Composition

Abstract

The present invention provides a casein micelle composition containing amorphous calcium phosphate (CaP), wherein the casein micelles contain at least one calcium-sensitive casein and do not contain κ-casein. The present invention also provides a method for producing the casein micelle composition. The present invention also provides a food product containing the casein micelle composition.

Description

Technical Field

[0001] Details of Priority This application claims priority from Australian Patent Application No. 2022901459, filed on May 30, 2022, the content of which is hereby incorporated by reference into this specification.

[0002] The present invention relates to a casein micelle composition and a method for producing the same. The present invention also relates to a food product containing the casein micelle composition.

Background Art

[0003] With global warming continuing and the world's population predicted to reach 10 billion by 2050, there is an urgent global need to reduce greenhouse gas emissions (GHGE) from animal production while sustainably producing high-quality protein.

[0004] Milk proteins are one of the most nutritious protein sources due to their amino acid profile, their innate ability to transport highly concentrated biologically available calcium and phosphates by protein aggregates known as casein micelles, and their digestibility in the stomach. The current main drawbacks of conventional dairy farming are the high environmental costs in milk production regarding land and water use and the amount of GHGE generated, particularly methane.

[0005] Plant-based dairy alternatives with lower GHGE and lower water usage do not match the nutrition, functionality, and taste of animal-based dairy products because they do not contain casein proteins, a class of proteins produced only in mammals. The protein component of mammalian milk consists of casein and whey protein. Casein is an intrinsically disordered protein (IDP), is sometimes glycosylated, and is often phosphorylated, while whey protein is more spherical and has a well-folded conformation. Casein is further divided into calcium-sensitive casein and casein insensitive to precipitation by calcium ions. Bovine calcium-sensitive casein is α S1 - , α S2 - , and β-casein, while κ-casein is insensitive to precipitation by calcium ions.

[0006] Cell agriculture is an alternative to conventional agriculture that involves the cultivation of engineered microorganisms that express muscle and fat cells to replace processed animal meat, as well as various food molecules, particularly animal proteins such as those found in dairy products and eggs. Using cell agriculture to produce casein proteins through genetic engineering of microorganisms is an approach that may mitigate the environmental drawbacks of animal agriculture. However, due to the fact that casein is an intrinsically disordered protein and its high level of post-translational modifications including phosphorylation and glycosylation, it is very difficult to produce them economically and to reconstruct them into native-like casein micelles, which are necessary for producing stable dairy products such as milk.

[0007] Native casein micelles are formed in the mammary gland. When combined with nanoclusters of amorphous calcium phosphate (CaP), they form stable polydisperse supramolecules. The white appearance of milk is due to intense light scattering by casein micelles and fat globules.

[0008] Milk and native casein micelles exhibit excellent stability compared to other biological fluids and globular proteins, respectively. In some species, milk can be stored in the mammary gland for days, weeks, or even months without aggregating or forming amyloid fibrils. Milk can withstand pasteurization and more severe heat treatments and can be dried and then reconstituted with water. Moreover, many milks contain high concentrations of calcium and phosphate that far exceed the solubility of calcium phosphate at the pH of milk, and usually none of these treatments result in the precipitation of calcium phosphate. These natural properties are utilized in the manufacture and storage of various liquid dairy products.

[0009] Casein micelles are stable at the pH of milk but readily aggregate to form gels at acidic pH. Similarly, the stability of casein micelles can be reduced or eliminated by limited proteolysis catalyzed by aspartic proteases such as chymosin or similar proteases. This ability is readily utilized in the manufacture of gel dairy products such as cheese and yogurt.

[0010] Among various species, there are large natural variations in the composition of casein micelles. For example, bovine milk contains secreted protein polymorphs derived from the four expressed casein genes κ-, β-, α s1 -, α s2 - in approximate ratios of about 1:4:4:1, respectively. In contrast, elephant milk contains expressed and secreted proteins derived from only two casein genes, κ- and β-casein, in ratios of about 1:8.5, respectively. In the milk of some other species such as rabbits, proteins derived from five casein genes are present. All milks that have been well characterized contain the form of κ-casein, but all other caseins may or may not be present in various mammalian species.

[0011] Naturally occurring casein micelles are diverse, but there are no known examples of casein micelles made from a single casein. Thus, currently, there are at least two casein proteins required to form casein micelles that are conserved. All naturally occurring casein micelles contain the type of κ-casein and one or more types of calcium-sensitive casein. The type of κ-casein is present in the milk of all mammalian species. Moreover, κ-casein is generally considered important for milk stability because it lacks sensitivity to precipitation by divalent cations such as Ca 2+ etc. The stability of casein micelles is widely accepted to result from the mechanism that stabilizes colloidal particles known as steric stabilization.

[0012] The proportion of κ-casein in total casein varies. Among equids, for example, low proportions of κ-casein are found, such as 1.8% in horse milk and 2.8% in donkey milk. This proportion can vary greatly between individuals and throughout the lactation period. For example, in a recent study of the variation in the lactation period of casein compositions in individual women, the proportion of κ-casein ranged from 11.5% to about 76% of total casein.

[0013] While there are limited examples in the prior art that claim to have produced a single casein micelle, to date, all examples examined have used commercially supplied casein proteins. Commercially available casein proteins are invariably contaminated with other casein proteins, especially κ-casein, which has previously been described as useful for micelle stability. Thus, there are no prior art examples of truly single casein micelles.

[0014] Artificial casein micelles are not inevitable. The various results that can occur when casein and salts are mixed are as shown in Figure 1. The results depend on several factors, among others, the final composition of the formulation, the order in which the ingredients are added, and the rate of addition. Moreover, the stability of the formulation can vary depending on how long after mixing the determination is made.

[0015] The hydrophobic substances such as various poorly soluble minerals identified in FIG. 1 can be stabilized in the colloidal state by the adsorption of a more hydrophilic coat. This phenomenon results in a coat-core structure, which can be kinetically stable and can persist over a long period under favorable circumstances. However, both the initially formed amorphous phase by CaP or CaCO3 and the casein-stabilized CaP to mature into a less soluble and more crystalline form, so there are two mechanisms of colloidal destabilization, both of which are affected by the concentration of casein and the affinity of casein to bind the amorphous phase to a more crystalline phase. Destabilization can also occur as a result of the loss of solubility of casein, for example, by salting out or a decrease in charge. By using the characterization of various methods, it is possible to determine the composition, size, and structure of the product, as well as its stability, in order to determine what has occurred as a result of a specific formulation and mixing procedure.

[0016] Confirmation of the formation of the casein micelle structure requires evidence from chemical analysis of the colloid and the continuous phase in which both calcium (Ca) and phosphate (P i ) are present in the colloid. Suitable chemical analyses include nanoparticle tracking analysis (NTA) size data, turbidity before and after centrifugation, transmission electron microscopy (TEM), and inorganic analysis. Evidence that the colloid contains CaP nanoclusters can also be shown, for example, using cryo-electron microscopy or small-angle scattering experiments with X-rays or neutrons.

Prior Art Documents

Non-Patent Documents

[0017]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0018] Therefore, there is still a need for improved artificial casein micelle compositions and methods for their preparation that at least partially address one or more of the disadvantages mentioned above or provide useful alternatives.

[0019] In particular, there is still a need for improved artificial casein micelle compositions that are similar to natural casein micelles of milk in their structure, composition, and physicochemical properties. These may include, for example, some of the following properties: (i) the white appearance of the solution and high calcium concentration; (ii) their stability at natural pH against heat treatment or drying; (iii) avoidance of amyloid fiber formation; (iv) gelation upon acidification; or (v) gelation after limited proteolysis.

[0020] References in this specification to patent documents and other matters given as prior art should not be taken as an admission that those patent documents and other matters are known or that the information they contain was part of common general knowledge at the priority date of any of the claims.

Means for Solving the Problems

[0021] In a first aspect, the present invention provides a composition comprising casein micelle particles containing amorphous calcium phosphate (CaP), wherein the casein micelle particles comprise at least one calcium-sensitive casein and the casein micelle does not contain κ-casein.

[0022] In different aspects of the present invention, the casein micelle particles comprise (i) only αs1-casein; (ii) only αs2-casein; (iii) only β-casein; (iv) a mixture of αs1-casein and β-casein; (v) a mixture of αs2-casein and β-casein; (vi) a mixture of αs1-casein and αs2-casein; or (vii) a mixture of αs1-casein, αs2-casein and β-casein.

[0023] Advantageously, the casein micelle composition of the present invention does not contain κ-casein, but exhibits similar stability and appearance as naturally occurring casein micelles. This makes it possible to produce casein micelle compositions containing only calcium-sensitive casein and, in some aspects, only one type of casein protein.

[0024] Furthermore, the casein micelles of the present invention can be produced more economically than current solutions. In a further aspect, the present invention relates to a method for producing the casein micelle compositions disclosed herein.

[0025] The food products of the present invention can advantageously provide improved plant-based dairy product alternatives. These food products provide improved nutrition, functionality, and / or taste while avoiding some or all of the drawbacks associated with both animal-based dairy products and current plant-based dairy product alternatives. In a further aspect, the present invention relates to food products comprising the casein micelle compositions disclosed herein.

[0026] Further aspects of the invention are shown in the detailed description of the invention below.

[0027] Embodiments of the present invention will be described only by way of examples with reference to the following accompanying drawings.

Brief Description of the Drawings

[0028]

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Best Mode for Carrying Out the Invention

[0029] Before explaining the present invention in detail, it should be understood that the technical terms used in this specification are for the purpose of explaining the embodiments only and are not intended to be limiting.

[0030] Definitions Unless otherwise specified, all technical and scientific terms used in this specification are to be construed as having the same meaning as commonly understood by one of ordinary skill in the art.

[0031] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0032] Throughout the description of this specification and the claims, the word "comprise" and variations of the word, such as "comprising" and "comprises", are not intended to exclude other additives, components, integers, or steps. As used in this specification, "comprises" means "includes". Thus, "comprising A or B" does not exclude additional elements and means "comprising A, B, or A and B".

[0033] The term "and / or" as used in this specification is to be understood as a specific disclosure of each of two specified features or components, with or without the other. For example, "A and / or B" is to be construed in this specification as (i) A, (ii) B, and (iii) A and B, as if each were individually presented.

[0034] As used herein, the term "food product" means a substance that can be used as food or can be prepared for use as food, which is any nutrient that humans or animals eat or drink to maintain life and growth.

[0035] As used herein, the term "calcium-sensitive casein" means a casein protein that is sensitive to precipitation by calcium ions. Similarly, the term "calcium-insensitive casein" means a casein protein that is insensitive to precipitation by calcium ions. Calcium-sensitive bovine casein is α s1 -, α s2 -, and β-casein, while κ-casein, on the other hand, is insensitive to precipitation by calcium ions. As used herein, the terms "α s1 -casein" and "alpha-S1 casein" are used interchangeably and mean all polymorphs of the CSN1S1 gene product that encodes alpha-S1 casein. As used herein, the terms "α s2 -casein" and "alpha-S2 casein" are used interchangeably and mean all polymorphs of the CSN1S2 gene product for alpha-S2 casein. As used herein, the terms "β-casein" and "beta casein" are used interchangeably and mean all polymorphs of the CSN2 casein gene product that encodes beta casein. As used herein, the terms "κ-casein" and "kappa casein" are used interchangeably and mean all polymorphs of the CSN3 gene product that encodes kappa casein.

[0036] Casein micelle composition The present invention relates to a casein micelle composition comprising casein micelle particles containing amorphous calcium phosphate (CaP), wherein the casein micelle particles contain at least one calcium-sensitive casein and do not contain κ-casein.

[0037] Casein micelles are colloidal particles formed by casein aggregates, which are dispersed in a liquid to form a colloidal suspension or composition. Usually, this liquid is water, but any suitable solvent can be used.

[0038] In different embodiments of the casein micelle composition of the present invention, the casein micelle particles are as follows: (i) α s1 -casein only; (ii) α s2 -casein only; (iii) β-casein only; (iv) α s1 -casein and β-casein mixture; (v) α s2 -casein and β-casein mixture; (vi) α s1 -casein and α s2 -casein mixture; or (vii) α s1 -casein and α s2 -casein and β-casein mixture comprise.

[0039] In one embodiment, the casein micelle composition contains CaP sufficient to bind to about 5% to 100% of the casein. The casein micelle composition may contain CaP sufficient to bind to at least 3% or about 5% of the casein. The casein micelle composition may contain CaP sufficient to bind to up to 100% of the casein. The casein micelle composition may contain CaP sufficient to bind to at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the casein.

[0040] In a further embodiment, the total casein concentration in the composition is from about 0.5 to 100 g / L. The total casein concentration in the composition can be at least about 0.5 g / L. The total casein concentration in the composition can be at most about 100 g / L. The total casein concentration in the composition can be 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / L. The total casein concentration in the composition can be about 0.5 - 5, 0.5 - 10, 0.5 - 15, 0.5 - 20, 0.5 - 25, 0.5 - 30, 0.5 - 35, 0.5 - 40, 0.5 - 45, 0.5 - 50, 0.5 - 55, 0.5 - 60, 0.5 - 65, 0.5 - 70, 0.5 - 75, 0.5 - 80, 0.5 - 85, 0.5 - 90, 0.5 - 95, 0.5 - 100, 1 - 5, 1 - 10, 1 - 15, 1 - 20, 1 - 25, 1 - 30, 1 - 35, 1 - 40, 1 - 45, 1 - 50, 1 - 55, 1 - 60, 1 - 65, 1 - 70, 1 - 75, 1 - 80, 1 - 85, 1 - 90, 1 - 95, 1 - 100, 2 - 5, 2 - 10, 2 - 15, 2 - 20, 2 - 25, 2 - 30, 2 - 35, 2 - 40, 2 - 45, 2 - 50, 2 - 55, 2 - 60, 2 - 65, 2 - 70, 2 - 75, 2 - 80, 2 - 85, 2 - 90, 2 - 95, 2 - 100, 3 - 5, 3 - 10, 3 - 15, 3 - 20, 3 - 25, 3 - 30, 3 - 35, 3 - 40, 3 - 45, 3 - 50, 3 - 55, 3 - 60, 3 - 65, 3 - 70, 3 - 75, 3 - 80, 3 - 85, 3 - 90, 3 - 95, or 3 - 100, 4 - 5, 4 - 10, 4 - 15, 4 - 20, 4 - 25, 4 - 30, 4 - 35, 4 - 40, 4 - 45, 4 - 50, 4 - 55, 4 - 60, 4 - 65, 4 - 70, 4 - 75, 4 - 80, 4 - 85, 4 - 90, 4 - 95, 4 - 100, 5 - 10, 5 - 15, 5 - 20, 5 - 25, 5 - 30, 5 - 35, 5 - 40, 5 - 45, 5 - 50, 5 - 55, 5 - 60, 5 - 65, 5 - 70, 5 - 75, 5 - 80, 5 - 85, 5 - 90, 5 - 95, 5 - 100, 10 - 15, 10 - 20, 10 - 25, 10 - 30, 10 - 35, 10 - 40, 10 - 45, 10 - 50, 10 - 55, 10 - 60, 10 - 65, 10 - 70, 10 - 75, 10 - 80, 10 - 85, 10 - 90, 10 - 95, 10 - 100, 15 - 10, 15 - 15, 15 - 20, 15 - 25, 15 - 30, 15 - 35,15 - 40, 15 - 45, 15 - 50, 15 - 55, 15 - 60, 15 - 65, 15 - 70, 15 - 75, 15 - 80, 15 - 85, 15 - 90, 15 - 95, 15 - 100, 20 - 25, 20 - 30, 20 - 35, 20 - 40, 20 - 45, 20 - 50, 20 - 55, 20 - 60, 20 - 65, 20 - 70, 20 - 75, 20 - 80, 20 - 85, 20 - 90, 20 - 95, 20 - 100, 25 - 30, 25 - 35, 25 - 40, 25 - 45, 25 - 50, 25 - 55, 25 - 60, 25 - 65, 25 - 70, 25 - 75, 25 - 80, 25 - 85, 25 - 90, 25 - 95, 25 - 100, 30 - 35, 30 - 40, 30 - 45, 30 - 50, 30 - 55, 30 - 60, 30 - 65, 30 - 70, 30 - 75, 30 - 80, 30 - 85, 30 - 90, 30 - 95, 30 - 100, 35 - 40, 35 - 45, 35 - 50, 35 - 55, 35 - 60, 35 - 65, 35 - 70, 35 - 75, 35 - 80, 35 - 85, 35 - 90, 35 - 95, 35 - 100, 40 - 45, 40 - 50, 40 - 55, 40 - 60, 40 - 65, 40 - 70, 40 - 75, 40 - 80, 40 - 85, 40 - 90, 40 - 95, 40 - 100, 45 - 50, 45 - 55, 45 - 60, 45 - 65, 45 - 70, 45 - 75, 45 - 80, 45 - 85, 45 - 90, 45 - 95, 45 - 100, 50 - 55, 50 - 60, 50 - 65, 50 - 70, 50 - 75, 50 - 80, 50 - 85, 50 - 90, 50 - 95, 50 - 100, 55 - 60, 55 - 65, 55 - 70, 55 - 75, 55 - 80, 55 - 85, 55 - 90, 55 - 95, 55 - 100, 60 - 65, 60 - 70, 60 - 75, 60 - 80, 60 - 85, 60 - 90, 60 - 95, 60 - 100, 65 - 70, 65 - 75, 65 - 80, 65 - 85, 65 - 90, 65 - 95, 65 - 100, 70 - 75, 70 - 80, 70 - 85, 70 - 90, 70 - 95, 70 - 100, 75 - 80, 75 - 85, 75 - 90, 75 - 95, 75 - 100, 80 - 85, 80 - 90, 80 - 95, 80 - 100, 85 - 90, 85 - 95, 85 - 100, 90 - 95, 90 - 100, or 95 - 100 g / L. Preferably, the total casein concentration in the composition can be about 5 - 50 g / L. More preferably, the total casein concentration in the composition can be about 30 g / L.,

[0041] In another embodiment, the pH of the composition can be from about pH 5.5 to about pH 8.0. The pH of the composition can be at least pH 5.5 or about 6.0. The pH of the composition can be at most about pH 8.0. The pH of the composition can be about pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, or pH 8.0. The pH of the composition can be from about 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.0, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.0, 7.0 to 7.5, 7.0 to 8.0, or 7.5 to 8.0. Preferably, the pH of the composition can be about pH 6.7.

[0042] In some embodiments, the casein micelle particles contain a mixture of α s1 -casein and β-casein. In some embodiments, the casein micelle particles contain a mixture of α s2 -casein and β-casein. In some embodiments, the casein micelle particles contain a mixture of α s1 -casein and α s2 -casein.

[0043] In some embodiments, the casein micelle particles contain a mixture of α s1 -casein and β-casein. The ratio of alpha S1 casein protein to beta casein protein in the casein micelle composition can be from about 1:15 to about 15:1. The ratio of alpha S1 casein protein to beta casein protein in the casein micelle composition can be about 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1.

[0044] In some embodiments, the casein micelle particles contain a mixture of α s2- It contains a mixture of casein and β-casein. The ratio of alpha S2-casein protein to beta-casein protein in the casein micelle composition can be from about 1:15 to about 15:1. The ratio of alpha S2-casein protein to beta-casein protein in the casein micelle composition can be 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1.

[0045] In some embodiments, the casein micelle particles are alpha s1 -casein and alpha s2 -casein. The ratio of alpha S1-casein protein to alpha S2-casein protein in the casein micelle composition can be from about 1:15 to about 15:1. The ratio of alpha S1-casein protein to alpha S2-casein protein in the casein micelle composition can be 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1.

[0046] The casein micelle particles are a mixture of two types of casein proteins (e.g., a mixture of alpha s1 -casein and beta-casein, or a mixture of alpha s2 -casein and beta-casein, or a mixture of alpha s1 -casein and alpha s2- In embodiments comprising a mixture with casein, etc., the ratio of another protein to the casein protein in the casein micelle composition can be from about 1:15 to about 15:1. For example, the ratio of another protein to the casein protein in the casein micelle composition can be 1:15 to 15:1; 1:14 to 15:1; 1:13 to 15:1; 1:12 to 15:1; 1:11 to 15:1; 1:10 to 15:1; 1:9 to 15:1; 1:8 to 15:1; 1:7 to 15:1; 1:6 to 15:1; 1:5 to 15:1; 1:4 to 15:1; 1:3 to 15:1; 1:2 to 15:1; 1:15 to 15:1; 1:1 to 15:1; 2:1 to 15:1; 3:1 to 15:1; 4:1 to 15:1; 5:1 to 15:1; 6:1 to 15:1; 7:1 to 15:1; 8:1 to 15:1; 9:1 to 15:1; 10:1 to 15:1; 11:1 to 15:1; 12:1 to 15:1; 13:1 to 15:1; 14:1 to 15:1; 1:15 to 14:1; 1:14 to 14:1; 1:13 to 14:1; 1:12 to 14:1; 1:11 to 14:1; 1:10 to 14:1; 1:9 to 14:1; 1:8 to 14:1; 1:7 to 14:1; 1:6 to 14:1; 1:5 to 14:1; 1:4 to 14:1; 1:3 to 14:1; 1:2 to 14:1; 1:15 to 14:1; 1:1 to 14:1; 2:1 to 14:1; 3:1 to 14:1; 4:1 to 14:1; 5:1 to 14:1; 6:1 to 14:1; 7:1 to 14:1; 8:1 to 14:1; 9:1 to 14:1; 10:1 to 14:1; 11:1 to 14:1; 12:1 to 14:1; 13:1 to 14:1; 1:15 to 13:1; 1:14 to 13:1; 1:13 to 13:1; 1:12 to 13:1; 1:11 to 13:1; 1:10 to 13:1; 1:9 to 13:1; 1:8 to 13:1; 1:7 to 13:1; 1:6 to 13:1; 1:5 to 13:1; 1:4 to 13:1; 1:3 to 13:1; 1:2 to 13:1; 1:15 to 13:1; 1:1 to 13:1; 2:1 to 13:1; 3:1 to 13:1; 4:1 to 13:1; 5:1 to 13:1; 6:1 to 13:1; 7:1 to 13:1; 8:1 to 13:1; 9:1 to 13:1; 10:1 to 13:1; 11:1 to 13:1; 12:1 to 13:1; 1:15 to 12:1; 1:14 to 12:1; 1:13 to 12:1; 1:12 to 12:1; 1:11 to 12:1; 1:10 to 12:1; 1:9 to 12:1; 1:8 to 12:1; 1:7 to 12:1;1:6~12:1;1:5~12:1;1:4~12:1;1:3~12:1;1:2~12:1;1:15~12:1;1:1~12:1;2:1~12:1;3:1~12:1;4:1~12:1;5:1~12:1;6:1~12:1;7:1~12:1;8:1~12:1;9:1~12:1;10:1~12:1;11:1~12:1;1:15~11:1;1:14~11:1;1:13~11:1;1:12~11:1;1:11~11:1;1:10~11:1;1:9~11:1;1:8~11:1;1:7~11:1;1:6~11:1;1:5~11:1;1:4~11:1;1:3~11:1;1:2~11:1;1:15~11:1;1:1~11:1;2:1~11:1;3:1~11:1;4:1~11:1;5:1~11:1;6:1~11:1;7:1~11:1;8:1~11:1;9:1~11:1;10:1~11:1;1:15~10:1;1:14~10:1;1:13~10:1;1:12~10:1;1:11~10:1;1:10~10:1;1:9~10:1;1:8~10:1;1:7~10:1;1:6~10:1;1:5~10:1;1:4~10:1;1:3~10:1;1:2~10:1;1:15~10:1;1:1~10:1;2:1~10:1;3:1~10:1;4:1~10:1;5:1~10:1;6:1~10:1;7:1~10:1;8:1~10:1;9:1~10:1;1:15~9:1;1:14~9:1;1:13~9:1;1:12~9:1;1:11~9:1;1:10~9:1;1:9~9:1;1:8~9:1;1:7~9:1;1:6~9:1;1:5~9:1;1:4~9:1;1:3~9:1;1:2~9:1;1:15~9:1;1:1~9:1;2:1~9:1;3:1~9:1;4:1~9:1;5:1~9:1;6:1~9:1;7:1~9:1;8:1~9:1;1:15~8:1;1:14~8:1;1:13~8:1;1:12~8:1;1:11~8:1;1:10~8:1;1:9~8:1;1:8~8:1;1:7~8:1;1:6~8:1;1:5~8:1;1:4~8:1;1:3~8:1;1:2~8:1;1:15~8:1;1:1~8:1;2:1~8:1;3:1~8:1;4:1~8:1;5:1~8:1;6:1~8:1;7:1~8:1;1:15~7:1;1:14~7:1;1:13~7:1;1:12~7:1;1:11~7:1;1:10~7:1;1:9~7:1;1:8~7:1;1:7~7:1;1:6~7:1;1:5~7:1;1:4~7:1;1:3~7:1;1:2~7:1;1:15~7:1;1:1~7:1;2:1~7:1;3:1~7:1;4:1~7:1;5:1~7:1;6:1~7:1;1:15~6:1;1:14~6:1;1:13~6:1;1:12~6:1;1:11~6:1;1:10~6:1;1:9~6:1;1:8~6:1;1:7~6:1;1:6~6:1;1:5~6:1;1:4~6:1;1:3~6:1;1:2~6:1;1:15~6:1;1:1~6:1;2:1~6:1;3:1~6:1;4:1~6:1;5:1~6:1;1:15~5:1;1:14~5:1;1:13~5:1;1:12~5:1;1:11~5:1;1:10~5:1;1:9~5:1;1:8~5:1;1:7~5:1;1:6~5:1;1:5~5:1;1:4~5:1;1:3~5:1;1:2~5:1;1:15~5:1;1:1~5:1;2:1~5:1;3:1~5:1;4:1~5:1;1:15~4:1;1:14~4:1;1:13~4:1;1:12~4:1;1:11~4:1;1:10~4:1;1:9~4:1;1:8~4:1;1:7~4:1;1:6~4:1;1:5~4:1;1:4~4:1;1:3~4:1;1:2~4:1;1:15~4:1;1:1~4:1;2:1~4:1;3:1~4:1;1:15~3:1;1:14~3:1;1:13~3:1;1:12~3:1;1:11~3:1;1:10~3:1;1:9~3:1;1:8~3:1;1:7~3:1;1:6~3:1;1:5~3:1;1:4~3:1;1:3~3:1;1:2~3:1;1:15~3:1;1:1~3:1;2:1~3:1;1:15~2:1;1:14~2:1;1:13~2:1;1:12~2:1;1:11~2:1;1:10~2:1;1:9~2:1;1:8~2:1;1:7~2:1;1:6~2:1;1:5~2:1;1:4~2:1;1:3~2:1;1:2~2:1;1:15~2:1;1:1~2:1;1:15~1:1;1:14~1:1;1:13~1:1;1:12~1:1;1:11~1:1;1:10~1:1;1:9~1:1;1:8~1:1;1:7~1:1;1:6 to 1:1; 1:5 to 1:1; 1:4 to 1:1; 1:3 to 1:1; 1:2 to 1:1; 1:15 to 1:2; 1:14 to 1:2; 1:13 to 1:2; 1:12 to 1:2; 1:11 to 1:2; 1:10 to 1:2; 1:9 to 1:2; 1:8 to 1:2; 1:7 to 1:2; 1:6 to 1:2; 1:5 to 1:2; 1:4 to 1:2; 1:3 to 1:2; 1:15 to 1:3; 1:14 to 1:3; 1:13 to 1:3; 1:12 to 1:3; 1:11 to 1:3; 1:10 to 1:3; 1:9 to 1:3; 1:8 to 1:3; 1:7 to 1:3; 1:6 to 1:3; 1:5 to 1:3; 1:4 to 1:3; 1:15 to 1:4; 1:14 to 1:4; 1:13 to 1:4; 1:12 to 1:4; 1:11 to 1:4; 1:10 to 1:4; 1:9 to 1:4; 1:8 to 1:4; 1:7 to 1:4; 1:6 to 1:4; 1:5 to 1:4; 1:15 to 1:5; 1:14 to 1:5; 1:13 to 1:5; 1:12 to 1:5; 1:11 to 1:5; 1:10 to 1:5; 1:9 to 1:5; 1:8 to 1:5; 1:7 to 1:5; 1:6 to 1:5; 1:15 to 1:6; 1:14 to 1:6; 1:13 to 1:6; 1:12 to 1:6; 1:11 to 1:6; 1:10 to 1:6; 1:9 to 1:6; 1:8 to 1:6; 1:7 to 1:6; 1:15 to 1:7; 1:14 to 1:7; 1:13 to 1:7; 1:12 to 1:7; 1:11 to 1:7; 1:10 to 1:7; 1:9 to 1:7; 1:8 to 1:7; 1:15 to 1:8; 1:14 to 1:8; 1:13 to 1:8; 1:12 to 1:8; 1:11 to 1:8; 1:10 to 1:8; 1:9 to 1:8; 1:15 to 1:9; 1:14 to 1:9; 1:13 to 1:9; 1:12 to 1:9; 1:11 to 1:9; 1:10 to 1:9; 1:15 to 1:10; 1:14 to 1:10; 1:13 to 1:10; 1:12 to 1:10; 1:11 to 1:10; 1:15 to 1:11; 1:14 to 1:11; 1:13 to 1:11; 1:12 to 1:11; 1:15 to 1:12; 1:14 to 1:12; 1:13 to 1:12; 1:15 to 1:13; 1:14 to 1:13; or may be 1:15 to 1:14;

[0047] In some embodiments, the casein micelle particles are α s1 -casein and α s2- It contains a mixture of casein and β-casein. In some preferred embodiments, each of the alpha S1-casein protein, alpha S2-casein protein, and beta-casein protein in the casein micelle composition can be present in an amount of 1 to 15 parts. The amounts of alpha S1-casein protein, alpha S2-casein protein, and beta-casein protein present in the casein micelle composition can be in any order, about 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:1:11, 1:1:12, 1:1:13, 1:1:14, 1:1:15, 1:2:2, 1:2:3, 1:2:4, 1:2:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, 1:2:10, 1:2:11, 1:2:12, 1:2:13, 1:2:14, 1:2:15, 1:3:3, 1:3:4, 1:3:5, 1:3:6, 1:3:7, 1:3:8, 1:3:9, 1:3:10, 1:3:11, 1:3:12, 1:3:13, 1:3:14, 1:3:15, 1:4:4, 1:4:5, 1:4:6, 1:4:7, 1:4:8, 1:4:9, 1:4:10, 1:4:11, 1:4:12, 1:4:13, 1:4:14, 1:4:15, 1:5:5, 1:5:6, 1:5:7, 1:5:8, 1:5:9, 1:5:10, 1:5:11, 1:5:12, 1:5:13, 1:5:14, 1:5:15, 1:6:6, 1:6:7, 1:6:8, 1:6:9, 1:6:10, 1:6:11, 1:6:12, 1:6:13, 1:6:14, 1:6:15, 1:7:7, 1:7:8, 1:7:9, 1:7:10, 1:7:11, 1:7:12, 1:7:13, 1:7:14, 1:7:15, 1:8:8, 1:8:9, 1:8:10, 1:8:11, 1:8:12, 1:8:13, 1:8:14, 1:8:15, 1:9:9, 1:9:10, 1:9:11, 1:9:12, 1:9:13, 1:9:14, 1:9:15, 1:10:10, 1:10:11, 1:10:12, 1:10:13, 1:10:14, 1:10:15, 1:11:11, 1:11:12, 1:11:13, 1:11:14, 1:11:15, 1:12:12, 1:12:13, 1:12:14, 1:12:15, 1:13:13, 1:13:14, 1:13:15, 1:14:14,1:14:15、1:15:15、2:2:3、2:2:4、2:2:5、2:2:6、2:2:7、2:2:8、2:2:9、2:2:10、2:2:11、2:2:12、2:2:13、2:2:14、2:2:15、2:3:3、2:3:4、2:3:5、2:3:6、2:3:7、2:3:8、2:3:9、2:3:10、2:3:11、2:3:12、2:3:13、2:3:14、2:3:15、2:4:4、2:4:5、2:4:6、2:4:7、2:4:8、2:4:9、2:4:10、2:4:11、2:4:12、2:4:13、2:4:14、2:4:15、2:5:5、2:5:6、2:5:7、2:5:8、2:5:9、2:5:10、2:5:11、2:5:12、2:5:13、2:5:14、2:5:15、2:6:6、2:6:7、2:6:8、2:6:9、2:6:10、2:6:11、2:6:12、2:6:13、2:6:14、2:6:15、2:7:7、2:7:8、2:7:9、2:7:10、2:7:11、2:7:12、2:7:13、2:7:14、2:7:15、2:8:8、2:8:9、2:8:10、2:8:11、2:8:12、2:8:13、2:8:14、2:8:15、2:9:9、2:9:10、2:9:11、2:9:12、2:9:13、2:9:14、2:9:15、2:10:10、2:10:11、2:10:12、2:10:13、2:10:14、2:10:15、2:11:11、2:11:12、2:11:13、2:11:14、2:11:15、2:12:12、2:12:13、2:12:14、2:12:15、2:13:13、2:13:14、2:13:15、2:14:14、2:14:15、2:15:15、3:3:4、3:3:5、3:3:6、3:3:7、3:3:8、3:3:9、3:3:10、3:3:11、3:3:12、3:3:13、3:3:14、3:3:15、3:4:4、3:4:5、3:4:6、3:4:7、3:4:8、3:4:9、3:4:10、3:4:11、3:4:12、3:4:13、3:4:14、3:4:15、3:5:5、3:5:6、3:5:7、3:5:8、3:5:9、3:5:10、3:5:11、3:5:12、3:5:13、3:5:14、3:5:15、3:6:6、3:6:7、3:6:8、3:6:9、3:6:10、3:6:11、3:6:12、3:6:13、3:6:14、3:6:15、3:7:7、3:7:8、3:7:9、3:7:10、3:7:11、3:7:12、3:7:13、3:7:14、3:7:15、3:8:8、3:8:9、3:8:10、3:8:11、3:8:12、3:8:13、3:8:14、3:8:15、3:9:9、3:9:10、3:9:11、3:9:12、3:9:13、3:9:14、3:9:15、3:10:10、3:10:11、3:10:12、3:10:13、3:10:14、3:10:15、3:11:11、3:11:12、3:11:13、3:11:14、3:11:15、3:12:12、3:12:13、3:12:14、3:12:15、3:13:13、3:13:14、3:13:15、3:14:14、3:14:15、3:15:15、4:4:5、4:4:6、4:4:7、4:4:8、4:4:9、4:4:10、4:4:11、4:4:12、4:4:13、4:4:14、4:4:15、4:5:5、4:5:6、4:5:7、4:5:8、4:5:9、4:5:10、4:5:11、4:5:12、4:5:13、4:5:14、4:5:15、4:6:6、4:6:7、4:6:8、4:6:9、4:6:10、4:6:11、4:6:12、4:6:13、4:6:14、4:6:15、4:7:7、4:7:8、4:7:9、4:7:10、4:7:11、4:7:12、4:7:13、4:7:14、4:7:15、4:8:8、4:8:9、4:8:10、4:8:11、4:8:12、4:8:13、4:8:14、4:8:15、4:9:9、4:9:10、4:9:11、4:9:12、4:9:13、4:9:14、4:9:15、4:10:10、4:10:11、4:10:12、4:10:13、4:10:14、4:10:15、4:11:11、4:11:12、4:11:13、4:11:14、4:11:15、4:12:12、4:12:13、4:12:14、4:12:15、4:13:13、4:13:14、4:13:15、4:14:14、4:14:15、4:15:15、5:5:6、5:5:7、5:5:8、5:5:9、5:5:10、5:5:11、5:5:12、5:5:13、5:5:14、5:5:15、5:6:6、5:6:7、5:6:8、5:6:9、5:6:10、5:6:11、5:6:12、5:6:13、5:6:14、5:6:15、5:7:7、5:7:8、5:7:9、5:7:10、5:7:11、5:7:12、5:7:13、5:7:14、5:7:15、5:8:8、5:8:9、5:8:10、5:8:11、5:8:12、5:8:13、5:8:14、5:8:15、5:9:9、5:9:10、5:9:11、5:9:12、5:9:13、5:9:14、5:9:15、5:10:10、5:10:11、5:10:12、5:10:13、5:10:14、5:10:15、5:11:11、5:11:12、5:11:13、5:11:14、5:11:15、5:12:12、5:12:13、5:12:14、5:12:15、5:13:13、5:13:14、5:13:15、5:14:14、5:14:15、5:15:15、6:6:7、6:6:8、6:6:9、6:6:10、6:6:11、6:6:12、6:6:13、6:6:14、6:6:15、6:7:7、6:7:8、6:7:9、6:7:10、6:7:11、6:7:12、6:7:13、6:7:14、6:7:15、6:8:8、6:8:9、6:8:10、6:8:11、6:8:12、6:8:13、6:8:14、6:8:15、6:9:9、6:9:10、6:9:11、6:9:12、6:9:13、6:9:14、6:9:15、6:10:10、6:10:11、6:10:12、6:10:13、6:10:14、6:10:15、6:11:11、6:11:12、6:11:13、6:11:14、6:11:15、6:12:12、6:12:13、6:12:14、6:12:15、6:13:13、6:13:14、6:13:15、6:14:14、6:14:15、6:15:15、7:7:8、7:7:9、7:7:10、7:7:11、7:7:12、7:7:13、7:7:14、7:7:15、7:8:8、7:8:9、7:8:10、7:8:11、7:8:12、7:8:13、7:8:14、7:8:15、7:9:9、7:9:10、7:9:11、7:9:12、7:9:13、7:9:14、7:9:15、7:10:10、7:10:11、7:10:12、7:10:13、7:10:14、7:10:15、7:11:11、7:11:12、7:11:13、7:11:14、7:11:15、7:12:12、7:12:13、7:12:14、7:12:15、7:13:13、7:13:14、7:13:15、7:14:14、7:14:15、7:15:15、8:8:9、8:8:10、8:8:11、8:8:12、8:8:13、8:8:14、8:8:15、8:9:9、8:9:10、8:9:11、8:9:12、8:9:13、8:9:14、8:9:15、8:10:10、8:10:11、8:10:12、8:10:13、8:10:14、8:10:15、8:11:11、8:11:12、8:11:13、8:11:14、8:11:15、8:12:12、8:12:13、8:12:14、8:12:15、8:13:13、8:13:14、8:13:15、8:14:14、8:14:15、8:15:15、9:9:10、9:9:11、9:9:12、9:9:13、9:9:14、9:9:15、9:10:10、9:10:11、9:10:12、9:10:13、9:10:14、9:10:15、9:11:11、9:11:12、9:11:13、9:11:14、9:11:15、9:12:12、9:12:13、9:12:14、9:12:15、9:13:13、9:13:14、9:13:15、9:14:14、9:14:15、9:15:15、10:10:11、10:10:12、10:10:13、10:10:14、10:10:15、10:11:11、10:11:12、10:11:13、10:11:14、10:11:15、10:12:12、10:12:13、10:12:14、10:12:15、10:13:13、10:13:14、10:13:15、10:14:14、10:14:15、10:15:15、11:11:12、11:11:13、11:11:14、11:11:15、11:12:12、11:12:13、11:12:14、11:12:15、11:13:13、11:13:14、11:13:15、11:14:14、11:14:15、11:15:15、12:12:13、12:12:14、12:12:15、12:13:13、12:13:14、12:13:15、12:14:14、12:14:15、12:15:15、13:13:14、13:13:15、13:14:14、13:14:15、13:15:15、14:14:15、It may be 14:15:15 or 15:15:15.

[0048] The diameter of the micelles (e.g., micelles in the casein micelles of the present invention, etc.) in the present specification can be from about 10 nm to about 500 nm. The diameter of the micelles in the present specification can be at least 10 nm. The diameter of the micelles in the present specification can be at most 500 nm. The diameter of the micelles in the present specification can be 10 nm - 20 nm, 10 nm - 50 nm, 10 nm - 100 nm, 10 nm - 150 nm, 10 nm - 200 nm, 10 nm - 250 nm, 10 nm - 300 nm, 10 nm - 350 nm, 10 nm - 400 nm, 10 nm - 450 nm, 10 nm - 500 nm, 20 nm - 50 nm, 20 nm - 100 nm, 20 nm - 150 nm, 20 nm - 200 nm, 20 nm - 250 nm, 20 nm - 300 nm, 20 nm - 350 nm, 20 nm - 400 nm, 20 nm - 450 nm, 20 nm - 500 nm, 50 nm - 100 nm, 50 nm - 150 nm, 50 nm - 200 nm, 50 nm - 250 nm, 50 nm - 300 nm, 50 nm - 350 nm, 50 nm - 400 nm, 50 nm - 450 nm, 50 nm - 500 nm, 100 nm - 150 nm, 100 nm - 200 nm, 100 nm - 250 nm, 100 nm - 300 nm, 100 nm - 350 nm, 100 nm - 400 nm, 100 nm - 450 nm, 100 nm - 500 nm, 150 nm - 200 nm, 150 nm - 250 nm, 150 nm - 300 nm, 150 nm - 350 nm, 150 nm - 400 nm, 150 nm - 450 nm, 150 nm - 500 nm, 200 nm - 250 nm, 200 nm - 300 nm, 200 nm - 350 nm, 200 nm - 400 nm, 200 nm - 450 nm, 200 nm - 500 nm, 250 nm - 300 nm, 250 nm - 350 nm, 250 nm - 400 nm, 250 nm - 450 nm, 250 nm - 500 nm, 300 nm - 350 nm, 300 nm - 400 nm, 300 nm - 450 nm, 300 nm - 500 nm, 350 nm - 400 nm, 350 nm - 450 nm, 350 nm - 500 nm, 400 nm - 450 nm, 400 nm - 500 nm, or 450 nm - 500 nm.The diameter of the micelles in the present specification can be about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or 500 nm. The diameter of the micelles in the present specification can be at least 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, or 450 nm. The diameter of the micelles in the present specification can be at most 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm.

[0049] In another embodiment, the casein micelle composition comprises (i) α s1 -casein only, and the calcium concentration in the composition is 1.48 to 150.00 mM, preferably 2.72 to 38.58 mM. The calcium concentration in the composition can be at least about 1.48 mM. The calcium concentration in the composition can be at most about 150.00 mM. The calcium concentration in the composition can be about 1.48, 1.85, 2.20, 2.55, 2.93, 3.30, 3.70, 4.1, 4.50, 4.80, 7.70, 14.30, 20.90, 27.50, 34.15, 40.8, 47.30, 54.00, 60.60, 67.20, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 105.0, 110.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, or 150.0 mM and can be. Preferably, the calcium concentration in the composition can be 1.48 to 150.00 mM, more preferably 2.72 to 38.58 mM.

[0050] In another embodiment, the casein micelle composition comprises (i) α s1- containing only casein, the concentration of phosphate in the composition is 23.5 - 58.40 mM, preferably 24.42 - 43.66 mM. The concentration of phosphate in the composition can be at least 23.5 mM. The concentration of phosphate in the composition can be up to about 58.5 mM. The concentration of phosphate in the composition can be about 23.30, 23.20, 23.00, 22.80, 22.60, 22.30, 22.20, 22.10, 22.00, 28.10, 31.80, 35.50, 39.2, 42.90, 46.60, 49.00, 52.00, 55.10, or 58.40 mM. Preferably, the concentration of phosphate in the composition is 23.5 - 58.40 mM, more preferably 24.42 - 43.66 mM.

[0051] In another embodiment, the casein micelle composition is (i) α s2 - containing only casein, the concentration of calcium in the composition is 1.48 - 150 mM, preferably 2.72 - 38.58 mM. The concentration of calcium in the composition can be at least about 1.48 mM. The concentration of calcium in the composition can be up to about 150.00 mM. The concentration of calcium in the composition can be about 1.48, 1.85, 2.20, 2.55, 2.93, 3.30, 3.70, 4.1, 4.50, 4.80, 7.70, 14.30, 20.90, 27.50, 34.15, 40.8, 47.30, 54.00, 60.60, 67.20, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 105.0, 110.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, or 150.0 mM. Preferably, the concentration of calcium in the composition is 1.48 - 150 mM, preferably 2.72 - 38.58 mM.

[0052] In another embodiment, the casein micelle composition is (i) α s2- It contains only casein, and the concentration of phosphate in the composition is 23.5 to 58.40 mM, preferably 24.42 to 43.66 mM. The concentration of phosphate in the composition can be at least 23.5 mM. The concentration of phosphate in the composition can be up to about 58.4 mM. The concentration of phosphate in the composition can be about 23.5, 23.3, 23.2, 23.0, 22.8, 22.6, 22.3, 22.2, 22.1, 22.0, 28.1, 31.8, 35.5, 39.21, 42.9, 46.6, 49.0, 51.5, 54.7, or 58.40 mM. Preferably, the concentration of phosphate in the composition is 23.5 to 58.40 mM, more preferably 24.42 to 43.66 mM.

[0053] In another embodiment, the casein micelle composition contains (i) only β-casein, and the concentration of calcium in the composition is 1.3 to 34.0 mM, preferably 1.81 to 7.69 mM. The concentration of calcium in the composition can be at least about 1.3 mM. The concentration of calcium in the composition can be up to about 35.00 mM. The concentration of calcium in the composition can be about 1.3, 1.5, 1.85, 2.2, 2.20, 2.55, 2.93, 3.30, 3.70, 4.1, 4.35, 4.50, 4.80, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 32.0, 33.0, 34.0, or 35.0 mM. Preferably, the concentration of calcium in the composition is 1.3 to 34.0 mM, more preferably 1.81 to 7.69 mM.

[0054] In another embodiment, the casein micelle composition contains only (i) β-casein, and the concentration of phosphate in the composition is 24.54 - 41.0 mM, preferably 24.54 - 36.1 mM. The concentration of phosphate in the composition can be at least 24.0 mM. The concentration of phosphate in the composition can be up to about 45.0 mM. The concentration of phosphate in the composition can be about 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, or 45.0 mM. Preferably, the concentration of phosphate in the composition is 24.54 - 41.0 mM, more preferably 24.54 - 36.1 mM.

[0055] In yet another further embodiment, the composition contains a mixture of calcium-sensitive caseins, and the concentrations of calcium and phosphate are a combination of ranges for the individual casein components, but are combined in proportion to the mole fraction of each casein in the mixture.

[0056] In some embodiments, the casein micelle composition of the present invention exhibited stability equivalent to that of naturally occurring micelle compositions. Ideally, the casein micelle particles maintain their dispersed state in their colloidal suspension or composition under conditions equivalent to those of naturally occurring micelle compositions. In both artificial micelle compositions and naturally occurring micelle compositions, under certain conditions, the dispersion of casein micelles will separate and the casein will precipitate.

[0057] In some embodiments, the casein micelle composition of the present invention maintains its dispersed state under centrifugation at 4000×g for 3 minutes. In some embodiments, the casein micelle composition of the present invention maintains its dispersed state under centrifugation at 3000×g for 5 minutes.

[0058] In some further embodiments, the casein micelle composition maintains a dispersed state when pasteurized at 72 °C for 15 seconds. In some further embodiments, the casein micelle composition maintains a dispersed state when pasteurized at 135 °C for 1-2 seconds.

[0059] In one embodiment of the present invention, the calcium-sensitive casein protein is recombinantly produced. The recombinant casein protein is recombinantly expressed in a host cell. As used herein, "host" or "host cell" means any protein-producing host selected or genetically engineered to produce a desired product. Exemplary hosts include fungi, bacteria, yeast, plants, insects, and mammalian cells. Preferably, the casein protein of the present invention is recombinantly produced from a host cell selected from the group consisting of bacteria, yeast, and fungi.

[0060] When the host cell is a bacterium, the bacterial host cell is preferably selected from the group consisting of Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus megaterium, Brevibacillus chohinensis, Mycobacterium smegmatic, Rhodococcus erythroplois, and Corynebacterium glutamicum, Lactobacillus sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis sp. 6803, and Escherichia coli (E. coli). Preferably, the bacterial host cell is Lactococcus lactis, Bacillus subtilis, or Escherichia coli (E. coli).

[0061] When the host cell is a yeast, the yeast host cell is preferably selected from the group consisting of Kluyveromyces sp., Pichia sp., Saccharomyces sp., Tetrahynena sp., Yarrowia sp., Hansenula sp., Blastobotrys sp., Candida sp., Zygosaccharomyces sp., or Debaryomyces sp.

[0062] When the host cell is a fungus, the fungal host cell is preferably selected from the group consisting of any Aspergillus species (e.g., Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, etc.), Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium granineum, Fusarium venenatum, Physcoinitrella patens, and Neurospora crassa.

[0063] In some embodiments, the casein protein comprises an amino acid sequence derived from any species. In particular, the casein protein generally comprises an amino acid sequence derived from any eutherian, metatherian, or monotreme species that contributes to the human diet, such as, for example, bovine, ovine, caprine, equine, or primate species. In some preferred embodiments, the casein protein comprises an amino acid sequence selected from the group consisting of bovine, caprine, ovine, buffalo, equine, bison, human, donkey, chimpanzee, rabbit, mouse, guinea pig, red fox, duckbill platypus, Australian wombat, wallaby, kobuushi, or mixtures thereof.

[0064] The calcium-sensitive casein protein of the present invention can be produced in the same host cell. Alternatively, the calcium-sensitive casein protein can be produced in different host cells.

[0065] Unlike casein proteins derived from animals, depending on the host cell used to express the casein protein, the casein protein can have a glycosylation or phosphorylation pattern (post-translational modification). In some cases, the casein protein does not contain post-translational modification (PTM). In some cases, the casein protein contains substantially reduced PTM. As used herein, substantially reduced PTM means at least a 50% reduction in one or more types of PTM compared to the amount of PTM in casein proteins derived from animals. For example, the casein protein can be post-translationally modified 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less than casein proteins derived from animals. Alternatively, the casein protein can contain PTM equivalent to that of animal-derived casein PTM.

[0066] PTM in casein proteins can be modified chemically or enzymatically. In some cases, the casein protein contains substantially reduced PTM or no PTM without chemical or enzymatic treatment. Casein micelles can be produced using casein proteins that have reduced PTM or no PTM, in which case the absence or reduction of PTM is not due to chemical or enzymatic treatment, for example, by producing recombinant casein proteins in a host where the casein protein is not post-translationally modified or the level of PTM is substantially reduced.

[0067] Glycosylation in casein proteins can be modified chemically or enzymatically. In some cases, casein proteins contain substantially reduced glycosylation or no glycosylation without chemical or enzymatic treatment. For example, casein proteins can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less glycosylated compared to casein from animals. Casein micelles can be produced using casein proteins with reduced glycosylation or no glycosylation, in which case the lack of glycosylation is not due to chemical or enzymatic treatment after recombinant production.

[0068] Phosphorylation in casein proteins can be modified chemically or enzymatically. In some cases, casein proteins contain substantially reduced phosphorylation or no phosphorylation without chemical or enzymatic treatment. For example, casein proteins can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less phosphorylated compared to casein from animals. Casein micelles can be produced using casein proteins with reduced phosphorylation or no phosphorylation, in which case the lack of phosphorylation is not due to chemical or enzymatic treatment, such as by producing recombinant proteins in a host where the casein proteins are not post-translationally modified or the level of PTM is substantially reduced.

[0069] The casein micelle composition of the present invention does not contain kappa-casein. Therefore, the proteins of the present invention do not contain protein sequences (SEQ ID NOs: 50 - 66) against kappa-casein.

[0070] In a preferred embodiment, the recombinantly produced casein protein has an amino acid sequence comprising SEQ ID NOs: 1 to 49 (as shown in Table 1) or variants thereof having at least 80% sequence homology. The protein may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NOs: 1 to 49 (as shown in Table 1).

[0071]

Table 1A

[0072]

Table 1B

[0073]

Table 1C

[0074]

Table 1D

[0075]

Table 1E

[0076]

Table 1F

[0077]

Table 1G

[0078]

Table 1H

[0079]

Table 1I

[0080]

Table 1J

[0081]

Table 1K

[0082]

Table 1L

[0083] In some embodiments of the present invention, the casein micelle composition further comprises one or more non-casein proteins. This additional one or more proteins can be any suitable protein. Preferably, the non-casein protein is selected from the group consisting of osteopontin, dentin matrix protein, matrix extracellular phosphoglycoprotein, bone sialoprotein, dentin sialophosphoprotein, amelogenin, statherin, starmaker or otolith matrix macromolecule-64 and related homologs, calcium-binding proteins, secreted calcium-binding phosphoprotein (SCPP), and mixtures thereof.

[0084] Method for producing a casein micelle composition The present invention also relates to a method for producing a casein micelle composition disclosed herein, the method comprising combining at least one calcium-sensitive casein protein with at least one salt under conditions such that the at least one calcium-sensitive casein protein forms casein micelle particles in a liquid colloid, wherein the casein micelle particles do not contain κ-casein protein.

[0085] Preferably, at least one salt is a calcium salt and / or a phosphate. More preferably, at least two salts are used, which are at least one calcium salt and at least one phosphate.

[0086] In some embodiments, the method further includes the addition of at least one additional salt. Any suitable salt such as calcium, phosphate, citrate, potassium, sodium, and / or chloride salts may be used. The calcium salt may be selected from calcium chloride, calcium carbonate, calcium citrate, calcium gluconate, calcium lactate, calcium gluconate, calcium acetate, and combinations thereof. The phosphate may be selected from orthophosphates such as monosodium phosphate, disodium phosphate, trisodium phosphate, monopotassium phosphate, dipotassium phosphate, tripotassium phosphate, etc.; pyrophosphates such as disodium or dipotassium pyrophosphate, trisodium or tripotassium pyrophosphate, tetrasodium or tetrapotassium pyrophosphate, etc.; polyphosphates such as sodium or potassium tripolyphosphate, sodium or potassium tetrapolyphosphate, sodium or potassium hexametaphosphate, etc. The citrate may be selected from calcium citrate, potassium citrate, sodium citrate, trisodium citrate, tripotassium citrate, or mixtures thereof. Preferably, at least one additional salt is selected from calcium, phosphate, citrate, sodium, chloride, potassium, metaphosphate, pyrophosphate, tripolyphosphate, longer polyphosphates, and mixtures thereof.

[0087] Micelle formation may require solubilization of the casein protein in a solvent such as water. Salt can be added after solubilization of the casein protein in the solvent. Alternatively, the salt and the casein protein may be added to the solution simultaneously. The salt can be added multiple times during micelle formation. For example, calcium salts, phosphates, and citrates can be added at regular intervals or in a continuous incremental addition process and mixed into the solution containing the casein protein until micelle formation is achieved. Different salts may be added at different times during the micelle formation process. For example, the calcium salt may be added before the addition of the phosphate and citrate, or the citrate may be added before the addition of the calcium and phosphate, or the phosphate may be added before the addition of the calcium and citrate. The preferred method is to add the salt incrementally to the protein solution while mixing and adjusting the pH to ensure that casein micelle particles are formed preferentially over the alternative results outlined in Figure 1.

[0088] For example, additional components such as lipids and saccharides may be added to the casein micelle composition. In some embodiments, a lipid is added to the casein micelle composition. In some embodiments, the lipid may not substantially contain animal-derived lipids. The lipids used herein may include plant-based lipids such as canola oil, sunflower oil, coconut oil, or combinations thereof. The concentration of the lipid can be from about 0% to about 5% in the casein micelle composition. The concentration of the lipid can be at least 0.5% or about 1%. The concentration of the lipid can be at most 5%. The concentration of the lipid can be about 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%. The concentration of the lipid can be from 0 to 0.5%, 0.5% to 1%, 1% to 3%, 1% to 4%, or 1% to 5%. The concentration of the lipid can be at most 2%, 3%, 4%, or 5%. The saccharides used herein may include plant-based disaccharides and / or oligosaccharides. Examples of saccharides include sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allose, tagatose, xylose, and arabinose.

[0089] In some embodiments of the method, the calcium-sensitive casein protein is preferably recombinantly produced from a host cell selected from the group consisting of bacteria, yeast, and fungi.

[0090] The calcium-sensitive casein protein of the present invention can be produced in the same host cell. Alternatively, the calcium-sensitive casein protein can be produced in different host cells.

[0091] The casein micelle composition of the present invention does not contain kappa-casein. Thus, the protein of the present invention does not contain the protein sequences (SEQ ID NOs: 50-66) for kappa-casein.

[0092] In a preferred embodiment, the recombinantly produced casein protein has an amino acid sequence comprising SEQ ID NOs: 1-49 (as shown in Table 1) or a variant thereof having at least 80% sequence homology. The protein can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NOs: 1-49 (as shown in Table 1).

[0093] The casein micelle composition of the present invention containing additional components can be produced by mixing different components at a temperature of 15°C to 45°C. For example, a casein micelle composition containing one or more recombinant proteins can be mixed with lipids and / or saccharides at a temperature of about 15°C, 17°C, 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, 40°C, 42°C, or 45°C.

[0094] Food product The present invention also relates to a food product comprising the casein micelle composition disclosed herein.

[0095] In some embodiments, the food product is a dairy product composition. Preferably, the food product is a cheese composition, a yogurt composition, or a milk composition.

[0096] The milk composition according to the present invention may contain other components such as saccharides, lipids, stabilizers, and flavorings. The texture of the milk composition described herein may be equivalent to that of milk derived from animals. The taste of the milk composition described herein may be equivalent to that of milk derived from animals.

[0097] The cheese composition according to the present invention may not contain components derived from animals. The cheese composition described herein may not contain dairy product-based components derived from animals, such as milk proteins derived from animals. The texture of the cheese composition described herein may be equivalent to that of a similar type of cheese produced using proteins derived from dairy products derived from animals, such as cheese made from animal milk. The taste of the cheese composition described herein may be equivalent to that of a similar type of cheese produced using milk proteins derived from animals.

[0098] The yogurt composition according to the present invention may contain other components such as saccharides, lipids, stabilizers, and flavorings. The texture of the yogurt composition described herein may be equivalent to that of yogurt derived from animals. The taste of the yogurt composition described herein may be equivalent to that of yogurt derived from animals.

Examples

[0099] The present invention will be further illustrated and exemplified by reference to the following non-limiting examples.

[0100] (Example 1) Expression of casein protein in E. coli Construct design, cloning, and transformation First, bovine kappa-casein (variant B) and beta-casein (variant A2) were codon-optimized for expression in E. coli, synthesized, and cloned into pET-26b(+) using NdeI / XhoI restriction sites by GenScript (registered trademark). These plasmids were then transformed into chemically competent E. coli T7 Express cells and grown overnight at 37 °C on LB agar supplemented with kanamycin for selection.

[0101] Protein Expression and Analysis Individual colonies were grown in liquid LB supplemented with kanamycin at 37 °C until an OD600 = 0.6 was reached. Expression of the target protein was then allowed to proceed for 2.5 hours by addition of 1 mM IPTG. Samples were prepared by pelleting 1 mL of the culture, and the pellet was lysed in 200 μL of NEB Express Lysis Reagent, and then an equal volume of Laemmli buffer (+ DTT) was added to the sample. Samples were analyzed using a BioRad Stain-free SDS-PAGE system, and protein identity was confirmed by MS / MS.

[0102] (Example 2) Expression of Casein Proteins in P. pastoris Construct Design, Cloning, and Transformation Genes corresponding to kappa-casein (variant B), beta-casein (variant A2), alpha-S1-casein (variant B), and alpha-S2-casein (variant A) were codon-optimized and synthesized by ATUM (California, USA). The genes were amplified by PCR using primers from IDT, assembled into the P. pastoris vector pD912-AA (ATUM) using NEBuilder® HiFi DNA Assembly mix, and transformed into DH5α competent cells. Minipreps of the plasmids were performed, and the vectors were confirmed by Sanger sequencing. Subsequently, chemically competent P. pastoris (PPS-9011 ATUM (California, USA)) was transformed and grown at 30 °C on YPD-agar containing Zeocin for selection.

[0103] Protein Expression and Analysis Stabilization stage 1: 96 clones were randomly selected and grown in 300 μL of YPD in a 96-well plate at 28 °C, 600 rpm, and 70% humidity for 24 h.

[0104] Stabilization stage 2: 3 μL of the previously grown strain was inoculated into YPD + Zeocin (250 μg / mL) and grown under the same culture conditions for 18 h.

[0105] Autoinduction stage: 3 μL of the strain in YPD + Zeo was inoculated into BMGM medium and grown under the same culture conditions for 72 h.

[0106] Next, the 96-well plates were centrifuged at 5000 × g for 30 min at 4 °C. Carefully, 10 μL of the supernatant was collected and analyzed using a BioRad Stain-free SDS-PAGE system, and the protein identity was confirmed by MS / MS.

[0107] (Example 3) Expression of Casein Proteins in Y. lipolytica Construct Design, Cloning, and Transformation The open reading frames (ORFs) corresponding to kappa-casein (variant B), beta-casein (variant A2), and bovine lactoferrin were codon-optimized using the Benchling (https: / / www.benchling.com / ) codon optimization tool. The Y. lipolytica LIP2 secretion signal sequence was added to the 5' end of each sequence. The codon-optimized sequences were synthesized and individually cloned into the pUC-GW-amp cloning vector by Azenta (Massachusetts, USA). The ORFs were released from each plasmid using PacI and SwaI restriction enzymes and cloned into the corresponding sites of the pHYL1001 vector after fragment isolation and purification. The pHYL1001 vector (synthesized by Azenta) was designed to enable the expression of the cloned ORFs using the Y. lipolytica TEF1 promoter, uracil auxotrophic selection, and zeta-site integration. Plasmids were enumerated in E. coli DH5α cells. Mini-preps of the plasmids were performed and the vectors were confirmed by Sanger sequencing. Subsequently, the plasmids were linearized by NotI restriction digestion and transformed into chemically competent Y. lipolytica PO1f (ATCC MYA-2613) cells. Transformants were selected using SD -ウラシル selective agar plates.

[0108] Protein expression and analysis Purification stage: Seven clones were randomly selected from each plate and streaked onto SD -ウラシル agar plates and incubated at 28 °C for 48 h.

[0109] Pre-culture stage: A single colony from each clone was inoculated into a 50 mL falcon tube containing 3 mL of buffered 2× SD -ウラシル (containing 2% glucose). These were grown at 28 °C for 24 h with shaking at 150 rpm.

[0110] Production stage: Using the pre-culture, up to an OD of 0.2, 10 mL of buffered 2× SD 600 was used.-ウラシル Seeded into a 250 mL flask containing (including 4% glucose). The cultures were grown at 28 °C for 72 h with shaking at 300 rpm at a continuous oxygen transfer rate while being monitored using a TOM fermentation system (Kuhner AG, Switzerland).

[0111] The optical density of each sample was measured and the supernatant was obtained by centrifugation at 8000×g for 5 min at 4 °C. 10 μL of the supernatant was taken and the secreted proteins were analyzed using a BioRad Stain-free SDS-PAGE system, and the protein identity was confirmed by MS / MS.

[0112] (Example 4) Expression of casein protein in Bacillus subtilis Construct design, cloning, and transformation Genes corresponding to kappa-casein (variant B), beta-casein (variant A2), alpha-S1-casein (variant B), and alpha-S2-casein (variant A) were codon-optimized using GenScript and synthesized by GenScript. The genes were amplified by PCR using primers from IDT, assembled into the Bacillus subtilis / Escherichia coli shuttle vector pTTB2 (MoBiTech, Germany) using NEBuilder® HiFi DNA Assembly mix, and transformed into DH5α competent cells. Mini-preps of the plasmids were performed and the vectors were confirmed by Sanger sequencing. The pTTB2 vector was linearized by EcoRI restriction digestion to remove all sequences necessary for E. coli growth. Subsequently, chemically competent Bacillus subtilis strain WEA (MoBiTech, Germany) was transformed and grown at 37 °C on 2×YT agar containing 1% xylose for selection.

[0113] Protein expression and analysis Pre-culture stage: 96 colonies were seeded into a deep-well plate containing 300 μL of LB medium and grown at 37 °C with shaking at 600 rpm for 18 h.

[0114] Self-induced stage: 3 μL of the preculture strain was seeded in LS medium and grown for 48 hours under the same culture conditions.

[0115] Subsequently, the 96-well plate was centrifuged at 5000×g for 30 minutes at 4°C. 10 μL of the supernatant was collected and analyzed using the BioRad Stain-free SDS-PAGE system, and the protein identity was confirmed by MS / MS.

[0116] (Example 5) Purification of bovine casein protein Confirmation of purity Bovine beta-casein (>98% PAGE purity), kappa-casein (>70% PAGE purity), and alpha-casein (>70% PAGE purity) proteins were purchased from Sigma Aldrich. These proteins were first analyzed by CE-SDS using Sciex Biophase 8800 to confirm purity and contamination, especially kappa-casein contamination in beta-casein and alpha-casein proteins. CE-SDS showed that beta-casein contained 87% beta-casein, 9% alpha-casein, and 3% kappa-casein (Figure 2), and kappa-casein contained 52% kappa-casein, 29% alpha-lactalbumin, 5% beta-lactoglobulin, 5% beta-casein, and 5% alpha-casein, and 4% others (Figure 3). Therefore, these proteins were subsequently purified using anion exchange chromatography. Anion exchange chromatography Buffer A: 3.3 M urea, 20 mM Tris, pH 8.0 Buffer B: 3.3 M urea, 20 mM Tris, 1 M NaCl, pH 8.0

[0117] 500 mg of sigma protein or sodium caseinate produced from raw milk was dissolved in 50 mL of buffer A containing 20 mM DTT, and then filtered through a 0.2 μm syringe filter. HiPrep Q HP 16 / 10 was connected to Akta Pure 150 and equilibrated with 5 CV of buffer A, after which the protein was loaded. The column was then washed with 4 CV of buffer A, and then a linear gradient to 40% buffer B over 10 CV was applied to the collected fractions. This provided very good separation of all the casein proteins, and when these proteins were reanalyzed by CE-SDS, they showed a purity of >99%.

[0118] (Example 6) Production and Analysis of β8κX Casein Micelles Using bovine beta-casein and kappa-casein purchased from Sigma Aldrich and re-purified, in the compositions shown in Table 2, while maintaining the beta-casein concentration at a constant 8 g / L -1 kappa-casein micelles were prepared by varying the kappa-casein concentration from 1 to 10 g / L.

[0119] [Table 2]

[0120] First, the protein was dissolved in 18.2 MΩ-cm ultra-high purity water, and then casein micelles were prepared by gradually adding inorganic substances to the protein solution. Sodium azide was added to stop bacterial growth for subsequent investigation of casein micelle stability.

[0121] After micelle formation, the solution was left as it was for an additional 12 hours to ensure the formation of stable micelles. The micelle solution is shown in Figure 4. Since the kappa-casein concentration is related to the casein micelle size and thus to light scattering, as can be seen from the figure, the whiteness of the solution decreases with an increase in the kappa-casein concentration, as expected for properly formed casein micelles. The turbidity of the solution was also measured before and after centrifugation at 1000×g for 3 minutes (Figure 5). When the casein micelles are properly formed, the measured turbidity values should be within about 50% of the readings before and after centrifugation. Turbidity was measured at 600 nm using a BMG Labtech CLARIOstar Plus microplate reader by diluting the sample four-fold in the ultrafiltrate of each sample. The ultrafiltrate was generated by centrifuging 6 mL of the casein micelle sample in a Sartorius Vivaspin 6 5,000 MWCO PES centrifugal filter. This same filtrate was used to dilute the sample for NanoSight particle tracking analysis (NTA).

[0122] Next, the size of the casein micelles in the solution was measured using a Malvern NanoSight NS300 equipped with a NanoSight Sample Assistant by first diluting the sample either 1000-fold (B8K2) or 10000-fold (B8K3 / 5 / 10) using the individual ultrafiltrate of each sample.

[0123] As can be seen from the figure, there is an inverse relationship between the amount of kappa-casein, the turbidity (Figure 5) and the size of the casein micelles (Figure 6).

[0124] Inorganic analysis using the ultrafiltration solution generated for NTA analysis was measured using a Perkin Elmer Nexion 259X Inductively Coupled Plasma Mass Spectrometer (ICPMS). Table 3 shows the concentrations of Pi and Ca measured in the ultrafiltration solutions of different micelle compositions, indicating that the micelles were correctly formed and bound to the rest of the Pi and Ca.

[0125]

Table 3

[0126] (Example 7) Stable artificial casein micelles prepared with bovine beta-casein without using kappa-casein Beta-casein micelle compositions were prepared, where the total protein concentration was maintained at 10 g / L and the pH was kept constant at 7.00, while the concentrations of calcium, phosphate, and NaCl were varied according to Table 4.

[0127]

Table 4

[0128] Samples were prepared by first dissolving beta-casein in 18.2 MΩ-cm ultra-high purity water and then performing a stepwise addition of the inorganic substances to achieve the desired final concentrations. Samples were mixed for 2 hours to equilibrate and then analyzed (Figure 7).

[0129] First, measurements were taken before and after centrifugation to analyze whether stable casein micelles were formed. As seen in Figure 8, stable casein micelles were formed when the turbidity was <50% before and after centrifugation. The turbidity increases with the increase in the Ca / Pi concentration.

[0130] Next, nanoparticle tracking analysis was used to measure the casein micelle size, which showed that the number of particles decreased with increasing Ca / Pi concentration, along with an increase in the micelle size (Figure 9).

[0131] Cryo-transmission electron microscopy (cryo-TEM) was used to visualize the formed casein micelles. Figure 10 shows the casein micelles of Sample 2, which indicates that the casein micelles are correctly formed by clearly visible calcium phosphate nanoclusters. These single casein protein micelles have an architecture significantly similar to that of natural bovine milk casein micelles in the literature such as Day, L., J.K. Raynes, A. Leis, L. H. Liu, and R.P.W. Williams. 2017. Food Hydrocolloids 69:150-163.

[0132] Inorganic analysis using the ultrafiltrate generated for NTA analysis was measured using a Perkin Elmer Nexion 259X Inductively Coupled Plasma Mass Spectrometer (ICPMS). Table 5 contains the concentrations of Pi and Ca measured in the ultrafiltrate of different micelle compositions, which indicates that the micelles are correctly formed and are bound to the remaining Pi and Ca.

[0133]

Table 5

[0134] (Example 8) Stable artificial casein micelles prepared with bovine alpha-S-casein without using kappa-casein Bovine alpha-S-casein, which contains both alpha-s1-casein and alpha-s2-casein, was purified from sodium caseinate using anion exchange chromatography as described above. Capillary tube electrophoresis was used to confirm the purity and ensure that kappa-casein was not included.

[0135] An alpha-s-casein micellar composition was prepared, where the total protein concentration was maintained at 10 g / L, the pH was kept constant at 7.00, while the concentrations of calcium, phosphate, and NaCl were varied according to Table 6.

[0136] [Table 6]

[0137] Samples were prepared by first dissolving alpha-s-casein in ultra-high purity water of 18.2 MΩ-cm and then performing a stepwise addition of inorganic substances to achieve the desired final concentrations. After mixing for 2 hours to equilibrate the samples, analysis was performed (Figure 11), and sodium caseinate micelles were prepared in parallel as a control.

[0138] First, measurements were taken before and after centrifugation to analyze and test whether stable casein micelles were formed. As seen in Figure 12, stable casein micelles were formed when the turbidity was <50% before and after centrifugation. The turbidity increases with the increase in Ca / Pi concentration.

[0139] Next, nanoparticle tracking analysis was used to measure the casein micelle size (Figure 13), which showed that the casein micelle size also increases with the increase in the concentrations of Ca and Pi.

[0140] (Anticipated Example 1) A milk beverage composition containing a single protein casein micelle Example compositions for milk beverages containing single casein micelles are shown in Table 7. Although only one composition per protein is represented here, it should be noted that, based on this patent application, many different compositions for milk beverages are possible, and those skilled in the art will recognize this. First, the casein protein powder is dissolved in the required amount of water, and then single casein micelles are formed by incrementally adding inorganic substances to this solution. After the micelles are formed, beta-lactoglobulin and maltose are added to the solution, and then the preheated lipid component is added. The solution is then homogenized, pasteurized, and bottled before consumption.

[0141] [Table 7]

[0142] (Predicted Example 2) Yogurt composition containing single protein casein micelles Example compositions for yogurt containing single casein micelles are shown in Table 8. Although only one composition per protein is represented here, it should be noted that, based on this patent application, many different compositions for yogurt are possible, and those skilled in the art will recognize this. First, the casein protein powder is dissolved in the required amount of water, and then single casein micelles are formed by incrementally adding inorganic substances to this solution. After the micelles are formed, beta-lactoglobulin and maltose are added to the solution, and then the preheated lipid component is added. Next, the milk base is heated to 85°C to denature the whey protein, and then, after cooling to 40°C, lactic acid starter cultures (e.g., Lactobacillus strains) are added. Then, after fermentation is allowed to proceed for 4 - 8 hours, it is cooled to 4°C and packaged.

[0143] [Table 8]

[0144] (Anticipated Example 3) Fresh cheese composition containing single protein casein micelles Example compositions for cheeses containing single casein micelles are shown in Table 9. Although only one composition per protein is represented here, it should be noted that based on this patent application, many different compositions for cheeses are possible and those skilled in the art will recognize this. First, the casein protein powder is dissolved in the required amount of water, and then single casein micelles are formed by gradually adding inorganic substances to this solution. After the micelles are formed, maltose is added to the solution, and then the preheated lipid component is added. Next, the milk base is warmed to 38°C, and based on the manufacturer's instructions, a rennet substitute (one that does not contain kappa-casein), such as microbial pepsin, etc., is added. Then, the milk is left to coagulate for 30 minutes. After coagulation, the curd is cut into approximately 2.5 cm cubes, gently scooped out with a spoon, and placed in a cheese mold to remove moisture.

[0145]

Table 9

[0146] In some embodiments, the present invention is provided according to the following numbered clauses. 1. A casein micelle composition containing amorphous calcium phosphate (CaP), wherein the casein micelles contain at least one calcium-sensitive casein and the casein micelles do not contain kappa-casein, a casein micelle composition. 2. The casein micelles are (i) α s1 -casein only; (ii) α s2 -casein only; (iii) β-casein only; (iv) α s1 -casein and β-casein mixture; (v) α s2 -casein and β-casein mixture; (vi) αs1 - Casein and α s2 - A mixture with casein; or (vii) α s1 - Casein and α s2 - A mixture of casein and β-casein The casein micelle composition according to claim 1, comprising 3. The casein micelle composition according to claim 2, containing sufficient CaP to bind to about 5 - 100% of the casein. 4. The casein micelle composition according to any one of claims 1 - 3, wherein the total casein concentration in the composition is about 0.5 - 100 g / L, preferably about 5 - 50 g / L, preferably about 30 g / L. 5. The casein micelle composition according to any one of claims 1 - 4, wherein the pH of the composition is about 5.5 - 8.0, preferably about pH 6.7. 6. When the casein micelle composition contains only (i) α s1 - casein, the calcium concentration in the composition is 1.48 - 150.00 mM, preferably 2.72 - 38.58 mM. The casein micelle composition according to any one of claims 2 - 5. 7. When the casein micelle composition contains only (i) α s1 - casein, the phosphate concentration in the composition is 23.5 - 58.40 mM, preferably 24.42 - 43.66 mM. The casein micelle composition according to any one of claims 2 - 5. 8. When the casein micelle composition contains only (i) α s2 - casein, the calcium concentration in the composition is 1.48 - 150.00 mM, preferably 2.72 - 38.58 mM. The casein micelle composition according to any one of claims 2 - 5. 9. When the casein micelle composition contains only (i) α s2 - casein, the phosphate concentration in the composition is 23.5 - 58.40 mM, preferably 24.42 - 43.66 mM. The casein micelle composition according to any one of claims 2 - 5. 10. The casein micelle composition according to any one of claims 2 to 5, wherein when the composition contains only (i) β-casein, the concentration of calcium in the composition is 1.3 to 34.0 mM, preferably 1.81 to 7.69 mM. 11. The casein micelle composition according to any one of claims 2 to 5, wherein when the composition contains only (i) β-casein, the concentration of phosphate in the composition is 24.54 to 41.0 mM, preferably 24.54 to 36.1 mM. 12. The casein micelle composition according to any one of claims 2 to 5, comprising a mixture of calcium-sensitive caseins, wherein the concentrations of calcium and phosphate are a combination within the ranges of the individual casein components, but are combined in proportion to the molar fraction of each casein in the mixture. 13. The casein micelle composition according to any one of claims 1 to 12, wherein the calcium-sensitive casein protein is recombinantly produced. 14. The casein micelle composition according to claim 13, wherein the recombinantly produced casein protein is produced from a host cell selected from the group consisting of bacteria, yeast, and fungi. 15. The casein micelle composition according to any one of claims 1 to 14, which maintains a dispersed state under centrifugation at 4000×g for 3 minutes. 16. The casein micelle composition according to any one of claims 1 to 14, which maintains a dispersed state when pasteurized at 72°C for 15 seconds. 17. The casein micelle composition according to any one of claims 1 to 14, which maintains a dispersed state when pasteurized at 135°C for 1 to 4 seconds. 18. The casein micelle composition according to any one of claims 13 to 17, wherein the recombinantly produced casein protein has an amino acid sequence comprising SEQ ID NOs: 1 to 49 or a variant thereof having at least 80% sequence homology. 19. The casein micelle composition according to any one of claims 14 to 18, wherein the bacterial host cell is selected from the group consisting of Lactococcus species, Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis, and Corynebacterium glutamicum, Lactobacillus species, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis species 6803, and Escherichia coli. 20. The casein micelle composition according to any one of claims 14 to 18, wherein the yeast host cell is selected from the group consisting of Kluyveromyces species, Pichia species, Saccharomyces species, Tetrahymena species, Yarrowia species, Hansenula species, Blastobotrys species, Candida species, Zygosaccharomyces species, or Debaryomyces species. 21. The casein micelle composition according to any one of claims 14 to 18, wherein the fungal host cell is selected from the group consisting of any Aspergillus species (e.g., Aspergillus pseudotuberculatus, Aspergillus niger, Aspergillus oryzae, etc.), Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium graminearum, Fusarium venenatum, Mucor miehei, and Rhizopus oryzae. 22. The casein micelle composition according to any one of claims 1 to 21, wherein the casein protein comprises the amino acid sequence of any one of the group consisting of bovine, goat, sheep, buffalo, camel, horse, bison, human, donkey, chimpanzee, rabbit, mouse, guinea pig, masked palm civet, Japanese weasel, Australian sea lion, wallaby, kob, or a mixture thereof. 23. The casein micelle composition according to any one of claims 1 to 22, wherein the casein micelle further comprises one or more non-casein proteins. 24. The casein micelle composition according to item 23, wherein the non-casein protein is selected from the group consisting of osteopontin, dentin matrix protein, matrix extracellular phosphoglycoprotein, bone sialoprotein, dentin sialophosphoprotein, amelogenin, statherin, starmaker or otolith matrix macromolecule-64 and related homologs, calcium-binding protein, secreted calcium-binding phosphoprotein (SCPP), and mixtures thereof. 25. A method for producing the casein micelle composition according to any one of items 1 to 24, comprising combining at least one calcium-sensitive casein protein with at least one salt under conditions where the at least one calcium-sensitive casein protein forms casein micelles in a liquid colloid, and the casein micelles do not contain κ-casein protein. 26. The method according to item 25, wherein the at least one salt is a calcium salt and / or a phosphate. 27. The method according to item 26, further comprising the addition of at least one further salt selected from calcium salts, phosphates, sodium chloride, potassium chloride, citrates, metaphosphates, pyrophosphates, tripolyphosphates, longer polyphosphates, and mixtures thereof. 28. A food product comprising the casein micelle composition according to any one of items 1 to 24. 29. The food product according to item 28, which is a dairy product composition, preferably a cheese composition, a yogurt composition, or a milk composition.

[0147] Those skilled in the art will understand that the present invention described herein can accept changes and modifications other than those clearly described. The present invention encompasses all such changes and modifications, and it should be understood that they are within the spirit and scope of the present invention.

Claims

1. A casein micelle composition comprising casein micelle particles containing amorphous calcium phosphate (CaP), wherein the casein micelle particles comprise at least one calcium-sensitive casein and do not contain κ-casein.

2. The casein micelle particles are (i) α s1 -casein only; (ii) α s2 -casein only; (iii) β-casein only; (iv) A mixture of α s1 -casein and β-casein; (v) A mixture of α s2 -casein and β-casein; (vi) A mixture of α s1 -casein and α s2 -casein; or (vii) A mixture of α s1 -casein, α s2 -casein, and β-casein The casein micelle composition according to claim 1, comprising

3. The casein micelle composition according to claim 2, containing sufficient CaP to bind to about 5 to 100% of the casein.

4. The casein micelle composition according to any one of claims 1 to 3, wherein the total casein concentration in the composition is about 0.5 to 100 g / L, preferably about 5 to 50 g / L, and preferably about 30 g / L.

5. The casein micelle composition according to any one of claims 1 to 4, wherein the pH of the composition is about 5.5 to 8.0, preferably about pH 6.

7.

6. The casein micelle particles are (i) α s1- When containing only casein, the concentration of calcium in the composition is 1.48 to 150.00 mM, preferably 2.72 to 38.58 mM, the casein micelle composition according to any one of claims 2 to 5.

7. The casein micelle particles are (i) α s1 - When containing only casein, the concentration of phosphate in the composition is 23.5 to 58.40 mM, preferably 24.42 to 43.66 mM, the casein micelle composition according to any one of claims 2 to 5.

8. The casein micelle particles are (ii) α s2 - When containing only casein, the concentration of calcium in the composition is 1.48 to 150.00 mM, preferably 2.72 to 38.58 mM, the casein micelle composition according to any one of claims 2 to 5.

9. The casein micelle particles are (ii) α s2 - When containing only casein, the concentration of phosphate in the composition is 23.5 to 58.40 mM, preferably 24.42 to 43.66 mM, the casein micelle composition according to any one of claims 2 to 5.

10. When the casein micelle particles contain only (iii) β-casein, the concentration of calcium in the composition is 1.3 to 34.0 mM, preferably 1.81 to 7.69 mM, the casein micelle composition according to any one of claims 2 to 5.

11. When the casein micelle particles contain only (iii) β-casein, the concentration of phosphate in the composition is 24.54 to 41.0 mM, preferably 24.54 to 36.1 mM, the casein micelle composition according to any one of claims 2 to 5.

12. The casein micelle particles contain a mixture of calcium-sensitive caseins, and the concentrations of calcium and phosphate are a combination within the ranges of the individual casein micelle particle components, but are combined in proportion to the molar fraction of each casein in the mixture, the casein micelle composition according to any one of claims 2 to 5.

13. The calcium-sensitive casein protein is recombinantly produced, and the casein micelle composition according to any one of claims 1 to 12.

14. The recombinantly produced casein protein is produced from a host cell selected from the group consisting of bacteria, yeasts, and fungi, and the casein micelle composition according to claim 13.

15. The casein micelle composition according to any one of claims 1 to 14, which maintains a dispersed state under centrifugation at 4000×g for 3 minutes.

16. The casein micelle composition according to any one of claims 1 to 14, which maintains a dispersed state when pasteurized at 72°C for 15 seconds and / or when pasteurized at 135°C for 1 to 4 seconds.

17. The casein micelle composition according to any one of claims 13 to 16, wherein the recombinantly produced casein protein has an amino acid sequence comprising SEQ ID NOs: 1 to 49 or a variant thereof having at least 80% sequence homology.

18. (a) When the host cell is a bacterial host cell, the bacterial host cell is selected from the group consisting of Lactococcus species, Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis, and Corynebacterium glutamicum, Lactobacillus species, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis species 6803, and Escherichia coli; (b) When the host cell is a yeast host cell, the yeast host cell is selected from the group consisting of Kluyveromyces species, Pichia species, Saccharomyces species, Tetrahymena species, Yarrowia species, Hansenula species, Blastobotrys species, Candida species, Zygosaccharomyces species, or Debaryomyces species; or (c) When the host cell is a fungal host cell, the fungal host cell is selected from the group consisting of any Aspergillus species (e.g., Aspergillus pseudotrichialis, Aspergillus niger, Aspergillus oryzae, etc.), Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium graminearum, Fusarium venenatum, Microdochium nivale, and Monascus purpureus, the casein micelle composition according to any one of claims 14 to 17.

19. The casein protein contains the amino acid sequence of any one of the group consisting of bovine, goat, sheep, buffalo, camel, horse, bison, human, donkey, chimpanzee, rabbit, mouse, guinea pig, red fox, duckbill platypus, Australian echidna, wallaby, kobushi, or a mixture thereof, the casein micelle composition according to any one of claims 1 to 18.

20. The casein micelle particles preferably further contain one or more non-casein proteins selected from the group consisting of osteopontin, dentin matrix protein, matrix extracellular phosphoglycoprotein, bone sialoprotein, dentin sialophosphoprotein, amelogenin, statherin, starmaker, or otolith matrix macromolecule-64 and related homologs, calcium-binding proteins, secreted calcium-binding phosphoprotein (SCPP), and mixtures thereof, the casein micelle composition according to any one of claims 1 to 19.

21. A method for producing a casein micelle composition according to any one of claims 1 to 20, comprising combining at least one calcium-sensitive casein protein with at least one salt under conditions such that the at least one calcium-sensitive casein protein forms casein micelle particles in a liquid colloid, wherein the casein micelles do not contain κ-casein protein. **Claim 22** The method according to claim 21, wherein the at least one salt is a calcium salt and / or a phosphate. **Claim 23** The method according to claim 22, further comprising the addition of at least one further salt selected from calcium salts, phosphates, sodium chloride, potassium chloride, citrates, metaphosphates, pyrophosphates, tripolyphosphates, longer polyphosphates, and mixtures thereof. **Claim 24** A food product comprising a casein micelle composition according to any one of claims 1 to 23. **Claim 25** The food product according to claim 24, which is a dairy product composition, preferably a cheese composition, a yogurt composition, or a milk composition.

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