POLYPHOSPHATE ACMs

Abstract

Provided herein are artificial casein micelles (ACMs), wherein the artificial casein micelles comprise one or more caseins, and a polyphosphate, optionally wherein the artificial casein micelles further comprise a divalent cation species; as well as processes for preparing such artificial casein micelles; with applicability to the manufacture of substitute dairy products, including from recombinantly produced caseins.

Description

POLYPHOSPHATE ACMs RELATED APPLICATIONS

[0000] The present application is an International application, filed under the provisions of the Patent Cooperation Treaty (PCT). The present application claims priority from Australian Provisional application number AU 2024901923, filed 24 June 2024. The contents of Australian Provisional application number AU 2024901923 are herein incorporated by reference in their entirety. TECHNICAL FIELD

[0001] The technology described herein resides in the field of protein-based food products and dairy substitutes. More specifically, the technology relates to processes for the production of artificial protein compositions comprising protein components derived from milk, or protein components that are identical to, or homologous to those derived from milk. BACKGROUND ART

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

[0003] With recent technological advancements, the production of recombinant food proteins such as caseins is becoming a feasible and sustainable alternative to conventional dairy production.

[0004] As of 5 July 2022, the global dairy market was valued at approximately 830 billion U.S. dollars, and it was predicted to grow to about 1130 billion U.S. dollars by 2026. Bovine milk holds the most significant share of this market, whilst plant-derived dairy alternatives, lactose-intolerant milk, milk products that are reduced in carbohydrates and enriched in proteins, and other modified milk products such as A1 beta-casein free milk (A2 milk) are increasingly prominent in an increasingly informed market of diet-conscious consumers.

[0005] Increasing awareness for sustainability and animal welfare draws a growing number of people to follow vegetarian or vegan diets. Consequently, the alternative protein industry is one of the fastest growing industries with a global market expected to grow fivefold by 2030. Many food manufacturers have developed plant-based alternatives to meet the increased consumer demand. However, plant-based dairy, and specifically plant-based cheese alternatives, often fail to meet consumer expectations with regards to sensory and nutritional qualities.

[0006] Mammalian-derived milk is a highly complex liquid composition comprising, aside from water, thousands of different compounds, from lipids, triglycerides, carbohydrates, saccharides, peptides, inorganic salts and other molecular entities. Although many consider mammalian- derived milk, including bovine milk, to be an ideal nutrition source, various alternatives to mammalian-derived milk are now successfully on the market, including plant- or nut-based milks, such as soy, almond, or coconut milk, and are accepted by consumers for reasons related to mammalian-derived milk's allergenicity, lactose intolerance of certain components, personal preference, or the perception of adverse environmental impacts arising from the dairy industry.

[0007] For example, the majority of mammalian-derived milk is sourced from ruminant animals including cows, buffalos, yaks, goats and sheep, as well as pseudo-ruminants such as camels, alpacas and llamas. Cattle-rearing and ruminant livestock agriculture in general produces more global warming greenhouse gases, as measured in carbon dioxide (CO2) equivalents, than transportation, according to a recent UN assessment. Ruminants are estimated to account for 10% of total greenhouse gas emissions in Australia. Ruminants produce methane (CH4) as a by- product of digestion via anaerobic microbial feed fermentation in the rumen and, to a lesser extent, the large intestine. This process is referred to as methanogenesis.

[0008] Methane absorbs solar infrared radiation efficiently, and it has a global warming potential 25 times that of CO2. The ruminal microbial population is made up of bacteria, protozoa, fungi, and bacteriophages, all of which work together to digest ingested organic matter and produce CO2, H2, volatile fatty acids, and formates. These end-products are used by methanogenic archaea in the rumen, which produces CH4. Although the generation of CH4lowers the partial pressure of H2, this has the potential to cause problems as it also limits the amount of energy and carbon available for the synthesis of volatile fatty acids, which are critical for ruminant nutrition and could otherwise restrict rumen fermentation. The majority of CH4 generated by ruminants is exhaled or discharged via the mouth, resulting in a waste of up to 12% of gross caloric intake in the ruminant diet. In addition, producing a single glass of dairy milk from cows consumes up to nine times more land, and significantly more water, than any of the plant-derived milk alternatives.

[0009] Attempts to address these environmental issues with plant-derived milks including soy, almond, or coconut milk, for example, fall short in both flavour and utility. In addition, a major portion of dairy milk's industrial and cultural value originates from its use in derivative goods such as cheese, yoghurt, cream, or butter. While dairy substitute plant-derived milks address some environmental and health problems (and provide sufficient flavour for a minor portion of the consumer population), when exposed to the same procedures as dairy milk, they virtually always fail to generate such derivative goods.

[0010] There is a need then, for an alternative dairy substitute or composition with desirable flavour and performance characteristics, such as a composition that replicates dairy flavours,whilst minimising foodborne pathogens, and that potentially has a lower environmental impact in production, while retaining the ability to be used for derivative or downstream applications of dairy milk and providing a nutritional profile similar to that of mammalian-derived milk.

[0011] The protein content of bovine milk required for most derivative products such as cheese is primarily comprised of four distinct caseins; αs1-casein, αs2-casein, β-casein and κ-casein. Cheese is the third most unsustainable animal product in the world (in terms of greenhouse gas emissions per kg of product), yet plant-based alternatives released onto the market in the previous decade have not decreased demand of dairy cheese. On the contrary, consumption of mozzarella cheese in the United States and other developing countries is increasing year after year. Due to a lack of casein proteins, current cheese replacements do not match the functionality (including melt behaviour and browning behaviour when cooked or grilled), texture, nutrition, and taste of dairy cheese. Meanwhile, human allergies to milk products are most often caused by the αs1- casein protein present in dairy milk.

[0012] Cheese is produced in a process whereby whey proteins are separated from dairy milk, leaving behind a suspension of casein micelles comprising αs1-casein, αs2-casein, β-casein and κ-casein. This suspension of casein micelles is then subjected to coagulation and treatment with rennet enzymes to form a curd, which is then aged to form cheeses of various types. Achieving a desirable texture, hardness, elasticity and other functional properties such as melt behaviour in the cheese and desirable browning behaviour when cooked or grilled, is highly dependent on micelle size and mineral content of the micelles in the suspension of casein micelles used to generate the curd. In general terms, if the micelles are not large enough, they tend not to form sufficiently firm curds when subjected to coagulation and rennetisation, resulting in undesirable texture, hardness, elasticity and other functional properties in the downstream products arising from coagulation and rennetisation. Furthermore, when the micelles are not large enough, they tend to be unable to entrap sufficient salts within their micellar structure to impart good flavour to the downstream products arising from coagulation and rennetisation.

[0013] One issue that many producers in the field of dairy substitutes have in common is the challenge in scaling at a rapid and cost-effective rate. Precision fermentation is a technology that has the potential to supply consumers with animal-free cheese products, that are of similar nutritional and sensorial quality as cheese made from bovine milk. With this technique, milk proteins, such as caseins, can be produced by genetically engineering microbial hosts (e.g. yeasts or bacteria). Precision fermentation promises to require less land, water and energy resources as microorganisms can be grown in large quantities in bioreactors. Decreasing production costs and recent advances in technology have made the mass production of these so- called recombinant food proteins become more realistic than ever.

[0014] Since the development and production of recombinant caseins can be costly and difficult, it would be desirable to produce artificial casein micelles, having similar structural and functional properties, including in terms of mineral content and micelle size, to those observed in dairy milk, but without necessarily requiring the presence of all four caseins present in dairy milk (αs1-casein, αs2-casein, β-casein and κ-casein), thereby greatly simplifying the production of such artificial casein micelles, especially where the casein proteins used are sourced from non-dairy origins such as via recombinant microorganisms.

[0015] One challenge in the use of recombinant caseins for cheese production is the assembly of the individual recombinant caseins into casein micelles, required as a precursor to coagulation and / or rennetisation to form the necessary curds for cheese production. The re-assembly of casein micelles with non-micellar bovine caseins, creating so-called artificial casein micelles (ACMs), has been achieved in vitro by progressively mixing salt solutions with a casein solution to form calcium phosphate nanoclusters, which initiates the formation of ACMs (Schmidt, D. G., Koops, J., & Westerbeek, D. (1977). Properties of artificial casein micelles.1. Preparation, size distribution and composition. Netherlands Milk and Dairy Journal, 31(4), 328–341).1The method developed by Schmidt involves utilising an array of pumps to mix a casein solution and three separate salt solutions (the first being a source of calcium and magnesium ions, the second providing phosphate ions and the third solution supplying citrate) by pumping them gradually into a central vessel that contains an initial amount of water over a time period of an hour. Concentrations were chosen to obtain a similar ion composition as in milk. The casein and salt solutions were mixed at 37°C in a controlled manner with fixed rate additions and a fixed pH of 6.7 by titration. ACM produced with this method were similar to natural casein micelles (CM) in morphology, size and functionality. Therefore, the ACM preparation method developed by Schmidt et al. (1977)1is most commonly used at the moment.

[0016] However, such methods for the production of artificial casein micelles (ACMs) are difficult to scale up and / or to speed up, and typically result in micelles of impaired functionality when produced at a larger scale due to uncontrolled calcium phosphate crystallization (Antuma et al., 2024),2especially when applied at increased preparation rates. Further challenges arise when attempting to implement the conventional methods of Schmidt et al. (1977)1with recombinantly produced caseins, since these lack the post translational modifications of phosphorylation and glycosylation, the former of which (i.e.; phosphorylation), has been shown to be necessary for the unimpaired formation of fully functional micelles having suitably acceptable rennet coagulation behaviour for the production of non-animal derived cheese products (Antuma et al., 2023)3.

[0017] There is a need to develop alternative processes for the formation of artificial casein micelles (ACMs), with the potential for application to the re-assembly of caseins, including recombinantly produced caseins, into fully functional ACMs, on an industrial scale.

[0018] It is against this background that the present invention has been developed. SUMMARY OF THE INVENTION

[0019] By developing a novel approach and process to (re)assemble non-micellar caseins into casein micelles, the present inventors have created artificial casein micelles (ACMs) with properties approaching natural bovine casein micelles in terms of micelle size and mineral content. These artificial micelles may also be coagulated by the action of rennet, leading to the prospect of creating the same cheese textures as those produced from bovine milk, thereby allowing for the complete replacement of the functionality of bovine casein micelles.

[0020] In one embodiment, the disclosure herein provides an artificial casein micelle, wherein the artificial casein micelle comprises one or more caseins, and a polyphosphate, optionally wherein the artificial casein micelle further comprises a divalent cation species.

[0021] In one embodiment, the disclosure herein provides a micellar solution comprising a plurality of the artificial casein micelles of the present invention.

[0022] In one embodiment, the disclosure herein provides a curds composition comprising the micellar solution of the present invention.

[0023] In one embodiment, the disclosure herein provides an edible composition comprising the curds composition of the present invention.

[0024] In some embodiments, the disclosure herein provides methods for producing one or more of the preceding embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

[0026] Figure 1 is a plot of the particle sizes of Artificial Casein Micelles (ACM) prepared with 30 mM calcium and varying concentrations of sodium hexametaphosphate (SHMP). Samples were measured in duplicate. The dotted line represents the value of the ACM control.

[0027] Figure 2 are plots of: Sedimentable casein content as percentage of total casein content (A); and apparent hydration of Artificial Casein Micelles (ACM) prepared with 30 mM calcium andvarying concentrations of sodium hexametaphosphate (SHMP) (B). Samples were measured in duplicate. The dotted line indicates the respective value of the ACM control.

[0028] Figure 3 are plots of: Sedimentable calcium content expressed as fraction of total calcium content (A); and sedimentable calcium content per gram sedimentable casein (B). Samples were measured in duplicate. The dotted line indicates the value of the ACM control.

[0029] Figure 4 is a plot of the coagulation behaviour during one hour of renneting of samples prepared with sodium caseinate, CaCl2 (C) and SHMP (S). The numbers after C and S indicate the concentration (mM). A control ACM sample was analyzed to compare preparation methods. Samples were analyzed in duplicate.

[0030] Figure 5 is a plot of the coagulation behaviour of samples prepared with sodium caseinate and varying calcium (C) concentrations, compared to the C30 S3 reference. The numbers after C indicate the concentration (mM). Samples were analyzed in duplicate.

[0031] Figure 6 is a plot of the coagulation behaviour of samples prepared with dephosphorylated sodium caseinate (DP), CaCl2(C) and SHMP (S). The numbers after C and S indicate the concentration (mM). A control ACM sample was analyzed to compare preparation methods. Samples were analyzed in duplicate.

[0032] Figure 7 is a plot of the coagulation behaviour of samples prepared from β and κ casein (DP), CaCl2 (C) and SHMP (S). The numbers after C and S indicate the concentration (mM). A control ACM sample was analyzed to compare preparation methods. Samples were analyzed in duplicate. β / κ C30 S7.5 was measured in singlicate.

[0033] Figure 8 is a schematic representation of the experimental set-up employed for the synthesis of ACMs from recombinantly produced caseins.

[0034] Figure 9 is a photograph of ACM suspensions with increasing CaCl₂ / SHMP concentrations from left to right: 3 / 0.3 mM, 15 / 1.5 mM, 22.5 / 2.25 mM, and 30 / 3.0 mM (samples S1.1–S1.4), photographed after 1 hour at room temperature (RT). DEFINITIONS

[0035] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0036] Unless expressly indicated as otherwise, the term “%” shall be understood throughout this specification as referring to weight %, or wt. %.

[0037] As used herein, the term “micelle”, and grammatical variations thereof, shall be understood to mean a generally (or roughly) spherical supramolecular structure that exists as a dispersion within a composition or solution. A micelle can have, e.g., a surface that is composed of a charged outer layer. A micelle can encapsulate one or more biomolecules. For example, a micelle can encapsulate two or more proteins (e.g., a β-casein protein and a κ-casein protein). A micelle can have diameter of between about 10 nm and about 500 nm. Additional aspects and characteristics of micelles are known in the art.

[0038] As used herein, the term “Artificial Casein Micelle” shall be used interchangeably with the acronym “ACM”, and the plural equivalents “Artificial Casein Micelles” and “ACMs”. Similarly, the term “CM” shall be understood to refer to one or more Casein Micelle(s).

[0039] As used herein, the term “non-micellar casein” shall be understood to mean any form of casein that is not part of a micelle structure, including any form of casein isolated from any source, including αs1-casein, αs2-casein, β-casein and κ-casein isolates of any mammalian species, as well as any synthetically or recombinantly produced αs1-casein, αs2-casein, β-casein or κ-casein, and including variants having at least 80% sequence homology with any mammalian αs1-casein, αs2-casein, β-casein or κ-casein sequence, and including such variants having at least 80% sequence homology with any mammalian αs1-casein, αs2-casein, β-casein or κ-casein sequence with or without post-translational modifications such as glycosylation and / or phosphorylation. Some embodiments of the artificial micelles of the present invention comprise synthetically or recombinantly produced αs1-casein, αs2-casein, β-casein or κ-casein variants possessing a sequence homology with any mammalian αs1-casein, αs2-casein, β-casein or κ-casein sequence, wherein the sequence homology with any mammalian αs1-casein, αs2-casein, β-casein or κ-casein sequence is selected from the group of sequence homologies consisting of; 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with any mammalian αs1-casein, αs2-casein, β-casein or κ-casein sequence, with or without post-translational modifications such as glycosylation and / or phosphorylation. It should also be understood that any of the aforementioned caseins include salts of said caseins.

[0040] As used herein, the term “animal-derived protein” shall be understood to mean any protein derived from any mammalian animal source. It shall further be understood that the term “animal- derived protein” does not include any proteins that are synthetically or recombinantly produced.

[0041] As used herein, the term “non-animal derived” as it applies to any of the constituents used in, or products or compositions derived from the processes of the present invention, will be understood to mean that the constituent or product or composition to which the term “non-animal derived” is applied, will not contain anything derived from an animal, whether the animal is amammal, a bird, a reptile, a fish, an insect, or another type of animal. However, as used herein, the term “non-animal derived” does include constituents or products or compositions derived from microbial species. That is to say, a plant fat or oil is a non-animal derived lipid, and a recombinantly derived β-casein is a non-animal derived β-casein. A plant protein is a non-animal derived protein, etc. In other words, the term “non-animal derived” includes any constituents or compositions or products derived from plant sources, or from synthetic sources, or microbial sources, or fungal sources, or recombinant sources.

[0042] As used herein, the term “phosphorylated” as it applies to the casein constituents utilized in the processes of the present invention, will be understood to mean that the constituent to which the term “phosphorylated” is applied, is at least partially phosphorylated, that is to say, a phosphorylated κ-casein will have at least one phosphate group attached to it, and may have a plurality of phosphate groups attached to it, and a phosphorylated β-casein will have at least one phosphate group attached to it, and may have a plurality of phosphate groups attached to it. In other words, the term “phosphorylated” will be understood to include partial phosphorylation as well as complete phosphorylation. Accordingly, the term “phosphorylated κ-casein” will be understood to include native κ-casein (i.e.; κ-casein isolated from mammalian milk without any dephosphorylation step), as well as partially dephosphorylated κ-casein, as well as recombinantly produced κ-casein that has been partially or completely phosphorylated. Similarly, the term “phosphorylated β-casein” will be understood to include native β-casein, (i.e.; β-casein isolated from mammalian milk without any dephosphorylation step), as well as partially dephosphorylated β-casein, as well as recombinantly produced β-casein that has been partially or completely phosphorylated.

[0043] As used herein, the term “non-phosphorylated” as it applies to the casein constituents utilized in the processes of the present invention, will be understood to mean that the constituent to which the term “non-phosphorylated” is applied, does not have any phosphate groups attached to it, that is to say, a non-phosphorylated κ-casein will have no phosphate groups attached to it, and a non-phosphorylated β-casein will have no phosphate groups attached to it. In other words, the term “non-phosphorylated” will be understood to include recombinant κ-casein and recombinant β-casein which have not been subjected to any phosphorylation, as well as mammalian-derived κ-casein and mammalian-derived β-casein which have been completely dephosphorylated.

[0044] As used herein, the term “dephosphorylated” as it applies to the casein constituents utilized in the processes of the present invention, will be understood to mean that the constituent to which the term “dephosphorylated” is applied, is at least partially dephosphorylated, that is to say, a dephosphorylated κ-casein may have no phosphate groups attached to it, or may have at least one phosphate group attached to it, or may have a plurality of phosphate groups attachedto it, and a dephosphorylated β-casein may have no phosphate groups attached to it, or may have at least one phosphate group attached to it, or may have a plurality of phosphate groups attached to it. In other words, the term “dephosphorylated” will be understood to include partial dephosphorylation as well as complete dephosphorylation.

[0045] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention provides a novel and advantageous approach to the production of Artificial Casein Micelles (ACMs) whereby non-micellar caseins are treated with a polyphosphate to induce the formation of colloidal aggregates herein referred to as ACMs.

[0047] Without wishing to be bound by theory, it is believed that such colloidal aggregates or ACMs of the present invention form in the presence of a polyphosphate due to the formation of polyphosphate-bridged crosslinks between adjacent casein molecules, wherein the negatively charged phosphate groups on the polyphosphate bond either to the positively-charged amino acids of caseins, or to divalent cations in solution, such as calcium, which are simultaneously bonded mainly to the negatively-charged phosphoserine residues or negatively charged amino acids (such as but not limited to Aspartic Acid or Glutamic Acid), of caseins, thus forming casein- divalent cation-polyphosphate complexes.

[0048] One example of a suitable polyphosphate is sodium hexametaphosphate (SHMP), which like other polyphosphates, due to its multiple negatively-charged binding sites, has the ability to simultaneously bind casein and polyphosphate-calcium complexes.

[0049] Scheme 1 depicts two types of potential binding sites of polyphosphates with casein, showing an exemplary negatively charged phosphate group forming a direct bond to a positively charged amino acid side chain (for example, arginine) on one casein molecule (left), and a further exemplary negatively charged phosphate group forming a bond through a divalent cation (for example, Ca2+) to a negatively charged amino acid side chain (for example, aspartate) on another casein molecule (right), thus forming casein-divalent cation-polyphosphate complex. Crosslinks may also occur due to bonding through divalent cations with phosphate groups present on caseins due to post translational phosphorylation (i.e.; via a phosphoserine side chain, for example; not depicted in Scheme 1):Scheme 1

[0050] Thus, in one embodiment, the disclosure herein provides an artificial casein micelle (ACM), or a solution comprising a plurality of such artificial casein micelles, wherein the or each artificial casein micelle comprises one or more caseins, and a polyphosphate, optionally wherein the or each artificial casein micelle further comprises a divalent cation species.

[0051] In some embodiments, the divalent cation species is present in the artificial casein micelle (ACM), or the solution comprising a plurality of such artificial casein micelles, at a molar ratio of divalent cation species to polyphosphate, selected from the group consisting of; 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1, 6:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7:1, 7.1:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1, 7.6:1, 7.7:1, 7.8:1, 7.9:1, 8:1, 8.1:1, 8.2:1, 8.3:1, 8.4:1, 8.5:1, 8.6:1, 8.7:1, 8.8:1, 8.9:1, 9:1, 9.1:1, 9.2:1, 9.3:1, 9.4:1, 9.5:1, 9.6:1, 9.7:1, 9.8:1, 9.9:1, 10:1, 10.1:1, 10.2:1, 10.3:1, 10.4:1, 10.5:1, 10.6:1, 10.7:1, 10.8:1, 10.9:1, 11:1, 11.1:1, 11.2:1, 11.3:1, 11.4:1, 11.5:1, 11.6:1, 11.7:1, 11.8:1, 11.9:1, 12:1, 12.1:1, 12.2:1, 12.3:1, 12.4:1, 12.5:1, 12.6:1, 12.7:1, 12.8:1, 12.9:1, 13:1, 13.1:1, 13.2:1, 13.3:1, 13.4:1, 13.5:1, 13.6:1, 13.7:1, 13.8:1, 13.9:1, 14:1, 14.1:1, 14.2:1, 14.3:1, 14.4:1, 14.5:1, 14.6:1, 14.7:1, 14.8:1, 14.9:1, 15:1, 15.1:1, 15.2:1, 15.3:1, 15.4:1, 15.5:1, 15.6:1, 15.7:1, 15.8:1, 15.9:1, 16:1, 16.1:1, 16.2:1, 16.3:1, 16.4:1, 16.5:1, 16.6:1, 16.7:1, 16.8:1, 16.9:1, 17:1, 17.1:1, 17.2:1, 17.3:1, 17.4:1, 17.5:1,17.6:1, 17.7:1, 17.8:1, 17.9:1, 18:1, 18.1:1, 18.2:1, 18.3:1, 18.4:1, 18.5:1, 18.6:1, 18.7:1, 18.8:1, 18.9:1, 19:1, 19.1:1, 19.2:1, 19.3:1, 19.4:1, 19.5:1, 19.6:1, 19.7:1, 19.8:1, 19.9:1, and 20:1.

[0052] Advantageously, the ACM solutions of the present invention possess suitable micelle sizes and micellar mineral salt content for applicability to downstream product manufacturing such as cheesemaking.

[0053] Advantageously, the protocols described herein are directly applicable to the preparation of artificial casein micelles from synthetically or recombinantly produced αs1-casein and / or αs2- casein and / or β-casein and / or κ-casein, whether or not the aforementioned caseins are glycosylated and / or phosphorylated. The protocols herein are also directly applicable to naturally derived caseins, including extracts of mammalian milk as well as native bovine sodium caseinate, for example.

[0054] In one embodiment, the disclosure herein provides a method for producing ACMs comprising combining a solution of one or more non-micellar caseins, with a solution of a polyphosphate, and optionally a solution of a divalent cation salt.

[0055] In preferred embodiments, the disclosure herein provides a method for producing ACMs comprising combining a solution of one or more non-micellar caseins, with a solution of a polyphosphate, and a solution of a divalent cation salt.

[0056] In some embodiments, the solution of a polyphosphate, and the solution of a divalent cation salt, are configured to provide, when combined with the solution of one or more non- micellar caseins, ACMs having a molar ratio of divalent cation species to polyphosphate, selected from the group consisting of; 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1, 6:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7:1, 7.1:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1, 7.6:1, 7.7:1, 7.8:1, 7.9:1, 8:1, 8.1:1, 8.2:1, 8.3:1, 8.4:1, 8.5:1, 8.6:1, 8.7:1, 8.8:1, 8.9:1, 9:1, 9.1:1, 9.2:1, 9.3:1, 9.4:1, 9.5:1, 9.6:1, 9.7:1, 9.8:1, 9.9:1, 10:1, 10.1:1, 10.2:1, 10.3:1, 10.4:1, 10.5:1, 10.6:1, 10.7:1, 10.8:1, 10.9:1, 11:1, 11.1:1, 11.2:1, 11.3:1, 11.4:1, 11.5:1, 11.6:1, 11.7:1, 11.8:1, 11.9:1, 12:1, 12.1:1, 12.2:1, 12.3:1, 12.4:1, 12.5:1, 12.6:1, 12.7:1, 12.8:1, 12.9:1, 13:1, 13.1:1, 13.2:1, 13.3:1, 13.4:1, 13.5:1, 13.6:1, 13.7:1, 13.8:1, 13.9:1, 14:1, 14.1:1, 14.2:1, 14.3:1, 14.4:1, 14.5:1, 14.6:1, 14.7:1, 14.8:1, 14.9:1, 15:1, 15.1:1, 15.2:1, 15.3:1, 15.4:1, 15.5:1, 15.6:1, 15.7:1, 15.8:1, 15.9:1, 16:1, 16.1:1, 16.2:1, 16.3:1, 16.4:1, 16.5:1, 16.6:1, 16.7:1, 16.8:1, 16.9:1, 17:1, 17.1:1, 17.2:1, 17.3:1, 17.4:1, 17.5:1, 17.6:1, 17.7:1, 17.8:1, 17.9:1, 18:1, 18.1:1, 18.2:1, 18.3:1, 18.4:1, 18.5:1, 18.6:1, 18.7:1, 18.8:1, 18.9:1, 19:1, 19.1:1, 19.2:1, 19.3:1, 19.4:1, 19.5:1, 19.6:1, 19.7:1, 19.8:1, 19.9:1, and 20:1.

[0057] In some embodiments, the divalent cation salt is selected from the group consisting of salts of Ca2+, salts of Mg2+, salts of Fe2+and salts of Zn2+.

[0058] In preferred embodiments, the divalent cation salt is CaCl2.

[0059] In some embodiments, a solution of one or more non-micellar caseins is combined with a solution of a polyphosphate, and a solution of a divalent cation salt, in a controlled fashion, at a suitable rate, and / or for a suitable period of time, at a suitable temperature, and a suitable pH, for inducing favourable ACM formation.

[0060] For example, without limitation, the solution of one or more non-micellar caseins, may be titrated into a titration vessel, simultaneously with the titration of the solution of a polyphosphate into the titration vessel, and simultaneously with the titration of the solution of a divalent cation salt into the titration vessel, wherein each of the three solutions may be independently titrated into the titration vessel at a rate selected from the group comprising; 1 mL / h, 2 mL / h, 3 mL / h, 4 mL / h, 5 mL / h, 6 mL / h, 7 mL / h, 8 mL / h, 9 mL / h, 10±5 mL / h, 20±5 mL / h, 30±5 mL / h, 40±5 mL / h, 50±5 mL / h, 60±5 mL / h, 70±5 mL / h, 80±5 mL / h, 90±5 mL / h, 100±5 mL / h, 110±5 mL / h, 120±5 mL / h, 130±5 mL / h, 140±5 mL / h, 150±5 mL / h, 160±5 mL / h, 170±5 mL / h, 180±5 mL / h, 190±5 mL / h, 200±5 mL / h, 210±5 mL / h, 220±5 mL / h, 230±5 mL / h, 240±5 mL / h, 250±5 mL / h, 260±5 mL / h, 270±5 mL / h, 280±5 mL / h, 290±5 mL / h, 300±5 mL / h, 310±5 mL / h, 320±5 mL / h, 330±5 mL / h, 340±5 mL / h, 350±5 mL / h, 360±5 mL / h, 370±5 mL / h, 380±5 mL / h, 390±5 mL / h, 400±5 mL / h, 410±5 mL / h, 420±5 mL / h, 430±5 mL / h, 440±5 mL / h, 450±5 mL / h, 460±5 mL / h, 470±5 mL / h, 480±5 mL / h, 490±5 mL / h, 500±5 mL / h, 510±5 mL / h, 520±5 mL / h, 530±5 mL / h, 540±5 mL / h, 550±5 mL / h, 560±5 mL / h, 570±5 mL / h, 580±5 mL / h, 590±5 mL / h, 600±5 mL / h, 610±5 mL / h, 620±5 mL / h, 630±5 mL / h, 640±5 mL / h, 650±5 mL / h, 660±5 mL / h, 670±5 mL / h, 680±5 mL / h, 690±5 mL / h, 700±5 mL / h, 710±5 mL / h, 720±5 mL / h, 730±5 mL / h, 740±5 mL / h, 750±5 mL / h, 760±5 mL / h, 770±5 mL / h, 780±5 mL / h, 790±5 mL / h, 800±5 mL / h, 810±5 mL / h, 820±5 mL / h, 830±5 mL / h, 840±5 mL / h, 850±5 mL / h, 860±5 mL / h, 870±5 mL / h, 880±5 mL / h, 890±5 mL / h, 900±5 mL / h, 910±5 mL / h, 920±5 mL / h, 930±5 mL / h, 940±5 mL / h, 950±5 mL / h, 960±5 mL / h, 970±5 mL / h, 980±5 mL / h, 990±5 mL / h, 1000±5 mL / h, 2 L / h, 3 L / h, 4 L / h, 5 L / h, 6 L / h, 7 L / h, 8 L / h, 9 L / h, 0±5 L / h, 20±5 L / h, 30±5 L / h, 40±5 L / h, 50±5 L / h, 60±5 L / h, 70±5 L / h, 80±5 L / h, 90±5 L / h, 100±5 L / h, 110±5 L / h, 120±5 L / h, 130±5 L / h, 140±5 L / h, 150±5 L / h, 160±5 L / h, 170±5 L / h, 180±5 L / h, 190±5 L / h, 200±5 L / h, 210±5 L / h, 220±5 L / h, 230±5 L / h, 240±5 L / h, 250±5 L / h, 260±5 L / h, 270±5 L / h, 280±5 L / h, 290±5 L / h, 300±5 L / h, 310±5 L / h, 320±5 L / h, 330±5 L / h, 340±5 L / h, 350±5 L / h, 360±5 L / h, 370±5 L / h, 380±5 L / h, 390±5 L / h, 400±5 L / h, 410±5 L / h, 420±5 L / h, 430±5 L / h, 440±5 L / h, 450±5 L / h, 460±5 L / h, 470±5 L / h, 480±5 L / h, 490±5 L / h, 500±5 L / h, 510±5 L / h, 520±5 L / h, 530±5 L / h, 540±5 L / h, 550±5 L / h, 560±5 L / h, 570±5 L / h, 580±5 L / h, 590±5 L / h, 600±5 L / h, 610±5 L / h, 620±5 L / h, 630±5 L / h, 640±5 L / h, 650±5 L / h, 660±5 L / h, 670±5 L / h, 680±5 L / h, 690±5 L / h, 700±5 L / h, 710±5 L / h, 720±5 L / h, 730±5 L / h, 740±5 L / h, 750±5 L / h, 760±5 L / h, 770±5 L / h,780±5 L / h, 790±5 L / h, 800±5 L / h, 810±5 L / h, 820±5 L / h, 830±5 L / h, 840±5 L / h, 850±5 L / h, 860±5 L / h, 870±5 L / h, 880±5 L / h, 890±5 L / h, 900±5 L / h, 910±5 L / h, 920±5 L / h, 930±5 L / h, 940±5 L / h, 950±5 L / h, 960±5 L / h, 970±5 L / h, 980±5 L / h, 990±5 L / h, and 1000±5 L / h.

[0061] Furthermore, for example, without limitation, the solution of one or more non-micellar caseins, may be titrated into a titration vessel, simultaneously with the titration of the solution of a polyphosphate into the titration vessel, and simultaneously with the titration of the solution of a divalent cation salt into the titration vessel, at a rate such that the total time for completing the titration of the three solutions may be independently selected from the group comprising; 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 1 hr, 1.1 hr, 1.2 hr, 1.3 hr, 1.4 hr, 1.5 hr, 1.6 hr, 1.7 hr, 1.8 hr, 1.9 hr, 2 hr, 2.1 hr, 2.2 hr, 2.3 hr, 2.4 hr, 2.5 hr, 2.6 hr, 2.7 hr, 2.8 hr, 2.9 hr, 3 hr, 3.1 hr, 3.2 hr, 3.3 hr, 3.4 hr, 3.5 hr, 3.6 hr, 3.7 hr, 3.8 hr, 3.9 hr, 4 hr, 4.1 hr, 4.2 hr, 4.3 hr, 4.4 hr, 4.5 hr, 4.6 hr, 4.7 hr, 4.8 hr, 4.9 hr, 5 hr, 5.1 hr, 5.2 hr, 5.3 hr, 5.4 hr, 5.5 hr, 5.6 hr, 5.7 hr, 5.8 hr, 5.9 hr, 6 hr, 6.1 hr, 6.2 hr, 6.3 hr, 6.4 hr, 6.5 hr, 6.6 hr, 6.7 hr, 6.8 hr, 6.9 hr, 7 hr, 7.1 hr, 7.2 hr, 7.3 hr, 7.4 hr, 7.5 hr, 7.6 hr, 7.7 hr, 7.8 hr, 7.9 hr, 8 hr, 8.1 hr, 8.2 hr, 8.3 hr, 8.4 hr, 8.5 hr, 8.6 hr, 8.7 hr, 8.8 hr, 8.9 hr, 9 hr, 9.1 hr, 9.2 hr, 9.3 hr, 9.4 hr, 9.5 hr, 9.6 hr, 9.7 hr, 9.8 hr, 9.9 hr, 10 hr, 10.1 hr, 10.2 hr, 10.3 hr, 10.4 hr, 10.5 hr, 10.6 hr, 10.7 hr, 10.8 hr, 10.9 hr, 11 hr, 11.1 hr, 11.2 hr, 11.3 hr, 11.4 hr, 11.5 hr, 11.6 hr, 11.7 hr, 11.8 hr, 11.9 hr, 12 hr, 12.1 hr, 12.2 hr, 12.3 hr, 12.4 hr, 12.5 hr, 12.6 hr, 12.7 hr, 12.8 hr, 12.9 hr, 13 hr, 13.1 hr, 13.2 hr, 13.3 hr, 13.4 hr, 13.5 hr, 13.6 hr, 13.7 hr, 13.8 hr, 13.9 hr, 14 hr, 14.1 hr, 14.2 hr, 14.3 hr, 14.4 hr, 14.5 hr, 14.6 hr, 14.7 hr, 14.8 hr, 14.9 hr, 15 hr, 15.1 hr, 15.2 hr, 15.3 hr, 15.4 hr, 15.5 hr, 15.6 hr, 15.7 hr, 15.8 hr, 15.9 hr, 16 hr, 16.1 hr, 16.2 hr, 16.3 hr, 16.4 hr, 16.5 hr, 16.6 hr, 16.7 hr, 16.8 hr, 16.9 hr, 17 hr, 17.1 hr, 17.2 hr, 17.3 hr, 17.4 hr, 17.5 hr, 17.6 hr, 17.7 hr, 17.8 hr, 17.9 hr, 18 hr, 18.1 hr, 18.2 hr, 18.3 hr, 18.4 hr, 18.5 hr, 18.6 hr, 18.7 hr, 18.8 hr, 18.9 hr, 19 hr, 19.1 hr, 19.2 hr, 19.3 hr, 19.4 hr, 19.5 hr, 19.6 hr, 19.7 hr, 19.8 hr, 19.9 hr, 20 hr, 20.1 hr, 20.2 hr, 20.3 hr, 20.4 hr, 20.5 hr, 20.6 hr, 20.7 hr, 20.8 hr, 20.9 hr, 21 hr, 21.1 hr, 21.2 hr, 21.3 hr, 21.4 hr, 21.5 hr, 21.6 hr, 21.7 hr, 21.8 hr, 21.9 hr, 22 hr, 22.1 hr, 22.2 hr, 22.3 hr, 22.4 hr, 22.5 hr, 22.6 hr, 22.7 hr, 22.8 hr, 22.9 hr, 23 hr, 23.1 hr, 23.2 hr, 23.3 hr, 23.4 hr, 23.5 hr, 23.6 hr, 23.7 hr, 23.8 hr, 23.9 hr, and 24 hr.

[0062] Furthermore, for example, without limitation, the temperature at which the solution of one or more non-micellar caseins, may be titrated into a titration vessel, simultaneously with the titration of the solution of a polyphosphate into the titration vessel, and simultaneously with the titration of the solution of a divalent cation salt into the titration vessel, may be independently selected from the group comprising; 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C,13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, and 55°C.

[0063] Furthermore, without limitation, the solution of one or more non-micellar caseins, may be titrated into a titration vessel, simultaneously with the titration of the solution of a polyphosphate into the titration vessel, and simultaneously with the titration of the solution of a divalent cation salt into the titration vessel, wherein the three solutions may each independently be provided at a pH selected from the group comprising; pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9, and pH 10.

[0064] Furthermore, without limitation, the solution of one or more non-micellar caseins, may be titrated into a titration vessel, simultaneously with the titration of the solution of a polyphosphate into the titration vessel, and simultaneously with the titration of the solution of a divalent cation salt into the titration vessel, wherein the three solutions may each independently be provided at a concentration selected from the group comprising; 1±0.5 mM, 2±1 mM, 3±1 mM, 4±1 mM, 5±1 mM, 6±1 mM, 7±1 mM, 8±1 mM, 9±1 mM, 10±1 mM, 12±1 mM, 14±1 mM, 16±1 mM, 18±1 mM, 20±1 mM, 22±1 mM, 24±1 mM, 26±1 mM, 28±1 mM, 30±1 mM, 32±1 mM, 34±1 mM, 36±1 mM, 38±1 mM, 40±1 mM, 42±1 mM, 44±1 mM, 46±1 mM, 48±1 mM, 50±1 mM, 52±1 mM, 54±1 mM, 56±1 mM, 58±1 mM, 60±1 mM, 62±1 mM, 64±1 mM, 66±1 mM, 68±1 mM, 70±1 mM, 72±1 mM, 74±1 mM, 76±1 mM, 78±1 mM, 80±1 mM, 82±1 mM, 84±1 mM, 86±1 mM, 88±1 mM, 90±1 mM, 92±1 mM, 94±1 mM, 96±1 mM, 98±1 mM, 100±1 mM, 102±1 mM, 104±1 mM, 106±1 mM, 108±1 mM, 110±1 mM, 112±1 mM, 114±1 mM, 116±1 mM, 118±1 mM, 120±1 mM, 122±1 mM, 124±1 mM, 126±1 mM, 128±1 mM, 130±1 mM, 132±1 mM, 134±1 mM, 136±1 mM, 138±1 mM, 140±1 mM, 142±1 mM, 144±1 mM, 146±1 mM, 148±1 mM, 150±1 mM, 152±1 mM, 154±1 mM, 156±1 mM, 158±1 mM, 160±1 mM, 162±1 mM, 164±1 mM, 166±1 mM, 168±1 mM, 170±1 mM, 172±1 mM, 174±1 mM, 176±1 mM, 178±1 mM, 180±1 mM, 182±1 mM, 184±1 mM, 186±1 mM, 188±1 mM, 190±1 mM, 192±1 mM, 194±1 mM, 196±1 mM, 198±1 mM, 200±1 mM, 202±1 mM, 204±1 mM, 206±1 mM, 208±1 mM, 210±1 mM, 212±1 mM, 214±1 mM, 216±1 mM, 218±1 mM, 220±1 mM, 222±1 mM, 224±1 mM, 226±1 mM, 228±1 mM, 230±1 mM, 232±1 mM, 234±1 mM, 236±1 mM, 238±1 mM, 240±1 mM, 242±1 mM, 244±1 mM, 246±1 mM, 248±1 mM, 250±1 mM, 252±1 mM, 254±1 mM, 256±1 mM, 258±1 mM, 260±1 mM, 262±1 mM, 264±1 mM, 266±1 mM, 268±1 mM, 270±1 mM, 272±1 mM, 274±1 mM, 276±1 mM, 278±1 mM, 280±1 mM, 282±1 mM, 284±1 mM, 286±1 mM, 288±1 mM, 290±1 mM, 292±1 mM, 294±1 mM, 296±1 mM, 298±1 mM, 300±1mM, 302±1 mM, 304±1 mM, 306±1 mM, 308±1 mM, 310±1 mM, 312±1 mM, 314±1 mM, 316±1 mM, 318±1 mM, 320±1 mM, 322±1 mM, 324±1 mM, 326±1 mM, 328±1 mM, 330±1 mM, 332±1 mM, 334±1 mM, 336±1 mM, 338±1 mM, 340±1 mM, 342±1 mM, 344±1 mM, 346±1 mM, 348±1 mM, 350±1 mM, 352±1 mM, 354±1 mM, 356±1 mM, 358±1 mM, 360±1 mM, 362±1 mM, 364±1 mM, 366±1 mM, 368±1 mM, 370±1 mM, 372±1 mM, 374±1 mM, 376±1 mM, 378±1 mM, 380±1 mM, 382±1 mM, 384±1 mM, 386±1 mM, 388±1 mM, 390±1 mM, 392±1 mM, 394±1 mM, 396±1 mM, 398±1 mM, 400±1 mM, 402±1 mM, 404±1 mM, 406±1 mM, 408±1 mM, 410±1 mM, 412±1 mM, 414±1 mM, 416±1 mM, 418±1 mM, 420±1 mM, 422±1 mM, 424±1 mM, 426±1 mM, 428±1 mM, 430±1 mM, 432±1 mM, 434±1 mM, 436±1 mM, 438±1 mM, 440±1 mM, 442±1 mM, 444±1 mM, 446±1 mM, 448±1 mM, 450±1 mM, 452±1 mM, 454±1 mM, 456±1 mM, 458±1 mM, 460±1 mM, 462±1 mM, 464±1 mM, 466±1 mM, 468±1 mM, 470±1 mM, 472±1 mM, 474±1 mM, 476±1 mM, 478±1 mM, 480±1 mM, 482±1 mM, 484±1 mM, 486±1 mM, 488±1 mM, 490±1 mM, 492±1 mM, 494±1 mM, 496±1 mM, 498±1 mM, and 500±1 mM.

[0065] In preferred embodiments, the solution of one or more non-micellar caseins, is titrated into the titration vessel, simultaneously with the titration of the solution of a polyphosphate into the titration vessel, and simultaneously with the titration of the solution of a divalent cation salt into the titration vessel, wherein the concentration of the solution of one or more non-micellar caseins is selected from the group consisting of; 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L, 86 g / L, 87 g / L, 88 g / L, 89 g / L, 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, 101 g / L, 102 g / L, 103 g / L, 104 g / L, 105 g / L, 106 g / L, 107 g / L, 108 g / L, 109 g / L, 110 g / L, 111 g / L, 112 g / L, 113 g / L, 114 g / L, 115 g / L, 116 g / L, 117 g / L, 118 g / L, 119 g / L, 120 g / L, 121 g / L, 122 g / L, 123 g / L, 124 g / L, 125 g / L, 126 g / L, 127 g / L, 128 g / L, 129 g / L, 130 g / L, 131 g / L, 132 g / L, 133 g / L, 134 g / L, 135 g / L, 136 g / L, 137 g / L, 138 g / L, 139 g / L, 140 g / L, 141 g / L, 142 g / L, 143 g / L, 144 g / L, 145 g / L, 146 g / L, 147 g / L, 148 g / L, 149 g / L, 150 g / L, 151 g / L, 152 g / L, 153 g / L, 154 g / L, 155 g / L, 156 g / L, 157 g / L, 158 g / L, 159 g / L, 160 g / L, 161 g / L, 162 g / L, 163 g / L, 164 g / L, 165 g / L, 166 g / L, 167 g / L, 168 g / L, 169 g / L, 170 g / L, 171 g / L, 172 g / L, 173 g / L, 174 g / L, 175 g / L, 176 g / L, 177 g / L, 178 g / L, 179 g / L, 180 g / L, 181 g / L, 182 g / L, 183 g / L, 184 g / L, 185 g / L, 186 g / L, 187 g / L, 188 g / L, 189 g / L, 190 g / L, 191 g / L, 192 g / L, 193 g / L, 194 g / L, 195 g / L, 196 g / L, 197 g / L, 198 g / L, 199 g / L, 200 g / L, 201 g / L, 202 g / L, 203 g / L, 204 g / L, 205 g / L, 206 g / L, 207 g / L, 208 g / L, 209 g / L, 210 g / L, 211 g / L, 212 g / L, 213 g / L, 214 g / L, 215 g / L, 216 g / L, 217 g / L, 218 g / L, 219 g / L, 220 g / L, 221 g / L, 222 g / L, 223 g / L, 224 g / L, 225 g / L, 226 g / L, 227 g / L, 228 g / L, 229 g / L, 230 g / L, 231 g / L, 232g / L, 233 g / L, 234 g / L, 235 g / L, 236 g / L, 237 g / L, 238 g / L, 239 g / L, 240 g / L, 241 g / L, 242 g / L, 243 g / L, 244 g / L, 245 g / L, 246 g / L, 247 g / L, 248 g / L, 249 g / L, 250 g / L, 251 g / L, 252 g / L, 253 g / L, 254 g / L, 255 g / L, 256 g / L, 257 g / L, 258 g / L, 259 g / L, 260 g / L, 261 g / L, 262 g / L, 263 g / L, 264 g / L, 265 g / L, 266 g / L, 267 g / L, 268 g / L, 269 g / L, 270 g / L, 271 g / L, 272 g / L, 273 g / L, 274 g / L, 275 g / L, 276 g / L, 277 g / L, 278 g / L, 279 g / L, 280 g / L, 281 g / L, 282 g / L, 283 g / L, 284 g / L, 285 g / L, 286 g / L, 287 g / L, 288 g / L, 289 g / L, 290 g / L, 291 g / L, 292 g / L, 293 g / L, 294 g / L, 295 g / L, 296 g / L, 297 g / L, 298 g / L, 299 g / L, 300 g / L, 301 g / L, 302 g / L, 303 g / L, 304 g / L, 305 g / L, 306 g / L, 307 g / L, 308 g / L, 309 g / L, 310 g / L, 311 g / L, 312 g / L, 313 g / L, 314 g / L, 315 g / L, 316 g / L, 317 g / L, 318 g / L, 319 g / L, 320 g / L, 321 g / L, 322 g / L, 323 g / L, 324 g / L, 325 g / L, 326 g / L, 327 g / L, 328 g / L, 329 g / L, 330 g / L, 331 g / L, 332 g / L, 333 g / L, 334 g / L, 335 g / L, 336 g / L, 337 g / L, 338 g / L, 339 g / L, 340 g / L, 341 g / L, 342 g / L, 343 g / L, 344 g / L, 345 g / L, 346 g / L, 347 g / L, 348 g / L, 349 g / L, 350 g / L, 351 g / L, 352 g / L, 353 g / L, 354 g / L, 355 g / L, 356 g / L, 357 g / L, 358 g / L, 359 g / L, 360 g / L, 361 g / L, 362 g / L, 363 g / L, 364 g / L, 365 g / L, 366 g / L, 367 g / L, 368 g / L, 369 g / L, 370 g / L, 371 g / L, 372 g / L, 373 g / L, 374 g / L, 375 g / L, 376 g / L, 377 g / L, 378 g / L, 379 g / L, 380 g / L, 381 g / L, 382 g / L, 383 g / L, 384 g / L, 385 g / L, 386 g / L, 387 g / L, 388 g / L, 389 g / L, 390 g / L, 391 g / L, 392 g / L, 393 g / L, 394 g / L, 395 g / L, 396 g / L, 397 g / L, 398 g / L, 399 g / L, 400 g / L, 401 g / L, 402 g / L, 403 g / L, 404 g / L, 405 g / L, 406 g / L, 407 g / L, 408 g / L, 409 g / L, 410 g / L, 411 g / L, 412 g / L, 413 g / L, 414 g / L, 415 g / L, 416 g / L, 417 g / L, 418 g / L, 419 g / L, 420 g / L, 421 g / L, 422 g / L, 423 g / L, 424 g / L, 425 g / L, 426 g / L, 427 g / L, 428 g / L, 429 g / L, 430 g / L, 431 g / L, 432 g / L, 433 g / L, 434 g / L, 435 g / L, 436 g / L, 437 g / L, 438 g / L, 439 g / L, 440 g / L, 441 g / L, 442 g / L, 443 g / L, 444 g / L, 445 g / L, 446 g / L, 447 g / L, 448 g / L, 449 g / L, 450 g / L, 451 g / L, 452 g / L, 453 g / L, 454 g / L, 455 g / L, 456 g / L, 457 g / L, 458 g / L, 459 g / L, 460 g / L, 461 g / L, 462 g / L, 463 g / L, 464 g / L, 465 g / L, 466 g / L, 467 g / L, 468 g / L, 469 g / L, 470 g / L, 471 g / L, 472 g / L, 473 g / L, 474 g / L, 475 g / L, 476 g / L, 477 g / L, 478 g / L, 479 g / L, 480 g / L, 481 g / L, 482 g / L, 483 g / L, 484 g / L, 485 g / L, 486 g / L, 487 g / L, 488 g / L, 489 g / L, 490 g / L, 491 g / L, 492 g / L, 493 g / L, 494 g / L, 495 g / L, 496 g / L, 497 g / L, 498 g / L, 499 g / L, and 500 g / L.

[0066] In some embodiments, the concentrations, rates of addition and total titration time for the processes of the present invention are selected so as to provide preferred final concentrations of the one or more caseins, polyphosphate and divalent cation upon completion of the titration into the titration vessel. For example, without limitation, the concentrations, rates of addition and total titration time for the processes of the present invention are selected so as to provide a final divalent cation concentration selected from the group comprising; 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM,81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM, 138 mM, 139 mM, 140 mM, 141 mM, 142 mM, 143 mM, 144 mM, 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, 161 mM, 162 mM, 163 mM, 164 mM, 165 mM, 166 mM, 167 mM, 168 mM, 169 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, 180 mM, 181 mM, 182 mM, 183 mM, 184 mM, 185 mM, 186 mM, 187 mM, 188 mM, 189 mM, 190 mM, 191 mM, 192 mM, 193 mM, 194 mM, 195 mM, 196 mM, 197 mM, 198 mM, 199 mM, and 200 mM.

[0067] Furthermore, for example, without limitation, the concentrations, rates of addition and total titration time for the processes of the present invention may be selected so as to provide a final polyphosphate concentration selected from the group comprising; 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM, 6.1 mM, 6.2 mM, 6.3 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM, 7 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM, 7.9 mM, 8 mM, 8.1 mM, 8.2 mM, 8.3 mM, 8.4 mM, 8.5 mM, 8.6 mM, 8.7 mM, 8.8 mM, 8.9 mM, 9 mM, 9.1 mM, 9.2 mM, 9.3 mM, 9.4 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 10.6 mM, 10.7 mM, 10.8 mM, 10.9 mM, 11 mM, 11.1 mM, 11.2 mM, 11.3 mM, 11.4 mM, 11.5 mM, 11.6 mM, 11.7 mM, 11.8 mM, 11.9 mM, 12 mM, 12.1 mM, 12.2 mM, 12.3 mM, 12.4 mM, 12.5 mM, 12.6 mM, 12.7 mM, 12.8 mM, 12.9 mM, 13 mM, 13.1 mM, 13.2 mM, 13.3 mM, 13.4 mM, 13.5 mM, 13.6 mM, 13.7 mM, 13.8 mM, 13.9 mM, 14 mM, 14.1 mM, 14.2 mM, 14.3 mM, 14.4 mM, 14.5 mM, 14.6 mM, 14.7 mM, 14.8 mM, 14.9 mM, 15 mM, 15.1 mM, 15.2 mM, 15.3 mM, 15.4 mM, 15.5 mM, 15.6 mM, 15.7 mM, 15.8 mM, 15.9 mM, 16 mM, 16.1 mM, 16.2 mM, 16.3 mM, 16.4 mM, 16.5 mM, 16.6 mM, 16.7 mM, 16.8 mM, 16.9 mM, 17 mM, 17.1 mM, 17.2 mM, 17.3 mM, 17.4 mM, 17.5 mM, 17.6 mM, 17.7 mM, 17.8 mM, 17.9 mM, 18 mM, 18.1 mM, 18.2 mM, 18.3 mM, 18.4 mM, 18.5 mM, 18.6 mM, 18.7 mM, 18.8 mM, 18.9 mM, 19 mM, 19.1 mM, 19.2 mM, 19.3 mM, 19.4 mM, 19.5 mM, 19.6 mM, 19.7 mM, 19.8 mM, 19.9 mM, 20 mM, 20.1 mM, 20.2 mM, 20.3 mM, 20.4 mM, 20.5 mM, 20.6 mM, 20.7 mM, 20.8 mM, 20.9 mM, 21 mM, 21.1 mM, 21.2 mM, 21.3 mM, 21.4 mM, 21.5 mM, 21.6 mM, 21.7 mM, 21.8 mM, 21.9 mM, 22 mM, 22.1 mM, 22.2 mM, 22.3 mM, 22.4 mM, 22.5 mM, 22.6 mM,22.7 mM, 22.8 mM, 22.9 mM, 23 mM, 23.1 mM, 23.2 mM, 23.3 mM, 23.4 mM, 23.5 mM, 23.6 mM, 23.7 mM, 23.8 mM, 23.9 mM, 24 mM, 24.1 mM, 24.2 mM, 24.3 mM, 24.4 mM, 24.5 mM, 24.6 mM, 24.7 mM, 24.8 mM, 24.9 mM, 25 mM, 25.1 mM, 25.2 mM, 25.3 mM, 25.4 mM, 25.5 mM, 25.6 mM, 25.7 mM, 25.8 mM, 25.9 mM, 26 mM, 26.1 mM, 26.2 mM, 26.3 mM, 26.4 mM, 26.5 mM, 26.6 mM, 26.7 mM, 26.8 mM, 26.9 mM, 27 mM, 27.1 mM, 27.2 mM, 27.3 mM, 27.4 mM, 27.5 mM, 27.6 mM, 27.7 mM, 27.8 mM, 27.9 mM, 28 mM, 28.1 mM, 28.2 mM, 28.3 mM, 28.4 mM, 28.5 mM, 28.6 mM, 28.7 mM, 28.8 mM, 28.9 mM, 29 mM, 29.1 mM, 29.2 mM, 29.3 mM, 29.4 mM, 29.5 mM, 29.6 mM, 29.7 mM, 29.8 mM, 29.9 mM, 30 mM, 30.1 mM, 30.2 mM, 30.3 mM, 30.4 mM, 30.5 mM, 30.6 mM, 30.7 mM, 30.8 mM, 30.9 mM, 31 mM, 31.1 mM, 31.2 mM, 31.3 mM, 31.4 mM, 31.5 mM, 31.6 mM, 31.7 mM, 31.8 mM, 31.9 mM, 32 mM, 32.1 mM, 32.2 mM, 32.3 mM, 32.4 mM, 32.5 mM, 32.6 mM, 32.7 mM, 32.8 mM, 32.9 mM, 33 mM, 33.1 mM, 33.2 mM, 33.3 mM, 33.4 mM, 33.5 mM, 33.6 mM, 33.7 mM, 33.8 mM, 33.9 mM, 34 mM, 34.1 mM, 34.2 mM, 34.3 mM, 34.4 mM, 34.5 mM, 34.6 mM, 34.7 mM, 34.8 mM, 34.9 mM, 35 mM, 35.1 mM, 35.2 mM, 35.3 mM, 35.4 mM, 35.5 mM, 35.6 mM, 35.7 mM, 35.8 mM, 35.9 mM, 36 mM, 36.1 mM, 36.2 mM, 36.3 mM, 36.4 mM, 36.5 mM, 36.6 mM, 36.7 mM, 36.8 mM, 36.9 mM, 37 mM, 37.1 mM, 37.2 mM, 37.3 mM, 37.4 mM, 37.5 mM, 37.6 mM, 37.7 mM, 37.8 mM, 37.9 mM, 38 mM, 38.1 mM, 38.2 mM, 38.3 mM, 38.4 mM, 38.5 mM, 38.6 mM, 38.7 mM, 38.8 mM, 38.9 mM, 39 mM, 39.1 mM, 39.2 mM, 39.3 mM, 39.4 mM, 39.5 mM, 39.6 mM, 39.7 mM, 39.8 mM, 39.9 mM, 40 mM, 40.1 mM, 40.2 mM, 40.3 mM, 40.4 mM, 40.5 mM, 40.6 mM, 40.7 mM, 40.8 mM, 40.9 mM, 41 mM, 41.1 mM, 41.2 mM, 41.3 mM, 41.4 mM, 41.5 mM, 41.6 mM, 41.7 mM, 41.8 mM, 41.9 mM, 42 mM, 42.1 mM, 42.2 mM, 42.3 mM, 42.4 mM, 42.5 mM, 42.6 mM, 42.7 mM, 42.8 mM, 42.9 mM, 43 mM, 43.1 mM, 43.2 mM, 43.3 mM, 43.4 mM, 43.5 mM, 43.6 mM, 43.7 mM, 43.8 mM, 43.9 mM, 44 mM, 44.1 mM, 44.2 mM, 44.3 mM, 44.4 mM, 44.5 mM, 44.6 mM, 44.7 mM, 44.8 mM, 44.9 mM, 45 mM, 45.1 mM, 45.2 mM, 45.3 mM, 45.4 mM, 45.5 mM, 45.6 mM, 45.7 mM, 45.8 mM, 45.9 mM, 46 mM, 46.1 mM, 46.2 mM, 46.3 mM, 46.4 mM, 46.5 mM, 46.6 mM, 46.7 mM, 46.8 mM, 46.9 mM, 47 mM, 47.1 mM, 47.2 mM, 47.3 mM, 47.4 mM, 47.5 mM, 47.6 mM, 47.7 mM, 47.8 mM, 47.9 mM, 48 mM, 48.1 mM, 48.2 mM, 48.3 mM, 48.4 mM, 48.5 mM, 48.6 mM, 48.7 mM, 48.8 mM, 48.9 mM, 49 mM, 49.1 mM, 49.2 mM, 49.3 mM, 49.4 mM, 49.5 mM, 49.6 mM, 49.7 mM, 49.8 mM, 49.9 mM, and 50 mM.

[0068] Furthermore, the concentrations, rates of addition and total titration time for the processes of the present invention may be selected so as to provide preferred ratios of polyphosphate to divalent cation upon completion of the titration into the titration vessel. For example, without limitation, the concentrations, rates of addition and total titration time for the processes of the present invention may be selected so as to provide a final ratio of polyphosphate to divalent cation selected from the group comprising; 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, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, and 30:1.

[0069] In some embodiments, the concentrations, rates of addition and total titration time for the processes of the present invention may be selected so as to provide preferred ratios of divalent cation species to polyphosphate upon completion of the titration into the titration vessel. For example, without limitation, the concentrations, rates of addition and total titration time for the processes of the present invention may be selected so as to provide a final ratio of divalent cation species to polyphosphate selected from the group consisting of; 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1, 6:1, 6.1:1, 6.2:1, 6.3:1, 6.4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7:1, 7.1:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1, 7.6:1, 7.7:1, 7.8:1, 7.9:1, 8:1, 8.1:1, 8.2:1, 8.3:1, 8.4:1, 8.5:1, 8.6:1, 8.7:1, 8.8:1, 8.9:1, 9:1, 9.1:1, 9.2:1, 9.3:1, 9.4:1, 9.5:1, 9.6:1, 9.7:1, 9.8:1, 9.9:1, 10:1, 10.1:1, 10.2:1, 10.3:1, 10.4:1, 10.5:1, 10.6:1, 10.7:1, 10.8:1, 10.9:1, 11:1, 11.1:1, 11.2:1, 11.3:1, 11.4:1, 11.5:1, 11.6:1, 11.7:1, 11.8:1, 11.9:1, 12:1, 12.1:1, 12.2:1, 12.3:1, 12.4:1, 12.5:1, 12.6:1, 12.7:1, 12.8:1, 12.9:1, 13:1, 13.1:1, 13.2:1, 13.3:1, 13.4:1, 13.5:1, 13.6:1, 13.7:1, 13.8:1, 13.9:1, 14:1, 14.1:1, 14.2:1, 14.3:1, 14.4:1, 14.5:1, 14.6:1, 14.7:1, 14.8:1, 14.9:1, 15:1, 15.1:1, 15.2:1, 15.3:1, 15.4:1, 15.5:1, 15.6:1, 15.7:1, 15.8:1, 15.9:1, 16:1, 16.1:1, 16.2:1, 16.3:1, 16.4:1, 16.5:1, 16.6:1, 16.7:1, 16.8:1, 16.9:1, 17:1, 17.1:1, 17.2:1, 17.3:1, 17.4:1, 17.5:1, 17.6:1, 17.7:1, 17.8:1, 17.9:1, 18:1, 18.1:1, 18.2:1, 18.3:1, 18.4:1, 18.5:1, 18.6:1, 18.7:1, 18.8:1, 18.9:1, 19:1, 19.1:1, 19.2:1, 19.3:1, 19.4:1, 19.5:1, 19.6:1, 19.7:1, 19.8:1, 19.9:1, and 20:1.

[0070] Whilst proof of principle of the processes of the present invention has been demonstrated using sodium hexametaphosphate as an exemplary polyphosphate, the person skilled in the art will understand that other polyphosphates may be equally applicable to the processes of the present invention. For example, without limitation, the formation of ACMs in accordance with the present invention may be facilitated with polyphosphates in general, including polyphosphates selected from the group consisting of; sodium polyphosphate, potassium polyphosphate, sodium calcium polyphosphate, calcium polyphosphate, sodium triphosphate, potassium triphosphate, sodium calcium triphosphate, calcium triphosphate, sodium trimetaphosphate, potassium trimetaphosphate, sodium calcium trimetaphosphate, calcium trimetaphosphate, sodium hexametaphosphate, potassium hexametaphosphate, sodium calcium hexametaphosphate, and calcium hexametaphosphate; preferably the polyphosphate is sodium hexametaphosphate.

[0071] Similarly, whilst proof of principle of the processes of the present invention has been demonstrated using Ca2+as an exemplary divalent cation species, the person skilled in the art will understand that other divalent cation species may be equally applicable to the processes of the present invention. For example, without limitation, the formation of ACMs in accordance with the present invention may be facilitated with divalent cation species in general, including divalent cation species selected from the group consisting of; Ca2+, Mg2+, Fe2+and Zn2+; preferably the divalent cation species is Ca2+.

[0072] In some embodiments, the Z-average diameter of the artificial casein micelles is greater than 30 nm. The Z-average diameter of the artificial casein micelles of the present invention may be determined, for example and without limitation, via dynamic light scattering measurements or via scanning electron microscopy (SEM). Without limitation, the Z-average diameter of the artificial casein micelles of the present invention may be greater than; 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, 110 nm, 111 nm, 112 nm, 113 nm, 114 nm, 115 nm, 116 nm, 117 nm, 118 nm, 119 nm, 120 nm, 121 nm, 122 nm, 123 nm, 124 nm, 125 nm, 126 nm, 127 nm, 128 nm, 129 nm, 130 nm, 131 nm, 132 nm, 133 nm, 134 nm, 135 nm, 136 nm, 137 nm, 138 nm, 139 nm, 140 nm, 141 nm, 142 nm, 143 nm, 144 nm, 145 nm, 146 nm, 147 nm, 148 nm, 149 nm, 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155 nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, 182 nm, 183 nm, 184 nm, 185 nm, 186 nm, 187 nm, 188 nm, 189 nm, 190 nm, 191 nm, 192 nm, 193 nm, 194 nm, 195 nm, 196 nm, 197 nm, 198 nm, 199 nm, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm, 244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, 290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm, 299 nm, or 300 nm.

[0073] In preferred embodiments, the Z-average diameter of the artificial casein micelles falls within the range of 40 to 500 nm. Without limitation, the Z-average diameter of the artificial casein micelles of particularly preferred embodiments may fall within any range selected from the group of ranges comprising; 40 to 500 nm, 45 to 500 nm, 50 to 500 nm, 55 to 500 nm, 60 to 500 nm, 65 to 500 nm, 70 to 500 nm, 75 to 500 nm, 80 to 500 nm, 85 to 500 nm, 90 to 500 nm, 95 to 500 nm, 100 to 500 nm, 105 to 500 nm, 110 to 500 nm, 115 to 500 nm, 120 to 500 nm, 125 to 500 nm, 130 to 500 nm, 135 to 500 nm, 140 to 500 nm, 145 to 500 nm, 150 to 500 nm, 155 to 500 nm, 160 to 500 nm, 165 to 500 nm, 170 to 500 nm, 175 to 500 nm, 180 to 500 nm, 185 to 500 nm,190 to 500 nm, 195 to 500 nm, 200 to 500 nm, 205 to 500 nm, 210 to 500 nm, 215 to 500 nm, 220 to 500 nm, 225 to 500 nm, 230 to 500 nm, 235 to 500 nm, 240 to 500 nm, 245 to 500 nm, 250 to 500 nm, 255 to 500 nm, 260 to 500 nm, 265 to 500 nm, 270 to 500 nm, 275 to 500 nm, 280 to 500 nm, 285 to 500 nm, 290 to 500 nm, 295 to 500 nm, 300 to 500 nm, 305 to 500 nm, 310 to 500 nm, 315 to 500 nm, 320 to 500 nm, 325 to 500 nm, 330 to 500 nm, 335 to 500 nm, 340 to 500 nm, 345 to 500 nm, 350 to 500 nm, 355 to 500 nm, 360 to 500 nm, 365 to 500 nm, 370 to 500 nm, 375 to 500 nm, 380 to 500 nm, 385 to 500 nm, 390 to 500 nm, 395 to 500 nm, 400 to 500 nm, 405 to 500 nm, 410 to 500 nm, 415 to 500 nm, 420 to 500 nm, 425 to 500 nm, 430 to 500 nm, 435 to 500 nm, 440 to 500 nm, 445 to 500 nm, 450 to 500 nm, 455 to 500 nm, 460 to 500 nm, 465 to 500 nm, 470 to 500 nm, 475 to 500 nm, 480 to 500 nm, 485 to 500 nm, 490 to 500 nm, and 495 to 500 nm.

[0074] In some embodiments, the micellar solutions described herein comprise a total casein concentration falling within the range of 10 g / L to 200 g / L. Without limitation, the total casein concentration in the micellar solutions of the present invention may be selected from the group comprising; 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L, 86 g / L, 87 g / L, 88 g / L, 89 g / L, 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, 101 g / L, 102 g / L, 103 g / L, 104 g / L, 105 g / L, 106 g / L, 107 g / L, 108 g / L, 109 g / L, 110 g / L, 111 g / L, 112 g / L, 113 g / L, 114 g / L, 115 g / L, 116 g / L, 117 g / L, 118 g / L, 119 g / L, 120 g / L, 121 g / L, 122 g / L, 123 g / L, 124 g / L, 125 g / L, 126 g / L, 127 g / L, 128 g / L, 129 g / L, 130 g / L, 131 g / L, 132 g / L, 133 g / L, 134 g / L, 135 g / L, 136 g / L, 137 g / L, 138 g / L, 139 g / L, 140 g / L, 141 g / L, 142 g / L, 143 g / L, 144 g / L, 145 g / L, 146 g / L, 147 g / L, 148 g / L, 149 g / L, 150 g / L, 151 g / L, 152 g / L, 153 g / L, 154 g / L, 155 g / L, 156 g / L, 157 g / L, 158 g / L, 159 g / L, 160 g / L, 161 g / L, 162 g / L, 163 g / L, 164 g / L, 165 g / L, 166 g / L, 167 g / L, 168 g / L, 169 g / L, 170 g / L, 171 g / L, 172 g / L, 173 g / L, 174 g / L, 175 g / L, 176 g / L, 177 g / L, 178 g / L, 179 g / L, 180 g / L, 181 g / L, 182 g / L, 183 g / L, 184 g / L, 185 g / L, 186 g / L, 187 g / L, 188 g / L, 189 g / L, 190 g / L, 191 g / L, 192 g / L, 193 g / L, 194 g / L, 195 g / L, 196 g / L, 197 g / L, 198 g / L, 199 g / L, 200 g / L.

[0075] In some embodiments, the hydration of the artificial casein micelles in the micellar solutions of the present invention, falls within the range of 1 to 8 (g water / g micellar protein). Without limitation, the hydration of the artificial casein micelles in the micellar solutions of the present invention, may be selected from the group comprising; 1 (g water / g micellar protein), 1.1 (g water / g micellar protein), 1.2 (g water / g micellar protein), 1.3 (g water / g micellar protein),1.4 (g water / g micellar protein), 1.5 (g water / g micellar protein), 1.6 (g water / g micellar protein), 1.7 (g water / g micellar protein), 1.8 (g water / g micellar protein), 1.9 (g water / g micellar protein), 2 (g water / g micellar protein), 2.1 (g water / g micellar protein), 2.2 (g water / g micellar protein), 2.3 (g water / g micellar protein), 2.4 (g water / g micellar protein), 2.5 (g water / g micellar protein), 2.6 (g water / g micellar protein), 2.7 (g water / g micellar protein), 2.8 (g water / g micellar protein), 2.9 (g water / g micellar protein), 3 (g water / g micellar protein), 3.1 (g water / g micellar protein), 3.2 (g water / g micellar protein), 3.3 (g water / g micellar protein), 3.4 (g water / g micellar protein), 3.5 (g water / g micellar protein), 3.6 (g water / g micellar protein), 3.7 (g water / g micellar protein), 3.8 (g water / g micellar protein), 3.9 (g water / g micellar protein), 4 (g water / g micellar protein), 4.1 (g water / g micellar protein), 4.2 (g water / g micellar protein), 4.3 (g water / g micellar protein), 4.4 (g water / g micellar protein), 4.5 (g water / g micellar protein), 4.6 (g water / g micellar protein), 4.7 (g water / g micellar protein), 4.8 (g water / g micellar protein), 4.9 (g water / g micellar protein), 5 (g water / g micellar protein), 5.1 (g water / g micellar protein), 5.2 (g water / g micellar protein), 5.3 (g water / g micellar protein), 5.4 (g water / g micellar protein), 5.5 (g water / g micellar protein), 5.6 (g water / g micellar protein), 5.7 (g water / g micellar protein), 5.8 (g water / g micellar protein), 5.9 (g water / g micellar protein), 6 (g water / g micellar protein), 6.1 (g water / g micellar protein), 6.2 (g water / g micellar protein), 6.3 (g water / g micellar protein), 6.4 (g water / g micellar protein), 6.5 (g water / g micellar protein), 6.6 (g water / g micellar protein), 6.7 (g water / g micellar protein), 6.8 (g water / g micellar protein), 6.9 (g water / g micellar protein), 7 (g water / g micellar protein), 7.1 (g water / g micellar protein), 7.2 (g water / g micellar protein), 7.3 (g water / g micellar protein), 7.4 (g water / g micellar protein), 7.5 (g water / g micellar protein), 7.6 (g water / g micellar protein), 7.7 (g water / g micellar protein), 7.8 (g water / g micellar protein), 7.9 (g water / g micellar protein), and 8 (g water / g micellar protein).

[0076] In some embodiments, the non-micellar caseins as a percentage of total caseins in the micellar solutions of the present invention, falls within the range of 5 to 20%. Without limitation, the non-micellar caseins as a percentage of total caseins in the micellar solutions of the present invention may be selected from the group comprising; 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%, 15.9%, 16%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18%,18.1%, 18.2%, 18.3%, 18.4%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19%, 19.1%, 19.2%, 19.3%, 19.4%, 19.5%, 19.6%, 19.7%, 19.8%, 19.9%, and 20%.

[0077] In one embodiment, the disclosure herein provides a curds composition comprising the micellar solution of the present invention, in coagulated form.

[0078] The curds composition may be a useful precursor for the manufacture of downstream products such as yogurt or cheese. The curds composition may be coagulated by the action of an acid or a renneting agent. Suitable acids for coagulation include, without limitation, citric acid vinegar, and lactic acid.

[0079] In some embodiments, a yogurt composition may be formed using the methods described herein. The yogurt may be formed using the micellar solution described herein. The method may comprise heating and then cooling the micellar solution and acidifying the micellar solution with an acid or a microorganism. The microorganism may comprise one or more of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, a lactobacilli, or a bifidobacteria.

[0080] In some embodiments, following acidification, a renneting agent may be added to form a renneted curd (coagulated curd matrix), which may then be used to make cheese. Micelles in a micellar solution, such as milk and also the micellar solution described herein, are stable and repel each other in colloidal suspension. In presence of renneting agents or milk-clotting enzymes, and when acidified, micelles are destabilized and attract each other, and thus coagulate. In presence of renneting agents or milk-clotting enzymes, cross-linked coagulated curd matrix is formed.

[0081] In some embodiments, the curds composition further comprises a renneting agent. Renneting agents suitable for performance of the present invention include, without limitation, protease enzymes, chymosin, pepsin, lipase, animal derived rennet, plant derived rennet (including extracts from Galium spp., dried caper leaves, nettles, thistles, mallow, Withania coagulans, ground ivy, Cynara, soy), calf rennet, kid goat rennet, fungi derived rennet, microbially derived rennet (e.g., extracts of Rhizomucor miehei) and recombinantly produced chymosin.

[0082] In a preferred embodiment, the curds composition has a Maximum G’ (storage modulus) falling within the range of 5 to 200 Pa, preferably after 1 hour incubation with rennet. Without limitation, the Maximum G’ of the curds composition of the present invention after any period of incubation with rennet selected from the group comprising; 0.1hr, 0.2hr, 0.3hr, 0.4hr, 0.5hr, 0.6hr, 0.7hr, 0.8hr, 0.9hr, 1hr, 1.1hr, 1.2hr, 1.3hr, 1.4hr, 1.5hr, 1.6hr, 1.7hr, 1.8hr, 1.9hr, 2hr, 2.1hr, 2.2hr, 2.3hr, 2.4hr, 2.5hr, 2.6hr, 2.7hr, 2.8hr, 2.9hr, 3hr, 3.1hr, 3.2hr, 3.3hr, 3.4hr, 3.5hr, 3.6hr, 3.7hr, 3.8hr, 3.9hr, 4hr, 4.1hr, 4.2hr, 4.3hr, 4.4hr, 4.5hr, 4.6hr, 4.7hr, 4.8hr, 4.9hr, 5hr, 5.1hr, 5.2hr, 5.3hr, 5.4hr, 5.5hr, 5.6hr, 5.7hr, 5.8hr, 5.9hr, and 6hr; may fall within any range selected from the groupof ranges comprising; 5 to 200 Pa, 10 to 200 Pa, 15 to 200 Pa, 20 to 200 Pa, 25 to 200 Pa, 30 to 200 Pa, 35 to 200 Pa, 40 to 200 Pa, 45 to 200 Pa, 50 to 200 Pa, 55 to 200 Pa, 60 to 200 Pa, 65 to 200 Pa, 70 to 200 Pa, 75 to 200 Pa, 80 to 200 Pa, 85 to 200 Pa, 90 to 200 Pa, 95 to 200 Pa, 100 to 200 Pa, 105 to 200 Pa, 110 to 200 Pa, 115 to 200 Pa, 120 to 200 Pa, 125 to 200 Pa, 130 to 200 Pa, 135 to 200 Pa, 140 to 200 Pa, 145 to 200 Pa, 150 to 200 Pa, 155 to 200 Pa, 160 to 200 Pa, 165 to 200 Pa, 170 to 200 Pa, 175 to 200 Pa, 180 to 200 Pa, 185 to 200 Pa, 190 to 200 Pa, and 195 to 200 Pa.

[0083] In a further embodiment, the disclosure herein provides an edible composition comprising the micellar solution of the present invention, or the curds composition of the present invention. Such edible compositions include, without limitation, yogurts, cheeses and milk substitutes.

[0084] In some embodiments, the edible composition does not contain any animal-derived protein.

[0085] In some embodiments of the method for producing an edible composition, the first condition is the addition of acid or acidification of the micellar solution with a microorganism.

[0086] In some embodiments of the method for producing an edible composition, the method further comprises subjecting the coagulates to a renneting agent to form a rennetted curd.

[0087] In some embodiments of the method for producing an edible composition, the method further comprises aging and / or maturing the rennetted curd to form a cheese composition. Renneted curd may be further treated to create a cheese or cheese like product. In some cases, such as a mozzarella product, the renneted curd may be heated and stretched. In other embodiments, the renneted curd is aged, such as for brie, camembert, feta, halloumi, gouda, edam, cheddar, manchego, swiss, colby, muenster, blue cheese or parmesan type cheese or cheese-like product.

[0088] In some embodiments, the micellar solution or renneted curd may be treated with hot water for the formation of cheese, such as for mozzarella-type cheese. Hot water treatment may be performed at a temperature of about 50°C to about 90°C. Hot water treatment may be performed at a temperature of at least 55°C. Hot water treatment may be performed at a temperature of at most 75°C. Hot water treatment may be performed at a temperature of 50°C to 55°C, 55°C to 60°C, 55°C to 65°C, 55°C to 70°C, 55°C to 75°C, 60°C to 65°C, 60°C to 70°C, 60°C to 75°C, 65°C to 70°C, 65°C to 75°C, 70°C to 75°C, 75°C to 80°C, 80°C to 85°C, or 85°C to 90°C. Hot water treatment may be performed at a temperature of about 50°C , about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C or about 90°C. Hot water treatment may be performed at a temperature of at least 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, or 85°C. Hot water treatment may be performed at a temperature of at most 55°C ,60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C. In some cases, after hot water treatment, the product is stretched into a cheese.

[0089] In some embodiments of the method for producing an edible composition, the edible composition does not contain any animal-derived protein.

[0090] Cheese compositions formed using the methods described herein optionally may not comprise any animal-derived components for example, where recombinantly derived casein proteins are utilised. Cheese compositions formed using the methods described herein may optionally not comprise any animal-derived dairy-based components, such as animal-derived dairy proteins. Cheese compositions formed using the methods described herein may optionally not comprise any whey proteins. Cheese compositions formed using the methods described herein may optionally not comprise any αs-casein proteins. Cheese compositions described herein may be pasta-filata like cheese such as mozzarella cheese. Soft cheeses such as paneer, cream cheese or cottage cheese may also be formed using the methods described herein. Other types of cheese such as aged and ripened cheeses may also be formed using the methods described herein, such as brie, camembert, feta, halloumi, gouda, edam, cheddar, manchego, swiss, colby, muenster, blue cheese and parmesan.

[0091] The texture of a cheese made by methods described herein may be comparable to the texture of a similar type of cheese made using animal-derived dairy derived proteins, such as cheese made from animal milk. Texture of a cheese may be tested using a trained panel of human subjects or machines such as a texture analyzer.

[0092] The taste of a cheese made by methods described herein may be comparable to a similar type of cheese made using animal-derived dairy proteins. Taste of a cheese may be tested using a trained panel of human subjects.

[0093] Cheese compositions described herein may have a browning ability which is comparable to a similar type of cheese made using animal-derived dairy proteins. Cheese compositions described herein may have a melting ability which is comparable to a similar type of cheese made using animal-derived dairy proteins.

[0094] The texture of a yogurt made by methods described herein may be comparable to the texture of a similar type of yogurt made using animal-derived dairy derived proteins, such as yogurt made from animal milk. Texture of a yogurt may be tested using a trained panel of human subjects or machines such as a texture analyzer.

[0095] The taste of a yogurt made by methods described herein may be comparable to a similar type of yogurt made using animal-derived dairy proteins. Taste of a yogurt may be tested using a trained panel of human subjects.

[0096] The following examples serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. EXAMPLES

[0097] Further features of the present invention are more fully described in the following non- limiting Examples. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad description of the invention as set out above. Example 1 – Synthesis, characterisation and coagulation behaviour of SHMP ACMs Materials

[0098] Bovine sodium caseinate (Lactonat EN, 89.8% protein, comprising 33% αs-casein, 47% β-casein, 20% κ-casein, and 14.5 mg / g sodium) was obtained from Lactoprot (Lactoprot Deutschland GmbH, Kaltenkirchen, Germany). Dephosphorylated casein was prepared by and according to Antuma et al. (2023),3with a protein purity of 96.1%. Purified β-and κ-casein fractions were produced in collaboration with the University of Hohenheim. The purified β-casein powder had a protein content of 91.9%, of which all was β-casein. The purified κ-casein powder had a protein content of 82.3%, comprising 67.8% κ-casein, 23.3% β-casein and 8.9% αs-casein.

[0099] Calcium chloride (C1016), magnesium chloride (M8266), potassium phosphate monobasic (P5379), sodium phosphate dibasic (S7907), citric acid (C0759), sodium hydroxide (221465), potassium hydroxide (1.0533), hydrochloric acid (1.13386), trisodium citrate dihydrate (S4641), nitric acid (1.00456), hydrogen peroxide (1.07209), sodium chloride (31434), lactic acid solution (252476), potassium chloride (1.04936), potassium carbonate (1.04928), potassium sulphate dibasic (1.05153), magnesium citrate nonahydrate (63067), sodium hexametaphosphate (71600), sodium acetate (71600), acetic acid (glacial) 100% (100063) and sodium phosphate monobasic dihydrate (71500) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Chymosin (CHY-MAX Plus, 3634543) was obtained from Christian Hansen Holding A / S (Hørsholm, Denmark). Tripotassium citrate monohydrate (6100-05-6) was obtained from VWR International bvba (Leuven, Belgium). Ultrapure water (MilliQ system, Merck KGaA, Darmstadt, Germany) was used for all experiments.Sample Preparation

[0100] Artificial casein micelles (ACM) were prepared according to Schmidt et al. (1977)1as control sample experiments. Sodium caseinate powder was dissolved in water at a concentration of 71.3 g / L, which amounted to 64.0 g casein / L. Three salt solutions were prepared. Solution I consisted of 445 mM CaCl2 and 75 mM MgCl2, solution II of 165 mM KH2PO4 and 165 mM Na2HPO4and solution III of 135 mM C6H8O7. The pH of solution I and III was adjusted to pH 6.70 with 0.1M HCl and 1M KOH, respectively. Controlled mixing of 60 mL of the caseinate solution and 10 mL of each salt solution was done in 60 min with syringe pumps (Harvard PHD2000, Harvard Apparatus, Massachusetts, USA and ProSense NE-1600, ProSense B.V., Oosterhout, The Netherlands). The mixing was done in a jacketed glass vessel at 37 °C, which contained a starting volume of 57 mL water under continuous mixing with a magnetic stirrer at speed 4. A pH of 6.70 was maintained during mixing and 20 min after mixing by titration with 1M NaOH (887 Titrino Plus, Metrohm AG, Barendrecht, The Netherlands). Typically, around 3 mL 1M NaOH was used. Total volume of the samples was finally adjusted to 150 mL with water.

[0101] Micelle formation with polyphosphate was tested using sodium hexametaphosphate (SHMP) in a set-up using the same apparatus as that according to Schmidt et al. (1977)1. Sample compositions and conditions were selected based on preliminary investigations, which showed potential micelle formation at simultaneous addition of SHMP and calcium at neutral pH. Sodium caseinate or dephosphorylated sodium caseinate powder was dissolved in water at 71.3 and 66.6 g / L, respectively. A solution of β- and κ-casein in a 70:30 ratio was prepared, which consisted of 41.8 g / L β-casein and 31.1 g / L κ-casein powder. In the latter solution, sodium citrate tribasic dihydrate was added at a 6.6 g / L concentration to aid in dissolving the casein. For all three types of casein solutions, this amounted to a total casein concentration of 64.0 g / L, to achieve a 25.6 g / L casein concentration in the ACM samples.

[0102] CaCl2 and SHMP solutions were used, instead of the three salt solutions employed in the Schmidt protocol. With the aim to resemble the control ACM prepared according to the protocol of Schmidt et al. (1977)1, a similar final calcium concentration of 30 mM was selected. Different ratios of calcium and SHMP were prepared, 15:1, 12:1, 10:1, 6:1, 4:1 and 3:1. This amounted to final SHMP concentrations of 1.5, 2.5, 3, 5, 7.5 and 10 mM, respectively. The controlled mixing of 60 mL casein solution, 10 mL CaCl2 solution and 10 mL SHMP solution was done in 30 min. The starting volume was 65 mL water. Control samples with 12, 21 and 30 mM CaCl2 without SHMP were prepared in a similar method. The starting volume was then 75 mL water. Two samples were prepared with magnesium chloride, instead of CaCl2; one with a 30 mM MgCl2and 3 mM SHMP concentration and one control sample without SHMP. An ACM control sample was prepared according to the protocol of Schmidt et al. (1977)1, with a preparation time of 30 minutes. The final volume of the samples was adjusted to 150 mL with water.

[0103] Sample names are coded by type of casein (sodium caseinate = NC, dephosphorylated casein = DP, β- and κ- casein = β / κ). The name is followed by the calcium (C), magnesium (M) and SHMP (S) concentration, with the number behind it representing the concentration in mM (e.g. DP C# S#). Characterization

[0104] Particle size and polydispersity were determined with a Zetasizer Ultra (Malvern Panalytical, Malvern, UK). Samples were equilibrated at room temperature for 1h before the measurement and subsequently diluted in a DTS0012, 10 mm square polystyrene cuvette. ACM formed with SHMP were diluted 100x in water. The refractive index of the casein micelles was set to 1.57 and that of the dispersant (water or SMUF) at 1.33. The temperature was set to 25°C. Samples were measured at a scattering angle of 173°and each measurement consisted of 5 sub- measurements. Samples were analyzed in duplicate. The ZS Xplorer software (version 2.3.1.4) provided the Z-average hydrodynamic diameter and polydispersity index.

[0105] Ultracentrifugation was done to separate the sedimentable and non-sedimentable fractions of the samples. Around 17 mL of sample was taken and equilibrated at room temperature for 1h. An Optima XE-90 ultracentrifuge was used (Beckman Coulter Inc., Woerden, The Netherlands) with a 70Ti rotor for 1h, set at 37000 rpm and 20°C. Around half of the supernatant volume was collected for further analyses. Samples were centrifuged in duplicate.

[0106] Casein micelle hydration was determined by analyzing the hydration of the ultracentrifugal pellets, which were assumed to consist of micelles. The pellets in centrifugation tubes were left upside down in a rack for at least 1h, to allow evaporation of residual supernatant. Pellets were weighed in aluminum pans and dried for a minimum of 2 days in a hot air oven at 105°C (Binder model E28, Binder GmbH, Tuttlingen Germany). After drying, the pans with the dried pellets were weighed again. Apparent casein micelle hydration was calculated in accordance with Equation (1): (1)

[0107] The casein content of samples and their centrifugal supernatants was determined with the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Ochten, The Netherlands). A six-point calibration range of 0-1.0 g casein / L was prepared from a 2 g casein / L sodium caseinate (NC) solution. Samples were first diluted to fall into the calibration range. Complete samples were diluted 50x, their supernatants 5 or 25x. Then, 50 µL of the diluted samples was taken and 1 mL of the working reagent was added, which consisted of reagent A and B in a 50:1 ratio. Samples were heated at 37°C for 30 min in a Thermomixer C (Eppendorf,Hamburg, Germany), refrigerated for 5 min and left at room temperature for 10 min. Immediately afterwards, the samples were measured in 1 mm cuvettes within 10 min, using a Hach DR6000 UV / VIS spectrophotometer at a wavelength of 562 nm (Hach Lange GmbH, Düsseldorf, Germany). Samples were analyzed in duplicate. The sedimentable (micellar) casein content was calculated by subtracting the casein content of the supernatants from the total casein content in the complete samples.

[0108] The mineral content of the SHMP-induced ACM samples prepared and their supernatants was analyzed to determine the mineral partitioning in the sedimentable (micellar) and serum (non-sedimentable) phase. The cation content (calcium, phosphorus, magnesium, sodium and potassium) was determined with inductively coupled plasma-optical emission spectrometry (ICP-OES). The anion content (chloride, phosphate and citrate) was determined with ion chromatography (IC).

[0109] For ICP-OES, 500 µL of ACM sample or supernatant was weighed, and 7.5 mL HCl, 2.5 mL HNO3and 1 mL H2O2were added for microwave-assisted wet digestion of the samples in an ETHOS EASY microwave digestion system (Milestone Srl, Sorisole, Italy) with operational conditions as specified by Guimarães et al. (2021). Digested samples were diluted ~200x in water. The exact dilution factor was calculated by weighing volumetric flasks before and after sample filling. Samples were prepared in duplicate. Analysis was done in an ICP-Optical Emission Spectrometer (Avio® 500, PerkinElmer, Massachusetts, USA). A six-point calibration range was prepared of 0-10 mg / L for standards of calcium, phosphorus, sodium and potassium and of 0.2-1 mg / L of magnesium in 10% aqua regia.

[0110] For IC, samples were diluted 500x and their supernatants 200x with water to dissociate the micelles. Samples were analyzed using a Dionex ICS-6000 ion chromatography system with conductivity detector and a 2 mm standard bore Dionex IonPac AS17-C column (Thermo Fisher Scientific B.V., Breda, The Netherlands) at 30 °C. The flow rate was set to 0.25 mL / min with an injection volume of 5 μL. Gradient elution was conducted with KOH, which was set to 5 mM for 10 min. Then, it was increased linearly to 40 mM within15 minutes, kept at 40 mM for 5 min and linearly decreased to 5 mM in 5 min.

[0111] Ion concentrations of supernatants were corrected for the excluded volume of the ultracentrifugal pellet. Correction factor K was calculated according to Equation 2, where P represents the sedimentable casein content (g / L) and W the apparent hydration in g water / g dry matter. Sedimentable mineral content was calculated by subtracting the non-sedimentable mineral content from the total mineral content: (2)

[0112] The coagulation firmness after rennet addition was monitored by oscillatory rheometry according to Antuma et al. (2023).3Samples were acidified to a pH of 6.3 below 10°C with a 10x diluted lactic acid solution. This was done the day before the measurement to account for the buffering ability of the solution and the pH was readjusted on the day of the measurement. Then, 20.0 g sample was weighed, to which 0.04% (v / w) CaCl2was added, using a 4% (w / v) CaCl2solution. Samples were heated while stirring and when 30°C was reached, 0.02% (v / w) chymosin was added. The samples were then transferred to a MCR502 rheometer (Anton Paar, Graz, Austria). A double-gap cup was used (DG26.7) to perform the 60-minute-long measurement. The strain amplitude was set to 0.001 and the frequency to 1 Hz. Measurements were done every 15 seconds. The temperature of the Peltier element was set to 30°C. Samples were analyzed in duplicate. Results and Discussion

[0113] The ability of sodium hexametaphosphate (SHMP) to induce micelle formation from sodium caseinate (NC) was explored, using 30 mM calcium (C30) and varying SHMP concentrations. A control of C30 without SHMP was also prepared. A reference sample of ACM was prepared in accordance with the protocol of Schmidt et al. (1977)1, to compare the ACM made by conventional crosslinking with calcium phosphate (CaP) nanoclusters and alternative crosslinking with SHMP. Samples prepared were: ACM control, C30, C30 S1.5, C30 S2.5, C30 S3, C30 S5, C30 S7.5, and C30 S10. The first five samples, from the ACM control to C30 S3 (30 mM calcium and 3 mM SHMP) had an opaque white appearance. Higher concentrations of SHMP, from C30 S5 onwards, appeared more yellow and transparent, similar to the NC solution. Visual observation suggested the presence of large, colloidal particles with the ability to scatter light. The observed white color and opaqueness were reduced in the three highest SHMP concentrations. Additional analyses were performed to clarify the interaction between caseins, calcium and SHMP. In the following section, sample characteristics will first be presented and then discussed altogether.

[0114] The average diameters of formed particles in samples were analyzed (Figure 1). The C30 control had the largest average diameter of 276.2 ± 12.2 nm. At a SHMP concentration below 5 mM, samples had a lower average particle size compared to the ACM control (203.6 ± 1.0 nm). The particle diameter of C30 S1.5 (164.4 ± 12.8 nm) was slightly larger than those of samples prepared with 2.5 to 5 mM SHMP, which had similar sizes. For higher SHMP concentrations (7.5 and 10 mM), larger particles than the ACM control were found of 242.8 ± 2.7 and 251.7 ± 1.0nm, respectively.

[0115] Furthermore, the sedimentable casein as fraction of total casein was determined (Figure 2A), as well as the hydration of ultracentrifuged pellets (Figure 2B). The highest observedsedimentable casein content was 88.2 ± 1.8% for the C30 control, which was closest to the ACM reference (91.6 ± 1.0%). All SHMP samples had less sedimentable casein compared to the C30 control, with an optimum of 85.4 ± 0.9% found for C30 S3. Lower and higher SHMP concentrations progressively reduced sedimentable casein, with the lowest sedimentable casein observed in C30 S10 (6.2 ± 11.5%). All SHMP samples were more hydrated than the C30 control, which had an apparent hydration of 3.4 ± < 0.1 g water per g sedimentable casein, though no clear trend was observed between hydration and SHMP concentration. All samples were more hydrated than the ACM reference (3.3 ± <0.1 g water per g sedimentable casein).

[0116] SHMP has a strong chelating action and was therefore expected to sequester calcium ions in the samples, so the partitioning of calcium between the serum and sedimentable phase was determined. The sedimentable calcium contents expressed as percentage of total calcium are displayed in Figure 3A. A similar trend in sedimentable casein content was found for SHMP samples, with an optimum of 48.2 ± <0.1% sedimentable calcium for C30 S3. C30 without SHMP had a sedimentable calcium content of 35.4 ± 0.2%. All samples had a markedly lower sedimentable casein fraction than the ACM control (68.0 ± 1.9%). When sedimentable calcium was expressed per gram of sedimentable casein (Figure 3B), the lowest value was found for C30 (0.49 ± 0.01mmol). The sedimentable casein content increased with higher SHMP concentrations. Values found for SHMP concentrations of 1.5 to 5 mM were then more similar to the ACM control (0.93 ± 0.02 mmol). For SHMP concentrations of 7.5 and 10 mM, the sedimentable calcium was nearly two times higher than the ACM control.

[0117] From the obtained data, it appears that micelle formation was induced by SHMP and calcium in samples C30 S1.5, -S2.5, -S3 and -S5. SHMP-calcium complexes were presumably bound to the anionic casein phosphate centres, through electrostatic attraction with the cationic calcium. At these SHMP concentrations, formed particles had the ability to scatter white light based on their white opaque appearance, despite the smaller diameters compared to the ACM reference (Figure 1). Furthermore, a considerable fraction of casein and calcium sedimented upon ultracentrifugation (>56.5 ± 2.3% casein; >38.7 ± 8.9% calcium), with the highest levels of both observed for C30 S3 (Figure 2A; Figure 3A). Ultracentrifuged pellets were more hydrated than the ACM control (Figure 2B). Combining this observation with the lower observed sedimentable casein and calcium contents of SHMP samples compared to the ACM control, it can be reasonably postulated that ACM formed with SHMP were likely more porous and that a lower number of ACM were formed than the reference ACM.

[0118] The aforementioned differences in micelle properties are most likely caused by a weaker bond strength of SHMP-calcium complexes to casein phosphate centres than the bond strength between calcium phosphate (CaP) nanoclusters and phosphate centres. In native casein micelles (CM), the surface of the CaP nanoclusters incorporate casein phosphate centres in theirbrushite-type crystal lattice. The SHMP-calcium complexes likely substituted the location of CaP nanoclusters, but the bond is expected to be through electrostatic attraction, which appears to be a much weaker bond than the direct incorporation of casein in CaP nanoclusters. Furthermore, the driving force of casein interaction with CaP nanoclusters is supersaturation of calcium phosphate (Antuma et al., 2024).2The formed SHMP-calcium complexes were assumed to be more water-soluble in the used concentrations than calcium phosphate. Hoppenreijs et al. (2024)4found that a calcium concentration of 150 mM was needed to precipitate 0.016 mM SHMP, so a larger calcium excess was needed to precipitate a lower SHMP concentration. The lack of driving force likely contributed to the lower bond strength of casein crosslinks formed by calcium-SHMP complexes. The weaker bond also likely contributes to the increased hydration and porosity of ACM formed with SHMP and calcium.

[0119] Sample characteristics also implied ACM formation in the C30 sample. ACM prepared with only calcium apparently contained more casein, but less calcium and were less hydrated, compared to ACM prepared with SHMP and calcium. The SHMP-calcium complexes presumably had a higher hydration ability than calcium on its own. Furthermore, without SHMP present, casein crosslinks likely consisted of only one divalent calcium ion as bridge between caseins, whereas an SHMP-casein crosslink likely consisted of at least two calcium ions, which could explain the lower sedimentable calcium content in the C30 sample. The lack of anionic SHMP could also have allowed more hydrophobic casein interactions and closer casein packing, as there was less electrostatic repulsion within the micelle. This could explain the larger particle size and lower hydration observed for the C30 control, compared to samples containing SHMP.

[0120] CM formation was unlikely to be observed in the C30 S7.5 and C30 S10 samples, considering the yellow, transparent appearance and low sedimentable casein contents (Figure 2A). The particle sizes of these two samples (Figure 1) were similar to values found for untreated sodium caseinate (NC) solution (~250 nm). If no CM were formed, the presence of loose self- associated caseins seems reasonable to assume. The fraction of sedimentable calcium was lowest for C30 S7.5 and C30 S10, compared to the other SHMP samples and the ACM control (Figure 3A). However, when expressed per g sedimentable casein, sedimentable calcium for both samples was around two times higher than the other samples (Figure 3B). SHMP consists of six negatively-charged phosphate groups and could therefore hypothetically bind six calcium ions, which in turn could bind six caseins. Previous studies found a SHMP-calcium binding ratio of 1:3, suggesting that one calcium ion bound two phosphates of SHMP (De Kort, 2012).5If complete calcium-sequestering by SHMP is assumed in a ratio of 1:3 (corresponding with C30 S10), no further binding can occur between calcium and casein. SHMP was assumed to be a hexaphosphate, though commercial SHMP may contain polyphosphates of varying lengths, so the exact binding ratio to calcium is difficult to predict. The inhibited casein crosslinking most likelyalready occurred from a SHMP concentration of 7.5 mM. Structures in the ultracentrifuged pellet are expected to be mainly large complexes of SHMP and calcium, with some caseins attached.

[0121] Altering the ratio of SHMP and calcium could improve micelle formation and properties, as indicated by visual appearance and an increase in sedimentable casein and calcium content (Figure 2A; Figure 3A). A calcium excess is likely needed to allow simultaneous calcium binding to SHMP and casein and excess calcium probably minimizes electrostatic repulsion between the two anionic compounds. Calcium was likely fully chelated at high concentrations of SHMP (> 5 mM), resulting in no detectable casein crosslinking and subsequent micelle formation. On the other hand, a higher SHMP concentration can form a larger number of SHMP-calcium complexes, which in turn determines how many casein crosslinks can exist, so a certain minimum concentration was required. This became apparent from the improved micelle properties at an SHMP concentration of 3 mM, compared to lower SHMP concentrations (1.5 and 2.5 mM SHMP). Therefore, the balance between SHMP and calcium appears important and can likely be employed to steer micelle formation and properties. The most favourable SHMP and calcium ratio observed in this initial study was found to be 1:10.

[0122] Sample characteristics suggested ACM formation in several SHMP samples. A key property of natural and artificial CM is their coagulation upon rennet addition, which can therefore be used as a method to verify the presence and functionality of ACM. Figure 4 shows the coagulation behaviour of several SHMP samples. The ACM control had the highest coagulation firmness, with a maximum G’ of 100.7 ± 1.6 Pa. The C30 control had a maximum G’ of 32.6 ± 0.3 Pa. The SHMP samples all showed lower coagulation firmness compared to the ACM control. The highest gel strength was observed for C30 S3, with a G’ of 51.4 ± 0.9 Pa. In the C30 S7.5 sample and samples with higher SHMP concentrations, no coagulation was observed upon rennet addition.

[0123] The samples in which SHMP addition yielded micelle-like particles (C30 control, S1.5, -S2.5, -S3 and -S5) all showed rennet-induced coagulation. Coagulation indicated destabilization of the κ-casein surface layer and subsequent flocculation of particles, which substantiated the notion that ACM were formed. The ratio between SHMP and calcium affected the coagulation behaviour, with the highest G’ found for C30 S3. A higher sedimentable casein content appeared to result in a stronger gel (Figure 2A). This reaffirmed the suggestion that ratio of SHMP and calcium of 1:10 appears to be most favourable for casein crosslinking and subsequent ACM formation, based in this initial study. The two samples with the highest SHMP concentrations, where SHMP likely sequestered most or all the calcium in solution, did not coagulate upon renneting, reaffirming the suggestion that micelles were likely not formed.

[0124] The reduced coagulation firmness of ACM formed with SHMP and calcium compared to the ACM control can be related back to their relative bond strength to casein phosphate centres. CM are the building blocks of the gel network and their internal integrity affected rennet-induced coagulation. Casein crosslinks formed by calcium-SHMP complexes could therefore be concluded to be weaker than crosslinks formed by CaP nanoclusters in ACM. This likely caused the lower coagulation firmness, which became apparent by the twofold decrease of G’ when comparing the control ACM to the C30 S3 sample. The C30 control had an even lower coagulation firmness than C30 S3. Calcium on its own was more loosely bound to casein compared to CaP nanoclusters. The calcium-casein bond was seemingly also weaker than the bond of SHMP-calcium complexes to casein. An additional, but less pronounced reason for the limited coagulation firmness of SHMP samples could be the lower sedimentable casein content, compared to the control ACM. A higher serum casein content could have obstructed coagulation by physical or steric hindrance between CM, which is similar to the adverse effect on coagulation that whey protein has, in the serum phase of milk.

[0125] A limitation in the interpretation of the results is the decreasing G’ in samples during incubation with rennet (after about 10 min for C30 S1.5 and 20 minutes for C30 S3 and C30). This unexpected behaviour was likely caused by syneresis, since a visible water layer was formed in the measuring cup. This syneresis was not observed for the ACM control. The lower G’ could be explained by two different factors, the first one being that the rheometer possibly partially measured water, which underestimated the G’ of only the gel. It was not possible to observe the gel inside the measuring cup. However, if the gel fractured or came loose from the wall, the system could have measured the slip, and in turn underestimated the coagulation firmness.

[0126] Secondly, syneresis is related to CM rearrangement into a more compact network during gel formation, with rearrangement occurring more readily when bonds are more mobile or weaker. Micelles fuse during rearrangement, creating pores in the gel and resulting in syneresis. SHMP-calcium complexes were concluded to form weaker crosslinks between caseins compared to CaP nanoclusters and therefore, rearrangement and subsequent syneresis were likely more prevalent. In addition, SHMP ACM were found to be more hydrated and more porous than the control ACM. Rearrangement probably resulted in denser aggregates and liquid was expelled towards larger pores. Syneresis is an innate property of CM during the aging stage of cheese production. Aging normally takes place over a longer time and different conditions (e.g., the cooking step in cheese-making) than in the oscillatory rheometry test, which could explain why it was not observed for ACM prepared with CaP nanoclusters. Because of the weaker casein crosslinks in SHMP ACM, the caseins rearranged and coalesced more easily, so syneresis occurred within the experimental timeframe.

[0127] A few limitations of this study were found in the determination of CM formation. Firstly, the sedimentable fraction is commonly regarded as, and referred to as micellar, because CM sediment upon ultracentrifugation. However, the term is mostly used when CM presence is known. For the SHMP samples with expected micelle formation, it cannot be concluded with full certainty that all sedimentable casein or calcium were incorporated into micelles. Since the exact structures are not known, a part of the sedimentable fraction could consist of large complexes of SHMP and calcium, with some caseins attached, which are non-micellar and sedimentable. Small angle X-ray scattering or scanning electron microscopy may be employed in further research to analyze the nanostructure and morphology of formed particles. Furthermore, no thresholds exist (yet) for minimum sedimentable casein and calcium contents and rennet-induced coagulation before a sample can be classified as micellar, which made it difficult to concretely define samples as ACM or not. Most likely, a mixture of ACM and non-micellar aggregates existed in solution. Another limitation was that SHMP was not measured as phosphate by ion chromatography, because the polyphosphates were likely too large for detection. Therefore, limited conclusions, and predominantly only assumptions could be made on the fraction of SHMP that sedimented with the casein and calcium.

[0128] The control sample prepared with only calcium and no added SHMP showed the presence of micelle-like particles which could be coagulated by rennet. This raises the question whether ACM formed with calcium-SHMP complexes were solely formed due to an excess of calcium ions, rather than the actual formation of calcium-SHMP complexes. In other words, did the added SHMP contribute to micelle formation or did it only have a calcium-sequestering action? To answer this question, two additional controls were prepared with calcium and without SHMP. The SHMP sample with the best coagulation behaviour (C30 S3) was chosen as reference. Calcium concentrations of samples without SHMP were selected based on the expected binding ratio of SHMP and calcium, which were 1:3 or 1:6. Taking S3, the expected calcium excess would be C21 and C12, respectively. Samples with these calcium concentrations and no added SHMP were prepared, both appeared white, with C12 less opaque. Samples were then coagulated with rennet, the results of which are presented in Figure 5. C12 and C21 had a maximum G’ of 0.2 ± <0.1 and 23.4 ± 0.4Pa, respectively.

[0129] Results show that a higher calcium concentration resulted in a higher coagulation firmness. Calcium addition presumably reduced electrostatic repulsion between caseins, allowing more hydrophobic interaction with the calcium ion itself acting as a crosslink between caseins. C30 S3 had the highest coagulation firmness, so SHMP appeared to contribute to and enhance micelle formation. SHMP-calcium complexes most likely formed casein crosslinks that were stronger than crosslinks formed by calcium alone. Secondly, SHMP addition could have delayed syneresis, so the gel had more time to form and strengthen. This was difficult to determine, asthe measured G’ was not fully reliable because of the measured syneresis. Nevertheless, it was clearly demonstrated that SHMP addition improved coagulation firmness, compared to calcium on its own.

[0130] After concluding that CM could be formed by controlled mixing of NC, calcium and SHMP, it was tested whether the same would apply to dephosphorylated sodium caseinate (DP). Samples were prepared with dephosphorylated sodium caseinate, 30 mM calcium and varying SHMP concentrations. DP C30 S3, -S5 and -S7.5 samples had a white opaque appearance. Both the DP control and DP C30 S2.5 samples showed phase separation and were therefore excluded from further analysis. The other three samples were further analyzed to determine if micelle-like structures had formed.

[0131] Particle size, sedimentable casein and calcium content as fraction of their respective total contents and apparent hydration of the DP samples are presented in Table 1: Table 1: Particle size, sedimentable casein and calcium content and hydration of samples prepared from dephosphorylated casein. Average ± standard deviation are based on duplicates.

[0132] The hydrodynamic diameters of DP samples were larger than control ACM (203.6 ±1.0 nm), with a two-fold increase in diameter observed for DP C30 S3. Sedimentable casein content was highest for DP C30 S3 and decreased with higher SHMP concentrations. The sedimentable calcium content increased with a higher SHMP concentration. Samples were less hydrated compared to control and SHMP ACM (Figure 2B), which could be caused by more hydrophobic casein interactions as the charged phosphate centers were removed. Similar as for the C30 control, increased hydrophobic attractions probably resulted in closer casein packing that excluded water.

[0133] The white opaque appearance and measured particle size indicated the presence of large colloidal particles (Table 1). Roughly half of total casein of DP samples sedimented upon ultracentrifugation, which suggested some binding of casein to SHMP-calcium complexes, though the other half remained soluble. Casein dephosphorylation prevented interaction of SHMP- calcium complexes to casein phosphate centres, which was the expected binding site in native casein. Electrostatic interactions between casein and SHMP-calcium complexes was thereforeonly possible at anionic amino acid residues of the casein or at leftover phosphoserine residues, which appeared to have occurred based on the sedimentable casein content. Multiple studies have previously reported calcium binding to dephosphorylated casein, although less calcium could be bound without phosphate centres.

[0134] The larger particle size and lower sedimentable casein content in DP samples indicated the formation of loose particles that were less hydrated, compared to the control ACM and SHMP samples with expected micelle formation (Figure 1; Figure 2A). Furthermore, a higher SHMP concentration resulted in reduced casein, but increased calcium sedimentation for DP samples. This contrasted NC samples, where both sedimentable casein and calcium decreased at higher SHMP concentrations (Figure 2A; Figure 3A). Moreover, it appeared that sedimentable casein content of DP samples was less affected by SHMP concentration; adjusting the SHMP concentration from S3 to S7.5 reduced the sedimentable casein of DP samples from 54.1 ± 1.4% to 37.9 ± 3.6%, whereas in NC samples sedimentable casein decreased from 85.40 ± 0.9% to 17.0 ± 4.6%. Without phosphate centres, increased hydrophobic casein interactions could have resulted in a higher sedimentable casein content. Additionally, calcium is known to precipitate dephosphorylated casein. At lower SHMP concentrations, more free calcium would be available to precipitate casein.

[0135] When SHMP concentration was increased, calcium was likely chelated by SHMP, resulting in less casein precipitation by calcium. The formed structures probably consisted mainly of larger SHMP-casein complexes with some casein dispersed throughout. Similar structures were assumed to have formed at high SHMP concentrations in NC samples. Antuma et al. (2023)3also found larger CaP nanoclusters and lower sedimentable casein content for ACM formed from dephosphorylated casein.

[0136] Reduced casein binding likely allowed for more growth of CaP nanoclusters and SHMP-calcium complexes. This could explain the increased calcium content and decreased casein content at higher SHMP concentrations. Based on the results of Table 1, it cannot be concluded whether these interactions yielded micelle-like structures or non-micellar aggregates. Further analysis of coagulation behaviour upon renneting was needed to determine the presence of a κ-casein surface layer, which is linked to CM formation.

[0137] All samples showed limited rennet-induced coagulation (Figure 6). Higher SHMP concentrations improved coagulation behaviour slightly, with the highest G’ of 5.4 ± 1.4 Pa found for DP C30 S5. An increased lag phase was observed for DP C30 S7.5, which had a maximum G’ of 3.1 ± 0.5 Pa. The DP control prepared with CaP nanoclusters was included for comparison and had the lowest G’ of 1.6± 0.1Pa.

[0138] The rennet-induced coagulation firmness of all DP samples decreased significantly with the largest decrease found for the conventional ACM preparation method: the G’ of DP control was 1.5 Pa, compared to 100.7 ± 1.6 Pa for the NC ACM control (Figure 4). Even though sedimentable casein content was roughly 50% for both NC C30 S5 and DP C30 S5, the maximum G’ decreased from 26.4 ± 2.8 Pa to 5.4 ± 1.4 Pa when using dephosphorylated casein (Figure 6). Binding with charged casein amino acid residues seemed unable to yield micelle-like structures with a κ-casein surface layer. Some CM-like structures were likely formed in the DP samples, as indicated by the limited rennet-induced coagulation, but the majority of casein was probably present as free casein or incorporated in large SHMP-calcium complexes.

[0139] It could therefore be concluded that SHMP-calcium complexes predominantly interacted with casein phosphate centers in the micelles prepared from native sodium caseinate, and not with anionic amino acid residues. The impaired coagulation when using dephosphorylated casein showed that casein phosphorylation is favourable for micelle formation, both for casein crosslinking with SHMP-calcium complexes and with CaP nanoclusters.

[0140] Additional micelles were prepared from purified β-and κ-casein powders. Preparing ACM from two casein families, instead of four, is beneficial for recombinant casein from a cost and production efficiency perspective. Two samples were prepared with a calcium concentration of 30 mM and an SHMP concentration of 3 or 7.5 mM. A control sample was prepared by the conventional method with CaP nanoclusters and used as reference. The β / κ control and β / κ C30 S3 appeared white and relatively opaque, but more transparent compared to NC samples. β / κ C30 S7.5 appeared slightly pink and resembled the β / κ-caseinate solution.

[0141] The hydrodynamic diameter, sedimentable casein and calcium contents and hydration of samples prepared from β-and κ-casein are presented in Table 2: Table 2: Particle size, sedimentable casein and calcium contents and hydration of samples

[0142] In previously-conducted preliminary research, β / κ ACM prepared by the conventional preparation method with CaP nanoclusters with a similar casein composition were found to be smaller and more hydrated than ACM from whole sodium caseinate (Antuma,unpublished). This was also observed in this study, with an average diameter of 143.5 ± 1.0 nm and hydration of 5.5 ± <0.1 g water per g sedimentable casein. The smaller micelles were a result of the relatively high amount of κ-casein in solution that limited further micelle growth (30% of total casein in β / κ samples, compared to 20% in whole NC). Sedimentable casein content of the β / κ control was 75.2 ± 1.8%, so less casein was present in the micellar phase compared to ACM from NC (91.6 ± 1.0%). The increased hydration indicated that more porous ACM were formed from β- and κ-casein than from sodium caseinate. This was also reported by Schmidt et al. (1974):6electron-micrographs showed that ACM from β-and κ-casein were fewer in number, looser and more irregularly shaped, compared to ACM formed of all four casein types.

[0143] All β / κ samples had lower sedimentable casein content compared to their native NC equivalents (Figure 2A). Casein-casein interactions could occur through hydrophobic interaction or through casein crosslinking by CaP nanoclusters or SHMP-calcium complexes. β- and κ-casein possess one and zero phosphate centers, respectively, and were therefore unable to crosslink multiple CaP nanoclusters or SHMP-calcium complexes. This missing crosslinking ability presumably resulted in the more porous structure of ACM formed from β- and κ-casein. It appeared that without the presence of αs-caseins, the overall binding of caseins to SHMP-calcium complexes was reduced. The multiple phosphate centres of αs-caseins probably increased the chance and the affinity of binding to SHMP-calcium complexes.

[0144] When comparing the β / κ control with samples crosslinked by SHMP, a similar white appearance and similar average diameter were found for β / κ C30 S3 (145.7 ± 0.8 nm), although the sedimentable casein content was nearly four times lower (18.1 ± 3.6%). Sedimentable calcium content also decreased more than thrice, from 62.1 ± 0.2% for the β / κ control to 16.3 ± 1.4% for β / κ C30 S3. The majority of casein and calcium was thus soluble, but a low number of micelles could have formed. Particles in β / κ C30 S3 were assumed to be more porous, but less hydrated than ACM formed in the β / κ control.

[0145] The β / κ C30 S7.5 appeared pinker than the other two β / κ samples. The particle size of β / κ C30 S7.5 of 251.6 ± 0.5 nm was in the same range as NC samples without micelle formation (Figure 1). This indicated the formation of large, loose casein aggregates, rather than micelles in β / κ C30 S7.5. Furthermore, sedimentable casein content decreased to 2.9 ± 6.3% for β / κ C30 S7.5, whereas sedimentable calcium content slightly increased to 20.6 ± 1.5%, compared to β / κ C30 S3. The sedimentable fraction likely comprised of predominantly SHMP-calcium complexes with some caseins attached, similar to NC samples prepared with a high SHMP concentration.

[0146] β / κ samples were coagulated by renneting to further clarify if CM formation had occurred. The highest G’ of 43.9 ± 5.3 Pa was observed for the β / κ control, which had expectedCM formation. For β / κ C30 S3 a G’ of 17.7 ± 0.3 Pa was found, indicating micelle formation to a lesser extent than the β / κ control. No coagulation occurred for β / κ C30 S7.5, confirming the assumption that no CM were formed (Figure 7).

[0147] The maximum G’ of the β / κ control (43.9 ± 5.3 Pa) had a more than two-fold decrease compared to the NC ACM control (100.7 ± 1.6 Pa). αs-Caseins were reported to be the backbone of casein micelles, by their ability to crosslink and be crosslinked by CaP nanoclusters and engage in hydrophobic casein-casein interactions, simultaneously (Sharma et al., 2001).7β- and κ-casein could not crosslink multiple CaP nanoclusters, due to their single or lacking phosphate centre, resulting in weaker internal bonds. Furthermore, β / κ ACM appeared to be more hydrated and less dense and the increased serum casein content could have obstructed gel formation. These factors probably all contributed to the lower G’. In this regard, it is expected that micelles from αs-casein and κ-casein would improve micelle formation and coagulation firmness, which could be tested in further studies.

[0148] Crosslinking by SHMP further reduced rennet-induced coagulation, which was likely caused by the four fold decrease in sedimentable casein content when comparing the β / κ control and β / κ C30 S3. This indicated that less micelles were formed, so the resulting gel in β / κ C30 S3 was likely comprised of less building blocks. Furthermore, weaker bonds were concluded to be formed by SHMP-calcium complexes compared to CaP nanoclusters, which also contributed to the lower G’.

[0149] The presence of the single phosphate center of β-casein already improved micelle formation, even without the ability of casein to crosslink CaP nanoclusters or SHMP-calcium complexes. Micelle formation induced by SHMP and calcium was better achievable from β- and κ-casein than from dephosphorylated casein. In DP samples, the complete lack of phosphate centres resulted in an even more impaired micelle formation and renneting functionality. The best samples of both are compared to illustrate, which are DP C30 S5 and β / κC30 S3. Despite the lower sedimentable casein content of β / κ C30 S3 (18.1 ± 3.6% vs.52.3 ± 0.7% for DP C30 S5), the observed coagulation firmness (17.7 ± 0.3 Pa) was more than three times higher than that of DP C30 S5 (4.9 ± 0.7 Pa). Once again, the favourability of casein phosphate centres for CM formation, integrity and functionality was observed. Conclusions

[0150] Inducing micelle formation with SHMP was first attempted with native sodium caseinate. It was found that ACM could be formed by addition of both SHMP and calcium, where the cationic calcium likely acted as a bridge between the anionic casein and SHMP. An excess of calcium was needed, with the best results observed for the ratio of C30 S3. Too much SHMPsequestered all calcium and additional binding of calcium to caseins was then prevented. Calcium on its own could form micelles, but the combination of SHMP and calcium resulted in increased CM hydration and a higher coagulation firmness. SHMP-calcium complexes were believed to substitute the CaP nanoclusters within the ACM, although intramicellar bonds formed by SHMP- calcium crosslinking were much weaker than crosslinking by CaP nanoclusters. Subsequent experiments with dephosphorylated caseinate confirmed that SHMP-calcium complexes primarily bind to casein phosphate centres, since rennet-induced coagulation was mostly impaired. This was reaffirmed when attempting to form CM from purified β- and κ-casein fractions.

[0151] Inducing micelle formation from sodium caseinate was possible with SHMP and calcium. SHMP-calcium complexes were able to crosslink caseins predominantly through casein phosphate centres and acted as a substitute of CaP nanoclusters. Compared to ACM produced only with calcium and no added SHMP, ACM with SHMP-calcium complexes showed comparable levels of sedimentable casein, yet increased hydration and improved coagulation behaviour. Altering the ratio between SHMP and calcium appeared to improve or deteriorate ACM formation, with visual appearance, sedimentable casein content and rennet-induced coagulation as key indicators. However, crosslinks in ACM with SHMP-calcium complexes were weaker compared to the strong crosslinks between caseins and CaP nanoclusters. As a result, sedimentable casein levels and coagulation behaviour similar to ACM formed with CaP nanoclusters could not yet be achieved by SHMP and calcium. Nonetheless, the results demonstrate that SHMP does contribute favourably to the formation of functional ACMs.

[0152] Alternative casein crosslinking was achieved, and intramicellar SHMP-calcium crosslinks mainly interacted with casein phosphate centres. Casein phosphate centres appeared to be a favourable feature in the well-established balance of interactions within the casein micelle, and could therefore not be easily replaced. Further research on controlling crosslinking, electrostatic and hydrophobic casein interactions is required to obtain a similar balance of interactions that will result in comparable gelation properties to native casein micelles, both for inducing micelle formation and strengthening micelles, allowing the use of recombinant casein for the production of future animal-free cheese without compromises on nutritional quality and product functionality. Example 2 – Extending the protocol to recombinant caseins

[0153] To investigate extending the above described protocol to the synthesis of SHMP ACMs from recombinantly produced casein, ACMs were prepared according to the method outlined in Figure 8. Four different concentrations of CaCl2and SHMP were tested (Table 3; Samples S1.1 – S1.4), with the goal of producing turbid ACM solutions containing particles withhydrodynamic diameters in the range of 100 - 300 nm, and capable of forming visible precipitates upon ultracentrifugation. Table 3: ACM sample overview: CaCl2and SHMP concentrations relate to the final concentration achieved at the end of the ACM formation process. Sample No 1.1 1.2 1.3 1.4 1.5.1 - 1.5.3 50 25CM formation was repeated in triplicate (Samples S1.5.1 – S1.5.3) to assess repeatability. The hydrodynamic diameter and polydispersity index (PDI) of the micelles were determined using dynamic light scattering (DLS). Sedimentable casein and calcium content were calculated based on the difference in total nitrogen and calcium concentrations in the ACM solution before and after centrifugation. Materials

[0155] Sodium hexametaphosphate (SHMP), Sigma-Aldrich

[71600] ; Calcium Chloride (CaCl2), Supelco [1.02378]; Recombinant ɑs1-CN (bovine), Tiaris 1 FO2, middle fraction, ground (purity HPLC >98%, DUMAS 92,53%); Tri-Sodium Citrate Dihydrate, VWR

[0101] ; 0.1 M HCl (diluted 37% HCl, VWR

[20252] ); 1.0 M NaOH, Carl Roth [K021]; Sodium azide (NaN3), Carl Roth

[4221] . Consumables

[0156] 50 mL Syringes with Luer-Lock Tip, Becton Dickinson [309653]; 200 µL, 1000 µL pipette tips. Equipment

[0157] 150 mL, 250 mL glass beakers; 50 mL, 250 mL Duran bottles with lids; 40 mL Ultra centrifuge tubes (Beckman Coulter); Silicone tubes (PharMed BPT, ID 0,09, WALL 0,033) with connectors FILL TO 1 / 8 BARB CONN KYNAR PK, Harvard Apparatus [72-1458]); Magnetic stirring bar (x-shape);200 µL, 1000 µL pipettes (Transferpette S, BRAND); Heating plate (C-MAG HS 7, IKA) with temperature probe (ETS-D5, IKA); pH-Meter (Orion Dual Star, Thermo Scientific); Syringe Press (PHD Ultra, Harvard Apparatus); Zetasizer Pro, Malvern; Dumatherm, Gerhardt; Centrifuge (Avanti J-E Centrifuge, Beckman Coulter) with rotor (JA-25.50, Beckman Coulter). Preparation of Calcium Chloride (CaCl2) Stock Solution

[0158] Calculation Concentration: 450 mM; Volume: 200 mL; pH: 6.7; Molecular weight of CaCl₂ (anhydrous) = 110.99 g / mol; 0.450 mol / L * 0.2 L = 0.090 mol; 0.090 mol * 110.99 g / mol = 9.989 g.

[0159] To prepare the stock solution of calcium chloride, 9.99 ±0.01 g CaCl₂ was weighed into a 250 ml beaker. Approximately 160 mL deionised water was added, and the CaCl₂ was dissolved therein, using a magnetic stirrer. The resultant solution was added to a 200 mL volumetric flask, and filled up to the 200 mL mark with deionised water. The solution from the volumetric flask was then transferred back into a 250 mL beaker, and adjusted to pH 6.70 using 0.1 M HCl. The final pH adjusted solution was stored in a 250 mL Duran flask until use. Preparation of Sodium Hexametaphosphate (SHMP) Stock Solution

[0160] Calculation Concentration: 45 mM; Volume: 200 mL; pH: 6.7; Assumed molecular weight of SHMP = 611.77 g / mol; 0.045 mol / L * 0.2 L = 0.009 mol; 0.009 mol * 611.77 g / mol = 5.506 g.

[0161] To prepare the stock solution of sodium hexametaphosphate, 5.50 ±0.01 g SHMP was weighed into a 250 ml beaker. Approximately 160 mL deionised water was added, and the SHMP was dissolved therein, using a magnetic stirrer. The resultant solution was added to a 200 mL volumetric flask, and filled up to the 200 mL mark with deionised water. The solution from thevolumetric flask was then transferred back into a 250 mL beaker, and adjusted to pH 6.70 using 0.1 M NaOH. The final pH adjusted solution was stored in a 250 mL Duran flask until use. Preparation of Sodium Azide Stock Solution

[0162] Calculation Concentration: 20 g / L; Volume: 50 mL; Purity ≥ 98 %; 20 g / L * 1.00 * 0.05 L = 1.000 g.

[0163] To prepare the stock solution of sodium azide, 1.00 ±0.01 g sodium azide was weighed into a 50 mL Duran flask.50 mL deionised water was added, and the sodium azide was dissolved therein, using a magnetic stirrer. The resultant solution was stored in the capped Duran flask, under refrigeration, until use. Preparation of Recombinant ɑs1-Casein (rCN) Stock Solution

[0164] Calculation Protein Concentration: 96 g / L; Volume: 180 mL; pH: 6.7; Purity = 92.58 %; 96 g / L * 0.9258 * 0.18 L = 15.998 g.

[0165] Calculation Citrate Concentration: 33.75 mM; Volume: 90 mL; pH: 6.7; Molecular weight of Na₃C₆H₅O₇· 2H₂O = 294.10 g / mol; 0.03375 mol / L * 0.09 L = 0.003 mol; 0.003 mol * 294.10 g / mol = 0.893 g.

[0166] To prepare the stock solution of recombinant ɑs1-Casein (rCN), 7.99 ±0.01 g rCN was weighed into a 150 mL beaker, 80 g of deionised water was added, and the mixture was stirred with a magnetic stirrer. To this mixture was then added 0.89 ±0.01 g citrate, with continued stirring and adjustment of the pH to 6.7 with additions of small amounts of 1 M aqueous NaOH. At 1 hour, and at 2 hours, after the citrate addition, the mixture was subjected to homogenisation with an Ultra TurraxTMhomogenizer, operated for 1 minute, at 10,000 RPM. Finally, 900 µLsodium azide solution (2 % w / v) was added, and the mixture was stored under refrigeration overnight. Preparation of Artificial Casein Micelles (ACMs) Comprising Recombinant ɑs1-Casein (rCN)

[0167] To prepare the ACMs comprising rCN, the following protocol, in accordance with the Schematic of Figure 8, was followed: 1) To a 250 mL beaker, add 75 mL deionised water, a stirring bar (x-shape) and heat up to 37 °C using a water bath; a) Heating plate: 60 °C; b) Stirring speed: 2.0; 2) Dilute CaCl2 and SHMP stock solutions according to Table 4; 3) Fill the plastic syringes with 20 mL (S1.1 - 1.4) or 40 mL (S1.5.1 - 1.5.3) of the corresponding liquid (4x rCN, 1x SHMP, 1x CaCl2) using a short plastic pipe. Hold the syringe upright to let any air bubbles out. Mount the syringes in syringe pump mounts, and manually empty 5 mL to ensure there is no air left in the pipes; 4) Inject liquids at 20 mL / h for 30 min, closely monitoring the pH and maintaining it at 6.7 by adding 1 M NaOH (adding 20 µL whenever the pH reaches 6.6); 5) After 30 minutes, let the pH stabilise for 20 minutes; 6) Add 13.9 mL deionised water (minus the volume of NaOH added); 7) Add 1.1 mL sodium azide solution to reach a final volume of 150 mL and store refrigerated until further analysis. Table 4: Dilution of CaCl2 or SHMP stock solutions Sample No 1.1 1.2 1.3 1.4 1.5.1 - 1.5.3 4 L L LCharacterization of Artificial Casein Micelles (ACMs) Comprising Recombinant ɑs1-Casein (rCN)

[0168] Hydrodynamic Diameter (HDM) and Polydispersity Index (PDI)

[0169] The hydrodynamic diameter and polydispersity index were measured using the Zetasizer. Samples were measured right after ACM formation, after 48 hrs of storage in the fridge and after ultracentrifugation (53,300 x g for 2 hrs).1 mL of undiluted supernatant was used from each sample for analysis.

[0170] Sedimentable Casein Content

[0171] The percentage of sedimentable casein was calculated based on the difference in nitrogen concentrations in the ACM solution before and after ultracentrifugation (53,300 x g for 2 hrs). Samples were analysed in duplicate using Dumatherm.

[0172] Sedimentable Calcium Content

[0173] The percentage of sedimentable calcium was calculated based on the difference in calcium concentrations in the ACM solution before and after ultracentrifugation (53,300 x g for 2 hrs). Samples were sent to SGS for external calcium analysis using ICP-AES according to DIN EN 15621:2017-10.

[0174] Results & Discussion Visual Observations

[0175] Increasing turbidity was observed for ACM solutions (Figure 9) with increasing CaCl2 / SHMP concentration (S1.2 to S1.4). For the ACM solution with the highest concentration of CaCl / SHMP (S1.1), strong precipitation and no turbidity were observed.

[0176] S1.5.1 - 1.5.3 showed similar turbidity as S1.4 before centrifugation. After centrifugation, a pellet was formed, and the solution cleared. Sedimentable Casein and Calcium

[0177] In sample S1.5, 1.9 wt% (±2.7) of casein was sedimented during the ultracentrifugation (Table 5). However, due to the generally low nitrogen concentrations in all ACM suspensions (<0.35 wt%) and the average analytical error of the sample set (±0.002 wt%), no reliable quantification was possible for samples S1.1–S1.4. For these samples, no biologicalreplicates were included, and the measured differences between centrifuged and uncentrifuged samples fell within the range of expected method variability. Table 5: Mean values and standard deviations for sedimentable casein and calcium. For samples S1.1–S1.4, values are based on technical duplicates. For sample S1.5, values represent biological triplicates, each measured in technical duplicates. Sample No Mean SD Sedimentable Sedimentable Hydrodynamic Dia

[0178] The hydrodynamic diameter (Z-average) of all samples (S1.1–S1.5) was measured before and after centrifugation. Among these, only samples S1.4 and S1.5 exhibited Z-average values exceeding 100 nm prior to centrifugation, suggesting the presence of larger particles, consistent with micelles. Freshly prepared samples showed a mean Z-average of 263.4 nm, which decreased by approximately 3% after 48 hours of refrigerated storage (Table 6). This reduction was accompanied by a decrease in the mean polydispersity index (PDI) from 0.273 to 0.238, indicating a narrower particle size distribution. This observation may suggest increased stabilisation and maturation of the ACM structures during cold storage. Table 6: Mean values and standard deviations for hydrodynamic diameter (z-average) and polydispersity index (PDI) for sample 1.5 before and after refrigeration for 48 hrs. Values represent biological triplicates, each measured in technical triplicates. Sample Description Mean SD Mean SDConclusions

[0179] This example demonstrates that ACMs can be successfully formed from rCN using CaCl₂ and SHMP. The most favourable results were achieved in the present study at a CaCl₂ to SHMP molar ratio of 10:1. The resulting ACMs exhibited a stable hydrodynamic diameter of 255.6 ±8.0 nm at room temperature, and were sedimentable by centrifugation at 15,300 × g for 2 hours. These findings show that the present protocols for preparing ACMs are directly applicable to producing fully functional ACMs from recombinantly produced caseins. General

[0180] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compositions referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0181] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0182] Any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0183] It should be appreciated that throughout this specification, any reference to any prior publication, including prior patent publications and non-patent publications, is not an acknowledgment or admission that any of the material contained within the prior publication referred to was part of the common general knowledge as at the priority date of the application.

[0184] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0185] The invention described herein may include one or more range(s) of values. A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in thespecification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Hence “about 80 %” means “about 80 %” and also “80 %”. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0186] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0187] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.REFERENCES 1. Schmidt, D. G., Koops, J., & Westerbeek, D. (1977). Properties of artificial casein micelles.1. Preparation, size distribution and composition. Netherlands Milk and Dairy Journal, 31(4), 328–341 2. Antuma, L. J., Stadler, M., Garamus, V. M., Boom, R. M., & Keppler, J. K. (2024). Casein micelle formation as a calcium phosphate phase separation process: Preparation of artificial casein micelles through vacuum evaporation and membrane processes. Innovative Food Science & Emerging Technologies, 92, 103582. https: / / doi.org / 10.1016 / j.ifset.2024.103582 3. Antuma, L. J., Steiner, I., Garamus, V. M., Boom, R. M., & Keppler, J. K. (2023). Engineering artificial casein micelles for future food: Is casein phosphorylation necessary? Food Research International, 173, 113315. https: / / doi.org / 10.1016 / j.foodres.2023.113315 4. Hoppenreijs, L. J. G., Annibal, A., Vreeke, G. J. C., Boom, R. M., & Keppler, J. K. (2024). Food proteins from yeast-based precision fermentation: Simple purification of recombinant β-lactoglobulin using polyphosphate. Food Research International, 176. https: / / doi.org / 10.1016 / j.foodres.2023.113801 5. De Kort, E. J. P. (2012). Influence of calcium chelators on concentrated micellar casein solutions: From micellar structure to viscosity and heat stability [PhD Thesis]. Wageningen University. 6. Schmidt, D. G., van der Spek, C. A., Buchheim, W., & Hinz, A. (1974). On the formation of artificial casein micelles. Neth. Milk Dairy Journal, 31, 342–351. 7. Sharma, R., Lorenzen, P. C., & Qvist, K. B. (2001). Influence of transglutaminase treatment of skim milk on the formation of e-(g-glutamyl)lysine and the susceptibility of individual proteins towards crosslinking. In International Dairy Journal (Vol.11).

Claims

CLAIMS 1. An artificial casein micelle, wherein the artificial casein micelle comprises one or more caseins, and a polyphosphate, optionally wherein the artificial casein micelle further comprises a divalent cation species.

2. The artificial casein micelle of any preceding claim, wherein the one or more caseins are selected from the group consisting of αs-caseins, β-caseins and κ-caseins.

3. The artificial casein micelle of any preceding claim, wherein the one or more caseins are non-animal derived caseins, or synthetic caseins, or recombinantly produced caseins.

4. The artificial casein micelle of any preceding claim, wherein the polyphosphate is hexametaphosphate, or a salt thereof.

5. The artificial casein micelle of any preceding claim, wherein the divalent cation species is present, and is selected from the group consisting of; Ca2+, Mg2+, Fe2+and Zn2+.

6. A micellar solution comprising a plurality of the artificial casein micelles of any preceding claim.

7. A curds composition comprising the micellar solution of claim 6, in coagulated form.

8. The curds composition of claim 7, further comprising a renneting agent.

9. An edible composition comprising the curds composition of claim 7 or claim 8, the micellar solution of claim 6, or the artificial casein micelle of any one of claims 1 to 5.

10. The edible composition of claim 9, wherein the edible composition does not contain any animal-derived protein.

11. A method for producing the micellar solution of claim 6, or the artificial casein micelle of any one of claims 1 to 5, comprising combining a solution of one or more non-micellar caseins, with a solution of a polyphosphate, and optionally a solution of a divalent cation salt.

12. The method of claim 11 wherein the one or more non-micellar caseins are selected from the group consisting of αs-caseins, β-caseins and κ-caseins.

13. The method of claim 11 or claim 12, wherein the one or more non-micellar caseins are non- animal derived caseins, or synthetic caseins, or recombinantly produced caseins.

14. The method of any one of claims 11 to 13, wherein the polyphosphate is hexametaphosphate, or a salt thereof, preferably wherein the polyphosphate is sodium hexametaphosphate (SHMP).

15. The method of any one of claims 11 to 13, wherein the divalent cation salt is present, and is selected from the group consisting of; salts of Ca2+, salts of Mg2+, salts of Fe2+and salts of Zn2+, preferably wherein the divalent cation salt is CaCl2.

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