Composition

Abstract

The present invention provides casein colloidal particle compositions comprising amorphous calcium phosphate (CaP) wherein the casein colloidal particles comprise at least one calcium ion sensitive casein, and wherein the casein colloidal particles do not comprise kappa-casein. The invention also provides a method for preparing the casein colloidal particle composition. The invention also provides a food product containing the casein colloidal particle composition.

Description

Priority details

[0001] This application claims priority to Australian Patent No. 2022901459 filed on May 30, 2022, the contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to a casein micelle composition and a preparation method thereof. The present invention also relates to a food containing the casein micelle composition. Background Art

[0003] With global warming continuing and the world population expected to reach 10 billion by 2050, there is an urgent need for high-quality protein that is produced sustainably while reducing greenhouse gas emissions (GHGE) generated by animals.

[0004] Milk protein is one of the most nutritious sources of protein due to its amino acid profile, its inherent ability to transport high concentrations of bioavailable calcium and phosphate via protein assemblies called casein micelles, and its easy digestibility in the stomach. The main drawbacks of current conventional dairy farming are the high environmental cost of land and water used to produce milk and the amount of greenhouse gases, especially methane, generated.

[0005] Plant-based milk alternatives, while having lower GHGE and lower water usage, differ in nutrition, functionality and taste from animal-based milks because they do not contain casein, a protein found only in mammals. The protein fraction of mammalian milk consists of casein and whey protein. Casein is an intrinsically disordered protein (IDP), sometimes glycosylated and often phosphorylated, while whey protein is more globular with a defined folded conformation. Casein is further divided into calcium-sensitive casein and casein that is insensitive to calcium ion precipitation. The calcium-sensitive caseins in cows are α S1 -Casein, α S2 -casein and β-casein, while κ-casein is not sensitive to calcium ion precipitation.

[0006] Cellular agriculture is an alternative form of traditional agriculture, involving the cultivation of muscle and fat cells to replace slaughtered animals for meat, and engineering microorganisms to express a range of food molecules, particularly animal proteins in milk and eggs. Using cellular agriculture to produce casein through genetic engineering of microorganisms is a promising approach to reduce the environmental disadvantages of animal agriculture. However, since casein is an inherently disordered protein with highly phosphorylated and glycosylated post-translational modifications, it is extremely difficult to produce it efficiently and prepare it into natural casein micelles required for the production of stable dairy products such as liquid milk.

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

[0008] Cow's milk and natural casein micelles exhibit great 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 to form amyloid fibrils. Cow's milk can withstand pasteurization and more extreme heat treatments and can be dried and reconstituted in water. Furthermore, although many milks contain high concentrations of calcium and phosphate, far exceeding the solubility of calcium phosphate at milk pH, these treatments generally do not produce precipitation of calcium phosphate. These natural properties are exploited in the widespread production and storage of liquid dairy products.

[0009] Although casein micelles are stable at the pH of milk, they rapidly polymerize to form gels at acidic pH. Also, the stability of casein micelles may be reduced or eliminated by limited proteolysis catalyzed by aspartic proteases such as chymosin or similar proteases. This property has been rapidly exploited in the production of gelled dairy products such as cheese and yogurt.

[0010] The composition of casein micelles varies greatly from species to species. For example, in cow milk there are polymorphic secretory proteins expressed by four casein genes: κ-casein, β-casein, α-casein, s1 -Casein and α s2 In contrast, elephant milk contains only two casein genes expressed and secreted proteins, κ-casein and β-casein in a ratio of approximately 1:4:4:1, respectively. In contrast, elephant milk contains only two casein genes expressed and secreted proteins, κ-casein and β-casein in a ratio of approximately 1:8.5, respectively. In the milk of other species, such as rabbits, there are proteins produced by 5 casein genes. All well-characterized milks contain the κ-casein form, but all the remaining caseins may or may not be present in different mammalian species.

[0011] Although naturally occurring caseins vary widely, there are no known examples of casein micelles formed from a single casein. Thus, at least two caseins are conservatively required to form casein micelles. All naturally occurring casein micelles contain one type of κ-casein and one or more types of calcium-sensitive caseins. One type of κ-casein is present in the milk of all mammals. Furthermore, it is generally believed that κ-casein is important for milk stability because it lacks sensitivity to divalent cations such as Ca. 2+ Susceptibility to precipitation. It is generally believed that casein micelle stability is due to a stabilizing colloidal mechanism known as steric stabilization.

[0012] The proportion of kappa-casein in total casein varies widely. Low proportions of kappa-casein are found in equines, such as 1.8% in horse milk and 2.8% in donkey milk, and this proportion varies widely between individuals and during lactation. For example, a recent investigation of lactational changes in casein composition in individual women showed that the proportion of kappa-casein in total casein ranged from 11.5% to nearly 76%.

[0013] Although there are limited examples in the prior art that claim to have obtained single casein micelles, all the examples examined so far have used commercially sourced casein. Commercially available casein is always adulterated with other caseins, especially the aforementioned kappa-casein that contributes to the stability of the micelles. Therefore, true single casein micelles do not exist in the prior art.

[0014] Artificial casein micelles are not a necessary result. Figure 1 As shown, there are a variety of possible outcomes when casein is mixed with salts. These outcomes depend on several factors, including the final composition of the formulation, the order in which the ingredients are added, and the rate of addition. In addition, the stability of the formulation depends on how soon after mixing the determination is made.

[0015] Hydrophobic substances, such as Figure 1 As pointed out in the literature, various low-solubility substances can be stabilized in a colloidal state by adsorbing a more hydrophilic outer shell. This phenomenon results in a shell-core structure that is kinetically stable and can survive for a long time under favorable conditions. However, in the case of CaP or CaCO3, the first formed amorphous phase is stabilized by casein, preventing it from maturing into the less soluble and more crystalline CaP form, so there are two mechanisms of colloidal destabilization, both of which are affected by the casein concentration and the affinity of casein to bind to the amorphous phase and the more crystalline phase. Decreased casein solubility can also lead to destabilization, such as salting out and charge reduction. In order to determine the results of a specific formulation and mixing step, various characterization methods can be used to determine the composition, particle size and substructure of the product and its stability.

[0016] Determining the structure of the formed casein micelles requires chemical analysis of the colloid and the continuous phase of calcium and phosphorus present in the colloid. Applicable chemical analyses include nanoparticle tracking analysis (NTA) particle size data, turbidity before and after centrifugation, transmission electron microscopy (TEM) and inorganic analysis. The micelles can be demonstrated to contain CaP nanoclusters by using, for example, cryo-electron microscopy, small angle X-ray scattering or neutron small angle scattering experiments.

[0017] Therefore, there is a need to continue to improve the artificial casein micelle composition and the preparation method thereof, at least partially solving one or more of the above-mentioned shortcomings or providing a practical alternative.

[0018] In particular, there is a need for improved artificial casein micelle compositions whose structure, composition and physico-chemical properties are similar to those of natural casein micelles in milk. Such compositions may include, for example, the following properties: (i) white appearance and high calcium concentration in solution; (ii) stability to heat treatment and drying at neutral pH; (iii) avoidance of amyloid fibril formation; (iv) acid-promoted gelation; or (v) limited protease-promoted gelation.

[0019] The reference herein to a patent document or other matter given as prior art is not an admission that the document or matter was known or that the information it contained was part of the common general knowledge at the priority date of any claim. Summary of the invention

[0020] In a first aspect, the present invention provides a composition comprising casein micelle particles, the casein micelle composition comprising amorphous calcium phosphate (CaP), wherein the casein micelle particles comprise at least one calcium-sensitive casein, and wherein the casein micelles do not comprise κ-casein.

[0021] According to another aspect of the present invention, casein micelle particles comprise: (i) only α S1 -casein; (ii) only α S2 -casein; (iii) β-casein only; (iv) α S1 A mixture of α-casein and β-casein; (v) α S2 A mixture of α-casein and β-casein; (vi) S1 -Casein and α S2 - a mixture of caseins or (vii) α S1 -Casein, α S2 -A mixture of casein and beta-casein.

[0022] Advantageously, the casein micelle compositions of the present invention do not contain kappa-casein and exhibit stability and appearance similar to naturally occurring casein micelles, which allows the casein micelle compositions to contain only calcium-sensitive caseins, and in certain aspects, only one type of casein.

[0023] Furthermore, the casein micelles of the present invention can be produced more economically than prior art solutions.In other aspects, the present invention relates to methods for preparing the casein micelle compositions of the present disclosure.

[0024] The food products to which the present invention relates can advantageously provide improved plant-based milk substitutes. These foods avoid some or all of the disadvantages associated with animal-based dairy products and current plant-based milk substitutes while providing improved nutrition, functionality and / or taste. In yet another aspect, the present invention relates to a food product comprising the casein micelle composition of the present disclosure.

[0025] Other aspects of the invention are presented in the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0027] Figure 1 The possible consequences of mixing casein with salts are shown, which lead to differences in structure and stability compared to native casein micelles.

[0028] Figure 2 The electrophoretic patterns of commercial β-casein from Sigma Aldrich and in-house purified β-casein were shown in the capillary electrophoresis apparatus.

[0029] Figure 3 The electrophoretic patterns of commercial κ-casein from Sigma Aldrich and in-house purified κ-casein were shown in the capillary electrophoresis instrument.

[0030] Figure 4 Schematic representation of β-casein micelle solutions prepared with increasing κ-casein concentrations.

[0031] Figure 5 Graph showing the turbidity of the casein micelle solution of β-casein with increasing κ-casein concentration before and after centrifugation.

[0032] Figure 6 Schematic diagram showing the relationship between the hydrodynamic radius and particle concentration of B8KX series samples measured by nanoparticle tracer analysis.

[0033] Figure 7 Schematic diagram of β-casein micelles and caseinate casein micelles control solutions at different inorganic concentrations.

[0034] Figure 8 Graph showing turbidity measurements of micelles containing only β-casein before and after centrifugation.

[0035] Fig. 9 Schematic diagram showing the relationship between the hydrodynamic radius and the particle concentration of micelle samples 1, 2 and 3 containing only β-casein measured by nanoparticle tracking analysis.

[0036] Fig.10 is a cryo-transmission electron micrograph of casein micelles containing only β-casein from sample 2.

[0037] Fig.11 Schematic diagram of α-s-casein micelles and caseinate micelles control solutions at different inorganic concentrations.

[0038] Fig.12 is a graph showing the turbidity of the α-s-casein micelle solution before and after centrifugation.

[0039] Fig.13 Schematic diagram showing the relationship between the hydrodynamic radius and particle concentration of micelle samples 1 and 2 containing only α-s-casein measured by nanoparticle tracing analysis. DETAILED DESCRIPTION OF THE INVENTION

[0041] Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting.

[0042] definition

[0043] Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meanings as commonly understood by one of ordinary skill in the art.

[0044] As used in this specification and the appended claims, plural references are not used unless the content clearly dictates otherwise.

[0045] Throughout the specification and claims, the word "comprise" and variations of the word, such as "include" and "comprising", are not intended to exclude other additives, components, integers or steps. As used herein, "comprising" means "including". Therefore, "comprising A or B" means "including A, B or A and B, without excluding other elements.

[0046] As used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components, whether or not the other feature or component is present. For example, "A and / or B" is considered to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each was listed individually herein.

[0047] As used herein, the term "food" refers to a substance that can be used or prepared as food, which is any nutritious substance eaten or drunk by humans or animals to maintain life and growth.

[0048] As used herein, the term "calcium-sensitive casein" refers to casein that is sensitive to calcium ion precipitation. Similarly, the term "non-calcium-sensitive casein" refers to casein that is not sensitive to calcium ion precipitation. Calcium-sensitive bovine casein is α S1 -Casein, α S2 -casein and β-casein, while κ-casein is not sensitive to calcium ion precipitation. As used herein, the term "α-casein" S1"-casein" and "α-S1 casein" are used interchangeably to refer to all polymorphs of the CSN1S1 gene product encoding α-S1 casein. As used herein, the term "α S2 As used herein, the terms "α-casein" and "α-S2 casein" are used interchangeably and refer to all polymorphs of the CSN1S2 gene product encoding α-S2 casein. As used herein, the terms "β-casein" and "β casein" are used interchangeably and refer to all polymorphs of the CSN2 gene product encoding β-casein. As used herein, the terms "κ-casein" and "κ casein" are used interchangeably and refer to all polymorphs of the CSN3 gene product encoding κ-casein.

[0049] Casein micelle composition

[0050] The present invention relates to a casein micelle composition comprising casein micelle particles, wherein the casein micelle composition comprises amorphous calcium phosphate (CaP), and wherein the casein micelle particles comprise at least one calcium-sensitive casein, and wherein the casein micelle particles do not comprise kappa-casein.

[0051] Casein micelles are colloidal particles formed by casein polymers, dispersed in a liquid to form a colloidal suspension or composition. Typically the liquid is water, but any suitable solvent may be used.

[0052] In various embodiments of the casein micelle composition of the present invention, the casein micelle particles comprise:

[0053] (i) Only α S1 -Casein;

[0054] (ii) Only α S2 -Casein;

[0055] (iii) β-casein only;

[0056] (iv) α S1 - A mixture of casein and beta-casein;

[0057] (v) α S2 - A mixture of casein and beta-casein;

[0058] (vi) α S1 -Casein and α S2 - a mixture of caseins; or

[0059] (vii) α S1 -Casein, α S2 -A mixture of casein and beta-casein.

[0060] In one embodiment, described casein micelle composition contains enough CaP to combine about 5% to 100% casein.This casein micelle composition can contain enough CaP to combine at least 3% or about 5% casein.This casein micelle composition can contain enough CaP to combine up to 100% casein.This casein micelle composition can contain enough CaP to combine at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% casein.

[0061] In another embodiment, the total casein concentration in the composition is about 0.5 g / L 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 about 100 g / L at most. The total casein concentration in the composition can be 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or 100 g / L. The total casein concentration in the composition can be 0.5 g / L to 5 g / L, 0.5 g / L to 10 g / L, 0.5 g / L to 15 g / L, 0.5 g / L to 20 g / L, 0.5 g / L to 25 g / L, 0.5 g / L to 30 g / L, 0.5 g / L to 35 g / L, 0.5 g / L to 40 g / L, 0.5 g / L to 45 g / L, 0.5 g / L to 50 g / L, 0.5 g / L to 55 g / L, 0.5 g / L to 60 g / L, 0.5 g / L to 65 g / L, 0.5 g / L to 70g / L, 0.5g / L to 75g / L, 0.5g / L to 80g / L, 0.5g / L to 85g / L, 0.5g / L to 90g / L, 0.5g / L to 95g / L, 0.5g / L to 100g / L, 1g / L to 5g / L, 1g / L to 10g / L, 1g / L to 15g / L, 1g / L to 20g / L, 1g / L to 25g / L, 1g / L to 30g / L, 1g / L to 35g / L, 1g / L to 40g / L, 1g / L to 45g / L, 1g / L to 50g / L, 1g / L to 55g / L, 1g / L to 60g / L, 1g / L to 65g / L, 1g / L to 70g / L, 1g / L to 75g / L, 1g / L to 80g / L, 1g / L to 85g / L, 1g / L to 90g / L, 1g / L to 95g / L, 1g / L to 100g / L, 2g / L to 5g / L, 2g / L to 10g / L, 2g / L to 15g / L, 2g / L to 20g / L, 2g / L to 25g / L, 2g / L to 30g / L, 2g / L to 35g / L, 2g / L to 40g / L, 2g / L to 45g / L, 2g / L to 50g / L, 2g / L to 55g / L, 2g / L to 60g / L, 2g / L to 65g / L, 2g / L to 70g / L, 2g / L to 75g / L, 2g / L to 80g / L, 2g / L to 85g / L, 2g / L to 90g / L, 2g / L to 95g / L, 2g / L to 100g / L, 3g / L to 5g / L, 3g / L to 10g / L, 3g / L to 15g / L,3g / L to 20g / L, 3g / L to 25g / L, 3g / L to 30g / L, 3g / L to 35g / L, 3g / L to 40g / L, 3g / L to 45g / L, 3g / L to 50g / L, 3g / L to 55g / L, 3g / L to 60g / L, 3g / L to 65g / L, 3g / L to 70g / L, 3g / 3g / L to 75g / L, 3g / L to 80g / L, 3g / L to 85g / L, 3g / L to 90g / L, 3g / L to 95g / L, or 3g / L to 100g / L, 4g / L to 5g / L, 4g / L to 10g / L, 4g / L to 15g / L, 4g / L to 20g / L, 4g / L to 25g / L, 4g / L to 30g / L, 4g / L to 35g / L, 4g / L to 40g / L, 4g / L to 45g / L, 4g / L to 50g / L, 4g / L to 55g / L, 4g / L to 60g / L, 4g / L to 65g / L, 4g / L to 70g / L, 4g / L to 75g / L, 4g / L to 80g / L, 4g / L to 85g / L, 4g / L to 90g / L, 4g / L to 95g / L, 4g / L to 100g / L, 5g / L to 10g / L, 5g / L to 15g / L, 5g / L to 20g / L, 5g / L to 25g / L, 5g / L to 30g / L, 5g / L to 35g / L, 5g / L to 40g / L, 5g / L to 45g / L , 5g / L to 50g / L, 5g / L to 55g / L, 5g / L to 60g / L, 5g / L to 65g / L, 5g / L to 70g / L, 5g / L to 75g / L, 5g / L to 80g / L, 5g / L to 85g / L, 5g / L to 90g / L, 5g / L to 95g / L, 5g / L to 100g / L, 1 0g / L to 15g / L, 10g / L to 20g / L, 10g / L to 25g / L, 10g / L to 30g / L, 10g / L to 35g / L, 10g / L to 40g / L, 10g / L to 45g / L, 10g / L to 50g / L, 10g / L to 55g / L, 10g / L to 60g / L, 10g / L to 65g / L, 10g / L to 70g / L, 10g / L to 75g / L, 10g / L to 80g / L, 10g / L to 85g / L, 10g / L to 90g / L, 10g / L to 95g / L, 10g / L to 100g / L, 15g / L to 10g / L, 15g / L to 15g / L, 15g / L to 20 g / L, 15g / L to 25g / L, 15g / L to 30g / L, 15g / L to 35g / L, 15g / L to 40g / L, 15g / L to 45g / L, 15g / L to 50g / L, 15g / L to 55g / L, 15g / L to 60g / L, 15g / L to 65g / L, 15g / L to 70g / L,15g / L to 75g / L, 15g / L to 80g / L, 15g / L to 85g / L, 15g / L to 90g / L, 15g / L to 95g / L, 15g / L to 100g / L, 20g / L to 25g / L, 20g / L to 30g / L, 20g / L to 35g / L, 20g / L to 40g / L, 20 g / L to 45g / L, 20g / L to 50g / L, 20g / L to 55g / L, 20g / L to 60g / L, 20g / L to 65g / L, 20g / L to 70g / L, 20g / L to 75g / L, 20g / L to 80g / L, 20g / L to 85g / L, 20g / L to 90g / L, 20g / L to 95g / L, 20g / L to 100g / L, 25g / L to 30g / L, 25g / L to 35g / L, 25g / L to 40g / L, 25g / L to 45g / L, 25g / L to 50g / L, 25g / L to 55g / L, 25g / L to 60g / L, 25g / L to 65g / L, 25g / L to 70g / L, 25g / L to 75g / L, 25g / L to 80g / L, 25g / L to 85g / L, 25g / L to 90g / L, 25g / L to 95g / L, 25g / L to 100g / L, 30g / L to 35g / L, 30g / L to 40g / L, 30g / L to 45g / L, 30g / L to 50g / L, 30g / L to 55g / L, 30g / L to 60g / L, 30g / L to 65g / L, 30g / L to 70g / L, 30g / L to 75g / L, 30g / L to 80g / L, 30g / L to 85g / L, 30g / L to 90g / L, 30g / L to 95g / L, 30g / L to 100g / L, 35 g / L to 40g / L, 35g / L to 45g / L, 35g / L to 50g / L, 35g / L to 55g / L, 35g / L to 60g / L, 35g / L to 65g / L, 35g / L to 70g / L, 35g / L to 75g / L, 35g / L to 80g / L, 35g / L to 85g / L, 35g / L to 90g / L, 35g / L to 95g / L, 35g / L to 100g / L, 40g / L to 45g / L, 40g / L to 50g / L, 40g / L to 55g / L, 40g / L to 60g / L, 40g / L to 65g / L, 40g / L to 70g / L, 40g / L to 75g / L, 40g / L to 80g / L, 40g / L to 85g / L, 40g / L to 90g / L, 40g / L to 95g / L, 40g / L to 100g / L, 45g / L to 50g / L, 45g / L to 55g / L, 45g / L to 60g / L, 45g / L to 65g / L, 45g / L to 70g / L, 45g / L to 75g / L,45g / L to 80g / L, 45g / L to 85g / L, 45g / L to 90g / L, 45g / L to 95g / L, 45g / L to 100g / L, 50g / L to 55g / L, 50g / L to 60g / L, 50g / L to 65g / L, 50g / L to 70g / L, 50g / L to 75g / L, 50g / L to 80g / L, 50g / L to 85g / L, 50g / L to 90g / L, 50g / L to 95g / L, 50g / L to g / L, 55g / L to 60g / L, 55g / L to 65g / L, 55g / L to 70g / L, 55g / L to 75g / L, 55g / L to 80g / L, 55g / L to 85g / L, 55g / L to 90g / L, 55g / L to 95g / L, 55g / L to 100g / L, 60g / L to 65g / L, 60g / L to 70g / L, 60g / L to 75g / L, 60g / L to 80g / L, 60g / L to 85g / L g / L, 60g / L to 90g / L, 60g / L to 95g / L, 60g / L to 100g / L, 65g / L to 70g / L, 65g / L to 75g / L, 65g / L to 80g / L, 65g / L to 85g / L, 65g / L to 90g / L, 65g / L to 95g / L, 65g / L to 100g / L, 70g / L to 75g / L, 70g / L to 80g / L, 70g / L to 85g / L, 70g / L to 90g / L, g / L, 70 g / L to 95 g / L, 70 g / L to 100 g / L, 75 g / L to 80 g / L, 75 g / L to 85 g / L, 75 g / L to 90 g / L, 75 g / L to 95 g / L, 75 g / L to 100 g / L, 80 g / L to 85 g / L, 80 g / L to 90 g / L, 80 g / L to 95 g / L, 80 g / L to 100 g / L, 85 g / L to 90 g / L, 85 g / L to 95 g / L, 85 g / L to 100 g / L, 90 g / L to 95 g / L, 90 g / L to 100 g / L or 95 g / L to 100 g / L. Preferably, the total casein concentration in the composition may be about 5 g / L to 50 g / L. More preferably, the total casein concentration in the composition may be about 30 g / L.

[0062] In another embodiment, the pH of the composition can be about pH 5.5 to pH 8.0. The pH of the composition can be about at least pH 5.5 or about pH 6.0. The pH of the composition can be about pH 8.0 at most. 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 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 6.7.

[0063] In some embodiments, the casein micelle particles comprise α S1 -casein and β-casein mixture. In some embodiments, the casein micelle particles contain α S2 -casein and β-casein mixture. In some embodiments, the casein micelle particles contain α S1 -Casein and α S2 - A mixture of casein proteins.

[0064] In some embodiments, the casein micelle particles comprise α S1 -casein and β-casein mixture. The casein micelle composition contains α S1 The ratio of casein to β casein can be about 1:15 to 15:1. S1 The ratio of casein to beta casein may be about 1:15; 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.

[0065] In some embodiments, the casein micelle particles comprise α S2-a mixture of casein and beta-casein. The ratio of α S2 casein to beta casein in the casein micelle composition may be about 1:15 to about 15:1. The ratio of α S2 casein to beta casein in the casein micelle composition may 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.

[0066] In some embodiments, the casein micelle particles comprise α S1 -Casein and α S2 - a mixture of caseins. The ratio of α S1 casein to α S2 casein in the casein micelle composition may be about 1:15 to about 15:1. The ratio of α S1 casein to α S2 casein in the casein micelle composition may 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.

[0067] In an embodiment, a mixture of two caseins (e.g., α S1 - A mixture of casein and beta-casein, or alpha S2 - A mixture of casein and beta-casein, or alpha S1 -Casein and α S2-casein mixture), the ratio of one casein to another casein in the casein micelle composition can be about 1:15 to about 15:1. For example, the ratio of one casein to another casein in the casein micelle composition can be about 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 5:1; 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 to 12:1; 1:5 to 12:1; 1:4 to 12:1;1:3 to 12:1; 1:2 to 12:1; 1:15 to 12:1; 1:1 to 12:1; 2:1 to 12:1; 3:1 to 12:1; 4:1 to 12:1; 5:1 to 12:1; 6:1 to 12:1; 7:1 to 12:1; 8:1 to 12:1; 9:1 to 12:1; 10:1 to 12:1; 11:1 to 12:1; 1:15 to 11:1; 1:14 to 11:1; 1:13 to 11:1; 1:12 to 11:1; 1:11 to 11:1; 1:10 to 11:1; 1:9 to 11:1; 1:8 to 11:1; 1:7 to 11:1; 1:6 to 11:1; 1:5 to 11:1; 1:4 to 11:1; 1:3 to 1 1:1; 1:2 to 11:1; 1:15 to 11:1; 1:1 to 11:1; 2:1 to 11:1; 3:1 to 11:1; 4:1 to 11:1; 5:1 to 11:1; 6:1 to 11:1; 7:1 to 11:1; 8:1 to 11:1; 9:1 to 11:1; 10:1 to 11:1; 1:15 to 10:1; 1:14 to 10:1; 1:13 to 10:1; 1:12 to 10:1; 1:11 to 10:1; 1:10 to 10:1; 1:9 to 10:1; 1:8 to 10:1; 1:7 to 10:1; 1:6 to 10:1; 1:5 to 10:1; 1:4 to 10:1; 1:3 to 10:1; 1:2 to 10:1; 1:1 5 to 10:1; 1:1 to 10:1; 2:1 to 10:1; 3:1 to 10:1; 4:1 to 10:1; 5:1 to 10:1; 6:1 to 10:1; 7:1 to 10:1; 8:1 to 10:1; 9:1 to 10:1; 1:15 to 9:1; 1:14 to 9:1; 1:13 to 9:1; 1:12 to 9:1; 1:11 to 9:1; 1:10 to 9:1; 1:9 to 9:1; 1:8 to 9:1; 1:7 to 9:1; 1:6 to 9:1; 1:5 to 9:1; 1:4 to 9:1; 1:3 to 9:1; 1:2 to 9:1; 1:15 to 9:1; 1:1 to 9:1; 2:1 to 9:1; 3:1 to 9:1; 4:1 to 9:1; 5: 1 to 9:1; 6:1 to 9:1; 7:1 to 9:1; 8:1 to 9:1; 1:15 to 8:1; 1:14 to 8:1; 1:13 to 8:1; 1:12 to 8:1; 1:11 to 8:1; 1:10 to 8:1; 1:9 to 8:1; 1:8 to 8:1; 1:7 to 8:1; 1:6 to 8:1; 1:5 to 8:1; 1:4 to 8:1; 1:3 to 8:1; 1:2 to 8:1; 1:15 to 8:1; 1:1 to 8:1; 2:1 to 8:1; 3:1 to 8:1; 4:1 to 8:1; 5:1 to 8:1; 6:1 to 8:1; 7:1 to 8:1; 1:15 to 7:1; 1:14 to 7:1; 1:13 to 7:1; 1:12 to 7:1;1:11 to 7:1; 1:10 to 7:1; 1:9 to 7:1; 1:8 to 7:1; 1:7 to 7:1; 1:6 to 7:1; 1:5 to 7:1; 1:4 to 7:1; 1:3 to 7:1; 1:2 to 7:1; 1:15 to 7:1; 1:1 to 7:1; 2:1 to 7:1; 3:1 to 7:1; 4:1 to 7:1; 5 :1 to 7:1; 6:1 to 7:1; 1:15 to 6:1; 1:14 to 6:1; 1:13 to 6:1; 1:12 to 6:1; 1:11 to 6:1; 1:10 to 6:1; 1:9 to 6:1; 1:8 to 6:1; 1:7 to 6:1; 1:6 to 6:1; 1:5 to 6:1; 1:4 to 6:1; 1:3 to 6:1; 1:2 to 6:1; 1:15 to 6:1; 1:1 to 6:1; 2:1 to 6:1; 3:1 to 6:1; 4:1 to 6:1; 5:1 to 6:1; 1:15 to 5:1; 1:14 to 5:1; 1:13 to 5:1; 1:12 to 5:1; 1:11 to 5:1; 1:10 to 5:1; 1:9 to 5:1; 1:8 to 5 :1; 1:7 to 5:1; 1:6 to 5:1; 1:5 to 5:1; 1:4 to 5:1; 1:3 to 5:1; 1:2 to 5:1; 1:15 to 5:1; 1:1 to 5:1; 2:1 to 5:1; 3:1 to 5:1; 4:1 to 5:1; 1:15 to 4:1; 1:14 to 4:1; 1:13 to 4:1; 1:12 to 4 :1; 1:11 to 4:1; 1:10 to 4:1; 1:9 to 4:1; 1:8 to 4:1; 1:7 to 4:1; 1:6 to 4:1; 1:5 to 4:1; 1:4 to 4:1; 1:3 to 4:1; 1:2 to 4:1; 1:15 to 4:1; 1:1 to 4:1; 2:1 to 4:1; 3:1 to 4:1; 1:15 to 3 :1; 1:14 to 3:1; 1:13 to 3:1; 1:12 to 3:1; 1:11 to 3:1; 1:10 to 3:1; 1:9 to 3:1; 1:8 to 3:1; 1:7 to 3:1; 1:6 to 3:1; 1:5 to 3:1; 1:4 to 3:1; 1:3 to 3:1; 1:2 to 3:1; 1:15 to 3:1; 1:1 to 3:1; 2:1 to 3:1; 1:15 to 2:1; 1:14 to 2:1; 1:13 to 2:1; 1:12 to 2:1; 1:11 to 2:1; 1:10 to 2:1; 1:9 to 2:1; 1:8 to 2:1; 1:7 to 2:1; 1:6 to 2:1; 1:5 to 2:1; 1:4 to 2:1; 1:3 to 2:1; 1: 2 to 2:1; 1:15 to 2:1; 1:1 to 2:1; 1:15 to 1:1; 1:14 to 1:1; 1:13 to 1:1; 1:12 to 1:1; 1:11 to 1:1; 1:10 to 1:1; 1:9 to 1:1; 1:8 to 1:1; 1:7 to 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; 1:1 5 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 1:15 to 1:14. ;

[0068] In some embodiments, the casein micelle particles comprise α S1 -Casein, α S2In some preferred embodiments, the content of each of α S1 casein, α S2 casein and β casein in the casein micelle composition may be present in an amount of 1 to 15 parts. The amounts of S2 casein and beta casein can be 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 :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, 11:12:1 3. 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, 14:15:15, or 15:15:15 in any order. ,

[0069] The diameter of the micelles of the present invention, such as the casein micelles of the present invention, can be from about 10 nm to about 500 nm. The diameter of the micelles of the present invention can be at least 10 nm. The diameter of the micelles of the present invention can be at most 500 nm. The diameter of the colloid particles of the present invention may be from 10 nm to 20 nm, 10 nm to 50 nm, 10 nm to 100 nm, 10 nm to 150 nm, 10 nm to 200 nm, 10 nm to 250 nm, 10 nm to 300 nm, 10 nm to 350 nm, 10 nm to 400 nm, 10 nm to 450 nm, 10 nm to 500 nm, 20 nm to 50 nm, 20 nm to 100 nm, 20 nm to 150 nm, 20 nm to 200 nm, 20 nm to 250 nm, 20 nm to 300 nm, 20 nm to 350 nm, 20 nm to 400 nm, 20 nm to 450 nm, 20 nm to 500 nm, 50 nm to 100 nm, 50 nm to 150 nm, 50 nm to 200 nm, 50 nm to 250 nm, 50 nm to 300 nm, 50 nm to 350 nm 400 nm, 150 nm to 450 nm, 150 nm to 500 nm, 200 nm to 250 nm, 200 nm to 300 nm, 200 nm to 350 nm, 200 nm to 400 nm, 200 nm to 450 nm, 200 nm to 500 nm, 250 nm to 300 nm, 200 nm to 350 nm, 200 nm to 400 nm, 200 nm to 450 nm, 200 nm to 500 nm, 250 The present invention relates to a range of from 250 nm to 350 nm, 250 nm to 400 nm, 250 nm to 450 nm, 250 nm to 500 nm, 300 nm to 350 nm, 300 nm to 400 nm, 300 nm to 450 nm, 300 nm to 500 nm, 350 nm to 400 nm, 350 nm to 450 nm, 350 nm to 500 nm, 400 nm to 450 nm, 400 nm to 500 nm, or 450 nm to 500 nm.The diameter of the colloid particles of the present invention 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 about 500 nm. The diameter of the colloid particles of the present invention 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 colloid particles of the present invention 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.

[0070] In another embodiment, the casein micelles comprise (i) only α S1 - casein, the calcium concentration in the composition being between 1.48 mM and 150.00 mM, preferably between 2.72 mM and 38.58 mM. The calcium concentration in the composition may be at least 1.48 mM. The calcium concentration in the composition may be at most 150.00 mM. The calcium concentration in the composition can be about 1.48 mM, 1.85 mM, 2.20 mM, 2.55 mM, 2.93 mM, 3.30 mM, 3.70 mM, 4.1 mM, 4.50 mM, 4.80 mM, 7.70 mM, 14.30 mM, 20.90 mM, 27.50 mM, 34.15 mM, 40.8 mM, 47.30 mM, 54.00 mM, 60.60 mM, 67.20 mM, 70.0 mM, 75.0 mM, 80.0 mM, 85.0 mM, 90.0 Preferably, the calcium concentration in the composition may be 1.48 mM to 150.00 mM, more preferably 2.72 mM to 38.58 mM.

[0071] In another embodiment, the casein micelles comprise (i) only α S1-casein, wherein the phosphate concentration in the composition is between 23.5 mM and 58.40 mM, preferably between 24.42 mM and 43.66 mM. The phosphate concentration in the composition may be at least 23.5 mM. The phosphate concentration in the composition may be at most 58.5 mM. The phosphate concentration in the composition may be about 23.30 mM, 23.20 mM, 23.00 mM, 22.80 mM, 22.60 mM, 22.30 mM, 22.20 mM, 22.10 mM, 22.00 mM, 28.10 mM, 31.80 mM, 35.50 mM, 39.2 mM, 42.90 mM, 46.60 mM, 49.00 mM, 52.00 mM, 55.10 mM or 58.40 mM. Preferably, the phosphate concentration in the composition may be 23.5 mM to 58.40 mM, more preferably 24.42 mM to 43.66 mM.

[0072] In another embodiment, the casein micelles comprise (i) only α S2 -casein, wherein the calcium concentration in the composition is between 1.48 mM and 150 mM, preferably between 2.72 mM and 38.58 mM. The calcium concentration in the composition may be at least 1.48 mM. The calcium concentration in the composition may be at most 150.00 mM. The calcium concentration in the composition may be about 1.48 mM, 1.85 mM, 2.20 mM, 2.55 mM, 2.93 mM, 3.30 mM, 3.70 mM, 4.1 mM, 4.50 mM, 4.80 mM, 7.70 mM, 14.30 mM, 20.90 mM, 27.50 mM, 34.15 mM, 40.8 mM, 47.30 mM, 54.00 mM, 6 Preferably, the calcium concentration in the composition may be 1.48 mM to 150.00 mM, more preferably 2.72 mM to 38.58 mM.

[0073] In another embodiment, the casein micelles comprise (i) only α S2-casein, wherein the phosphate concentration in the composition is between 23.5 mM and 58.40 mM, preferably between 24.42 mM and 43.66 mM. The phosphate concentration in the composition may be at least 23.5 mM. The phosphate concentration in the composition may be at most about 58.4 mM. The phosphate concentration in the composition may be about 23.5 mM, 23.3 mM, 23.2 mM, 23.0 mM, 22.8 mM, 22.6 mM, 22.3 mM, 22.2 mM, 22.1 mM, 22.0 mM, 28.1 mM, 31.8 mM, 35.5 mM, 39.21 mM, 42.9 mM, 46.6 mM, 49.0 mM, 51.5 mM, 54.7 mM or 58.40 mM. Preferably, the phosphate concentration in the composition may be 23.5 mM to 58.40 mM, more preferably 24.42 mM to 43.66 mM.

[0074] In another embodiment, the casein micelle comprises (i) only β-casein, and the calcium concentration in the composition is 1.3 mM to 34.0 mM, preferably 1.81 mM to 7.69 mM. The calcium concentration in the composition may be at least 1.3 mM. The calcium concentration in the composition may be at most 35.00 mM. The calcium concentration in the composition may be about 1.3 mM, 1.5 mM, 1.85 mM, 2.2 mM, 2.20 mM, 2.55 mM, 2.93 mM, 3.30 mM, 3.70 mM, 4.1 mM, 4.35 mM, 4.50 mM, 4.80 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 11.0 mM, 12.0 mM, 13.0 mM, 14.0 mM Preferably, the calcium concentration in the composition may be 1.3 mM to 34.0 mM, more preferably 1.81 mM to 7.69 mM.

[0075] In another embodiment, the casein micelle comprises (i) only β-casein, and the phosphate concentration in the composition is 24.54 mM to 41.0 mM, preferably 24.54 mM to 36.1 mM. The phosphate concentration in the composition may be at least 24.0 mM. The phosphate concentration in the composition may be at most 45.0 mM. The phosphate concentration in the composition may be about 24.0 mM, 25.0 mM, 26.0 mM, 27.0 mM, 28.0 mM, 29.0 mM, 30.0 mM, 31.0 mM, 32.0 mM, 33.0 mM, 32.0 mM, 33.0 mM, 34.0 mM, 35.0 mM, 36.0 mM, 37.0 mM, 38.0 mM, 39.0 mM, 40.0 mM, 41.0 mM, 42.0 mM, 43.0 mM, 44.0 mM or 45.0 mM. Preferably, the phosphate concentration in the composition may be 24.54 mM to 41.0 mM, more preferably 24.54 mM to 36.1 mM.

[0076] In another embodiment, the composition comprises a mixture of calcium sensitive proteins; and the calcium and phosphate concentrations are a combination of ranges for the various casein components, but are proportional to the mole fraction of each casein in the mixture.

[0077] In some embodiments, the casein micelle compositions of the present invention exhibit stability comparable to that of naturally occurring micelle compositions. Ideally, the casein micelles remain dispersed in a colloidal suspension or composition under conditions comparable to those of naturally occurring micelle compositions. For both artificial and naturally occurring micelle compositions, under certain conditions, the dispersion of the casein micelles dissociates and the casein precipitates.

[0078] In some embodiments, the casein micelle composition of the present invention is centrifuged at 4000×g for 3 minutes to keep it dispersed. In some embodiments, the casein micelle composition of the present invention is centrifuged at 3000×g for 5 minutes to keep it dispersed.

[0079] In other embodiments, the casein micelle composition of the present invention is pasteurized at 72°C for 15 seconds to maintain dispersion. In other embodiments, the casein micelle composition of the present invention is pasteurized at 135°C for 1 to 2 seconds to maintain dispersion.

[0080] In another embodiment of the present invention, the calcium sensitive casein is recombinantly produced. A recombinant casein is recombinantly expressed in a host cell. As used herein, "host" or "host cell" refers to any protein production host selected or genetically modified to produce a desired product. Typical hosts include fungi, bacteria, yeast, plants, insects and mammalian cells. The casein recombinantly produced by the host cell of the present invention is preferably selected from bacteria, yeast and fungi.

[0081] The host cell is a bacterial host cell 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, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus spp. plantarum), Synechocystis sp. 6803 and Escherichia coli. Preferably, the bacterial host cell is Lactococcus lactis, Bacillus subtilis or Escherichia coli.

[0082] The host cell is a yeast host cell, which is preferably selected from Kluyveromyces sp., Pichia sp., Saccharomyces sp., Tetrahymena sp., Yarrowia sp., Hansenula sp., Blastobotrys sp., Candida sp., Zygosaccharomyces sp. or Debaryomyces sp.

[0083] The host cell is a fungal host cell, which is preferably selected from any Aspergillus sp. (e.g. Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae), Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium granineum, Fusarium venenatum, Physcoinitrella patens and Neurospora crassa.

[0084] In some embodiments, the casein comprises an amino acid sequence from any species. In particular, the casein comprises an amino acid sequence from any eutherian, metatherian or monotreme species that is generally useful for human diet, such as cattle, sheep, goats, camels, horses or primate species. In some preferred embodiments, the casein composition comprises an amino acid sequence or a mixture thereof selected from the group consisting of cattle, goats, sheep, buffalo, camels, horses, bison, humans, donkeys, chimpanzees, rabbits, mice, guinea pigs, brushtail possums, platypuses, Australian echidnas, wallabies, zebu cattle.

[0085] The calcium-sensitive casein of the present invention can be produced in the same host cell. Alternatively, the calcium-sensitive casein can be produced in different host cells.

[0086] Depending on the host cell used to express casein, casein may have a glycosylation or phosphorylation pattern (post-translational modification) different from animal-derived casein. In some cases, casein does not include post-translational modification (PTM). In some cases, casein includes significantly reduced PTM. The significantly reduced PTM used in the present invention refers to one or more types of PTMs reduced by at least 50% compared to the amount of PTM in animal-derived casein. For example, compared with animal-derived casein, the post-translational modification of casein can reduce 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99%. Alternatively, casein can include PTMs comparable to animal-derived casein PTMs.

[0087] PTMs in casein can be modified chemically or enzymatically. In some cases, casein without chemical or enzymatic treatment comprises significantly reduced PTMs or is free of PTMs. Casein micelles can be produced by casein with reduced or no PTMs, wherein the lack or reduction of PTMs is not due to chemical or enzymatic treatment, such as by producing recombinant casein without post-translational modification or significantly reduced PTM levels in a host.

[0088] Glycosylation in casein can be modified chemically or enzymatically. In some cases, casein that has not been treated chemically or enzymatically contains significantly reduced glycosylation or is free of glycosylation. For example, the degree of glycosylation of casein can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% lower than casein from animal sources. Casein with reduced glycosylation or free of glycosylation can be used to produce casein micelles, wherein the lack of glycosylation is not due to chemical or enzymatic treatment after recombinant production.

[0089] Phosphorylation in casein can be modified by chemical method or enzymatic method.In some cases, casein without chemical or enzyme treatment comprises significantly reduced phosphorylation or does not contain phosphorylation.For example, compared with casein of animal origin, the phosphorylation degree of casein can be low by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99%.Phosphorylation can be used to reduce or do not contain phosphorylated casein to produce casein micelles, wherein lack of phosphorylation is not due to chemical or enzyme treatment, for example, by producing recombinant casein that is not significantly reduced by post-translational modification or PTM level in a host.

[0090] The casein micelle composition of the present invention does not contain kappa casein. Therefore, the protein of the present invention does not contain the protein sequence of kappa casein (SEQ ID NO: 50 to SEQ ID NO: 66).

[0091] In a preferred embodiment, the recombinantly produced casein has an amino acid sequence comprising SEQ ID NO.1 to SEQ ID NO.49 (as shown in Table 1) or a variant thereof having at least 80% sequence identity thereto. The protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.1 to SEQ ID NO.49 (as shown in Table 1).

[0092] Table 1: Bold indicates the amino acid sequence of proteins containing signal peptides. The mature protein does not contain a signal peptide.

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] In some embodiments of the present invention, the casein micelle composition further comprises one or more than one non-casein protein. Such additional one or more than one protein may be any suitable protein. The non-casein protein is preferably selected from osteopontin, dentin matrix protein, extracellular matrix phosphoglycoprotein, bone sialoprotein, dentin sialophosphoprotein, amelogenin, salivary statherin, starmaker or otolith matrix macromolecule-64 and related homologues, calcium binding protein, secretory calcium binding phosphoprotein (SCPP) and mixtures thereof.

[0105] Method for preparing casein micelle composition

[0106] As disclosed herein, the present invention also relates to a method for preparing a casein micelle composition, the method comprising: combining at least one calcium-sensitive casein with at least one salt under conditions such that at least one calcium-sensitive casein in a liquid colloid forms casein micelle particles, wherein the casein micelles do not contain κ-casein.

[0107] Preferably, at least one salt is a calcium salt and / or a phosphate salt. More preferably, at least two salts are used and the two salts are at least one calcium salt and one phosphate salt.

[0108] In some embodiments, the method further comprises adding at least one other salt. Any suitable salt may be used, including calcium salts, phosphates, citrates, potassium salts, sodium salts and / or chloride salts. The calcium salt may be selected from calcium chloride, calcium carbonate, calcium citrate, calcium gluconate, calcium lactate, calcium gluconate, calcium acetate and combinations thereof. Phosphates may be selected from: orthophosphates such as monosodium phosphate, disodium phosphate, trisodium phosphate, monopotassium phosphate, dipotassium phosphate, tripotassium phosphate; pyrophosphates such as disodium pyrophosphate or dipotassium pyrophosphate, trisodium pyrophosphate or tripotassium pyrophosphate, tetrasodium pyrophosphate or tetrapotassium pyrophosphate; polyphosphates such as sodium tripolyphosphate or potassium tripolyphosphate, sodium tetrapolyphosphate or potassium tetrapolyphosphate, sodium hexametaphosphate or potassium hexametaphosphate. Citrates may be selected from calcium citrate, potassium citrate, sodium citrate, trisodium citrate, tripotassium citrate or mixtures thereof. Preferably, the at least one other salt is selected from the group consisting of calcium salts, phosphates, citrates, sodium salts, chlorides, potassium salts, metaphosphates, pyrophosphates, tripolyphosphates, longer polyphosphates, and mixtures thereof.

[0109] Mice formation requires that the casein be dissolved in a solvent, such as water. The salts may be added after the casein is dissolved in the solvent. Alternatively, the salts and casein may be added to the solution simultaneously. During the micelle formation process, the salts may need to be added multiple times. For example, calcium salts, phosphates, and citrates may be added periodically or in a continuous titration process and mixed with the solution containing the casein until micelles are formed. Different salts may be added at different times during the micelle formation process. For example, calcium salts may be added before phosphates and citrates, or citrates may be added before calcium and phosphates, or phosphates may be added before calcium and citrates. A preferred method is to gradually add the salts to the protein solution by mixing and adjusting the pH to ensure that the casein micelles are formed prior to the formation of the micelles. Figure 1 The alternative outcomes outlined in .

[0110] Other components can be added to the casein micelle composition, such as fats and sugars. In some embodiments, fats are added to the casein micelle composition. In some embodiments, the fats can be fats that are substantially free of animal origin. The fats of the present invention can include vegetable fats, such as canola oil, sunflower oil, coconut oil or a combination thereof. The fat content in the casein micelle composition is about 0% to about 5%. The fat content in the casein micelle composition can be at least about 0.5% to about 1%. The fat content in the casein micelle composition can be about 5% at most. The fat content in the casein micelle composition can be about 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5%. The fat content in the casein micelle composition can be about 0% to 0.5%, 0.5% to 1%, 1% to 3%, 1% to 4%, or 1% to 5%. The fat content in the casein micelle composition can be about 2%, 3%, 4% or 5% at most. The sugars of the present invention may include plant-based disaccharides and / or oligosaccharides, such as sucrose, glucose, fructose, galactose, lactose, maltose, mannose, psicose, tagatose, xylose, and arabinose.

[0111] In some embodiments of the method, the calcium-sensitive casein is produced recombinantly and the host cell is preferably selected from bacteria, yeast and fungi.

[0112] The calcium-sensitive casein of the present invention can be produced in the same host cell. Alternatively, the calcium-sensitive casein can be produced in different host cells.

[0113] The casein micelle composition of the present invention does not contain κ-casein. Therefore, the protein of the present invention does not contain the protein sequence of κ-casein (SEQ ID NO: 50 to SEQ ID NOs: 66).

[0114] In a preferred embodiment, the recombinantly produced casein has an amino acid sequence comprising SEQ ID NO.1 to SEQ ID NO.49 (as shown in Table 1) or a variant thereof having at least 80% sequence identity thereto. The protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.1 to SEQ ID NO.49 (as shown in Table 1).

[0115] The casein micelle composition containing other components of the present invention can be produced by mixing the different components at a temperature of 15° C. to 45° C. For example, a casein micelle composition containing one or more than one recombinant protein can be mixed with fats and / or sugars 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.

[0116] food

[0117] The present invention also relates to a food product comprising the casein micelle composition disclosed herein.

[0118] In some embodiments, the food is a dairy composition, preferably a cheese composition, a yogurt composition or a liquid dairy composition.

[0119] The liquid milk composition according to the present invention may contain other components, such as sugars, fats, stabilizers and flavoring agents. The texture of the liquid milk composition may be comparable to that of liquid milk of animal origin. The taste of the liquid milk composition may be comparable to that of liquid milk of animal origin.

[0120] The cheese composition according to the present invention may not contain any animal-derived components. The cheese composition according to the present invention may not contain any animal-derived dairy ingredients, such as animal-derived milk proteins. The texture of the cheese composition may be comparable to the texture of similar types of cheese made using animal-derived milk proteins, such as cheese made from animal milk. The taste of the cheese composition may be comparable to the taste of similar types of cheese made using animal-derived milk proteins.

[0121] The yogurt composition according to the present invention may contain other components, such as sugars, fats, stabilizers and flavoring agents. The texture of the yogurt composition may be comparable to the texture of yogurt from animal sources. The taste of the yogurt composition may be comparable to the taste of yogurt from animal sources. Example

[0122] The present invention will now be further explained and illustrated with reference to the following non-limiting examples.

[0123] Example 1: Expression of casein in E. coli

[0124] Vector design, cloning and transformation

[0125] First, bovine κ-casein (variant B) and β-casein (variant A2) were codon optimized for expression in E. coli, and the relevant sequences were synthesized by GenScript® using Ndel / Xhol restriction enzyme sites and cloned into pET-26b(+). These plasmids were then transformed into chemically competent E. coli T7 expression cells and cultured overnight at 37°C in LB agar medium supplemented with kanamycin for screening.

[0126] Protein expression and analysis

[0127] Single clones were grown overnight in LB liquid medium supplemented with kanamycin to OD600 = 0.6. Target protein expression was then performed by adding 1 mM IPTG for 2.5 hours. Samples were prepared by pelleting 1 mL of culture, lysing the pellet in 200 μL NEB expression lysis reagent, and then adding an equal volume of Laemmli buffer (+DTT) to the sample. Samples were analyzed using the BioRad stain-free SDS-PAGE system, and protein identity was determined by MS / MS.

[0128] Example 2: Expression of casein in Pichia pastoris

[0129] Vector design, cloning and transformation

[0130] The genes corresponding to κ-casein (variant B), β-casein (variant A2), α-S1-casein (variant B) and α-S2-casein (variant A) are codon optimized and synthesized by ATUM (California, the United States). These genes are amplified by PCR using primers from IDT, and assembled into the Pichia vector pD912-AA (ATUM) using NEBuilder® high-fidelity DNA assembly premix, and then the vector is transformed into DH5α competent cells. Plasmids are prepared in small quantities, and the vector is verified by Sanger sequencing. It is then transformed into chemical competent Pichia (PPS-9011 ATUM (California, the United States)) and cultured at 30°C on YPD agar medium supplemented with bleomycin for screening.

[0131] Protein expression and analysis

[0132] Stabilization phase 1: 96 clones were randomly selected and grown in 96-well plates in 300 μL YPD at 28 °C, 600 rpm, 70% humidity for 24 h.

[0133] Stabilization phase 2: 3 μL of the above growing strain was inoculated into YPD+zeocin (250 ug / ml) medium and grown under the same conditions for 18 hours.

[0134] Auto-induction stage: 3 μL of the above strain in YPD+zeocin was inoculated into BMGM medium and grown under the same culture conditions for 72 h.

[0135] The 96-well plate was centrifuged at 5000 × g for 30 min at 4°C. 10 μL of the supernatant was carefully removed and analyzed using the BioRad stain-free SDS-PAGE system, and protein identity was confirmed by MS / MS.

[0136] Example 3: Expression of casein in Yarrowia lipolytica

[0137] Vector design, cloning and transformation

[0138] The open reading frames (ORFs) corresponding to κ-casein (variant B), β-casein (variant A2), and bovine lactoferrin were identified by Benchling ( https: / / www.benchling.com / ) codon optimization tool. The Yarrowia lipolytica LIP2 signal peptide sequence was added to the 5' end of each sequence. The optimized codon sequences were synthesized and cloned individually into the pUC-GW-amp cloning vector using Azenta (MA, USA). The ORFs were obtained from each vector using PacI and SwaI restriction enzymes and cloned into the corresponding sites in the pHYL1001 vector after fragment separation and purification. The pHYL1001 vector (synthesized by Azenta) was designed using the Yarrowia lipolytica TEF1 promoter, uracil auxotrophy selection, and ζ-site integration to obtain expression of the cloned ORFs. Plasmids were counted in Escherichia coli DH5α cells. Plasmid minipreps were performed and the vectors were verified by Sanger sequencing. Subsequently, the plasmids were linearized by NotI restriction enzyme digestion and transformed into chemically competent Yarrowia lipolytica PO1f (ATCC MYA-2613) cells. Using SD -尿嘧啶 Transformants were screened on screening agar plates.

[0139] Protein expression and analysis

[0140] Purification stage: 7 clones were randomly selected from each plate and cultured in SD -尿嘧啶 Streak on agar plates and incubate at 28 °C for 48 h.

[0141] Pre-culture stage: a single colony of each clone was inoculated into 3 mL of 2×SD -尿嘧啶 The cells were incubated in a 50 mL falcon centrifuge tube containing 2% glucose buffer and shaken at 150 rpm at 28°C for 24 hours.

[0142] Production phase: inoculate the pre-culture into 10 mL 2×SD -尿嘧啶 The culture was incubated in a 250 mL culture flask containing 4% glucose buffer until OD600 = 0.2. The culture was shaken at 300 rpm at 28 °C for 72 h, and the oxygen transfer rate was continuously monitored using the TOMs fermentation system (Kuhner AG, Switzerland).

[0143] The optical density of each sample and the supernatant obtained by centrifugation at 8000 for 5 minutes at 4°C was measured. 10 μL of the supernatant was removed and analyzed for secreted proteins using the BioRad stain-free SDS-PAGE system, and protein identity was determined by MS / MS.

[0144] Example 4: Expression of casein in Bacillus subtilis

[0145] Vector design, cloning and transformation

[0146] The corresponding genes for κ-casein (variant B), β-casein (variant A2), α-S1-casein (variant B), and α-S2-casein (variant A) were codon-optimized and synthesized by GenScript. These genes were amplified by PCR using primers from IDT and assembled into the Bacillus subtilis / Escherichia coli shuttle vector pTTB2 (MoBiTech, Germany) using NEBuilder® high-fidelity DNA assembly premix, and then the vector was transformed into DH5α competent cells. Plasmid miniprep was performed and the vector was verified by Sanger sequencing. Subsequently, the pTTB2 plasmid was linearized by EcoRI restriction enzyme digestion to remove all sequences required for E. coli proliferation. The plasmid was then transformed into the chemically competent Bacillus subtilis strain WEA (MoBiTech, Germany) and cultured at 37°C on 2×YT agar plates containing 1% xylose for screening.

[0147] Protein expression and analysis

[0148] Purification stage:

[0149] Pre-culture stage: 96 clones were selected and inoculated into a deep-well plate containing 300 μL LB medium and cultured at 37°C with shaking at 600 rpm for 18 hours.

[0150] Automatic induction stage: 3 μL of the above pre-cultured strain was inoculated into LS medium and grown under the same culture conditions for 48 h.

[0151] The 96-well plate was centrifuged at 5000 × g for 30 min at 4°C. 10 μL of the supernatant was analyzed using the BioRad stain-free SDS-PAGE system and protein identity was determined by MS / MS.

[0152] Example 5: Purification of bovine casein

[0153] Purity testing

[0154] Bovine β-casein (>98% PAGE purity), κ-casein (>70% PAGE purity), and α-S1-casein (>70% PAGE purity) proteins were purchased from Sigma Aldrich. The proteins were first analyzed by CE-SDS using a Sciex Biophase 8800 system to detect purity and contamination, especially κ-casein contamination in β-casein and α-casein. CE-SDS showed that β-casein contained 87% β-casein, 9% α-casein, and 3% κ-casein ( Figure 2 ), κ-casein contains 52% κ-casein, 29% α-lactalbumin, 5% β-lactoglobulin, 5% β-casein and 5% α-casein and 4% other substances ( Figure 3 ). Therefore, the protein was subsequently purified using anion exchange chromatography.

[0155] Anion exchange chromatography

[0156] Buffer A: 3.3 M urea, 20 mM Tris, pH 8.0

[0157] Buffer B: 3.3 M urea, 20 mM Tris, 1 M NaCl, pH 8.0

[0158] 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 subsequently filtered through a 0.2 μm syringe filter. The Hi Prep Q HP 16 / 10 was connected to the Äkta Pure150 and equilibrated with 5 CV of buffer A before protein loading. The column was then washed with 4 CV of buffer A and eluted with a linear gradient of buffer B to 40% over 10 CV for fixed volume collection. This method provided good separation of all caseins, and their purity was reanalyzed by CE-SDS and showed > 99% purity.

[0159] Example 6: Preparation and analysis of β8κX casein micelles

[0160] Casein micelles were prepared using repurified bovine β-casein and κ-casein from Sigma Aldrich, keeping the β-casein concentration constant at 8 g / L and varying the concentration of κ-casein in the composition from 1 g / L to 10 g / L, as shown in Table 2.

[0161] Table 2: Casein micelle composition

[0162]

[0163] Casein micelles were prepared by first dissolving the protein in 18.2 MΩ-cm ultrapure water and then titrating the inorganics into the protein solution. For subsequent casein micelle stability studies, sodium azide was added to prevent bacterial growth.

[0164] After the formation of the colloid particles, the solution was allowed to stand for another 12 hours to ensure the formation of stable colloid particles. Figure 4In the micellar solution shown, as expected for the correct formation of casein micelles, the whiteness of the solution decreases as the κ-casein concentration increases. Since the κ-casein concentration is related to the casein micelle size, its concentration is related to light scattering. The turbidity of the solution was measured before and after centrifugation at 1000×g for 5 minutes ( Figure 5 ). If the casein micelles are formed correctly, the turbidity should be within 50% of the measured values ​​for both pre- and post-centrifugation readings. The ultrafiltrate of each sample was diluted 4-fold and the turbidity was measured at 600 nm using a BMG Labtech CLARIOstar Plus High Flexibility Multi-Purpose Microplate Reader. ® The ultrafiltrate was obtained after centrifugation in a 6,5000 MWCO PES ultrafiltration tube. The same ultrafiltrate was used to dilute the samples for nanoparticle tracer analysis (NTA).

[0165] Subsequently, the particle size of the casein micelles in solution was determined using a Malvern NanoSight NS300 equipped with the NanoSight Sample Assistant by first diluting the samples 1000× (B8K2) or 10000× (B8K3 / 5 / 10) from a single ultrafiltrate of each sample.

[0166] As can be seen from the figure, κ-casein content, turbidity ( Figure 5 ) and casein micelle size ( Figure 6 ) are inversely proportional to each other.

[0167] The ultrafiltrates used for NTA analysis were analyzed for inorganics using a Perkin Elmer Nexion 259X Inductively Coupled Plasma Mass Spectrometer (ICPMS). Table 3 lists the Pi and Ca concentrations measured in the ultrafiltrates of the different micelle compositions, indicating that the micelles were properly formed and incorporated with the remaining Pi and Ca.

[0168] Table 3: Ultrafiltrate (UF) inorganic analysis by ICPMS

[0169]

[0170] Example 7: Preparation of Stable Artificial Casein Micelle Free of κ-Casein from Bovine β-Casein

[0171] β-casein micelle compositions were prepared in which the total protein concentration was maintained at 10 g / L, the pH was constant at 7.00, and the concentrations of calcium, phosphate, and NaCl were varied according to Table 4.

[0172] Table 4. Composition of micelles containing only β-casein

[0173]

[0174] The samples were prepared by first dissolving β-casein in 18.2 MΩ-cm ultrapure water and then titrating the inorganics to the desired final concentration. The samples were allowed to equilibrate for 2 h by mixing before analysis ( Figure 7 ).

[0175] First, the stability of the formed casein was analyzed by measuring before and after centrifugation. Figure 8 As shown in Figure 2, stable casein micelles are formed when the turbidity before and after centrifugation is < 50%. The turbidity increases with the increase of Ca / Pi concentration.

[0176] The particle size of casein micelles was then determined by nanoparticle tracking analysis, and the results showed that as the Ca / Pi concentration increased, the particle size increased as the number of particles decreased ( Fig. 9 ).

[0177] The formed casein micelles were observed using cryo-transmission electron microscopy (cryo-TEM). Fig.10 Sample 2 casein micelles are shown, forming correct casein micelles with clearly visible calcium-phosphate nanoclusters. These single casein micelles have a very similar structure to natural liquid milk casein micelles in the literature, e.g., Day, L., JK Raynes, A.Leis, LH Liu, and RPW Williams. 2017. Food Hydrocolloids 69:150–163.

[0178] The ultrafiltrates used for NTA analysis were analyzed for inorganics using a Perkin Elmer Nexion 259X Inductively Coupled Plasma Mass Spectrometer (ICPMS). Table 5 lists the Pi and Ca concentrations measured in the ultrafiltrates of the different micelle compositions, indicating that the micelles were properly formed and bound the remaining Pi and Ca.

[0179] Table 5: Inorganic analysis of ultrafiltrate (UF) containing only β-casein by ICPMS

[0180]

[0181] Example 8: Preparation of Stable Artificial Casein Micelle Free of κ-Casein from Bovine α-s-Casein

[0182] Bovine α-S-casein containing α-S1-casein and α-S2-casein was purified from sodium tyrosinate by anion exchange chromatography as described previously. Capillary electrophoresis was used to confirm purity and ensure the absence of κ-casein.

[0183] α-s-casein micelle compositions were prepared in which the total protein concentration was maintained at 10 g / L, the pH was constant at 7.00, and the concentrations of calcium, phosphate, and NaCl were varied according to Table 6.

[0184] Table 6. Composition of micelles containing only α-s-casein

[0185]

[0186] The samples were prepared by first dissolving α-s-casein in 18.2 MΩ-cm ultrapure water and then titrating the inorganics to the desired final concentration. The samples were allowed to equilibrate for 2 h ( Fig.11 ), sodium tyrosine micelles were used as parallel control.

[0187] First, the stability of the formed casein was analyzed by measuring before and after centrifugation. Fig.12 As shown in Figure 2, stable casein micelles are formed when the turbidity before and after centrifugation is < 50%. The turbidity increases with the increase of Ca / Pi concentration.

[0188] The casein micelle size was then determined by nanoparticle tracking analysis ( Fig.13 ), the results showed that the particle size increased with the increase of Ca / Pi concentration.

[0189] Prognostic Example 1: Milk beverage composition containing single protein casein micelles

[0190] Table 7 shows an example composition of a milk beverage containing single casein micelles. It should be noted that only a single composition of each protein is described here, but based on this patent application, milk beverages may contain many different components, and those skilled in the art will recognize this. First, casein powder is dissolved in the required volume of water, and then inorganic substances are titrated into the solution to form single casein micelles. After the micelles are formed, beta-lactoglobulin and maltose are added to the solution, followed by the preheated fat component. The solution is homogenized, pasteurized and filled before consumption.

[0191] Table 7. Milk drink composition

[0192]

[0193] * The oil can be any mixture of vegetable oils, including but not limited to coconut oil, canola oil, sunflower oil, monoglycerides and diglycerides, tributyric acid, butyric acid and caproic acid, phospholipids

[0194] Prognostic Example 2: Yogurt Composition Containing Single Protein Casein Micelle

[0195] Table 8 shows an example composition of a yogurt containing single casein micelles. It should be noted that only a single composition of each protein is described here, but based on this patent application, yogurt may contain many different components, and those skilled in the art will be able to recognize this. First, casein powder is dissolved in the required volume of water, and then inorganic matter is titrated into the solution to form a single casein micelle. After the micelles are formed, β-lactoglobulin and maltose are added to the solution, followed by the preheated fat component. The liquid milk base is then heated to 85°C to denature the whey protein, then cooled to 40°C, and then a lactic acid starter (e.g., a lactobacillus strain) is added. Fermentation is carried out for 4 to 8 hours, and then cooled to 4°C for packaging.

[0196] Table 8. Yogurt composition

[0197]

[0198] * The oil can be any mixture of vegetable oils, including but not limited to coconut oil, canola oil, sunflower oil, monoglycerides and diglycerides, tributyric acid, butyric acid and caproic acid, phospholipids

[0199] Prognostic Example 3: Fresh cheese composition containing single protein casein micelles

[0200] Table 9 shows an exemplary composition of fresh cheese containing single casein micelles. It should be noted that only a single composition of each protein is described here, but based on this patent application, fresh cheese may contain many different components, and those skilled in the art will be able to recognize this. First, casein powder is dissolved in the required volume of water, and then the inorganic substance is titrated into the solution to form a single casein micelle. After the micelles are formed, maltose is added to the solution, and then the preheated fat component is added. The liquid milk base is then heated to 38°C and a rennet substitute (not containing κ casein), such as microbial pepsin, is added according to the manufacturer's instructions. The liquid milk is then left for 30 minutes to coagulate. After coagulation, the curd is cut into approximately 2.5 cm cubes and gently scooped out and placed in a cheese mold to remove moisture.

[0201] Table 9. Cheese composition

[0202]

[0203] * The oil can be any mixture of vegetable oils, including but not limited to coconut oil, canola oil, sunflower oil, monoglycerides and diglycerides, tributyric acid, butyric acid and caproic acid, phospholipids

[0204] In some embodiments, the invention provides according to the following numbered clauses:

[0205] 1. A casein micelle composition comprising amorphous calcium phosphate (CaP), wherein the casein micelle comprises at least one calcium-sensitive casein, and wherein the casein micelle does not comprise kappa-casein.

[0206] 2. The casein micelle composition according to clause 1, wherein the casein micelle comprises:

[0207] (i) Only α S1 -Casein;

[0208] (ii) Only α S2 -Casein;

[0209] (iii) β-casein only;

[0210] (iv) α S1 - A mixture of casein and beta-casein;

[0211] (v) α S2 - A mixture of casein and beta-casein;

[0212] (vi) α S1 -Casein and α S2 - a mixture of caseins; or

[0213] (vii) α S1 -Casein, α S2 -A mixture of casein and beta-casein.

[0214] 3. The casein micelle composition according to clause 2, wherein the casein micelle comprises sufficient CaP to bind about 5% to 100% of the casein.

[0215] 4. The casein micelle composition according to any one of clauses 1 to 3, wherein the total casein concentration is about 0.5 g / L to 100 g / L, preferably about 5 g / L to 50 g / L, preferably about 30 g / L.

[0216] 5. The casein micelle composition according to any one of clauses 1 to 4, wherein the composition has a pH of about 5.5 to 8.0, preferably a pH of about 6.7.

[0217] 6. A casein micelle composition according to any one of clauses 2 to 5, wherein the casein micelle comprises (i) only α S1 - casein, the calcium concentration in the composition ranges from 1.48 mM to 150.00 mM, preferably from 2.72 mM to 38.58 mM.

[0218] 7. A casein micelle composition according to any one of clauses 2 to 5, wherein the casein micelle comprises (i) only αS1 - casein, the phosphate concentration in the composition ranges from 23.5 mM to 58.40 mM, preferably from 24.42 mM to 43.66 mM.

[0219] 8. A casein micelle composition according to any one of clauses 2 to 5, wherein the casein micelle comprises (i) only α S2 - casein, the calcium concentration in the composition is 1.48 mM to 150.00 mM, preferably 2.72 mM to 38.58 mM.

[0220] 9. A casein micelle composition according to any one of clauses 2 to 5, wherein the casein micelle comprises (i) only α S2 - casein, the phosphate concentration in the composition ranges from 23.5 mM to 58.40 mM, preferably from 24.42 mM to 43.66 mM.

[0221] 10. The casein micelle composition according to any one of clauses 2 to 5, wherein the casein micelle comprises (i) only β-casein, and the calcium concentration in the composition ranges from 1.3 mM to 34.0 mM, preferably from 1.81 mM to 7.69 mM.

[0222] 11. The casein micelle composition according to any one of clauses 2 to 5, wherein the casein micelle comprises (i) only β-casein, and the phosphate concentration in the composition ranges from 24.54 mM to 41.0 mM, preferably from 24.54 mM to 36.1 mM.

[0223] 12. A casein micelle composition according to any one of clauses 2 to 5, wherein the composition comprises a mixture of calcium sensitive caseins, the calcium and phosphate concentrations being a combination of ranges for the various casein components but proportional to the mole fraction of each casein in the mixture.

[0224] 13. The casein micelle composition according to any one of clauses 1 to 12, wherein the calcium-sensitive casein is recombinantly produced.

[0225] 14. The casein micelle composition according to clause 13, wherein the recombinantly produced casein is produced from a host cell selected from bacteria, yeast and fungi.

[0226] 15. The casein micelle composition according to any one of clauses 1 to 14, wherein the casein micelle composition remains dispersed after centrifugation at 4000 xg for 3 minutes.

[0227] 16. The casein micelle composition according to any one of clauses 1 to 14, wherein the casein micelle composition remains dispersed when pasteurized at 72°C for 15 seconds.

[0228] 17. The casein micelle composition according to any one of clauses 1 to 14, wherein the casein micelles remain dispersed when pasteurized at 135°C for 1 to 4 seconds.

[0229] 18. The casein micelle composition according to any one of clauses 13 to 17, wherein the recombinantly produced casein has an amino acid sequence comprising SEQ ID NO. 1 to SEQ ID NO. 49 or a variant thereof having at least 80% sequence identity thereto.

[0230] 19. A casein micelle composition according to any one of clauses 14 to 18, wherein when the host cell is a bacterial host cell, the bacterial host cell is selected from Lactococcus, Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Bacillus brevis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacillus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis 6803 and Escherichia coli.

[0231] 20. The casein micelle composition according to any one of clauses 14 to 18, wherein when the host cell is a yeast host cell, the yeast host cell is selected from Kluyveromyces, Pichia pastoris, Saccharomyces cerevisiae, Tetrahymena, Yarrowia, Hansenula, Botrytis cinerea, Candida, Zygosaccharomyces or Debaryomyces.

[0232] 21. The casein micelle composition according to any one of clauses 14 to 18, wherein when the host cell is a fungal host cell, the fungal host cell is selected from any Aspergillus (such as Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae), Trichoderma reesei, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venezuelae, Physcomitrella patens and Neurospora crassa.

[0233] 22. A casein micelle composition according to any one of clauses 1 to 21, wherein the casein comprises an amino acid sequence selected from the group consisting of cattle, goats, sheep, buffalo, camels, horses, bison, humans, donkeys, chimpanzees, rabbits, mice, guinea pigs, brushtail possums, platypuses, Australian echidnas, wallabies, zebu or mixtures thereof.

[0234] 23. A casein micelle composition according to clauses 1 to 22, wherein the casein micelle further comprises one or more than one non-casein protein.

[0235] 24. The casein micelle composition according to claim 23, wherein the non-casein protein is selected from osteopontin, dentin matrix protein, extracellular matrix phosphoglycoprotein, bone sialoprotein, dentin sialophosphoprotein, amelogenin, salivary statherin, starmaker or otolith matrix macromolecule-64 and related homologues, calcium binding protein, secretory calcium binding phosphoprotein (SCPP) and mixtures thereof.

[0236] 25. A method for preparing a casein micelle composition according to any one of clauses 1 to 24, comprising combining at least one calcium-sensitive casein with at least one salt in a liquid colloid under conditions where the at least one calcium-sensitive casein forms casein micelles, wherein the casein micelles do not comprise kappa-casein.

[0237] 26. The method according to clause 25, wherein at least one salt is a calcium salt and / or a phosphate salt.

[0238] 27. The method according to clause 26, further comprising adding at least one additional salt selected from calcium salts, phosphates, sodium chloride, potassium chloride, citrates, metaphosphates, pyrophosphates, tripolyphosphates, longer polyphosphates, and mixtures thereof.

[0239] 28. A food product comprising the casein micelle composition according to any one of clauses 1 to 24.

[0240] 29. The food product according to clause 28, which is a dairy composition, preferably a cheese composition, a yoghurt composition or a liquid dairy composition.

[0241] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the invention includes all variations and modifications that fall within the spirit and scope of the invention.

Claims

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

2. The casein micelle composition according to claim 1, wherein the casein micelle particles comprise: (i) Only α S1 -Casein; (ii) Only α S2 -Casein; (iii) β-casein only; (iv) α S1 - A mixture of casein and beta-casein; (v) α S2 - A mixture of casein and beta-casein; (vi) α S1 -Casein and α S2 - a mixture of caseins; or (vii) α S1 -Casein, α S2 -A mixture of casein and beta-casein.

3. The casein micelle composition of claim 2, wherein the casein micelle composition comprises sufficient CaP to bind from 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 g / L to 100 g / L, preferably about 5 g / L to 50 g / L, 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 composition according to any one of claims 2 to 5, wherein when the casein micelle particles comprise (i) only α S1 When the composition contains casein, the calcium concentration is 1.48 mM to 150.00 mM, preferably 2.72 mM to 38.58 mM.

7. The casein micelle composition according to any one of claims 2 to 5, wherein when the casein micelle particles comprise (i) only α S1 When the composition comprises casein, the phosphate concentration in the composition is 23.5 mM to 58.40 mM, preferably 24.42 mM to 43.66 mM.

8. The casein micelle composition according to any one of claims 2 to 5, wherein when the casein micelle particles comprise (ii) only α S2 -casein, the calcium concentration in the composition is 1.48 mM to 150.00 mM, preferably 2.72 mM to 38.58 mM.

9. The casein micelle composition according to any one of claims 2 to 5, wherein when the casein micelle particles comprise (ii) only α S2 When the composition contains casein, the phosphate concentration is 23.5 mM to 58.40 mM, preferably 24.42 mM to 43.66 mM.

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

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

12. A casein micelle composition according to any one of claims 2 to 5, wherein the casein micelle particles comprise a mixture of calcium-sensitive caseins; and the calcium and phosphate concentrations are a combination of ranges of the various casein micelle particle components, but are combined in proportion to the mole 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 is recombinantly produced.

14. The casein micelle composition according to claim 13, wherein the recombinantly produced casein 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, wherein the casein micelle composition remains dispersed after centrifugation at 4000 xg for 3 minutes.

16. The casein micelle composition according to any one of claims 1 to 14, wherein the casein micelle composition remains dispersed when pasteurized at 72°C for 15 seconds and / or at 135°C for 1 second to 4 seconds.

17. The casein micelle composition according to any one of claims 13 to 16, wherein the recombinantly produced casein has an amino acid sequence comprising SEQ ID NO.1 to SEQ ID NO.49 or a variant thereof having at least 80% sequence identity thereto.

18. The casein micelle composition according to any one of claims 14 to 17, wherein: (a) when the host cell is a bacterial host cell, the bacterial host cell is selected from Lactococcus, Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Bacillus brevis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacillus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis 6803 and Escherichia coli; (b) when the host cell is a yeast host cell, the yeast host cell is selected from Kluyveromyces, Pichia pastoris, Saccharomyces cerevisiae, Tetrahymena, Yarrowia, Hansenula, Botrytis cinerea, Candida, Zygosaccharomyces or Debaryomyces; or (c) When the host cell is a fungal host cell, the fungal host cell is selected from any Aspergillus (such as Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae), Trichoderma reesei, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venezuelae, Physcomitrella patens and Neurospora crassa.

19. The casein micelle composition according to any one of claims 1 to 18, wherein the casein comprises an amino acid sequence of any one of the group consisting of cattle, goats, sheep, buffalo, camels, horses, bison, humans, donkeys, chimpanzees, rabbits, mice, guinea pigs, brushtail possums, platypuses, Australian echidnas, wallabies, zebu or mixtures thereof.

20. A casein micelle composition according to any one of claims 1 to 19, wherein the casein micelle particles further comprise one or more non-casein proteins, preferably selected from osteopontin, dentin matrix proteins, extracellular matrix phosphoglycoproteins, bone sialoproteins, dentin sialophosphoproteins, amelogenins, salivary statherins, astrogenic proteins or otolith matrix macromolecule-64 and related homologues, calcium binding proteins, secretory calcium binding phosphoproteins (SCPPs) and mixtures thereof.

21. A method for preparing a casein micelle composition according to any one of claims 1 to 20, comprising combining at least one calcium-sensitive casein with at least one salt in a liquid colloid under conditions where the at least one calcium-sensitive casein forms casein micelle particles, wherein the casein micelles do not contain kappa-casein.

22. The method of claim 21, wherein at least one salt is a calcium salt and / or a phosphate salt.

23. The method of claim 22, further comprising adding at least one additional salt selected from the group consisting of calcium salts, phosphates, sodium chloride, potassium chloride, citrates, metaphosphates, pyrophosphates, tripolyphosphates, longer polyphosphates, and mixtures thereof.

24. A food product comprising the casein micelle composition according to any one of claims 1 to 23.

25. The food product according to claim 24, wherein the food product is a dairy composition, preferably a cheese composition, a yoghurt composition or a liquid dairy composition.

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