emulsion in an amount of 0.2-2 mM / g protein.

[0298] Optionally, sodium chloride is added in an amount of 1 to 2% by weight of the emulsion.

[0299] Optionally, a polyphosphate (such as sodium polyphosphate) is added in an amount of 0.3% by weight of the emulsion.

[0300] Optionally, the method of the present invention comprises mixing non-phosphorylated casein or caseinate with transglutaminase to form a polymeric protein, adding fat to the polymeric protein to form an emulsion, and then performing at least one of the following steps to form a treated emulsion: i) cooling the emulsion to a temperature below 10°C; and / or ii) adding a calcium salt to the emulsion, and / or iii) adjusting the pH of the emulsion to a pH of 5 to 6 or adding rennet to the emulsion.

[0301] Optionally, the method of the present invention comprises mixing non-phosphorylated casein or caseinate with transglutaminase to form a polymeric protein, adding fat to the polymeric protein to form an emulsion, and subsequently adding rennet and / or acid to the emulsion to form a mixture, and further comprises at least one of the following processing steps: a) incubating the mixture, optionally performing conventional cleavage; and / or b) whey drainage; and / or c) plasticizing and shaping to produce cheese products; and / or d) cooling the cheese product.

[0302] Excessive kneading of the product should be avoided during the shaping step as this will reduce the moisture content of the final product.

[0303] As used herein, the term "high-moisture cheese product" refers to any cheese food product having a moisture content exceeding 60% by weight. Moisture content can be determined using an infrared dryer (MA 30, Sartorius). For example, a specific amount of grated pasta filata cheese can be weighed into an aluminum pan and heated using infrared radiation to a temperature of 105°C until a constant weight is reached, and the moisture content can be calculated from the weight loss.

[0304] The process can be carried out at standard atmospheric pressure, thus avoiding any expensive pressurization steps.

[0305] In another aspect, the present invention provides a pasta filata-style (e.g., mozzarella-style) cheese product formed using the methods described above. Because dephosphorylated casein / caseinate is used to form the pasta filata-style (e.g., mozzarella-style) cheese product, the cheese product is formed using dephosphorylated casein / caseinate proteins, as compared to traditional mozzarella cheese where only the casein is present in phosphorylated form.

[0306] Thus, the present invention provides pasta filata-style (eg, mozzarella-style) cheese products formed using dephosphorylated αs1-casein, αs2-casein, β-casein, and / or κ-casein.

[0307] Preferred or alternative features of each aspect or embodiment of the invention apply mutatis mutandis to every other aspect or embodiment of the invention (unless the context requires otherwise).

[0308] Example

[0309] Material

[0310] Bovine sodium caseinate (Lactonat EN, 90.2% protein, of which 41% is α s Casein, 42% β-casein, and 17% κ-casein) was donated by Lactoprot (Lactoprot Deutschland GmbH, Kaltenkirchen, Germany).

[0311] Tris(hydroxymethyl)aminomethane (Tris; 252859), bovine intestinal mucosal alkaline phosphatase (P7640), calcium chloride (C1016), magnesium chloride (M8266), potassium dihydrogen phosphate (P5379), disodium hydrogen phosphate (S7907), citric acid (C0759), potassium hydroxide (1.05033), sodium hydroxide (221465), potassium chloride (1.04936), potassium carbonate (1.04928), potassium sulfate (1.05153), trisodium citrate dihydrate (S4641), magnesium citrate nonahydrate (63067), bovine α-casein (C6780), bovine β-casein (C6905), bovine κ-casein (C 0406), hydrochloric acid (1.13386), nitric acid (1.00456), hydrogen peroxide (1.07209), calcium standard (1.19778), phosphorus standard (1.70340), magnesium standard (1.70331), sodium standard (1.70353), potassium standard (1.70230), sodium chloride (31434), disodium hydrogen phosphate dihydrate (1.06580), citric acid monohydrate (1.00244), anhydrous ethanol (1.00983), guanidine hydrochloride (50950), L-dithiothreitol (D9760), and lactic acid solution (252476) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

[0312] Osmium tetroxide (19134), 50% glutaraldehyde solution (16316-10), and carbon adhesive sheet (77825-12) were purchased from ESM (Electron Microscopy Sciences, Hatfield, PA, USA). Hydrochloric acid solution (7647-01-0) and acetonitrile ULC-MS (75-05-8) were purchased from Actu-All (Actu-All Chemicals BV, Oss, The Netherlands).

[0313] Tripotassium citrate monohydrate (6100-05-6) was purchased from VWR (VWR International bvba, Leuven, Belgium). Trifluoroacetic acid (44630) was purchased from Alfa Aesar (Thermo Fisher Scientific, Kandel, Germany). Recombinant chymosin (CHY-MAX Plus, batch number 3634543) was obtained from Chr. Hansen Holding A / S ( Ultrapure water (MilliQ system, Merck KGaA, Darmstadt, Germany) was used in all experiments.

[0314] Purification and isolation of β-casein and κ-casein

[0315] According to Schubert et al. (2018) 1 The methods and steps described herein purify and separate bovine beta-casein and kappa-casein from bovine sodium caseinate.

[0316] Example 1.1: Preparation of artificial casein micelles

[0317] By comparing Schmidt et al. (1977) 2 Artificial casein micelles ("ACM") were prepared by modifying the method of β-casein and κ-casein in various ratios (b:k; 70:30, 75:25, 80:20, 85:15 and 95:5) in water containing 22.5 mM sodium citrate and stirred at 60°C for 30 minutes to a total protein concentration of 64.1 g L -1 Two salt solutions were prepared: solution I contained 325 mM CaCl2 and 61.2 mM MgCl2, adjusted to pH 6.70 with 0.1 M HCl, and solution II contained 155 mM KH2PO4 and 155 mM Na2HPO4. Caseinate solution (60 mL) and salt solution (10 mL each) were carefully pumped into a jacketed glass vessel containing a starting volume of 66 mL of water at 37°C over 60 min to give final concentrations of 30 mM calcium, 22 mM phosphate, 9 mM citrate, 5 mM magnesium, and 25.6 g L -1 Casein. Stir the solution continuously and vigorously using a magnetic stirrer.

[0318] The addition of solution was controlled by using a syringe pump (Harvard PHD2000, Harvard Apparatus, Holliston, MA, USA and ProSense NE-1600, ProSense BV, Oosterhout, The Netherlands). The pH was maintained at 6.70 with 1M NaOH by titration (877 Titrino Plus, Metrohm AG, Barendrecht, The Netherlands), and titration was continued for 20 minutes to balance the pH after the desired micelle composition was achieved. In a typical experiment, approximately 3 mL of 1M NaOH was used. The total volume of the solution was adjusted to 150 mL with water. All samples were prepared in triplicate.

[0319] Example 2.1: Particle size analysis

[0320] The hydrodynamic diameter and polydispersity of casein micelles were analyzed using a Malvern Zetasizer Ultra (Malvern Panalytical Ltd, Worcestershire, UK) with a He-Ne laser at 633 nm. The samples were placed in a 400 nm sieve according to Jenness and Koops (1962). 3 The prepared simulated milk ultrafiltrate was diluted 100 times, where Dumpler et al. (2017) 4 Improvements were made in DTS0012 cells and measurements were performed at 25°C and a fixed scattering angle of 173°. The refractive index of the dispersant was set to 1.33, its viscosity was 0.8872 mPa.s, and the refractive index of the casein micelles was 1.57 (Griffin & Griffin, 1985). 5 Two measurements were performed for each sample, each consisting of 5 sub-measurements.

[0321] Table 1 below lists dynamic light scattering results comparing the size (Z-average diameter in nm; intensity-weighted mean hydrodynamic size, as defined in ISO 13321) of "normal" artificial casein micelles (ACM) produced from bovine sodium caseinate, and ACM produced according to the present invention from only isolated bovine β-casein and bovine κ-casein fractions, showing comparable diameters and polydispersity indices.

[0322] Table 1. Dynamic light scattering results showing the diameter (nm) of ACM prepared from bovine caseinate (containing all four caseins) and ACM prepared according to the present invention from only β-casein and κ-casein.

[0323]

[0324] Figure 2.1 The data in the literature show that the size of the obtained micelle particles is within the size range of natural bovine casein micelles (50-400 nm, average 200 nm; De Kruif (1998) 6 ), and the size of the particles increases with increasing β-casein levels (and therefore decreasing κ-casein levels). The number average diameter of the micelles observed in the micellar solutions of the present invention ranges from about 125 nm (at higher κ-casein ratios) to about 280 nm (at higher β-casein ratios) ( Figure 2.1 ).

[0325] Example 3.1: Scanning Electron Microscopy

[0326] By using Magellan 400 microscope (FEI Company, Hillsboro, OR, the U.S.), the micellar morphology was studied with a scanning electron microscope (SEM). A drop of sample was pipetted onto a 12 mm poly-L-lysine slide (Corning Inc., Corning, NY, the U.S.) and allowed to adhere for 30 minutes. Subsequently, the slide was washed twice with 0.1 M phosphate / citrate buffer (pH 7.2) and fixed with 2.5% glutaraldehyde for one hour after removing the buffer. Then, the fixative was removed and the slide was washed six times with phosphate / citrate buffer. The sample was then fixed with 1% osmium tetroxide solution for one hour. The fixative was removed again and the slide was washed three times with water and dehydrated using a gradient ethanol series (5 minutes 30%, 5 minutes 50%, 5 minutes 70%, 5 minutes 80%, 5 minutes 90%, 5 minutes 96%, 10 minutes 100% and another 10 minutes 100% EtOH). Subsequently, the samples were critical point dried with CO using a Leica EM CPD 300 (Leica Biosystems GmbH, Nussloch, Germany). The sputter-coated glass slides were mounted on aluminum sample stubs using carbon adhesive sheets. The mounted samples were coated with a 12 nm tungsten layer in a Leica EM SCD 500 sputter coater. Images were taken at 100,000X magnification.

[0327] The SEM images provide evidence for the presence of spherical micelles in the micellar solutions of the present invention. Figure 3.1 The distribution of micelle sizes is shown, and when the ratio of κ-casein is high, the micelle sizes are mostly smaller [ Figure 3.1 (1)ACM b:k 70:30, Figure 3.1 (2)ACM b:k 75:25, Figure 3.1 (3)ACM b:k 80:20], while larger micelles were observed when the ratio of β-casein was higher [ Figure 3.1 (4) ACM b:k 85:15], confirming the dynamic light scattering results presented above. For comparison, Figure 4.1 SEM images of bovine skim milk are provided, which have Figure 3.1 Same size scale. Figure 3.1 The micelles present in the micellar solution of (4) are similar in size to Figure 4.1 The micelles were comparable to those observed in bovine skim milk.

[0328] Example 4.1: Ultracentrifugation

[0329] Approximately 17 mL of sample was equilibrated at room temperature for 1 hour and then ultracentrifuged at 100,000 x g and 20° C. for 1 hour in a Beckman Coulter Optima XE-90 ultracentrifuge (Beckman Coulter Inc., Woerden, The Netherlands) equipped with a 70Ti rotor. All samples were centrifuged in duplicate. Approximately half of each supernatant was collected and the supernatants of the corresponding samples were combined.

[0330] Example 5.1: Mineral partitioning analysis

[0331] The samples and their supernatants were analyzed for cation content (calcium, phosphorus, magnesium, sodium, and potassium). 0.5 mL of the sample was mixed with aqua regia and H2O2. The digested material was diluted approximately 200-fold with water. Samples were prepared in duplicate. An Avio 500 ICP-OES system (Perkin Elmer Nederland BV, Groningen, the Netherlands) was used for analysis by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0332] Anion analysis was performed by ion chromatography (IC) on a Dionex ICS-6000 liquid chromatography system (Thermo Fisher Scientific BV, Breda, The Netherlands) equipped with a 2 mm standard pore Dionex IonPac AS17-C column to analyze the anion content (chloride, phosphate, and citrate) in the samples and their supernatants. The samples were diluted 200 times, and the supernatants were diluted 500 times in water. Peak detection was performed using a conductivity detector. The flow rate was set to 0.25 mL min -1 The injection volume was 5 μL and the column temperature was 30°C. Gradient elution with KOH was performed, initially set at 5 mM for 10 minutes, then linearly increased to 40 mM over 15 minutes, isocratic elution at 40 mM for 6 minutes, and linearly decreased to 5 mM over 5 minutes. Samples were analyzed in duplicate.

[0333] According to Pierre and Brule (1981) 7 The calculated correction factor K corrects all supernatant concentrations of ionic species for the excluded volume:

[0334] Where P is the measured micelle protein content (in g L -1 ), W is the micellar hydration measured (in g water g -1 dry matter).

[0335] When studying casein micelles, it is common practice to assume that all material that precipitates during ultracentrifugation is micelles and constitutes the micellar phase, while all material that does not precipitate (cannot precipitate) is the serum phase. The data in Table 2 show that the total concentration of ionic species (**) is stable across the sample. In addition, the distribution of ionic species (*) between the micellar and serum phases is comparable.

[0336] Table 2. Comparison of the composition of ionic species of the samples prepared in the present invention and natural bovine casein micelles

[0337] Compared with casein micelles from bovine skim milk, the partitioning of minerals between the micellar and serum phases showed that the formed artificial micelles “encapsulated” a similar amount of calcium phosphate nanoclusters as natural bovine casein micelles, indicating a similar structure of the micelles.

[0338] Example 6.1: Analysis of Micellar Casein Composition

[0339] According to Schubert et al. (2018) 1 The total casein content and non-precipitable casein content in the ultracentrifugation supernatant of the samples were determined using a modified reverse-phase high-performance liquid chromatography (RP-HPLC; Dionex UltiMate 3000 system, Thermo Fisher Scientific BV, Breda, The Netherlands) using a VDSpher OptiBio Pur 300C4-SE column (VDS Optilab, Berlin, Germany). The precipitable or micellar casein content was inferred by subtracting the non-precipitable casein content from the total casein content.

[0340] The eluent compositions during the analysis are shown in Table 3, where eluent A consisted of 1% (v / v) acetonitrile (ACN) and 0.1% (v / v) trifluoroacetic acid (TFA) in water, and eluent B consisted of 1% (v / v) water and 0.072% (v / v) TFA in ACN. Only linear gradients were used. The flow rate was set at 1.0 mL min -1 The injection volume was 10 μL, the column temperature was 30°C, and the detection wavelength was 214 nm. Samples were diluted 5:1, and supernatants were diluted 2:1 in a buffer solution of 6 M guanidine hydrochloride, 20 mM dithiothreitol, and 5 mM sodium citrate. The samples were incubated at room temperature for at least one hour before analysis. All analyses were performed in duplicate.

[0341] Table 3. Eluent composition during RP-HPLC analysis.

[0342] Time (min) Eluent A (%) Eluent B (%) 0.0 72.0 28.0 21.5 62.4 37.6 22.5 62.4 37.6 26.0 54.0 46.0 28.0 0.0 100.0 29.0 0.0 100.0 30.0 72.0 28.0 35.0 72.0 28.0

[0343] The total casein content of the prepared samples is shown in Table 4 below.

[0344] Table 4. Casein composition of prepared samples.

[0345]

[0346] abbreviation: ACM=Artificial Casein Micelles CN = Casein The target total casein concentration during the preparation of artificial casein micelles (ACM) was 25.6 g L -1 , and considering that there is a small amount of α s -Casein impurities, the total concentration measured in the prepared ACM was close to this concentration. β-casein (β-CN) concentration increased from 16.5 to 21.8 g L -1 , the concentration of κ-casein (κ-CN) decreased from 8.1 to 2.6 g L -1 Due to the presence of trace amounts of α s Casein, the resulting β:κ-casein ratio is shown in the last column and ranges from 63% β-casein and 31% κ-casein (thus 6% α s -casein) to 88% beta-casein and 11% kappa-casein (thus 2% alpha s -casein).

[0347] The micellar casein content or precipitable casein content of the micellar solution is plotted on Figure 5.1 The range was from about 84% (ACM b:k 70:30) to greater than 90% (ACM b:k 95:5). This data indicates that about 10-15% of the casein did not precipitate during ultracentrifugation and can therefore be considered as non-micellar casein present in the serum phase. This is consistent with the level of micellar casein in bovine casein micelles, which is typically around 90% (Cerbulis, 1969). 8 ; Huppertz et al., 2021 9 ; Reiter et al., 2022 10 ).

[0348] Example 7.1: Determination of Casein Micellar Hydration

[0349] The ultracentrifuged pellet was weighed in an aluminum crucible and dried in a hot air oven (Binder model E 28, Binder GmbH, Tuttlingen, Germany) at 105 °C for approximately 48 h. 11The method calculates casein micellar hydration by dividing the moisture content of the pellet by the dry matter of the pellet.

[0350] Figure 6.1 The data in show that the micelles obtained are highly hydrated (2.5->4.0 g water / g protein), which is characteristic of casein micelles (Huppertz et al., 2017) 11 The higher the proportion of β-casein present in the micelles, the less hydrated the micelles are.

[0351] Example 8.1: Rheological Characterization

[0352] After the rennet coagulation, oscillatory rheometry was performed. The samples were adjusted to pH 5.8 or 6.3 by acidification with dilute lactic acid at a temperature below 10°C, and the pH of some samples was maintained at pH 6.7 without adjustment. Subsequently, 0.05% or 0.10% (v / v) of 40% (w / v) calcium chloride solution was added, and the samples were heated to 30°C while stirring. The samples were then coagulated for one hour with 0.02% (v / v) rennet (added in the form of a 10% (v / v) solution) and transferred to a rheometer (Physica MCR 302, Anton Paar, Graz, Austria) equipped with a double gap device (DG26.7). A strain amplitude of 0.001 and a frequency of 1 Hz were applied, while the temperature was set to 30°C. The maximum G' (storage modulus; a measure of curd firmness) was measured in duplicate.

[0353] The results are listed in Table 5: Table 5. Maximum G' (storage modulus; a measure of firmness) of curds produced after rennet-induced coagulation.

[0354]

[0355] Figure 7.1 Data are presented for curds produced by rennet-induced coagulation of samples at pH 6.3 with the addition of 0.10% CaCl2, demonstrating that micelles can coagulate and form curds. Curd firmness decreases almost linearly with increasing β:κ ratios. Micelles with a β:κ ratio of 63:31 produce the strongest curds, with a storage modulus of approximately 45 Pa. In cheese production, curds are cut when they reach sufficient firmness and then further processed into cheese by aging or drying.

[0356] The present invention's method, intended for use in preparing artificial casein micelles from only β- and κ-caseins, surprisingly, unexpectedly, and advantageously produces highly hydrated, spherical particles of similar size to bovine casein micelles, capable of coagulation to a certain extent, and exhibiting microscopic micelle-like properties. The micelles and micellar solutions prepared from these two caseins according to the present invention also have a favorable mineral salt content, comparable to that of bovine casein micelles, and are suitable for producing cheese and other downstream products with favorable flavor characteristics, texture, and other functional properties.

[0357] Example 0.2A: Production of phosphorylated kappa-casein (native KCN)

[0358] Micellar casein (MCN) with reduced β-casein was obtained according to the protocol of Schaefer et al. 12 and modified by performing two additional cold filtration steps to further reduce the β-casein content.

[0359] According to Schubert et al. 13 The protocol for processing β-casein-reduced MCNs, wherein calcium precipitation and subsequent isolation are performed 1.

[0360] The collected supernatant containing phosphorylated κ-casein was filtered through a 1 kDa membrane and diafiltered with a filtration coefficient of 6 to remove minerals and residual lactase, and then concentrated for spray drying. The collected concentrate containing phosphorylated κ-casein was spray dried.

[0361] The total protein concentration of the resulting powder was 80%.

[0362] Example 0.2B: Production of dephosphorylated kappa-casein (DPKCN) and dephosphorylated beta-casein (DPBCN)

[0363] A 2.5% protein solution of phosphorylated κ-casein was prepared using 10mM TRIS buffer at pH 8.0. The hydrated protein solution was heated to 37°C and 10mg / 100mL alkaline phosphatase was added while mixing. The incubation conditions were 37°C for 8 hours with gentle mixing. The incubation was further extended to overnight at room temperature. The phosphate groups that were cut were removed by diafiltration using a nanofiltration (10kDa) membrane. The collected dephosphorylated κ-casein (retentate from the diafiltration process) was spray dried.

[0364] To generate dephosphorylated β-casein, first the protein was purified according to Schaefer et al. 12The method described herein obtains a natural β-casein concentrate. Micellar casein concentrate is obtained from skim milk by a warm microfiltration (MF) method at 50°C (0.1 μm pore size, ceramic membrane). The concentrate is stored at 2-3°C for about 40 h to induce temperature-dependent dissociation of β-casein from the casein micelles. β-casein is separated from the refrigerated concentrate using microfiltration (MF, 0.3 μm pore size, organic membrane) at ≤5°C. The β-casein permeate is heated to 50°C to cause self-association of β-casein micelles and then ultrafiltered at 50°C (10 kDa cut-off, organic membrane). The obtained natural β-casein concentrate has a purity of 92-93% and a yield of up to 18%, and is dephosphorylated by the same method as the above-mentioned κ-casein dephosphorylation.

[0365] A 2.5% protein solution of phosphorylated β-casein was prepared using 10mM TRIS buffer at pH 8.0. The hydrated protein solution was heated to 37°C and 10mg / 100mL alkaline phosphatase was added while mixing. The incubation conditions were 37°C for 8 hours with gentle mixing. The incubation was further extended to overnight at room temperature. The phosphate groups that were cut were removed by diafiltration using a nanofiltration (10kDa) membrane. The collected dephosphorylated β-casein (retentate from the diafiltration process) was spray dried.

[0366] Example 1.2: Production of mozzarella cheese using phosphorylated κ-casein (native KCN)

[0367] Phosphorylated kappa-casein (native KCN, 5% total protein concentration) was hydrated for 60 minutes at room temperature. To prepare the emulsion, a fat to protein ratio of approximately 1:1 was used. Once the vegetable-based fat (rapeseed oil) was added, the mixture was pre-homogenized using an ultraturrax at 10,000 rpm and room temperature for 3 minutes. The mixture was further emulsified using a two-stage homogenizer at 200 / 50 bar at room temperature. Approximately 10 mM CaCl2 was added to the emulsion and the emulsion was acidified to pH 5.2 with 1 M hydrochloric acid. The acidified mixture was incubated at 37°C for 30 minutes. The resulting gel phase (curd) was separated from the whey. Figure 1.2 ,a),1.]. The separated curd is textured in hot water at a curd to water ratio of 1:2 at 75-85°C. The curd is kneaded and stretched. Figure 1.2 ,a),2.] and folded into balls and then cooled in water. The final product was then stored in a 0.1% lactic acid solution. The melting test showed the typical melting behavior of mozzarella cheese [ Figure 1.2 ,a),3.].

[0368] Example 2.2: Production of Mozzarella Cheese Using Dephosphorylated κ-Casein (DPKCN)

[0369] Dephosphorylated kappa-casein (total protein concentration 5%) was hydrated for 60 min at room temperature. To prepare the emulsion, a fat to protein ratio of approximately 1:1 was used. Once the vegetable-based (rapeseed oil) fat was added, the mixture was pre-homogenized using an ultraturrax at 10,000 rpm and room temperature for 3 min. The mixture was further emulsified using a two-stage homogenizer at 200 / 50 bar at room temperature. Approximately 10 mM CaCl2 was added to the emulsion and acidified to pH 5.2 with 1 M hydrochloric acid. The acidified mixture was incubated at 37°C for 30 min. The resulting gel phase (curd) was separated from the whey. Figure 1.2 ,b),1.]. The separated curd is textured in hot water at a curd to water ratio of 1:2 at 75-85°C. The curd is kneaded and stretched. Figure 1.2 ,b),2.] and folded into balls and then cooled in water. The final product was then stored in a 0.1% lactic acid solution. The melting test showed the typical melting behavior of mozzarella cheese [ Figure 1.2 ,b),3.].

[0370] Example 3.2: Production of β-casein using phosphorylated κ-casein (native KCN) and dephosphorylated β-casein (DPBCN) Raw mozzarella cheese

[0371] As a protein source, dephosphorylated β-casein (DPBCN) and phosphorylated κ-casein (native KCN) were mixed in a 3:1 ratio to achieve a final protein concentration of 5%. The protein mixture was hydrated at room temperature for 60 minutes. To prepare the emulsion, a fat to protein ratio of 1:1 was used. Once the vegetable-based fat (rapeseed oil) was added, the mixture was pre-homogenized using an Ultraturrax at 10,000 rpm and room temperature for 3 minutes. The mixture was further emulsified using a two-stage homogenizer at 200 / 50 bar at room temperature. The resulting emulsion was subjected to the following five protocols for curd production and downstream processing.

[0372] Option 1: No heat treatment, no transglutaminase treatment

[0373] 10 mM CaCl2 was added to the emulsion and the emulsion was acidified to pH 5.2 using 1 M HCl. The acidified mixture was incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 2.2 ,a),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 2.2 ,a),2.], folded into balls and cooled in water. The final product is then stored in a 0.1% lactic acid solution. This method produces fine, non-cohesive curds that retain their shape well when lightly pressed. During texturization, the curds are very soft and elastic.

[0374] Option 2: Heat treatment before adding calcium

[0375] The emulsion was heated at 45-70°C for 30 min and then cooled back to room temperature. 10 mM CaCl2 was then added to the emulsion and the emulsion was acidified to pH 5.2 with 1 M HCl. The acidified mixture was incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 2.2 ,b),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 2.2 ,b),2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. Melting tests showed the typical melting behavior of mozzarella cheese [ Figure 2.2 ,b),3.]. Compared to Scheme 1, this scheme produced larger aggregates and better whey separation during curd formation, as well as increased extensibility during texturization.

[0376] Option 3: Heat treatment after calcium addition

[0377] 10 mM CaCl2 was added to the emulsion and the emulsion was heated at 45-70°C for 30 min. The emulsion was then allowed to cool back to room temperature and acidified to pH 5.2 using 1 M HCl. The acidified mixture was incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 2.2 ,c),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 2.2 ,c),2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. Melting tests showed the typical melting behavior of mozzarella cheese [ Figure 2.2 ,c),3.]. Compared to Scheme 1, this scheme produced a very fine, soft and non-cohesive gel. The gel could be slightly squeezed to remove the whey, then retained its shape and showed increased stretchability during texturing.

[0378] Option 4: Transglutaminase treatment followed by heat treatment before calcium addition

[0379] To investigate the effect of cross-linking with transglutaminase (TG), the emulsion was incubated with 0.1% (or a 2:100 enzyme / substrate ratio) TG at 50°C for 30 min. The emulsion was then heated at 50-70°C for 30 min, cooled back to room temperature, and then 10 mM CaCl2 was added to the emulsion, which was acidified to pH 5.2 using 1 M HCl. The acidified mixture was incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 2.2 ,d),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 2.2 ,d),2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. The resulting curd was very similar to that produced in Option 2, but with slightly reduced extensibility during texturization.

[0380] Protocol 5: Transglutaminase treatment followed by calcium addition and heat treatment

[0381] 10 mM CaCl2 was added to the emulsion. To investigate the effect of cross-linking with transglutaminase (TG), the emulsion was incubated with 0.1% (or a 2:100 enzyme / substrate ratio) TG at 50°C for 30 min. The emulsion was then heated at 50-70°C for 30 min, cooled back to room temperature, and then acidified to pH 5.2 using 1 M HCl. The acidified mixture was incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 2.2 ,e),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Figure 2.2 ,e),2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. The resulting curd was very similar to that produced in Option 3, but with a slightly increased yield and the least stretchability during texturization of any of the five options in Example 3.2.

[0382] Example 4.2: Rennet treatment

[0383] Scheme I – Production of mozzarella cheese using phosphorylated kappa-casein (native KCN) and dephosphorylated beta-casein (DPBCN) with heat treatment before calcium addition and rennet treatment after calcium addition

[0384] In this exemplary embodiment, a 3:1 emulsion of dephosphorylated β-casein (DPBCN) and phosphorylated κ-casein (native KCN) prepared according to Example 3.2 was used. The emulsion was heated at 45-70°C for 30 min and then cooled back to room temperature. 10 mM CaCl2 was then added to the emulsion and the emulsion was acidified to pH 5.2 with 1 M HCl. Rennet was added to the acidified mixture and incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 3.2 ,a),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 3.2,a),2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. Compared to Example 3.2, Option 2, rennet treatment resulted in a lower yield. Rennet treatment produced a soft / pasty curd that exhibited good stretchability during texturization.

[0385] Scheme II - Production of mozzarella cheese using phosphorylated kappa-casein (native KCN) and dephosphorylated beta-casein (DPBCN) with heat treatment after calcium addition followed by rennet treatment.

[0386] In this exemplary embodiment, a 3:1 emulsion of dephosphorylated β-casein (DPBCN) and phosphorylated κ-casein (native KCN) prepared according to Example 3.2 was used. 10 mM CaCl2 was added to the emulsion and the emulsion was heated at 45-70°C for 30 min. The emulsion was then allowed to cool back to room temperature and acidified to pH 5.2 using 1 M HCl. Rennet was added to the acidified mixture and incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 3.2 ,b),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 3.2 ,b), 2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. Compared to Example 3.2, Option 3, rennet treatment resulted in a lower yield. Rennet treatment produced a soft / pasty curd that exhibited good stretchability during texturization.

[0387] Scheme III - Production of mozzarella cheese using dephosphorylated kappa-casein (DPKCN) with heat treatment before calcium addition and rennet treatment after calcium addition.

[0388] In this exemplary embodiment, a dephosphorylated κ-casein (DPKCN) emulsion prepared according to Example 2.2 was used. The emulsion was heated at 45-70°C for 30 min and then cooled back to room temperature. 10 mM CaCl2 was then added to the emulsion and the emulsion was acidified to pH 5.2 with 1 M HCl. Rennet was added to the acidified mixture and incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 3.2 ,c),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 3.2,c), 2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. Compared to Example 2.2, heat treatment and rennet treatment resulted in a lower yield. Rennet treatment produced a soft / pasty curd that exhibited good stretchability during texturization.

[0389] Scheme IV – Production of mozzarella cheese using dephosphorylated κ-casein (DPKCN) with heat treatment after calcium addition and subsequent rennet treatment

[0390] In this exemplary embodiment, a dephosphorylated κ-casein (DPKCN) emulsion prepared according to Example 2.2 was used. 10 mM CaCl2 was added to the emulsion and the emulsion was heated at 45-70°C for 30 min. The emulsion was then allowed to cool back to room temperature and acidified to pH 5.2 using 1 M HCl. Rennet was added to the acidified mixture and incubated at 37°C for 30 min. The gel phase (curd) was then separated from the whey. Figure 3.2 ,d),1.] and texturize the curd in hot water at 75-85°C with a curd to water ratio of 1:2. Knead and stretch the curd. Figure 3.2 ,d), 2.], folded into balls and cooled in water. The final product was then stored in a 0.1% lactic acid solution. Compared to Example 2.2, heat treatment and rennet treatment resulted in a lower yield. Rennet treatment produced a soft / pasty curd that exhibited good stretchability during texturization.

[0391] Example 1.3: Emulsions, curd compositions and cheeses containing non-animal source protein

[0392] Zein protein (Zein F4400C - Food grade Flo corporation: Supplier: AF Suttor & Co Ltd) was used as an exemplary non-animal source protein in the aforementioned examples. Commercially available zein was deodorized / decolorized according to the method described in US Pat. No. 7,939,633.

[0393] Prepare 100 mL of 8% zein solution containing 65% ethanol and mix at room temperature for 30 minutes. In a 50 mL syringe, place about one inch thick cotton wool, followed by a layer of Whatman filter paper, and place 25 g of molecular sieves (MS) on top of the filter paper. Pass 40 mL of 65% ethanol through the filled syringes to wet the MS. Add 50 mL of 8% zein solution to each syringe and pass it through the MS, collecting the filtrate in a beaker. Heat is observed to be released during the filtration process. The MS is then washed twice with 50 mL of 65% ethanol and the filtrate is collected in the same beaker.

[0394] In another 50 mL syringe, place approximately one inch thick cotton wool, followed by a layer of Whatman filter paper, and place 12.25 g of activated carbon on top of the filter paper. The filtrate collected in the previous step is added to each syringe and passed through the activated carbon. The filtrate is collected in an oil-coated beaker. The resulting twice-filtered zein solution is then dried at 50°C to obtain a zein film. The zein film is then scraped from the beaker, ground, and stored in an airtight container for use in the previous examples.

[0395] NMC or β-CN alone is used as the protein source. However, the methods used to produce mozzarella cheese are almost similar. Therefore, the protein source is referred to as casein herein.

[0396] To produce zein:casein-based mozzarella cheese, approximately 25-40% of the casein is replaced with zein. The fat-to-protein ratio and lactose used in the production process are 1.35 and 3.1%, respectively. Zein protein exhibits high hydrophobicity, which hinders hydration in distilled water at its natural pH. Therefore, different methods are used to introduce zein protein into the premix. These methods are described below.

[0397] Table 1.3 shows the recipes used for the production of mozzarella cheese containing either sodium caseinate and zein or beta-casein and zein. Approximately 25% of the total protein content has been replaced by zein.

[0398] Table 1.3

[0399] Method 1: Hydration at a Highly Alkaline pH

[0400] The pH of the distilled water was adjusted to 12.5. According to the recipe, the zein protein and alkalized water were mixed at room temperature for 30 minutes. Casein was then added to the zein mixture and allowed to hydrate at room temperature for 30 minutes at the same pH. Once the protein was hydrated, the pH was adjusted to 7.0, and fat and lactose were added according to the recipe. The premix was then subjected to high shear at 16,000 rpm for 5 minutes, followed by homogenization at 200 / 50 bar at room temperature.

[0401] Method 2: Enzymatic hydrolysis of zein

[0402] The pH of distilled water was adjusted to 12.5. According to the recipe, zein protein and alkalized water were mixed at room temperature for 30 min. The pH was then adjusted to 9.0, and the temperature was raised to 50 ° C. In order to perform enzymatic hydrolysis, partial to complete hydrolysis (using alcalase 2.4L FG, enzyme / substrate ratio of 12.5: 100 or 25: 100) was performed. Protein hydrolysis was performed at 50 ° C for 1 h. The pH of the protein mixture was then reduced to 7.0, and the enzyme was inactivated at 95 ° C for 10 min. In addition, casein was added to the zein mixture and hydrated at room temperature for 30 min at the same pH. The premix was then subjected to high shear for 5 minutes at 16000 rpm and subsequently homogenized at room temperature at 200 / 50 bar.

[0403] Method 3: Adding Zein to the Premix by High Shear

[0404] Initially, casein was hydrated in distilled water at room temperature for 30 min (NMC) or 180 min (β-CN). Subsequently, lactose and fat were added and mixed for 5 min. The premix was then subjected to high shear treatment at 16,000 rpm for 10 min while zein was slowly added. This was followed by homogenization at room temperature at 200 / 50 bar.

[0405] Mozzarella cheese production: The emulsion is cooled to 5-10°C. 30mM CaCl2 is then added and directly acidified to pH 5.2 using a 30% lactic acid solution. The acidified emulsion temperature is further increased to 37°C to perform rennet-induced coagulation (100 μL / 100mL) at 37°C for 30min. When β-CN is used as a casein source, rennet coagulation is not performed. By passing the coagulum through a sieve and a cheesecloth layer, the gel-like solid phase is separated from the liquid phase. The curd is then textured in a hot water bath (70-85°C). The curd is stretched and kneaded for 2-3min as performed in a conventional mozzarella cheese production process. The ball-like portion is then cooled and stored in a cold 0.1% lactic acid solution.

[0406] The cheese according to this embodiment is shown in Figure 2.3 middle.

[0407] Example 1.4: Formation of Mozzarella-like Cheese Using Dephosphorylated Sodium Caseinate

[0408] Dephosphorylated sodium caseinate (DPNaCN) is produced by dephosphorylating sodium caseinate with alkaline phosphatase. This is used to simulate non-phosphorylated casein produced by recombinant expression using bacterial fermentation.

[0409] Transglutaminase (TG) treatment was performed as follows: a 5% (by weight) DPNaCN solution was prepared. TG (4 U / g protein) was then added, and the resulting mixture was incubated at 50°C for 30 minutes to induce protein polymerization. The enzyme was then inactivated by high temperature treatment (80°C for 2 minutes).

[0410] To test the coagulation and gelling properties of TG-treated DPNaCN, emulsions were prepared by adding edible vegetable oil to a protein solution at a fat-to-protein ratio of 1:1 (by weight) and a homogenization step. Gel formation was then induced by slow acidification or by a combination of acidification and rennet as described below.

[0411] Example 2.4: Inducing gelation by slow acidification

[0412] Before gelation, 4 mmol of CaCl2 was added to the emulsion. Gel formation was induced by slowly acidifying the emulsion with 1% glucono-delta-lactone at 36°C for 2 h.

[0413] The texture of the produced gels was analyzed by texture analysis and the curd yield was measured after acidification. The acidic gels based on untreated DPNaCN were softer and more brittle than the gels based on TG-treated DPNaCN and showed lower cohesion and higher syneresis (see Figure 1.4 and 2.4 ). Figure 3.4 It was shown that gel firmness increased with increasing TG concentration during DPNaCN treatment. Curd yield also appeared to increase slightly ( Figure 4.4 ). The curd is shown in Figure 5.4 middle.

[0414] Example 3.4: Combined acid and rennet-induced gelation

[0415] Prior to gelation, 4 mmol CaCl2 was added to the emulsion and the pH was adjusted to 5.6 by adding 30% lactic acid to the cold emulsion (5°C). The acidified emulsion was coagulated by adding rennet and incubating at 36°C for 30 min. After incubation, the coagulated gel was cut into 1-2 cm cubes to allow the release of whey. The curd was separated from the whey and allowed to drain for 30 min. During the whey draining, the curd was gently mixed every 5 min to remove any excess whey. The curd was then textured by adding 80°C hot water in a 1:1 curd to water ratio and kneading and stretching by hand. Finally, the textured curd was molded into balls and cooled in water. The results are shown in FIG. Figure 6.4 shown.

[0416] Overview

[0417] Each document, reference, patent application or patent cited herein is expressly incorporated herein by reference in its entirety, which means that the reader should read and consider it as part of this document. Documents, references, patent applications or patents cited herein are not repeated herein solely for reasons of brevity.

[0418] It should be appreciated that any reference throughout this specification to any prior publication (including prior patent publications and non-patent publications) is not an acknowledgement or admission that any of the material contained in the referenced prior publication was part of the common general knowledge as at the priority date of the application.

[0419] Manufacturer's instructions, descriptions, product specifications, and product data sheets for any products mentioned herein, or any products mentioned in any documents cited herein, are incorporated herein by reference and may be used in the practice of the present invention.

[0420] The invention described herein may include one or more numerical ranges (e.g., size, displacement, field strength, etc.). A numerical range will be understood to include all values ​​within that range, including the values ​​defining the range, as well as values ​​adjacent to the range that result in the same or substantially the same results as the values ​​immediately adjacent to the boundaries defining the range.

[0421] The scope of the present invention is not limited to any specific embodiments described herein. These embodiments are for illustrative purposes only. As described herein, functionally equivalent products, formulations, and methods are clearly within the scope of the present invention.

[0422] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The present invention includes all such variations and modifications. The present invention also includes all steps, features, formulations, and compounds referred to or indicated in this specification, whether singly or collectively, and any and all combinations of any two or more of said steps or features.

[0423] References

[0424] 1.Schubert,T.,Meric,A.,Boom,R.M.,Hinrichs,J.,&Atamer,Z.(2018).Application of a deca nter centrifuge for casein fractionation on pilotscale:Effect of operational parameters on total solid,purity and yield insolid discharge.International Dairy Journal,84,6–14.https: / / doi.org / 10.1016 / j.idairyj.2018.04.002

[0425] 2.Schmidt,D.G.,Koops,J.,&Westerbeek,D.(1977).Properties of artificialcasein micelles.1.Preparation,size distribution and composition.NetherlandsMilk and Dairy Journal,31(4),328–341.

[0426] 3.Jenness,R.,&Koops,J.(1962).Preparation and properties of a saltsolution which simulates milk ultrafiltrate.Netherlands Milk and DairyJournal,16,153–164.

[0427] 4.Dumpler,J.,Kieferle,I., H.,&Kulozik,U.(2017).Milkultrafiltrate analysis byion chromatography and calcium activity for SMUFpreparation for different scientific purposes and prediction of itssupersaturation.International Dairy Journal,68,60–69.https: / / doi.org / 10.1016 / j.idairyj.2016.12.009

[0428] 5.Griffin,M.C.A.,&Griffin,W.G.(1985).A simple turbidimetric methodfor the determinati on of the refractive index of large colloidal particlesapplied to casein micelles.Journalo fColoid And Interface Science,104(2),409–415.https: / / doi.org / 10.1016 / 0021-9797(85)90049-9

[0429] 6.De Kruif,C.G.(1998).Supra-aggregates of casein micelles as aprelude to coagulation.Journ al of Dairy Science,81(11),3019–3028.https: / / doi.org / 10.3168 / jds.S0022-0302(98)75866-7

[0430] 7.Pierre,A.,&Brule,G.(1981).Mineral and protein equilibria betweenthe colloidal and soluble phases of milk at low temperature.Journalo fDairyResearch,48(3),417–428.https: / / doi.org / 10.1017 / S0022029900021890

[0431] 8.Cerbulis,J.(1969).Influence of dispersing agents on micelle contentof milk and lipid content of casein fractions.Journal o fAgricultural andFood Chemistry,17(5),1085–1088.https: / / doi.org / 10.1021 / jf60165a024

[0432] 9.Huppertz,T.,Heck,J.,Bijl,E.,Poulsen,N.A.,&Larsen,L.B.(2021).Variation in casein distribution and mineralisation in the milk fromHolstein-Friesian cows.International Dairy Journal,119,105064.https: / / doi.org / 10.1016 / j.idairyj.2021.105064

[0433] 10.Reiter,M.,Reitmaier,M.,&Kulozik,U.(2022).Compositional changes ofcasein micelles induced by calcium or chelatant addition at threefold andnatural casein concentration.International Dairy Journal,130,105365.https: / / doi.org / 10.1016 / j.idairyj.2022.105365

[0434] 11.Huppertz,T.,Gazi,I.,Luyten,H.,Nieuwenhuijse,H.,Alting,A.,&Schokker,E.(2017).Hydration of casein micelles and caseinates:Implicationsfor casein micelle structure.International Dairy Journal,74,1-11.https: / / doi.org / 10.1016 / j.idairyj.2017.03.006

[0435] 12.Schaefer,J.,Schubert,T.,&Atamer,Z.,“Pilot-scale b-casein depletionfrom micellar casein via cold microfiltration in diafiltration mode”,International Dairy Journal,2019,97,222e229.

[0436] 13.Schubert,T.,Ergin,I.,Panetta,F.,Hinrichs,J.,Atamer,Z.,“Applicationof a temperature-controlled decanter centrifuge for the fractionation of αs-,β-and κ-casein on pilot scale”,International Dairy Journal,2021,122,105148.

Claims

1. A micellar solution comprising a plurality of artificial casein micelles, wherein the artificial casein micelles comprise isolated non-human β-casein and isolated non-human κ-casein, and wherein the artificial casein micelles are substantially free of α-casein. s -Casein.

2. The micellar solution according to claim 1, wherein The Z-average diameter of the artificial casein micelles is greater than 30 nm.

3. A micellar solution according to claim 1 or claim 2, wherein the micellar calcium content is at least 70% of the total calcium in the micellar solution.

4. The micellar solution according to any one of claims 1 to 3, wherein the micellar magnesium content is at least 30%, preferably at least 35%, of the total magnesium in the micellar solution.

5. A micellar solution according to any one of claims 1 to 4 comprising less than 7 wt% of α-casein, calculated as a percentage of the total casein present in the micellar solution. s -Casein.

6. The micellar solution according to any one of claims 1 to 5, comprising a wt% ratio of non-human β-casein to non-human κ-casein falling within the range of 10:90 to 90:

10.

7. The micellar solution according to any one of claims 1 to 6, comprising a total casein concentration falling within the range of 10 g / L to 95 g / L.

8. The micellar solution according to any one of claims 1 to 7, comprising a total non-human β-casein concentration falling within the range of 1 g / L to 90 g / L.

9. The micellar solution according to any one of claims 1 to 8, comprising a total non-human kappa-casein concentration falling within the range of 1 g / L to 90 g / L.

10. The micellar solution according to any one of claims 1 to 9, comprising α s1 -Casein concentration.

11. The micellar solution according to any one of claims 1 to 10, comprising α s2 -Casein concentration.

12. The micellar solution according to any one of claims 1 to 11, wherein the Z-average diameter of the artificial casein micelles falls within the range of 40 to 500 nm.

13. The micellar solution according to any one of claims 1 to 12, wherein the hydration of the artificial casein micelles falls within the range of 1 to 8 (g water / g micellar protein).

14. Micellar solution according to any one of claims 1 to 13, comprising non-micellar casein as a percentage of total casein falling within the range of 5 to 20%.

15. The micellar solution according to any one of claims 1 to 14, wherein the micellar inorganic phosphate content is at least 50% of the total inorganic phosphate in the micellar solution.

16. The micellar solution according to any one of claims 1 to 15, wherein the micellar citrate content is at least 5% of the total citrate in the micellar solution.

17. A curd composition comprising the micellar solution of any one of claims 1 to 16 in coagulated form. The curd composition according to claim 17 , further comprising a curdling agent.

19. A coagulation composition according to claim 17 or claim 18, which preferably has a maximum G' (storage modulus) falling within the range of 5 to 200 Pa after incubation with rennet for 1 hour.

20. An edible composition comprising the micellar solution of any one of claims 1 to 16 or the curd composition of any one of claims 17 to 19.

21. An edible composition according to claim 20, wherein the edible composition does not contain any protein of animal origin.

22. A method for producing an edible composition comprising: The isolated non-human β-casein, the isolated non-human κ-casein, and at least one salt are combined under conditions where the β-casein and κ-casein form a micellar solution, wherein the micellar solution is substantially free of α-casein. s - casein; and The micellar solution is subjected to a first condition to form a coagulum.

23. The method according to claim 22, wherein the first condition is the addition of acid or the acidification of the micellar solution with a microorganism.

24. The method of claim 22 or 23, wherein the method further comprises subjecting the coagulum to a coagulant to form a coagulant curd.

25. The method of claim 24, wherein the method further comprises aging and / or ripening the curdled curd to form a cheese composition.

26. A method according to any one of claims 22 to 25, wherein the edible composition does not contain any protein of non-animal origin.

27. A method for preparing a curd composition, wherein the method comprises the following steps: a) preparing an emulsion comprising: (i) phosphorylated κ-casein; or (ii) dephosphorylated or non-phosphorylated κ-casein; or (iii) phosphorylated κ-casein and dephosphorylated or non-phosphorylated κ-casein; or (iv) phosphorylated kappa-casein, dephosphorylated or non-phosphorylated kappa-casein, and dephosphorylated or non-phosphorylated beta-casein; or (v) dephosphorylated or non-phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; or (vi) phosphorylated kappa-casein and dephosphorylated or non-phosphorylated beta-casein; and (vii) optionally, a lipid source; b) adding a calcium salt to the emulsion; and c) causing the emulsion to coagulate.

28. The method according to claim 27, wherein step c) comprises adding acid; and / or adding rennet; and / or adding chymosin to provide a coagulated emulsion.

29. A method according to claim 27 or claim 28, further comprising the additional steps of heating the emulsion prior to step b); and / or heating the emulsion prior to step c); maintaining the emulsion at a suitable temperature for a suitable time; preferably, wherein the temperature falls within the range of 45°C to 70°C; preferably, wherein the time period falls within the range of 1 min to 180 min.

30. The method according to any one of claims 27 to 29, further comprising the additional step of treating the emulsion with a transglutaminase before step b); and / or treating the emulsion with a transglutaminase before step c); preferably, wherein the additional step of treating the emulsion with a transglutaminase comprises adding transglutaminase to the emulsion and incubating at a suitable temperature for a suitable period of time; preferably, wherein the temperature falls within the range of 45°C to 60°C; preferably, wherein the time period falls within the range of 1 min to 180 min.

31. The method according to any one of claims 27 to 30, wherein the emulsion comprises a total protein concentration falling within the range of 2 wt.% to 20 wt.%; preferably, wherein the emulsion comprises a total protein concentration falling within the range of 3 wt.% to 10 wt.%; most preferably, wherein the emulsion comprises a total protein concentration falling within the range of 4 wt.% to 8 wt.%.

32. A method according to any one of claims 27 to 31 , wherein the emulsion comprises a fat to protein ratio falling within the range of fat free to 2:0.5; preferably, wherein the emulsion comprises a fat to protein ratio falling within the range of 1 :1.5 to 1.5:1 ; most preferably, wherein the emulsion comprises a fat to protein ratio of about 1 :

1.

33. The method according to any one of claims 27 to 32, wherein the calcium salt is added to bring the concentration of calcium in the emulsion to a concentration falling within the range of 2 mM to 20 mM; preferably, wherein the calcium salt is added to bring the concentration of calcium in the emulsion to a concentration falling within the range of 5 mM to 15 mM; most preferably, wherein the calcium salt is added to bring the concentration of calcium in the emulsion to a concentration falling within the range of 8 mM to 12 mM.

34. The method according to any one of claims 27 to 33, wherein the calcium salt is selected from the group consisting of calcium chloride, calcium hydroxide, calcium carbonate, calcium citrate, calcium phosphate, calcium stearate, calcium malate, calcium glycerophosphate, calcium lactate and calcium gluconate or mixtures thereof.

35. The method according to any one of claims 27 to 34, wherein the lipid source is selected from the group consisting of non-animal fats or oils, vegetable fats or oils, microbial fats or oils, fungal fats or oils, recombinantly produced fats or oils, or mixtures of any of the foregoing lipid sources; preferably, wherein the lipid source is an edible vegetable oil selected from the group consisting of peanut oil, soybean oil, sunflower oil, safflower oil, rapeseed oil, corn oil, avocado oil, almond oil, olive oil, cottonseed oil, coconut oil, sesame oil, chia (Salvia Hispanica L.) seed oil, wheat germ oil, grapeseed oil, rice bran oil, linseed oil, mustard oil, palm oil, castor oil, hydrogenated castor oil, hemp seed oil, and any mixtures thereof.

36. The method according to any one of claims 27 to 35, wherein step c) of coagulating the emulsion comprises acidifying the emulsion by adding acid, or by treating it with a microbial acidifier, or by a fermentation process, or by hydrolysis or cleavage of glucono-delta-lactone.

37. The method according to any one of claims 27 to 36, wherein step c) of coagulating the emulsion comprises acidifying the emulsion to a pH falling within the range of pH 4.2 to pH 6.5; preferably, wherein coagulating the emulsion comprises acidifying the emulsion to a pH falling within the range of pH 4.8 to pH 5.8; most preferably, wherein acidifying the emulsion comprises bringing the pH of the emulsion to about 5.

2.

38. The method according to any one of claims 27 to 37, further comprising the steps of: d) incubating the coagulated emulsion produced in step c) at a suitable temperature for a period of time to produce a coagulum comprising a crude curd and a liquid phase; preferably, wherein the period of time falls within the range of 10 min to 180 min; most preferably, wherein the period of time falls within the range of 20 min to 60 min; preferably, wherein the temperature falls within the range of 18° C. to 50° C.; most preferably, wherein the temperature falls within the range of 25° C. to 40° C.

39. The method according to claim 38, further comprising the steps of: e) removing the crude curd of the coagulant produced in step d) from the liquid phase of the coagulant produced in step d) to provide a curd composition.

40. The method according to claim 39, further comprising the steps of: f) texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey, at elevated temperature to provide a textured curd composition.

41. The method according to claim 40, wherein step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises an elevated temperature falling within the range of 60°C to 95°C; preferably an elevated temperature falling within the range of 70°C to 90°C; most preferably an elevated temperature falling within the range of 75°C to 85°C.

42. The method according to claim 40 or claim 41, wherein step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises texturizing at a ratio of curd composition to water falling within the range of 0.5:3 to 3:0.5; preferably at a ratio of curd composition to water falling within the range of 1:2 to 2:1; most preferably at a ratio of curd composition to water of 1:

2.

43. The method according to any one of claims 40 to 42, wherein step f) of texturizing the curd composition produced in step e) in water, or brine, or water comprising whey, or brine comprising whey at elevated temperature comprises texturizing by kneading and / or stretching and / or folding the curd composition, and optionally forming the curd composition into balls.

44. The method according to any one of claims 40 to 43, further comprising the steps of: g) Cooling the textured curd composition; preferably in brine or water.

45. Method according to any one of claims 40 to 44, wherein the textured curd composition provided by step f) is a cheese product of non-animal origin; preferably a mozzarella-type cheese product of non-animal origin.

46. ​​A food product produced by the method of any one of claims 27 to 45 comprising casein.

47. The food product according to claim 46, wherein the food product is a cheese product of non-animal origin.

48. The food product according to claim 46 or 47, wherein the food product is a mozzarella-type cheese product of non-animal origin.

49. A method of forming a cheese product, wherein the method comprises the step of mixing non-phosphorylated casein or caseinate with transglutaminase to form a polymerized protein.

50. The method according to claim 49, wherein the non-phosphorylated casein or caseinate is recombinantly produced.

51. The method according to claim 49, wherein the non-phosphorylated casein or caseinate is formed by dephosphorylating native casein or caseinate.

52. The method according to any one of claims 49 to 51, wherein the transglutaminase is added in an amount of at least 0.5 U / g protein.

53. The method according to any one of claims 49 to 52, wherein the method further comprises forming an emulsion by emulsifying the polymerized protein with fat.

54. The method according to claim 53, wherein the emulsifying step occurs at a temperature between ambient temperature and 60°C.

55. The method according to any one of claims 53 and 54, wherein i) rennet is added to the emulsion in an amount of 5 to 15 mg rennet per 100 g emulsion, and / or ii) acid or D-(+)-glucono-delta-lactone is added to the emulsion.

56. A method according to claim 55, wherein the emulsion is cooled to a temperature of 10°C or less before adding rennet or acid.

57. A method according to claim 56, wherein the emulsion is cooled to a temperature of 4 to 8°C.

58. A method according to any one of claims 55 to 57, wherein the pH of the emulsion is acidified to a pH of 5.0 to 6.0 prior to adding rennet.

59. The method according to claim 58, wherein the emulsion is first cooled to a temperature of 10°C or less to form a cooled emulsion, and the pH of the cooled emulsion is acidified to a pH of 5.0 to 6.0 before adding rennet.

60. The process according to any one of claims 56 to 59, wherein hydrogen chloride, lactic acid, citric acid, acetic acid and / or D-(+)-glucono-delta-lactone are used for acidification of the emulsion.

61. A method according to any one of claims 56 to 60, wherein a bacterial starter culture is used for acidification of the emulsion.

62. A process according to any one of claims 56 to 61, wherein the acidification step is carried out at a temperature of 5 to 10°C.

63. A method according to any one of claims 56 to 62, wherein the method further comprises adding a calcium salt to the emulsion prior to the addition of rennet and any acidification step.

64. The method according to claim 63, wherein the emulsion is first cooled to a temperature of 10°C or less to form a cooled emulsion prior to adding the calcium salt.

65. The method according to any one of claims 63 and 64, wherein the emulsion is first cooled to a temperature of 10°C or less to form a cooled emulsion, a calcium salt is added, and then the emulsion is acidified to a pH of 5.0 to 6.0 to form an acidified emulsion.

66. A method according to any one of claims 63 to 65, wherein the calcium salt is added to the emulsion in an amount of 0.2 to 2 mM / g protein.

67. The method according to any one of claims 63 to 66, wherein the calcium salt is calcium chloride, calcium hydroxide, calcium carbonate, calcium citrate, calcium phosphate, calcium stearate, calcium malate, calcium glycerophosphate, calcium lactate, calcium gluconate or a mixture thereof.

68. The composition of claim 67, wherein the calcium salt is calcium chloride.

69. The method according to any one of claims 56 to 68, wherein the addition of the rennet or acid is followed by at least one of the following processing steps: a) incubation of the mixture and periodic cutting; and / or b) whey drainage; and / or c) plasticizing and shaping to produce cheese products; and / or d) cooling the cheese product.

70. A cheese product formed using the method of any one of claims 49 to 69.

71. The cheese product of claim 70 which is a pasta filata style cheese product.

72. A cheese product according to any one of claims 70 and 71 which is a mozzarella style cheese product.

73. A high moisture cheese product comprising dephosphorylated αs1-casein, αs2-casein, β-casein and / or κ-casein.

74. The high moisture cheese product according to claim 73 which is a pasta filata cheese product.

75. A cheese product according to any one of claims 73 and 74 which is a mozzarella style cheese product.

76. A method of forming an emulsified composition, wherein the method comprises the following steps: i) preparing a mixture of casein and zein proteins; ii) adding one or more lipids; and iii) emulsifying the mixture to obtain an emulsified composition.

77. The method of claim 76, wherein the casein is non-micellar casein.

78. The method of claim 76 or claim 77, wherein step i) comprises preparing a zein solution at an alkaline pH and then adding casein to the alkaline zein solution.

79. The method of claim 76 or claim 77, wherein step i) comprises preparing an aqueous solution of the casein and then adding zein to the aqueous casein solution.

80. The method according to any one of claims 76 to 79, wherein step iii) comprises homogenizing the mixture to obtain the emulsified composition.

81. The method according to any one of claims 76 to 80, wherein step ii) further comprises adding sugar.

82. The method according to any one of claims 76 to 81, wherein step i) further comprises adjusting the pH of the mixture of casein and zein proteins to a neutral pH of about pH 6.5 to about pH 7.

5.

83. A method according to any one of claims 76 to 82, wherein the zein is enzymatically hydrolysed prior to being combined with the casein.

84. The method according to any one of claims 76 to 83, wherein the method further comprises the steps of: iv) acidifying the emulsion; and / or v) adding calcium salt, to form a treated emulsion.

85. The method according to claim 84, further comprising reducing the temperature of the treated emulsion.

86. A method according to claim 84 or claim 85, further comprising incubating the treated emulsion with an enzyme.

87. A method according to any one of claims 84 to 86, further comprising texturising the treated emulsion to provide a cheese product, and optionally aging the cheese product.

88. The method according to claim 87, wherein the cheese product is a pasta filata or mozzarella style cheese product.

89. A cheese product obtainable using the method of any one of claims 76 to 88.

90. An emulsified composition comprising one or more lipid components and a protein component, wherein the protein component has a protein content of at least 80% protein by weight, wherein about 25-40% by weight of the protein content comprises zein protein and the remainder is casein.

91. The emulsified composition of claim 90, wherein the casein is non-micellar casein.

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