Dairy-like compositions and related methods

Abstract

Provided herein are dairy-like analog compositions and methods for making same using recombinant single variants of casein proteins, including, for example, recombinant single variants of alpha-casein protein. The compositions can be consumable compositions, and the recombinant single variants provide the consumable composition with at least one dairy-like property selected from the group consisting of adhesion, extensibility, texture, mouthfeel, melting, browning, hardness, creaminess, taste, odor, and softness.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 234,193, filed August 17, 2021, and U.S. Utility Patent Application No. 17 / 829,951, filed June 1, 2022, both of which are incorporated by reference in their entireties herein. [Background technology]

[0002] background The clean food industry is comprised of both plant-based and cell-based foods. Cell-based foods is a broad, umbrella term that includes culturing muscle and fat cells to replace slaughtered meat, as well as culturing bioengineered organisms expressing recombinant animal proteins to replace other animal products, such as dairy and eggs. The need to find alternative sources of animal protein arises from the inefficiency and unsustainability of current animal food production.

[0003] Cheese is the third most unsustainable animal product globally (measured in greenhouse gas emissions per kg of product), and dairy cheese consumption has not slowed due to plant-based alternatives introduced to the market over the last decade. Conversely, mozzarella cheese consumption has increased year-over-year in the United States and developing markets. Current cheese alternatives cannot match the functionality, nutrition, and taste of dairy cheese due to their lack of casein protein. Summary of the Invention [Means for solving the problem]

[0004] Abstract Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature, and not restrictive.

[0005] In some aspects, consumable compositions are provided herein. In some embodiments, the consumable compositions can include recombinant single variants of alpha casein protein. In some embodiments, the single variants provide the consumable compositions with at least one dairy-like property selected from the group consisting of adhesion, stretchiness, texture, mouthfeel, melting, browning, hardness, creaminess, taste, odor, and softness. In some embodiments, the recombinant single variants may not be animal-derived casein and are not physically dissociated from casein micelles. In some embodiments, the dairy-like properties can be substantially provided by recombinant single variant casein protein; the consumable compositions can be comparable in at least one of the dairy-like properties to dairy-derived consumable compositions.

[0006] In some embodiments, the consumable composition may comprise one or more dairy-like properties selected from the group consisting of adhesiveness, extensibility, firmness, consistency, cohesiveness, chewiness, resilience, elasticity, mouthfeel, melting, hardness, creaminess and softness.

[0007] In some embodiments, the casein content of the consumable composition may substantially comprise a recombinant single variant of alpha-casein protein.

[0008] In some embodiments, the casein content of the consumable composition may include only a recombinant single variant of alpha-casein protein.

[0009] In some embodiments, the recombinant single variant of alpha-casein protein may constitute at least 95% or at least 97% of the casein content in the consumable composition.

[0010] In some embodiments, the recombinant single variant of alpha-casein protein may constitute at least 99% of the casein content in the consumable composition.

[0011] In some embodiments, the consumable composition lacks any additional casein other than the recombinant single variant of alpha-casein protein.

[0012] In some embodiments, the single variant of alpha casein protein may not be derived from a caseinate.

[0013] In some embodiments, the composition may not include any animal-produced proteins.

[0014] In some embodiments, the composition is devoid of any other animal-derived dairy proteins.

[0015] In some embodiments, the dairy-like properties may be comparable to or improved compared to a milk-derived consumable composition; the milk-derived consumable composition may be identical in all components to the consumable composition, except that the milk-derived consumable composition may contain milk, one or more milk-derived proteins or milk-derived ingredients instead of or in addition to the recombinant single variant of alpha-casein protein.

[0016] In some embodiments, the dairy-like properties may be comparable to or improved compared to a micellar casein composition, which may be identical in all components to the consumable composition, except that the micellar casein composition may contain micellar casein isolated from milk instead of or in addition to recombinant single variants of alpha-casein protein.

[0017] In some embodiments, the recombinant single variant may be an alpha-casein protein that may include at least one non-native post-translational modification.

[0018] In some embodiments, the recombinant single variant of alpha-casein protein may further comprise at least one native post-translational modification.

[0019] In some embodiments, the recombinant single variant may be an alpha-casein protein that lacks one or more post-translational modifications of a native alpha-casein protein.

[0020] In some embodiments, the single variant of alpha-casein protein may further comprise at least one non-native post-translational modification.

[0021] In some embodiments, the single variant of alpha-casein protein may not be post-translationally modified.

[0022] In some embodiments, the single variant of alpha casein protein may not be phosphorylated.

[0023] In some embodiments, the single variant of alpha casein protein may be alpha-s1 casein protein. In some embodiments, the single variant of alpha casein protein may be alpha-s2 casein protein. In some embodiments, the composition may include full-length alpha casein protein.

[0024] In some embodiments, a single variant of alpha casein protein may comprise any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56.

[0025] In some embodiments, a single variant of alpha casein protein may comprise the amino acid sequence of bovine, caprine or ovine alpha casein protein, or any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56, or a sequence having at least 70%, 80%, 85% or 90% identity to any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56.

[0026] In some embodiments, a single variant of alpha casein protein may include one or more non-native amino acids at the N-terminus.

[0027] In some embodiments, a single variant of the alpha casein protein may include a non-native methionine at the N-terminal position.

[0028] In some embodiments, the single variant of alpha casein protein may not be derived from casein micelles.

[0029] In some aspects, what is described herein can be a dairy analog. The dairy analog can include any of the consumable compositions provided herein, and the analog can be selected from the group consisting of a cheese analog, a yogurt analog, a cream analog, and an ice cream analog.

[0030] In some embodiments, the dairy analog may comprise recombinant single variant alpha casein.

[0031] In some embodiments, the dairy analog may further comprise fat or oil from a non-animal source. In some embodiments, the dairy analog lacks any animal-derived dairy protein. In some embodiments, the dairy analog lacks any other casein protein. In some embodiments, the recombinant single variant of alpha casein protein may not be included in the dairy analog in micellar form.

[0032] In some embodiments, the dairy analog can include one or more of: (a) oil derived from a plant; (b) starch derived from a plant; (c) sugar; and (d) salt.

[0033] In some embodiments, the dairy analog can be a cheese analog. In some embodiments, the cheese analog can be a mozzarella analog, a cheddar analog, or a parmesan analog. In some embodiments, the cheese analog can be a low moisture cheese analog. In some embodiments, the cheese analog can be a soft cheese analog. In some embodiments, the cheese analog can be a hard cheese analog.

[0034] In some embodiments, the cheese analog may be a mozzarella analog, and the single variant of casein protein may be alpha casein. In some embodiments, the alpha casein may be alpha S1 casein. In some embodiments, the alpha S1 casein may be bovine alpha S1 casein. In some embodiments, the alpha S1 casein may be full-length casein. In some embodiments, the alpha casein may be alpha S2 casein. In some embodiments, the alpha S2 casein may be bovine alpha S2 casein. In some embodiments, the alpha S2 casein may be full-length casein.

[0035] In some embodiments, the extensibility of the cheese analog may be comparable to or improved relative to the extensibility of dairy-derived cheese or dairy-derived cheese analogs.

[0036] In some embodiments, the cheese analog has a comparable or greater melting area / time compared to the melting area / time of a dairy-derived cheese or a dairy-derived cheese analog.

[0037] In some embodiments, the spreadability of the cheese analog may be comparable to or improved compared to the spreadability of the dairy-derived cheese or dairy-derived cheese analog. In some embodiments, the texture of the cheese analog may be comparable to or improved compared to the texture of the dairy-derived cheese or dairy-derived cheese analog. In some embodiments, the adhesiveness of the cheese analog may be reduced compared to the adhesiveness of the dairy-derived cheese or dairy-derived cheese analog.

[0038] In some embodiments, the dairy-derived cheese or dairy-derived cheese analog may comprise micellar casein. In some embodiments, the stretchability of the cheese analog may be improved compared to the stretchability of the plant-derived cheese analog. In some embodiments, the melting of the cheese analog may be improved compared to the melting of the plant-derived cheese analog. In some embodiments, the texture of the cheese analog may be improved compared to the texture of the plant-derived cheese analog. In some embodiments, the adhesiveness of the cheese analog may be reduced compared to the adhesiveness of the plant-derived cheese analog.

[0039] In some embodiments, the cheese analog may comprise at least 5% of a single variant of casein protein w / w. In some embodiments, the cheese analog may comprise from about 5% to about 30% of a single variant of casein protein w / w. In some embodiments, the cheese analog may comprise from about 10% to about 25% of a single variant of casein protein w / w. In some embodiments, the cheese analog may comprise from about 15% to about 25% of a single variant of casein protein w / w.

[0040] In some embodiments, the cheese analog can include at most 25 mg calcium per gram casein. In some embodiments, the cheese analog can include 0 mg to 25 mg calcium per gram casein. In some embodiments, the cheese analog can include 0 mg to 20 mg calcium per gram casein. In some embodiments, the cheese analog can include 0 mg to 10 mg calcium per gram casein. In some embodiments, the cheese analog can include 5 mg to 15 mg calcium per gram casein. In some embodiments, the cheese analog can include about 0 mg calcium per gram casein. In some embodiments, the cheese analog can include about 10 mg calcium per gram casein.

[0041] In some embodiments, the cheese analog can include about 15% to about 30% fat w / w. In some embodiments, the cheese analog can include about 18% to about 28% fat w / w. In some embodiments, the cheese analog can include about 20% to about 25% fat w / w.

[0042] In some embodiments, the cheese analog may comprise from about 0.5% to about 4% starch w / w. In some embodiments, the cheese analog may comprise from about 1% to about 3% starch w / w. In some embodiments, the cheese analog may comprise from about 2% to about 3% starch w / w. In some embodiments, the cheese analog may comprise at most 10% starch w / w. In some embodiments, the cheese analog may comprise at most 5% starch w / w.

[0043] In some embodiments, the ratio of recombinant single variant alpha casein to emulsifying salts may be from 12: 1 to 6: 1. In some embodiments, the cheese analog does not contain any emulsifiers other than emulsifying salts.

[0044] In some embodiments, the dairy analog can be a yogurt analog. In some embodiments, the single variant of casein protein can be alpha casein. In some embodiments, the alpha casein can be alpha S1 casein. In some embodiments, the alpha S1 casein can be bovine alpha S1 casein. In some embodiments, the alpha S1 casein can be full-length casein. In some embodiments, the alpha casein can be alpha S2 casein. In some embodiments, the alpha S2 casein can be bovine alpha S2 casein. In some embodiments, the alpha S2 casein can be full-length casein.

[0045] In some embodiments, the emulsification of the yogurt analog may be comparable to or improved compared to the emulsification of a dairy-based yogurt or a dairy-based yogurt analog. In some embodiments, the firmness, adhesion or viscosity of the yogurt analog may be comparable to or improved compared to a dairy-based yogurt or a dairy-based yogurt analog. In some embodiments, the adhesiveness of the yogurt analog may be reduced compared to a dairy-based yogurt or a dairy-based yogurt analog. In some embodiments, the dairy-based yogurt or a dairy-based yogurt analog may comprise micellar casein.

[0046] In some embodiments, dairy-derived yogurt or dairy-derived yogurt analogs may be identical to dairy analogs in all ingredients, except that dairy-derived yogurt or dairy-derived yogurt analogs may contain micellar casein instead of recombinant single variants of alpha casein protein. In some embodiments, the emulsification of the yogurt analogs may be comparable to or improved compared to the emulsification of plant-derived yogurt analogs. In some embodiments, the firmness, adhesion or viscosity of the yogurt analogs may be comparable to or improved compared to plant-derived yogurt analogs. In some embodiments, the adhesiveness of the yogurt analogs may be reduced compared to plant-derived yogurt analogs.

[0047] In some embodiments, the plant-based yogurt analog lacks any dairy proteins.

[0048] In some embodiments, the yogurt analog may comprise about 1% to about 4% of a single variant of casein protein w / w. In some embodiments, the yogurt analog may comprise about 2% to about 4% of a single variant of casein protein w / w. In some embodiments, the yogurt analog may comprise about 2% to about 6% of fat w / w. In some embodiments, the yogurt analog may comprise about 4% to about 8% of carbohydrates w / w.

[0049] In some embodiments, the dairy analog may be a beverage. In some embodiments, the single variant of casein protein may be alpha casein. In some embodiments, the smoothness of the beverage may be comparable to or improved compared to the smoothness of a dairy-derived beverage. In some embodiments, the texture of the beverage may be comparable to or improved compared to the texture of a dairy-derived beverage. In some embodiments, the emulsification of the beverage may be comparable to or improved compared to a dairy-derived beverage. In some embodiments, the beverage may comprise about 0.5% to about 10% single variant of casein protein w / w. In some embodiments, the beverage may comprise about 0.1% to about 6% fat w / w. Incorporation by Reference

[0050] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0051] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 illustrates recombinantly produced full-length alpha S1 casein protein and two occurring truncated forms of alpha S1 casein.

[0053] [Diagram 2] FIG. 2 illustrates a comparison of the stretching ability (extensibility) between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0054] [Diagram 3] FIG. 3 illustrates a comparison of texture (on a logarithmic scale) between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0055] [Figure 4] FIG. 4 illustrates a comparison of the melting profiles between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0056] [Diagram 5] FIG. 5 illustrates a comparison of stretching ability (extensibility) between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0057] [Figure 6] FIG. 6 illustrates a comparison of melting between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0058] [Figure 7] FIG. 7 illustrates a comparison of stretching ability (extensibility) between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0059] [Figure 8] FIG. 8 illustrates a comparison of texture (on a logarithmic scale) between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0060] [Figure 9] FIG. 9 illustrates a comparison of the melting profiles between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0061] [Figure 10] FIG. 10 illustrates a comparison of stretching ability (extensibility) between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0062] [Figure 11] FIG. 11 illustrates a comparison of the melting profiles between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0063] [Figure 12] FIG. 12 illustrates a comparison of the extensibility profiles between analog cheeses made using the methods described herein and several commercially available cheeses and cheese analogs.

[0064] [Figure 13] FIG. 13 illustrates yogurt made using the methods described herein and yogurt from milk.

[0065] [Figure 14] FIG. 14 illustrates yogurt drinks made using the methods described herein and milk-derived yogurt drinks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Detailed Description of the Invention While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0067] The dairy industry is worth $330 billion, but research needs to be done for clean dairy solutions using recombinant dairy proteins. Because dairy cheese is the least efficient dairy product in terms of resources required per gram, as well as the most difficult dairy product to accurately replicate from plant-based ingredients alone, methods and compositions for dairy-like products made using recombinant proteins are presented herein.

[0068] The components that give dairy cheese its unique characteristics are casein proteins. When milk or milk-derived ingredients are used in dairy products, casein is found in micelles. Micelles are protein colloids, typically found in cow's milk, and are composed of four casein proteins (alpha S1 casein, alpha S2 casein, beta casein and kappa casein) that interact with insoluble calcium phosphate in the colloid center. When chymosin is added to milk, the micelles in the milk attract each other. This forms curd, which is then used to make 99% of all cheese. In the case of yogurt, acidification of the micelles containing liquid colloids can be done using starter cultures of bacteria known for yogurt production. The present disclosure is based on the discovery that a recombinant, non-naturally occurring single variant of casein can be used to produce dairy or dairy-like products without the presence of other caseins and without the formation of micelles. While those skilled in the art have attempted to isolate different casein proteins from milk or milk micelles to produce dairy-like products, the inventors of the present application have discovered for the first time that recombinant forms, and in some embodiments single variant caseins lacking or having different post-translational modifications (PTMs) than native casein, can provide dairy-like characteristics without being present in micellar form and without association with or presence of other caseins or other dairy proteins.

[0069] Those skilled in the art know that micelles are complex structures of multiple proteins and would not expect that a single casein protein can be used to form dairy-like products, such as cheese, cheese analogs, yogurt and other dairy products. The present disclosure is based on the surprising discovery made by the present inventors that a single recombinantly produced casein protein, a single variant of casein, such as alpha casein, can form consumables without forming or being incorporated into micelles. The present disclosure also describes recombinantly produced dairy products, such as cheese analogs and other dairy analog products, and powders using the compositions formed by the methods described herein.

[0070] Those skilled in the art know that micelles are complex structures of multiple proteins with multiple post-translational modifications and would therefore not expect that a single recombinant casein protein can be used to form dairy-like products with comparable or improved dairy-like properties, such as cheese, cheese analogs, yogurt and other dairy products. The present disclosure is based on the surprising discovery made by the inventors that a single recombinant casein protein, a single variant of casein, such as alpha casein, can form consumables with comparable or improved dairy-like properties despite differences in post-translational modifications and despite not being incorporated into a micelle.

[0071] The present disclosure also describes consumable compositions incorporating recombinantly produced truncated forms of a single variant of alpha-casein protein. The compositions described herein can include different truncated forms of casein protein.

[0072] The consumable compositions described herein are formed from recombinant single casein variants, e.g., alpha casein variants. Recombinant casein proteins can be expressed in microbial organisms, e.g., bacteria, e.g., the gram-positive bacteria Lactococcus lactis and Bacillus subtilis, and the gram-negative model organism E. coli, as well as other host organisms, e.g., yeast, fungi, and plants. These recombinant casein proteins can be combined with other components (e.g., minerals, fats, sugars, and vitamins) to create dairy-like products, e.g., cheeses that behave, smell, taste, look, and feel like dairy cheeses derived from animals. Such dairy-like products may be free of i) lactose, ii) cholesterol, iii) saturated fats derived from animals, iv) whey proteins derived from milk; and / or v) casein proteins derived from milk.

[0073] In some embodiments, the method includes producing a single variant of casein protein in a bacterial host cell such that such protein is secreted from the cell into the surrounding medium. In some examples, the method includes producing a single variant of alpha-casein protein in a bacterial host cell such that such protein is secreted from the cell into the surrounding medium. In some embodiments, the method includes producing a recombinant protein in a bacterial host cell such that such protein is intracellular. The recombinant protein can then be isolated, purified or partially purified and used in a method to make a composition that can be used as a dairy ingredient or emulsified with plant-based fats and other nutrients to form milk, cheese, yogurt or other dairy-like analog products.

[0074] In some embodiments, the method includes using a single casein from a variety of different species to produce the composition. In some examples, the method includes using a single alpha casein variant from a variety of different species to produce the composition. The casein can be from human, Bovinae (cattle, bison, buffalo), Caprinae (sheep and goat), Equine (horse, zebra) and Camelus (camel). The single casein variant can be modified compared to native alpha casein, for example, a truncated form of native casein. The compositions described herein can be produced in some examples without beta casein or kappa casein.

[0075] In some embodiments, the recombinant casein may be isolated, purified or partially purified from a genetically modified microorganism or its culture broth.

[0076] The term "about" as used herein can mean within 1 or 2 standard deviations. Alternatively, "about" can mean a range of up to 10%, up to 5%, or up to 1% of a given value. For example, about can mean up to ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a given value.

[0077] The term "dairy protein," as used herein, means a protein having an amino acid sequence derived from a protein found in milk, including variants thereof.

[0078] The term "dairy protein of animal origin" as used herein means a protein derived from milk, such as a protein obtained and / or isolated from the milk of milk-producing organisms, including but not limited to dairy cows, sheep, goats, humans, bison, buffalo, camels and horses. "Casein protein of animal origin" means a casein protein obtained and / or isolated from the milk of milk-producing organisms.

[0079] The term "recombinant dairy protein" as used herein means a protein expressed in a heterologous or recombinant organism having an amino acid sequence derived from a protein (including variants thereof) found in milk. "Recombinant casein protein" means casein produced by a recombinant organism or in a heterologous host cell.

[0080] The term "single variant of casein" (also referred to as "single variant of casein protein"), as used herein, can refer to a composition that includes or is created from one variant of the casein protein amino acid sequence. For example, a composition that includes a single variant of casein includes only alpha casein. In some cases, the term "single variant of casein protein" can refer to a composition in which a single casein protein provides one or more dairy-like properties to the composition, independent of the presence of other caseins.

[0081] Compositions comprising a single variant of casein can be created from only one casein protein amino acid sequence, but can include truncated forms of the protein sequence instead of or in addition to the full-length version of the protein.

[0082] The term "single variant of alpha casein" (also referred to as "single variant alpha casein protein"), as used herein, can refer to a composition that includes or is created from one variant of the alpha casein protein amino acid sequence. For example, a composition that includes a single variant of alpha casein includes only one of alpha S1 or alpha S2 casein. A composition that includes a single variant of alpha casein can be created from only one alpha casein protein amino acid sequence, but can include truncated forms of the protein sequence instead of or in addition to the full-length version of the protein. For example, a composition that includes a single variant of alpha casein protein can include a mixture of full-length alpha S1 casein protein and its truncated forms. A composition that includes a single variant of alpha casein can include only full-length alpha S1 casein protein, or only truncated forms of alpha S1 casein protein, or only a mixture of truncated forms of alpha S1 protein.

[0083] Percentage of "sequence identity", as used herein in the context of polynucleotide or polypeptide (amino acid) sequences, refers to the percentage of residues in two sequences that are the same when the sequences are aligned for maximum correspondence. There are several different algorithms known in the art that can be used to measure polynucleotide or polypeptide sequence identity. For example, sequences can be compared using FASTA (e.g., using its default parameters as provided in the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, WI), Gap (e.g., using its default parameters as provided in the Wisconsin Package version 10.0, GCG, Madison, WI), Bestfit, ClustalW (e.g., using the default parameters of version 1.83), or BLAST (e.g., using reciprocal BLAST, PSI-BLAST, BLASTP, BLASTN) (see, e.g., Pearson. 1990. Methods Enzymol. 183:63; Altschul et al. 1990. J. Mol. Biol. 215:403). Compositions containing casein A. Casein Protein

[0084] Traditionally, cheese begins with milk from animals. The process of animal-derived cheese production involves precipitating micellar forms from milk, which are most often complex protein mixtures (including multiple types of casein proteins, such as alpha, beta, and kappa proteins). The milk is acidified to cause slight shrinkage and dissociation of the micelles, and then the milk is renneted to form curds, which become cheese. Cheese analogs can be created from animal milk by first precipitating the casein micelles from the milk using one of the following methods: 1) sodium salt to create sodium caseinate, 2) acid to create acid casein, or 3) enzymatic coagulation with rennet to create rennet casein. The casein thus precipitated from the milk is then further treated with fat to create the cheese analog.

[0085] Provided herein is a consumable composition having a recombinantly produced single variant of casein that is not in micellar form and is not derived from milk or milk casein. In some aspects, the consumable composition of the present disclosure is not only made from a single variant of casein, but can also provide dairy-like properties to food and beverage products that are not derived from a micellar structure or from a starting material that has a micellar structure. In some aspects, the consumable composition of the present disclosure is not only made using a single variant of casein, but a single variant of casein can also provide desired or improved dairy-like properties without the presence of other caseins and without the complexity of casein structure derived from milk. In some aspects, the consumable composition of the present disclosure includes a single variant of alpha casein. In some alternatives, the consumable composition of the present disclosure includes a single variant of beta or kappa casein.

[0086] In some embodiments, the compositions herein (and products made therefrom) do not contain any dairy protein other than a single variant of casein protein. In some cases, the single variant of casein protein is a single variant of alpha casein protein. In some cases, the compositions herein (and products made therefrom) do not contain any whey protein or any whey protein derived from milk. In some embodiments, the compositions herein (and products made therefrom) do not contain any dairy protein derived from animals. The compositions herein do not contain any casein protein isolated from any animal-derived product or micelle.

[0087] The compositions described herein include single variants of casein produced by recombinant production.In some cases, the single variant casein in the consumable composition can be modified casein protein compared to native casein protein.The modification in the single variant of casein protein can include one or more amino acid insertions, deletions or substitutions compared to wild-type or native casein protein.In some cases, the single variant casein protein is a single variant of alpha casein protein.In some examples, the casein protein can be beta or kappa or gamma alpha casein protein.

[0088] The single variant of alpha casein protein can be a recombinant protein that is a truncated alpha casein protein compared to wild type or native alpha casein protein. The truncation can resemble a truncation found in nature (e.g., have a common number of amino acids). The truncation can be a non-naturally occurring truncation of alpha casein protein. The single variant of alpha casein protein can have an N-terminal truncation compared to wild type or native alpha casein protein. The single variant of alpha casein protein can have a C-terminal truncation compared to wild type or native alpha casein protein. The single variant of alpha casein protein can have an N-terminal truncation and a C-terminal truncation compared to wild type or native alpha casein protein.

[0089] In some embodiments, the single variant of alpha casein in the consumable composition is alpha S1 casein.In such compositions, the alpha S1 casein can include modified alpha S1 casein, such as modified with post-translational modifications (phosphorylation, glycosylation, the position of such modifications or the content of such modifications).In some cases, the alpha S1 casein can be full-length alpha S1 casein.In some cases, the composition comprising alpha S1 casein protein is devoid of any animal-derived protein.

[0090] In some embodiments, the single variant of alpha casein in the consumable composition is alpha S2 casein.In such compositions, the alpha S2 casein can include modified alpha S2 casein, such as modified with post-translational modifications (phosphorylation, glycosylation, the position of such modifications or the content of such modifications).In some cases, the alpha S2 casein can be full-length alpha S2 casein.In some cases, the composition comprising alpha S2 casein protein is devoid of any animal-derived protein.

[0091] Compositions herein comprising a single variant of alpha-casein protein are recombinant proteins and do not include alpha-casein protein isolated from casein micelles, alpha-casein protein isolated from any naturally occurring micellar form, or micelles or products containing micellar forms.

[0092] In some embodiments, the consumable compositions described herein include a single variant of casein protein, such as alpha casein protein. In some cases, the casein content of the consumable composition substantially includes a single variant of casein protein. In some cases, the casein content of the consumable composition includes only a single variant of casein protein. In some cases, the single variant of casein protein constitutes at least 95% or at least 97% of the casein content in the consumable composition. In some cases, the single variant of casein protein constitutes at least 99% of the casein content in the consumable composition. In some cases, the consumable composition lacks any additional casein other than the single variant of casein protein. In a preferred embodiment, the single variant of casein is alpha casein protein.

[0093] The single variant of alpha casein protein can be derived from a ruminant species. The single variant of alpha casein protein can be bovine alpha casein protein. The single variant of alpha casein protein can be caprine alpha casein protein. The single variant of alpha casein protein can be ovine alpha casein protein. The single variant of alpha casein protein can be equine alpha casein protein. The single variant of alpha casein protein can be camelid or camelid alpha casein protein. The single variant of alpha casein protein can be human alpha casein protein.

[0094] The single variant of alpha casein protein may be a mature form of alpha casein (lacking a signal sequence, such as exemplified in SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56) or a truncated form thereof (exemplified as SEQ ID NOs: 4-12, 16-24). The single variant of alpha casein protein may be a casein protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to bovine alpha casein protein, for example, SEQ ID NOs: 1-3, 28-30, 39-41 or 48-50, or a truncated form thereof. The single variant of alpha casein may be an ovine alpha casein protein, e.g., a casein protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 13-15 or 42-44, or a truncated form thereof. The single variant of alpha casein protein may be a caprine alpha casein protein, e.g., a casein protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 25-27 or 45-47, or a truncated form thereof. The single variant of alpha casein protein may be an equine alpha casein protein, e.g., a casein protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 31-33 or 51-53, or a truncated form thereof. The single variant of alpha casein protein may be a camel alpha casein protein, e.g., a casein protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 34-36 or 54-56, or a truncated form thereof.A single variant of alpha casein protein can be a human alpha casein protein, for example a casein protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 37-38, or a truncated form thereof.

[0095] The single variant of alpha casein protein in the consumable composition can be alpha S1 casein protein. The alpha S1 casein protein can be a full-length alpha S1 casein protein. In some cases, the alpha S1 protein is a truncated alpha S1 protein compared to the wild-type or native alpha S1 casein protein. In some cases, the alpha S1 casein protein has an N-terminal truncation compared to the wild-type or native alpha S1 casein protein. In some cases, the alpha S1 casein protein has a C-terminal truncation compared to the wild-type or native alpha S1 casein protein. In some cases, the alpha S1 casein can have an N-terminal truncation and a C-terminal truncation compared to the wild-type or native alpha S1 casein. In some cases, the alpha S1 protein lacks between 1-59 N-terminal amino acids. In some cases, the alpha S1 protein lacks between 1-5, 1-10, 1-20, 1-30, 1-50, 1-59 N-terminal amino acids. In some cases, a bovine alphaS1 protein having SEQ ID NO:2 lacks between 1 and 59 N-terminal amino acids. In some cases, a bovine alphaS1 protein having SEQ ID NO:2 lacks 22, 23, 24 or 25 N-terminal amino acids (see examples such as SEQ ID NOs:4-12). In some cases, an ovine alphaS1 protein having SEQ ID NO:14 lacks between 1 and 59 N-terminal amino acids (see examples such as SEQ ID NOs:16-24). In some cases, an ovine alphaS1 protein having SEQ ID NO:14 lacks 22, 23, 24 or 25 N-terminal amino acids.

[0096] In some cases, the alpha S1 casein is a mixture of full length alpha S1 casein and one or more truncated forms of alpha S1 casein protein, such as any one or more of the truncated forms described herein.

[0097] In some cases, the compositions herein made from a single variant of alpha casein comprise a mixture of full-length and one or more truncated forms of alpha casein protein, and in such compositions, the total alpha casein may comprise up to 20% wt / wt of one or more truncated forms of alpha S1 casein.The truncated forms may be any truncated forms of full-length single variant alpha S1 casein protein, examples of which are provided elsewhere herein.In some cases, the compositions herein made from a single variant of alpha casein comprise only truncated forms of alpha S1 casein protein.

[0098] In some cases, the single variant of alpha casein in the composition (such as a cheese analog) comprises more than 0% of the truncated form, for example, 0.1%, 0.2%, 0.5%, 0.8% wt / wt of the single variant of alpha casein is a truncated form(s) of alpha S1 protein. In some embodiments, the single variant of alpha S1 casein comprises at least 1% wt / wt of one or more truncated forms of alpha S1 casein. In some cases, the single variant of alpha S1 casein comprises at most 20% wt / wt of one or more truncated forms of alpha S1 casein. In some cases, the single variant of alpha S1 casein comprises 1%-3%, 1%-5%, 1%-7%, 1%-10%, 1%-12%, 1%-15%, 1%-20%, 3%-5%, 3%-7%, 3%-10%, 3%-12%, 3%-15%, 3%-20%, 5%-7%, 5%-10%, 5%-12%, 5%-15%, 5%-20%, 7%-10%, 7%-12%, 7%-15%, 7%-20%, 10%-12%, 10%-15%, 10%-20%, 12%-15%, 12%-20% or 15%-20% wt / wt of one or more truncated forms of alpha S1 casein. In some cases, the single variant of alpha S1 casein comprises about 1%, 3%, 5%, 7%, 10%, 12%, 15% or 20% wt / wt of one or more truncated forms of alpha S1 casein. In some cases, the single variant of alpha S1 casein comprises at least 1%, 3%, 5%, 7%, 10%, 12% or 15% wt / wt of one or more truncated forms of alpha S1 casein. In some cases, the single variant of alpha S1 casein comprises at most 3%, 5%, 7%, 10%, 12%, 15% or 20% wt / wt of one or more truncated forms of alpha S1 casein. In such compositions, the remaining percentage of alpha S1 casein in the single variant of alpha casein is the full-length form of alpha S1 casein. B. Post-translational Modifications

[0099] Depending on the host organism used to express casein, a single variant of casein protein, such as alpha casein protein, can have a different glycosylation or phosphorylation pattern (post-translational modification) from animal-derived casein protein. In some cases, a single variant of casein protein, such as alpha casein protein, does not contain post-translational modifications (PTMs). In some cases, a single variant of casein protein, such as alpha casein protein, contains substantially reduced PTMs. As used herein, substantially reduced PTMs refers to at least 50% reduction in one or more types of PTMs compared to the amount of PTMs in animal-derived casein protein. For example, a single variant of alpha casein protein can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less post-translationally modified compared to animal-derived alpha casein. In some cases, the post-translationally modified casein may lack one or more sites of post-translational modification found in animal-derived casein proteins.

[0100] In some cases, a single variant of a casein protein, such as an alpha-casein protein, contains one or more PTMs that differ from a casein protein derived from an animal, e.g., a modification at an amino acid in the single variant of an alpha-casein protein that is not modified in an alpha-casein protein derived from an animal, or a modification that has a different chemical structure compared to an alpha-casein protein derived from an animal, such as a different phosphorylation structure.

[0101] Alternatively, a single variant of casein protein, such as alpha casein protein, can contain PTMs comparable to animal-derived casein PTMs. In some cases, a single variant of casein protein, such as alpha casein protein, contains substantially increased PTMs. As used herein, substantially increased PTMs refers to at least a 5% increase in one or more types of PTMs compared to the amount of PTMs in animal-derived casein protein. For example, a single variant of alpha casein protein can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% more post-translationally modified compared to animal-derived alpha casein.

[0102] The PTMs in casein proteins, such as alpha casein proteins, may be chemically or enzymatically modified. In some cases, a single variant of a casein protein, such as alpha casein protein, contains substantially reduced PTMs or no PTMs without chemical or enzymatic treatment. The composition can be produced using a single variant of a casein protein, such as alpha casein protein, with reduced or no PTMs, where the lack of PTMs is not due to chemical or enzymatic treatment of the protein, such as producing a single variant of alpha casein protein by recombinant production where the recombinant protein lacks PTMs.

[0103] The phosphorylation in the single variant of casein protein, such as alpha casein protein, may be modified chemically or enzymatically. In some cases, the single variant of casein protein has substantially reduced or no phosphorylation without chemical or enzymatic treatment. For example, the single variant of alpha casein protein may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less phosphorylated compared to animal-derived alpha casein. The composition may be produced using a single variant of casein protein, such as alpha casein protein, with reduced or no phosphorylation, where the lack of phosphorylation is not due to chemical or enzymatic treatment, such as when recombinant production provides a single variant of casein protein with reduced or no phosphorylation.

[0104] The composition can be produced using a single variant of casein, such as alpha casein protein, which is a mixture of caseins with different PTMs. For example, the composition can include a single variant of alpha casein, which can include a mix of alpha caseins with no PTMs, with reduced PTMs, with native-like (or native-like) PTMs, and / or with increased PTMs. In some cases, the composition can include a single variant of casein protein, such as alpha casein protein with native (natural-like or animal-derived) PTMs, casein protein lacking one or more types of PTMs and / or no PTMs. Alternatively, the composition can include a single variant of casein protein, such as alpha casein protein with uniform PTMs. The PTM structure in such cases can include reduced PTMs, PTMs lacking one or more types of PTMs. For example, the consumable composition can include only a single variant of alpha casein with reduced phosphorylation. Consumable Composition A. Cheese and Cheese-like Analogues

[0105] The compositions of single variant caseins described in this application can be used to create cheese analogs. Cheese analogs can include ingredients in addition to the recombinantly produced single variant of casein protein. Cheese analogs can include ingredients in addition to the recombinantly produced single variant of alpha casein protein. In some cases, cheese analogs can include solvents, such as water, fat, salt, starch, sugar, flavorings, acids, pH stabilizers, carbohydrates, and the like. Cheese analogs can include proteins other than single variants of casein. For example, other proteins can include proteins (other than casein) found in dairy products of animal origin. Alternatively, cheese analogs can include proteins not found in dairy products of animal origin, examples of which include, but are not limited to, plant and / or microbial proteins.

[0106] The cheese analogs described herein can contain from 5% w / w to about 30% w / w of a recombinant single variant of casein. In some cases, the cheese analogs can contain at least 5% w / w of a recombinant single variant of casein, such as any of the single variants of casein described herein. In some cases, the cheese analogs can contain at most 30% w / w of a recombinant single variant of casein. In some cases, the cheese analog can comprise 5%-7%, 5%-10%, 5%-15%, 5%-20%, 5%-25%, 5%-30%, 7%-10%, 7%-15%, 7%-20%, 7%-25%, 7%-30%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 20%-25%, 20%-30% or 25%-30% w / w of a recombinant single variant of casein. In some cases, the cheese analog can comprise about 5%, 7%, 10%, 15%, 20%, 25% or 30% w / w of a recombinant single variant of casein.

[0107] The cheese analogs described herein can contain from 5% w / w to about 30% w / w of a recombinant single variant of alpha-casein. In some cases, the cheese analogs can contain at least 5% w / w of a recombinant single variant of alpha-casein, such as any of the single variants of alpha-casein described herein. In some cases, the cheese analogs can contain at most 30% w / w of a recombinant single variant of alpha-casein. In some cases, the cheese analog can comprise 5%-7%, 5%-10%, 5%-15%, 5%-20%, 5%-25%, 5%-30%, 7%-10%, 7%-15%, 7%-20%, 7%-25%, 7%-30%, 10%-15%, 10%-20%, 10%-25%, 10%-30%, 15%-20%, 15%-25%, 15%-30%, 20%-25%, 20%-30% or 25%-30% w / w of the recombinant single variant of alpha casein. In some cases, the cheese analog can comprise about 5%, 7%, 10%, 15%, 20%, 25% or 30% w / w of the recombinant single variant of alpha casein. In some preferred cases, the cheese analog may contain 10%-25%, 15%-25%, 18%-25%, 20-25%, 10%-20%, 15%-20%, 18%-20% w / w of recombinant single variant alpha-casein.

[0108] In some cases, the cheese analogs can include 5% w / w to 40% w / w fat. Examples of fats that can be added to the cheese analogs include coconut, canola, high oleic sunflower, and palm oil. Other examples are provided elsewhere herein. In some cases, the cheese analogs can include at least 5% w / w fat. In some cases, the cheese analogs can include at most 40% w / w fat. In some cases, the cheese analogue may be 5% w / w to 10% w / w, 5% w / w to 15% w / w, 5% w / w to 20% w / w, 5% w / w to 25% w / w, 5% w / w to 30% w / w, 5% w / w to 40% w / w, 10% w / w to 15% w / w, 10% w / w to 20% w / w, 10% w / w to 25% w / w, 10% w / w to 30% w / w, 10% w / w to 40% w / w, % w / w, 15% w / w to 20% w / w, 15% w / w to 25% w / w, 15% w / w to 30% w / w, 15% w / w to 40% w / w, 20% w / w to 25% w / w, 20% w / w to 30% w / w, 20% w / w to 40% w / w, 25% w / w to 30% w / w, 25% w / w to 40% w / w, or 30% w / w to 40% w / w fat. In some cases, the cheese analog can contain about 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, or 40% w / w fat.

[0109] In some cases, the cheese analogs can include 0% w / w to 50% w / w starch. Examples of starches that can be added to the cheese analogs include processed potato, corn. Other examples are provided elsewhere herein. In some cases, the cheese analogs can include at least 0% w / w starch. In some cases, the cheese analogs can include at most 50% w / w starch. In some cases, the cheese analog can contain between 0% w / w and 10% w / w, between 0% w / w and 20% w / w, between 0% w / w and 30% w / w, between 0% w / w and 40% w / w, between 0% w / w and 50% w / w, between 10% w / w and 20% w / w, between 10% w / w and 30% w / w, between 10% w / w and 40% w / w, between 10% w / w and 50% w / w, between 20% w / w and 30% w / w, between 20% w / w and 40% w / w, between 20% w / w and 50% w / w, between 30% w / w and 40% w / w, between 30% w / w and 50% w / w, or between 40% w / w and 50% w / w of starch. In some cases, the cheese analog can contain about 0% w / w, 10% w / w, 20% w / w, 30% w / w, 40% w / w or 50% w / w starch. In some preferred cases, the cheese analog can contain 0.5%-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 1%-2%, 1%-3%, 1%-4%, 2%-3%, 2%-4%, 3%-4% w / w starch.

[0110] Preferentially, the cheese analogue may comprise at most 30% w / w starch. In some cases, the cheese analogue may comprise 0% w / w to 30% w / w starch. In some cases, the cheese analogue may comprise at least 0% w / w starch. In some cases, the cheese analogue may comprise 0% w / w to 5% w / w, 0% w / w to 10% w / w, 0% w / w to 15% w / w, 0% w / w to 20% w / w, 0% w / w to 25% w / w, 0% w / w to 30% w / w, 5% w / w to 10% w / w, 5% w / w to 15% w / w, 5% w / w to 20% w / w, 5% w / w to 25% w / w, 5% w / w to 30% w / w , 10% w / w to 15% w / w, 10% w / w to 20% w / w, 10% w / w to 25% w / w, 10% w / w to 30% w / w, 15% w / w to 20% w / w, 15% w / w to 25% w / w, 15% w / w to 30% w / w, 20% w / w to 25% w / w, 20% w / w to 30% w / w, or 25% w / w to 30% w / w of starch. In some cases, the cheese analog can contain 0% w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, or 30% w / w of starch. In some cases, the cheese analog may contain at most 1% w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w or 30% w / w starch.

[0111] In some cases, the cheese analog may contain from 0% w / w to 16% w / w of salt, such as calcium salts, emulsifying salts, table salt, etc. Examples of such salts are also provided elsewhere herein.

[0112] In some cases, the cheese analog can include a calcium salt, e.g., calcium chloride. In some cases, the cheese analog can include 0.1%-6% w / w of a calcium salt. In some cases, the cheese analog can include at least 0.1% w / w of a calcium salt. In some cases, the cheese analog can include at most 6% w / w of a calcium salt. In some cases, the cheese analog can include 0.1%-1%, 0.1%-2%, 0.1%-3%, 0.1%-4%, 0.1%-5%, 0.1%-6%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%-6%, 2%-3%, 2%-4%, 2%-5%, 2%-6%, 3%-4%, 3%-5%, 3%-6%, 4%-5%, 4%-6%, or 5%-6% w / w of a calcium salt. In some cases, the cheese analog can include about 0.1%, 1%, 2%, 3%, 4%, 5% or 6% w / w of a calcium salt. In some cases, the cheese analog can include less than 0.1%, 1%, 2%, 3%, 4%, 5% or 6% w / w of a calcium salt. In some cases, the cheese analog can include more than 0.1%, 1%, 2%, 3%, 4% or 5% w / w of a calcium salt. In some cases, the cheese analog can include calcium ions. Calcium ions can be added to the cheese analog in the form of a calcium-based salt, e.g., calcium chloride.

[0113] In some cases, the cheese analog can contain 0%-0.6% calcium ion w / w. In some cases, the cheese analog can contain at least 0% calcium ion w / w. In some cases, the cheese analog can contain at most 0.6% calcium ion w / w. In some cases, the cheese analog can contain 0%-0.1%, 0%-0.2%, 0%-0.3%, 0%-0.4%, 0%-0.5%, 0%-0.6%, 0.1%-0.2%, 0.1%-0.3%, 0.1%-0.4%, 0.1%-0.5%, 0.1%-0.6%, 0.2%-0.3%, 0.2%-0.4%, 0.2%-0.5%, 0.2%-0.6%, 0.3%-0.4%, 0.3%-0.5%, 0.3%-0.6%, 0.4%-0.5%, 0.4%-0.6%, or 0.5%-0.6% w / w calcium ions. In some cases, the cheese analog can contain about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 0.6% calcium ion w / w. In some cases, the cheese analog can contain at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 0.6% calcium ion w / w. In some cases, the cheese analog can contain at most 0.1%, 0.2%, 0.3%, 0.4% or 0.5% calcium ion w / w.

[0114] In some cases, the cheese analog can include 0 mg to 30 mg of calcium ion per gram of casein. In some cases, the cheese analog can include at least 0 mg of calcium ion per gram of casein. In some cases, the cheese analog can include at most 30 mg of calcium ion per gram of casein. In some cases, the cheese analog can include 0 mg to 5 mg, 0 mg to 10 mg, 0 mg to 15 mg, 0 mg to 20 mg, 0 mg to 25 mg, 0 mg to 30 mg, 5 mg to 10 mg, 5 mg to 15 mg, 5 mg to 20 mg, 5 mg to 25 mg, 5 mg to 30 mg, 10 mg to 15 mg, 10 mg to 20 mg, 10 mg to 25 mg, 10 mg to 30 mg, 15 mg to 20 mg, 15 mg to 25 mg, 15 mg to 30 mg, 20 mg to 25 mg, 20 mg to 30 mg, or 25 mg to 30 mg of calcium ion per gram of casein. In some cases, the cheese analog can contain 0, 5, 10, 15, 20, 25, or 30 mg of calcium ion per gram of casein. In some cases, the cheese analog can contain at least 1, 5, 10, 15, 20, 25, or 30 mg of calcium ion per gram of casein. In some cases, the cheese analog can contain at most 1, 5, 10, 15, 20, 25, or 30 mg of calcium ion per gram of casein.

[0115] In some cases, the cheese analogs can include emulsifying salts, such as disodium phosphate, trisodium citrate, or other emulsifying salts. In some cases, the cheese analogs can include 0.1%-6% w / w of emulsifying salts. In some cases, the cheese analogs can include at least 0.1% w / w of emulsifying salts. In some cases, the cheese analogs can include at most 6% w / w of emulsifying salts. In some cases, the cheese analog can contain 0.1%-1%, 0.1%-2%, 0.1%-3%, 0.1%-4%, 0.1%-5%, 0.1%-6%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%-6%, 2%-3%, 2%-4%, 2%-5%, 2%-6%, 3%-4%, 3%-5%, 3%-6%, 4%-5%, 4%-6%, or 5%-6% w / w of emulsifying salt. In some cases, the cheese analog can contain about 0.1%, 1%, 2%, 3%, 4%, 5%, or 6% w / w of emulsifying salt. In some cases, the cheese analog can contain less than 0.1%, 1%, 2%, 3%, 4%, 5%, or 6% w / w of emulsifying salt. In some cases, the cheese analogs may contain more than 0.1%, 1%, 2%, 3%, 4% or 5% w / w emulsifying salts. Alternatively, in some cases, the cheese analogs do not contain any emulsifying salts. In some cases, the cheese analogs do not contain any emulsifiers other than emulsifying salts.

[0116] In some embodiments, the cheese analog can include a ratio of casein (e.g., recombinant single variant casein) to emulsifying salt. In some cases, the cheese analog can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of about 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. In some cases, the cheese analog can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of about 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. In some cases, the cheese analog can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of about 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1.

[0117] In some cases, the cheese analogs can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salts of between about 3:1 and 6:1. In some cases, the cheese analogs can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salts of between about 6:1 and 9:1. In some cases, the cheese analogs can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salts of between about 8:1 and 10:1. In some cases, the cheese analogs can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salts of between about 8:1 and 12:1. In some cases, the cheese analogs can have a ratio (weight / weight) of casein (e.g., recombinant single variant casein) to emulsifying salts of between about 8:1 and 15:1.

[0118] In some cases, the cheese analog can include table salt, e.g., sodium chloride salt. In some cases, the cheese analog can include 0.1%-4% w / w sodium chloride. In some cases, the cheese analog can include at least 0.1% w / w sodium chloride. In some cases, the cheese analog can include at most 4% w / w sodium chloride. In some cases, the cheese analog can include 0.1%-1%, 0.1%-2%, 0.1%-3%, 0.1%-4%, 1%-2%, 1%-3%, 1%-4%, 2%-3%, 2%-4%, or 3%-4% w / w sodium chloride. In some cases, the cheese analog can include about 0.1%, 1%, 2%, 3%, or 4% w / w sodium chloride. In some cases, the cheese analog can include less than 0.1%, 1%, 2%, 3%, or 4% w / w sodium chloride. In some cases, the cheese analog may contain more than 0.1%, 1%, 2% or 3% w / w sodium chloride.

[0119] In some examples, recombinantly produced casein, e.g., a single variant of alpha casein protein (5-30% w / w of the cheese analog) (exemplary optimal range 10-20% w / w), may be added in the form of water (30-65% w / w) (exemplary optimal range 45-55% w / w), fat (5-40% w / w) (exemplary optimal range 20-25% w / w), sodium chloride (salt) (0-4% w / w) (exemplary optimal range 0-1.5% w / w), calcium chloride (CaCl) (exemplary optimal range 0-1.5% w / w), calcium phosphate ... can be combined with sodium hydroxide (0-6% w / w) (exemplary optimum range 0-1.5% w / w), emulsifying salts (disodium phosphate, trisodium citrate) (0-6% w / w) (exemplary optimum range 0-3% w / w), starch (0-50% w / w) (exemplary optimum range 0-8% w / w), natural vegan flavors (0-5% w / w) (exemplary optimum range 0.5-1% w / w) and acids (0-5% w / w) (exemplary optimum range 0-1% w / w). Optional ingredients such as plant-based or other animal-free proteins (0-30%) (exemplary optimum range 0-8% w / w), hydrocolloids (0-5%) (exemplary optimum range 0-2%), sugars such as mono-, di- and oligosaccharides (0-5% w / w) (exemplary optimum range 0-2% w / w), emulsifying agents such as mono- and diglycerides (0-2% w / w) (exemplary optimum range 0-0.5% w / w), natural flavor maskers, color additives (0-5% w / w), preservatives (0-1% w / w), anti-caking agents (0-2% w / w) and micronutrients such as vitamins (0-1%) can be incorporated into the cheese analog as well.

[0120] In some embodiments, the following ingredients are premixed: recombinantly produced single variant of alpha casein, fat(s), water, starch, salt(s), e.g., sodium chloride. In some embodiments, pH adjustment is performed at this stage using a pH adjuster, e.g., sodium hydroxide (lye), to bring the composition to a neutral pH, 6.8-7.2. Optional ingredients, e.g., plant-based or other animal-free proteins, sugars, hydrocolloids, and emulsifying agents, can be added at this step or at a later stage. Premixing can be performed at ambient or elevated temperature (15-50°C). In some cases, the fat is premelted [30-70°C] (exemplary optimal range 40-50°C) and held at its melting temperature prior to incorporation. Calcium chloride and emulsifying salts can be added at the premix stage or at a later stage. Alternatively, calcium chloride and emulsifying salts can be added sequentially in any order: calcium chloride can be added before or after the emulsifying salts. For example, calcium chloride and emulsifying salts can be added in two stages over the course of 4 minutes to 1 hour (exemplary optimum range 10-20 minutes) with an incubation interval of 2 minutes to 30 minutes (exemplary optimum range 5-10 minutes) at ambient or elevated temperature. Alternatively, the cheese analog can be produced without calcium chloride or emulsifying salts. Calcium chloride can also be added at the end of the cheese analog making process, before or after acid addition.

[0121] The mixture can be heated over a temperature gradient from the pre-mix temperature (ambient or elevated) to 50-95°C (exemplary optimum range 75-90°C) over a gradient period of 1-30 minutes (exemplary optimum range 1-5 minutes) while being mechanically mixed. The heated mixture can then be held at the final gradient temperature for 0-20 minutes (exemplary optimum range 2-5 minutes) as the ingredients are mechanically incorporated to form the emulsion. Mechanical incorporation (mixing) can be accomplished using a variety of mixers, e.g., vertical cutter mixers or twin screw mixers.

[0122] The acidity of the mixture can be adjusted by incorporating an acid, e.g., lactic acid or citric acid, and continuing mixing for a short period of time to achieve a final pH of about 5-6.5 (exemplary optimum range 5.7-6.2). The acidity can also be adjusted by using glucono-delta-lactone early in the mixing process. The resulting mixture can then be placed into molds, other formed containers, or vacuum-sealed packaging. The resulting product can be cooled to 4°C immediately after division into molds to create a cheese analog. Such cheese analogs can then be used as food products, toppings, and incorporated into other food products.

[0123] In some embodiments, the amount of salt or minerals in the cheese can be altered to produce advantageous qualities. For example, in one example, the amount of calcium can be altered to improve melting, texture, spreadability, etc. In one example, the amount of calcium in the cheese analog can be reduced to improve the melting of the cheese analog. In another example, the amount of calcium in the cheese analog can be increased to improve the texture or spreadability of the cheese analog.

[0124] In some embodiments, the cheese analog comprises a single variant casein, where the single variant casein, e.g., alpha casein, provides the composition with one or more properties of cheese or a cheese analog.

[0125] The texture of a cheese analog made with a single variant of casein, such as alpha casein, by the methods described herein, can be comparable to the texture of a similar type of cheese made with dairy proteins of animal origin, such as cheese made from animal milk. The texture of a cheese analog made using the compositions described herein having a single variant of casein, such as alpha casein, can be comparable to the texture of a cheese or cheese analog made with a micellar form of casein, such as a cheese made from milk or a cheese analog made from caseinate or rennet casein. The texture of cheese analogs made using the compositions described herein having a single variant of casein, e.g., alpha casein, may be improved / more desirable when compared to the texture of cheese or cheese analogs made using micellar forms of casein, e.g., cheese made from milk or cheese analogs made from caseinate or rennet casein, or compared to plant-based cheese analogs lacking dairy proteins (i.e., cheese-like products made using plant-based proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or no protein (such as cheese-like products made primarily with starch)). The texture of the cheese analogs can be tested using a trained human subject population or using a machine, such as a texture analyzer.

[0126] The taste of a cheese analog made with a single variant of casein, e.g., alpha casein, such as by the methods described herein, can be comparable to the taste of a similar type of cheese made with an animal-derived dairy protein, e.g., cheese made from animal milk. The taste of a cheese analog made using the compositions described herein having a single variant of casein, e.g., alpha casein, can be comparable to the taste of a cheese or cheese analog made with a micellar form of casein, e.g., cheese made from milk or cheese analog made from caseinate or rennet casein. The taste of cheese analogs made using the compositions described herein having a single variant of casein, e.g., alpha-casein, may be improved when compared to the taste of cheeses or cheese analogs made using plant-derived cheese analogs lacking dairy proteins (i.e., cheese-like products made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other vegetable proteins as the sole / major protein source, or without protein (such as cheese-like products made primarily with starch)). Cheese taste can be tested using a trained human subject population.

[0127] The cheese analogue compositions described herein with a single variant of casein, e.g., alpha casein, can have a browning ability comparable to a similar type of cheese made using dairy proteins of animal origin, e.g., cheese made from animal milk. The cheese analogue compositions described herein with a single variant of casein, e.g., alpha casein, can have a browning ability comparable to a similar type of cheese or cheese analog made using micellar forms of casein, e.g., cheese made from milk or cheese analog made from caseinate or rennet casein. The cheese analogue compositions described herein with a single variant of casein, e.g., alpha casein, can have an improved browning ability when compared to a similar type of cheese or cheese analog made using a plant-derived cheese analog lacking dairy proteins (i.e., cheese-like products made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or without protein (such as cheese-like products made primarily with starch)). The browning ability of the cheese analogues can be examined using ovens and computer imaging.

[0128] The cheese analogue compositions described herein with a single variant of casein, e.g., alpha casein, can have a melting ability comparable to a similar type of cheese made using dairy proteins of animal origin, e.g., cheese made from animal milk. The cheese analogue compositions described herein with a single variant of casein, e.g., alpha casein, can have a melting ability comparable to a similar type of cheese or cheese analog made using micellar forms of casein, e.g., cheese made from milk or cheese analog made from caseinate or rennet casein. The cheese analogue compositions described herein with a single variant of casein, e.g., alpha casein, can have an improved melting ability when compared to a similar type of cheese or cheese analog made using a plant-derived cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or without protein (such as a cheese-like product made primarily with starch)). The melting ability of the cheese analogs can be tested using a modified Schreiber melting test and computer imaging. An exemplary assay for measuring this melting property is provided in the Examples section.

[0129] In some embodiments, the cheese or cheese analog is analyzed for melting characteristics upon heating, such as heating on a hot plate at 95° C. for 15 minutes, and melting is assessed by the ratio of melted area to unmelted area, where melting is defined as a ratio greater than or equal to 1. In some embodiments, the cheese analog comprises recombinant single variant alpha casein has a melt value of or greater than 1. In some embodiments, the cheese analog comprises recombinant single variant alpha casein has a melt value of greater than 1, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 or greater. In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have a melt value greater than 1, e.g., between 1.0-1.3, 1.3-1.5, 1.5-1.7, 1.7-1.9, 1.9-2.1, 2.1-2.3, or 2.3-2.5. In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have a melt value greater than 1, and the melted area retains an opaque appearance. In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have a melt value greater than the melt value of the plant-based cheese analog, e.g., the melt value of the cheese analogs comprising recombinant single variant alpha casein is 1.5x, 2x, 2.5x, or greater than 2.5x the melt value of the plant-based cheese analog.

[0130] The stretching capacity of cheese analogs made with a single variant of casein, e.g., alpha casein, such as by the methods described herein, can be comparable to that of a similar type of cheese made with dairy proteins of animal origin, e.g., cheese made from animal milk. The stretching capacity of cheeses made using the compositions described herein with a single variant of casein, e.g., alpha casein, can be comparable to that of cheeses or cheese analogs made with micellar forms of casein, e.g., cheeses made from milk or cheese analogs made from caseinate or rennet casein. The stretchability of cheese made using the compositions described herein having a single variant of casein, e.g., alpha casein, may be improved / more desirable when compared to the stretchability of cheese or cheese analogs made using micellar forms of casein, e.g., cheese made from milk or cheese analogs made from caseinate or rennet casein, or compared to plant-derived cheese analogs lacking dairy proteins (i.e., cheese-like products made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins, as the sole / major protein source, or no protein (such as cheese-like products made primarily with starch). The stretchability of cheese can be tested using a trained human subject population or using a machine, such as a texture analyzer. The stretchability of cheese analogs made using the methods described herein can exceed 2.5 cm when measured with a texture analyzer after cooking. An exemplary assay for measuring this stretchability property is provided in the Examples section.

[0131] In some embodiments, the cheese or cheese analogs are analyzed for extensibility using a texture analyzer, such as by heating the cheese in an oven at 90° C. for 10 minutes in an extensibility setup and measuring the extensibility on the texture analyzer as the distance to break (i.e., the distance at which all of the cheese strings break). In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have an extensibility value of about 200 mm (20 cm). In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have an extensibility of at least 40 mm, at least 50 mm, at least 60 mm, at least 80 mm, at least 100 mm, at least 120 mm, at least 140 mm, at least 160 mm, at least 180 mm, at least 190 mm, at least 200 mm, at least 210 mm, at least 220 mm, at least 230 mm, at least 240 mm, at least 250 mm, or greater than 250 mm. In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have an extensibility between 40-70 mm, 70-100 mm, 100-125 mm, 125-150 mm, 150-180 mm, 180-200 mm, 200-225 mm, 225-250 mm, or 250-300 mm. In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have an extensibility that exceeds that of a plant-based cheese analog, for example, the extensibility of the cheese analogs comprising recombinant single variant alpha casein is at least 2×, at least 3×, at least 4×, at least 5×, at least 7×, at least 10× that of a plant-based cheese analog. In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have an extensibility comparable to that of a cheese derived from low moisture milk (such as low moisture mozzarella) or a caseinate-containing cheese, for example, imitation mozzarella cheese. In some embodiments, a cheese analog comprising recombinant single variant alpha casein has an extensibility within 10-20%, 15-25%, 20-40% or 10-150% of the extensibility of a cheese derived from low moisture milk (such as low moisture Mozzarella) or a caseinate-containing cheese, e.g., imitation Mozzarella cheese.

[0132] The hardness of a cheese analog made with a single variant of casein, such as alpha casein, by the methods described herein, can be comparable to the hardness of a similar type of cheese made with dairy proteins of animal origin, such as cheese made from animal milk. The hardness of a cheese analog made using the compositions described herein having a single variant of casein, such as alpha casein, can be comparable to the hardness of a cheese or cheese analog made with a micellar form of casein, such as a cheese made from milk or a cheese analog made from caseinate or rennet casein. The firmness of cheese analogs made using the compositions described herein having a single variant of casein, e.g., alpha-casein, may be improved when compared to the firmness of cheeses or cheese analogs made using plant-derived cheese analogs lacking dairy proteins (i.e., cheese-like products made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or without protein (such as cheese-like products made primarily with starch). Cheese analog firmness can be tested using a trained human subject population or using a machine, such as a texture analyzer.

[0133] The adhesiveness of cheese analogs made with a single variant of casein, such as alpha casein, by the methods described herein, can be comparable to that of similar types of cheese made with dairy proteins from animals, such as cheese made from animal milk.The adhesiveness of cheese analogs made with the compositions described herein having a single variant of casein, such as alpha casein, can be comparable to that of cheese or cheese analogs made with a micellar form of casein, such as cheese made from milk or cheese made from caseinate or rennet casein.The adhesiveness of cheese analogs made by the methods described herein having a single variant of casein, such as alpha casein, can be reduced when compared to that of cheese or cheese analogs made with a micellar form of casein, such as cheese made from milk or cheese made from caseinate or rennet casein. The adhesiveness of cheese analogs made by the methods described herein having a single variant of casein, e.g., alpha casein, may be reduced when compared to the adhesiveness of plant-derived cheese analogs lacking dairy proteins (i.e., cheese-like products made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins, as the sole / major protein source, or no protein (such as cheese-like products made primarily with starch). The adhesiveness of cheese analogs can be tested using a trained human subject population or using a machine such as a texture analyzer. The adhesiveness of cheese analogs made using the methods described herein may be less than 2 (g*sec) in a texture analyzer test. An exemplary assay for measuring this adhesive property is provided in the Examples section.

[0134] The adhesion of the cheese analogs can be tested using a texture analyzer, for example, a TA.XT Plus Texture Analyzer with a TA-18 ½” ball probe, where adhesion represents the force required to remove the cheese from the probe. In some embodiments, the cheese analogs with recombinant single variant alpha casein have an adhesion between 0.1-3.0 g*sec. In some embodiments, the cheese analogs with recombinant single variant alpha casein have an adhesion of less than 3.0 g*sec, less than 2.5 g*sec, less than 2.0 g*sec, less than 1.5 g*sec, less than 1.0 g*sec or less than 0.5 g*sec. In some embodiments, the cheese analogues comprising recombinant single variant alpha casein have an adhesiveness of between 0.1-2.5 g*sec, 0.1-2.0 g*sec, 0.1-1.5 g*sec, 0.1-1.0 g*sec, 0.1-0.5 g*sec, 0.5-2.5 g*sec, 0.5-2.0 g*sec, 0.5-1.5 g*sec, 0.5-1.0 g*sec or 0.5-1.5 g*sec. ... In some embodiments, the adhesiveness of the cheese analogs comprising recombinant single variant alpha casein is reduced by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, or more than 20-fold, when compared to the adhesiveness of cheeses derived from low moisture milk (such as low moisture mozzarella) or caseinate-containing cheeses (such as imitation mozzarella). In some embodiments, the cheese analogs comprising recombinant single variant alpha casein have reduced adhesiveness compared to plant-based cheese analogs. In some embodiments, the adhesiveness of the cheese analogs comprising recombinant single variant alpha casein is reduced by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, or more than 20-fold, when compared to the adhesiveness of plant-based cheese analogs.

[0135] The creaminess of a cheese analog made with a single variant of casein, e.g., alpha casein, such as by the methods described herein, can be comparable to the creaminess of a similar type of cheese made with dairy proteins of animal origin, e.g., cheese made from animal milk. The creaminess of a cheese analog made using the compositions described herein having a single variant of casein, e.g., alpha casein, can be comparable to the creaminess of a cheese or cheese analog made with a micellar form of casein, e.g., cheese made from milk or cheese analog made from caseinate or rennet casein. The creaminess of a cheese analog made using the compositions described herein having a single variant of casein, e.g., alpha-casein, may be improved when compared to the creaminess of a cheese or cheese analog made using a plant-based cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or without protein (such as a cheese-like product made primarily with starch)).

[0136] The mouthfeel of a cheese analog made with a single variant of casein, such as alpha casein, by methods such as those described herein, can be comparable to the mouthfeel of a similar type of cheese made with dairy proteins of animal origin, such as cheese made from animal milk. The mouthfeel of a cheese analog made using the compositions described herein with a single variant of casein, such as alpha casein, can be comparable to the mouthfeel of a cheese or cheese analog made with a micellar form of casein, such as a cheese made from milk or a cheese analog made from caseinate or rennet casein. The mouthfeel of a cheese analog made using the compositions described herein having a single variant of casein, e.g., alpha-casein, may be improved when compared to the mouthfeel of a cheese or cheese analog made using a plant-derived cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or without protein (such as a cheese-like product made primarily with starch)).

[0137] The opacity of a cheese analog made with a single variant of casein, e.g., alpha casein, such as by the methods described herein, when heated (e.g., melted), can be comparable to the opacity of a similar type of cheese made with an animal-derived dairy protein, e.g., cheese made from animal milk. The opacity of a cheese analog made using the compositions described herein having a single variant of casein, e.g., alpha casein, when heated, can be comparable to the opacity of a cheese or cheese analog made with a micellar form of casein, e.g., cheese made from milk or cheese analog made from caseinate or rennet casein. The opacity of cheese analogs made using the compositions described herein having a single variant of casein, e.g., alpha-casein, when heated may be improved compared to the opacity of cheese or cheese analogs made using plant-based cheese analogs lacking dairy proteins (i.e., cheese-like products made using plant-derived proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or without protein (such as cheese-like products made primarily with starch)).

[0138] The cheese analogs or similar compositions described herein can be low moisture cheese analogs. For example, low moisture cheese analogs can contain 45-52% w / w moisture. Low moisture cheese analogs can contain less than 52% w / w moisture. B. Yogurt or yogurt-like substances

[0139] Single variant casein compositions, such as those comprising alpha casein described herein, can be used to produce consumable compositions, such as yogurt analog or yogurt-like compositions. For example, single variant alpha casein compositions can be used to form a yogurt analog product. The yogurt analog product can be formed without the formation of micelles or micellar-like compositions. Yogurt analog products made using the compositions described herein can provide similar or equivalent characteristics (such as texture, creaminess, firmness, adhesion, viscosity, odor and taste) to animal-derived dairy yogurt or dairy yogurt analogs made using micellar forms of casein, such as those made from caseinates, rennet casein or micellar casein. Yogurt analog products made using the compositions described herein can provide improved one or more characteristics (such as texture, creaminess, firmness, adhesion, viscosity, odor and taste) when compared to plant-based yogurt analogs lacking any dairy protein (i.e., yogurt-like products made without protein or using plant-based proteins, e.g., pea, chickpea, nut and / or other vegetable proteins, as the only protein source (meaning they do not contain any casein)).

[0140] The yogurt analogs described herein may contain from 1% w / w to about 20% w / w of a recombinant single variant of casein. In some cases, the yogurt analogs may contain at least 1% w / w of a recombinant single variant of casein, such as any of the single variants of casein described herein. In some cases, the yogurt analogs may contain at most 20% w / w of a recombinant single variant of casein, such as any of the single variants of casein described herein. In some cases, the yogurt analog may comprise 1%-2%, 1%-4%, 1%-6%, 1%-8%, 1%-10%, 1%-15%, 1%-20%, 2%-4%, 2%-6%, 2%-8%, 2%-10%, 2%-15%, 2%-20%, 4%-6%, 4%-8%, 4%-10%, 4%-15%, 4%-20%, 6%-8%, 6%-10%, 6%-15%, 6%-20%, 8%-10%, 8%-15%, 8%-20%, 10%-15%, 10%-20% or 15%-20% w / w of a recombinant single variant of casein, such as any of the single variants of casein described herein. In some cases, the yogurt analog may contain 1%, 2%, 4%, 6%, 8%, 10%, 15% or 20% w / w of a recombinant single variant of casein, such as any of the single variants of casein described herein, hi some preferred embodiments, the yogurt analog may contain 2%-5% w / w of a recombinant single variant of casein.

[0141] The yogurt analogs described herein may contain from 1% w / w to about 20% w / w of recombinant single variants of alpha-casein. In some cases, the yogurt analogs may contain at least 1% w / w of recombinant single variants of alpha-casein, such as any of the single variants of alpha-casein described herein. In some cases, the yogurt analogs may contain at most 20% w / w of recombinant single variants of alpha-casein, such as any of the single variants of alpha-casein described herein. In some cases, the yogurt analog may comprise 1%-2%, 1%-4%, 1%-6%, 1%-8%, 1%-10%, 1%-15%, 1%-20%, 2%-4%, 2%-6%, 2%-8%, 2%-10%, 2%-15%, 2%-20%, 4%-6%, 4%-8%, 4%-10%, 4%-15%, 4%-20%, 6%-8%, 6%-10%, 6%-15%, 6%-20%, 8%-10%, 8%-15%, 8%-20%, 10%-15%, 10%-20% or 15%-20% w / w of a recombinant single variant of alpha casein, such as any of the single variants of alpha casein described herein. In some cases, the yogurt analog may contain 1%, 2%, 4%, 6%, 8%, 10%, 15% or 20% w / w of a recombinant single variant of alpha-casein, such as any of the single variants of alpha-casein described herein, hi some preferred embodiments, the yogurt analog may contain 2%-5% w / w of a recombinant single variant of alpha-casein.

[0142] The yogurt analogs described herein may contain 0.5% to 20% w / w fat. The yogurt analogs described herein may contain at least 0.5% w / w fat. The yogurt analogs described herein may contain at most 20% w / w fat. The yogurt analogs described herein may contain 0.5% to 1%, 0.5% to 4%, 0.5% to 6%, 0.5% to 8%, 0.5% to 10%, 0.5% to 12%, 0.5% to 15%, 0.5% to 18%, 0.5% to 20%, 1% to 4%, 1% to 6%, 1% to 8%, 1% to 10%, 1% to 12%, 1% to 15%, 1% to 18%, 1% to 20%, 4% to 6%, 4% to 8%, 4% to 10%, 4% to 12%, 4% to 15 ... The yogurt analogs described herein may contain 0.5%, 1%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, or 20% w / w fat. The yogurt analogs described herein may contain 0.5%, 1%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, or 20% w / w fat. The yogurt analogs described herein may contain at most 0.5%, 1%, 4%, 6%, 8%, 10%, 12%, 15% or 18% w / w fat. The yogurt analogs described herein may contain at least 0.5%, 1%, 4%, 6%, 8%, 10%, 12%, 15% or 18% w / w fat. Alternatively, the yogurt analogs may not contain any fat.

[0143] The yogurt analogs described herein may comprise 0%-10% w / w starch. The yogurt analogs described herein may comprise at least 0.5% w / w starch. The yogurt analogs described herein may comprise at most 10% w / w starch. The yogurt analogs described herein may comprise 0.5%-1%, 0.5%-2%, 0.5%-4%, 0.5%-6%, 0.5%-8%, 0.5%-10%, 1%-2%, 1%-4%, 1%-6%, 1%-8%, 1%-10%, 2%-4%, 2%-6%, 2%-8%, 2%-10%, 4%-6%, 4%-8%, 4%-10%, 6%-8%, 6%-10% or 8%-10% w / w starch. The yogurt analogs described herein may contain 0.5%, 1%, 2%, 4%, 6%, 8% or 10% w / w starch. The yogurt analogs described herein may contain at most 0.5%, 1%, 2%, 4%, 6%, 8% or 10% w / w starch. The yogurt analogs described herein may contain at least 0.5%, 1%, 2%, 4%, 6%, 8% or 10% w / w starch.

[0144] The yogurt analogs described herein can include proteins other than a single variant of casein. For example, the other proteins can include proteins (other than casein) found in dairy products of animal origin. Alternatively, the yogurt analogs can include proteins not found in dairy products of animal origin, examples of which can include, but are not limited to, plant and / or microbial proteins.

[0145] In some embodiments, the yogurt analog comprises a single variant casein, where the single variant casein, e.g., alpha casein, provides the composition with one or more properties of the yogurt analog.

[0146] The texture of a yogurt analog made with a single variant of casein, such as alpha casein, by the methods described herein, may be comparable to the texture of a similar type of yogurt made with dairy proteins of animal origin, such as yogurt made from animal milk. The texture of a yogurt analog made using the compositions described herein having a single variant of casein, such as alpha casein, may be comparable to the texture of a yogurt or yogurt analog made with a micellar form of casein, such as a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein. The texture of a yogurt analog made using the compositions described herein having a single variant of casein, e.g., alpha casein, may be improved / more desirable when compared to the texture of a yogurt or yogurt analog made using micelles, e.g., a yogurt made from milk or a yogurt analog made from caseinate, micellar casein, or rennet casein, or compared to a plant-based cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-based proteins, e.g., pea, chickpea, nuts, and / or other plant proteins, as the sole / major protein source, or without protein (such as a cheese-like product made primarily with starch). The texture of the yogurt analog can be tested using a trained human subject population or using a machine, such as a texture analyzer. Texture properties can include, for example, one or more of firmness, adhesion, adhesiveness, and consistency of the yogurt or yogurt analog.

[0147] The smoothness or creaminess of a yogurt analog made with a single variant of casein, such as alpha casein, by the methods described herein, may be comparable to the smoothness of a similar type of yogurt made with dairy proteins of animal origin, such as yogurt made from animal milk. The smoothness of a yogurt analog made using the compositions described herein having a single variant of casein, such as alpha casein, may be comparable to the smoothness of a yogurt or yogurt analog made with a micellar form of casein, such as a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein. The smoothness of a yogurt analog made using the compositions described herein having a single variant of casein, e.g., alpha casein, may be improved / more desirable when compared to the smoothness of a yogurt or yogurt analog made using a micellar form of casein, e.g., a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein, or compared to a plant-based cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-based proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or no protein (such as a cheese-like product made primarily with starch)).

[0148] The odor of a yogurt analog made with a single variant of casein, such as alpha casein, by the methods described herein, may be comparable to the odor of a similar type of yogurt made with dairy proteins of animal origin, such as yogurt made from animal milk. The odor of a yogurt analog made using the compositions described herein having a single variant of casein, such as alpha casein, may be comparable to the odor of a yogurt or yogurt analog made with a micellar form of casein, such as a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein. The odor of a yogurt analog made using the compositions described herein having a single variant of casein, e.g., alpha-casein, may be improved / more desirable when compared to the odor of a yogurt or yogurt analog made using a micellar form of casein, e.g., a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein, or compared to a plant-based cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-based proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or no protein (such as a cheese-like product made primarily with starch)).

[0149] The taste of a yogurt analog made with a single variant of casein, such as alpha casein, by the methods described herein, may be comparable to the taste of a similar type of yogurt made with dairy proteins of animal origin, such as yogurt made from animal milk. The taste of a yogurt analog made using the compositions described herein having a single variant of casein, such as alpha casein, may be comparable to the taste of a yogurt or yogurt analog made with a micellar form of casein, such as a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein. The taste of a yogurt analog made using the compositions described herein having a single variant of casein, e.g., alpha casein, may be improved / more desirable when compared to the taste of a yogurt or yogurt analog made using a micellar form of casein, e.g., a yogurt made from milk or a yogurt analog made from caseinate, micellar casein or rennet casein, or compared to a plant-based cheese analog lacking dairy proteins (i.e., a cheese-like product made using plant-based proteins, e.g., pea, chickpea, nuts and / or other plant proteins as the sole / major protein source, or no protein (such as a cheese-like product made primarily with starch)). The taste of the yogurt analog can be tested using a trained human subject population. C.Beverage

[0150] Single variant casein compositions, such as compositions comprising alpha casein described herein, can be used to produce consumable compositions, such as beverages. For example, single variant alpha casein compositions can be used to form milk-like or yogurt-like drinks. Beverage products can be formed without the formation of micelles or micellar-like compositions. Beverage products made using the compositions described herein can provide similar or equivalent characteristics (such as texture, creaminess and taste) to animal-derived dairy beverages or dairy beverages made using micellar forms of casein, such as those made from caseinates or micellar casein. Beverage products made using the compositions described herein can provide improved one or more characteristics (such as texture, creaminess and taste) when compared to beverages made with plant-derived proteins.

[0151] In some cases, the beverage may be selected from the group consisting of juice products, broths, soups, sodas, soft drinks, nutritional drinks, energy drinks, sports drinks, recovery drinks, heated drinks, coffee-based drinks, tea-based drinks, milk-based drinks, yogurt-like drinks, shakes, non-dairy products, plant-based mild drinks, infant formula drinks, and meal replacement drinks. In some embodiments, the beverage includes carbonation.

[0152] The compositions described herein can be used to produce beverage compositions, such as milk or milk-like compositions. For example, a single variant of alpha casein can be used to form a milk-like analog product without the formation of micelles or micellar-like compositions. The milk-like analog products made using the compositions described herein can provide similar or equivalent characteristics (such as texture, creaminess, and taste) compared to milk-like analog products made using animal milk or micelles, such as those made from caseinate or rennet casein. The milk-like analog products made using the compositions described herein can provide improved one or more characteristics (such as texture, creaminess, and taste) compared to plant-derived milk analogs.

[0153] The beverages described herein may comprise from 0.5% w / w to about 10% w / w of a recombinant single variant of casein, e.g., alpha casein. In some cases, the beverages may comprise at least 0.5% w / w of a recombinant single variant of casein, e.g., any of the single variants of casein described herein. In some cases, the beverages may comprise at most 10% w / w of a recombinant single variant of casein, e.g., any of the single variants of casein, e.g., alpha casein, described herein. In some cases, the beverages described herein may comprise 0.5%-1%, 0.5%-2%, 0.5%-4%, 0.5%-6%, 0.5%-8%, 0.5%-10%, 1%-2%, 1%-4%, 1%-6%, 1%-8%, 1%-10%, 2%-4%, 2%-6%, 2%-8%, 2%-10%, 4%-6%, 4%-8%, 4%-10%, 6%-8%, 6%-10%, or 8%-10% w / w of a recombinant single variant of casein. The beverages described herein may comprise 0.5%, 1%, 2%, 4%, 6%, 8%, or 10% w / w of a recombinant single variant of casein, such as any of the single variants of casein described herein. In some preferred embodiments, the beverage may contain 0.5% to 5% w / w of a recombinant single variant of casein.

[0154] The beverages described herein may comprise from 0.5% w / w to about 10% w / w of a recombinant single variant of alpha casein. In some cases, the beverages may comprise at least 0.5% w / w of a recombinant single variant of alpha casein, such as any of the single variants of alpha casein described herein. In some cases, the beverages may comprise at most 10% w / w of a recombinant single variant of alpha casein, such as any of the single variants of alpha casein described herein. In some cases, the beverage may comprise 0.5%-1%, 0.5%-2%, 0.5%-4%, 0.5%-6%, 0.5%-8%, 0.5%-10%, 1%-2%, 1%-4%, 1%-6%, 1%-8%, 1%-10%, 2%-4%, 2%-6%, 2%-8%, 2%-10%, 4%-6%, 4%-8%, 4%-10%, 6%-8%, 6%-10%, or 8%-10% w / w of a recombinant single variant of alpha casein, such as any of the single variants of alpha casein described herein. In some cases, the beverage may comprise 0.5%, 1%, 2%, 4%, 6%, 8%, or 10% w / w of a recombinant single variant of alpha casein, such as any of the single variants of alpha casein described herein. In some preferred embodiments, the beverage may contain 0.5% to 5% w / w of recombinant single variant of alpha-casein.

[0155] In some embodiments, the beverage comprises a single variant casein, where the single variant casein, for example alpha casein, provides one or more properties of the beverage to the composition.

[0156] The texture of the beverages made with a single variant of casein, such as alpha casein, by the methods described herein, etc., can be comparable to the texture of similar types of beverages made with dairy proteins from animals, such as yogurt drinks made from animal milk. The texture of the beverages made with the compositions described herein having a single variant of casein, such as alpha casein, can be comparable to the texture of beverages made with micellar forms of casein, such as milk, yogurt, or beverages made from caseinate or micellar casein. The texture of the beverages made with the compositions described herein having a single variant of casein, such as alpha casein, can be improved / more desirable when compared to the texture of beverages made with micellar forms of casein, such as milk, yogurt, or beverages made from caseinate, micellar casein, or beverages made with plant-derived proteins. The texture of the beverages can be tested using a trained human subject population and using a machine, such as a viscometer for measuring viscosity. Texture characteristics can include, for example, consistency, smoothness, mouthfeel, graininess and creaminess.

[0157] The taste of the beverages made with a single variant of casein, such as alpha casein, by the methods described herein, etc., can be comparable to the taste of similar types of beverages made with dairy proteins from animals, such as yogurt drinks made from animal milk. The taste of the beverages made with the compositions described herein having a single variant of casein, such as alpha casein, can be comparable to the taste of beverages made with micellar forms of casein, such as beverages made from milk, yogurt, or caseinates, micellar casein, or rennet casein. The taste of the beverages made with the compositions described herein having a single variant of casein, such as alpha casein, can be improved / more desirable when compared to the taste of beverages made with micellar forms of casein, such as beverages made from milk, yogurt, or caseinates, micellar casein, or rennet casein, or when compared to beverages made with proteins from plants. The taste of the beverages can be tested using a trained human subject population. D. Other Consumable Compositions

[0158] In some embodiments, food, dairy or dairy-like product analogs can be produced using compositions comprising the single variants of alpha casein protein described herein. Dairy or dairy-like analog products that can be made using the compositions described herein can include milk, cream, milkshakes, creamers (liquid and powder form), ice cream, condensed milk, yogurt or cheese analogs. Cheese analogs or cheese-like products that are not made from real curd or by coagulation of liquid colloids can also be made using compositions comprising the single variants of alpha casein protein, including the full-length single variants of alpha casein and optionally its truncated forms, as described herein.

[0159] The single variant alpha casein compositions described herein can be used to produce consumable compositions, such as dairy cream analogs or cream-like compositions. For example, the single variant alpha casein compositions can be used to form dairy cream analog products. Dairy cream analog products can be formed without the formation of micelles or micelle-like compositions. Cream analog products made using the compositions described herein can provide similar or equivalent characteristics (such as texture, creaminess and taste) as animal-derived dairy creams or dairy cream analogs made using micelles, such as those made from caseinates or rennet caseins. Cream analog products made using the compositions described herein can provide improved one or more characteristics (such as texture, creaminess and taste) when compared to plant-derived cream analogs.

[0160] The compositions described herein can be used to generate consumable compositions, such as ice cream analog compositions. For example, single variant alpha casein compositions can be used to form ice cream analog products. Ice cream analog products can be formed without the formation of micelles or micelle-like compositions. Ice cream analog products made using the compositions described herein can provide similar or equivalent to animal-derived dairy ice cream analogs, or ice cream analogs made using micelles, such as those made from caseinates or rennet caseins. Ice cream analog products made using the compositions described herein can provide improved one or more characteristics, such as texture, creaminess, and taste, when compared to plant-derived ice cream analogs.

[0161] The compositions described herein can be used to produce a variety of consumable compositions, including, but not limited to, beverages (e.g., energy drinks, dairy-related drinks, etc.), salad dressings, baking ingredients, cooking ingredients, etc. For example, the single variant alpha casein compositions described herein can be used to produce yogurt drinks, ranch dressings, etc. As an additional example, the single variant alpha casein compositions described herein can be used to produce ingredients used for baking and cooking. E. Other components

[0162] The compositions described herein can be used as ingredients in producing consumable compositions, such as food products. Food products can include cheese analogs, yogurt analog products and other food products described elsewhere herein. Such consumable compositions can include one or more ingredients in addition to the single variant casein protein. Ingredients can include, but are not limited to, solvents, salts, sugars, fats, flavors, colorants, and the like.

[0163] Consumable compositions comprising single variant casein protein may include salts, such as calcium, phosphorous, citrate, potassium, sodium and / or chloride salts. Calcium salts may be selected from calcium chloride, calcium carbonate, calcium citrate, calcium glubionate, calcium lactate, calcium gluconate, calcium acetate, equivalents and / or combinations thereof. Phosphates may be selected from orthophosphates, such as monosodium (dihydrogen) phosphate, disodium phosphate, trisodium phosphate, monopotassium (dihydrogen) phosphate, dipotassium phosphate, tripotassium phosphate; pyrophosphates, such as disodium or dipotassium pyrophosphate, trisodium or tripotassium pyrophosphate, tetrasodium or tetrapotassium pyrophosphate; polyphosphates, such as pentosodium or potassium tripolyphosphate, sodium or potassium tetrapolyphosphate, sodium or potassium hexametaphosphate. Citrates may be selected from calcium citrate, potassium citrate, sodium citrate, trisodium citrate, tripotassium citrate or equivalents thereof. Consumable compositions may include combinations of salts. In some embodiments, the consumable composition comprises calcium, phosphate and citrate. In some embodiments, the consumable composition comprises calcium and phosphate. In some embodiments, the consumable composition comprises calcium and citrate. In some embodiments, the consumable composition comprises phosphate and citrate.

[0164] In some embodiments, fat is added to the consumable composition. In some cases, the fat may be essentially free of fat from animal origin. As used herein, fat can include plant-based fat, such as canola oil, sunflower oil, coconut oil, palm oil, or combinations thereof. As used herein, fat can include recombinant animal or vegetable fats produced by microorganisms. As used herein, fat can include recombinant animal or vegetable fats cultured in mammalian cells.

[0165] The consumable compositions described herein may further comprise sugar. As used herein, sugar may comprise plant-based monosaccharides, disaccharides and / or oligosaccharides. Examples of sugar include sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allulose, tagatose, xylose and arabinose.

[0166] Consumable food compositions made from single variant casein proteins as described herein and methods of making such compositions can include adding or mixing with one or more ingredients. For example, food additives can be added to or mixed with the composition. Food additives can add volume and / or mass to the composition. Food additives can improve functional performance and / or physical characteristics. For example, food additives can prevent gelling or increased viscosity due to lipid portions of lipoproteins during freeze-thaw cycles. Anti-caking agents (cellulose, potato starch, corn starch, starch blends) can be added to create free-flowing compositions. Carbohydrates can be added to increase resistance to heat damage, e.g., less protein denaturation upon drying and improve stability and flowability of the dried composition. Food additives include starches (e.g., potato, processed potato, corn, rice), food colorings, pH adjusters (e.g., glucono-delta-lactone, sodium hydroxide), natural flavors (e.g., mozzarella, parmesan, butter, cream, colby, provolone, asiago, etc.), artificial flavors, flavor enhancers, aroma maskers, batch markers, food acids (e.g., lactic acid, citric acid), fillers, anti-caking agents (e.g., sodium silicoaluminate), antigreening agents (e.g., sodium silicoaluminate), and antioxidants (e.g., sodium silicoaluminate). The additives may include, but are not limited to, fatty acids, antioxidants, acidity regulators, bulking agents, color fixing agents, whipping agents (e.g., ester-type whipping agents, triethyl citrate, sodium lauryl sulfate), emulsifiers (e.g., lecithin, monoglycerides, diglycerides), humectants, thickeners, pharmaceutical excipients, solid diluents, nutrients, sweeteners, glazing agents, preservatives (e.g., sorbic acid, nisin), vitamins (e.g., vitamin B, vitamin D, vitamin A), dietary elements, carbohydrates, polyols, gums, starches, flours, oils, and bran. In some cases, the flavoring may include mozzarella flavoring, cheddar flavoring, parmesan flavoring, or other similar cheese flavoring.

[0167] Food colorants include, but are not limited to, FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Red No. 40, FD&C Red No. 3, FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 3, carotenoids (e.g., saffron, beta-carotene), annatto, betanin, butterfly pea, caramel color, chlorophyllin, elderberry juice, lycopene, carmine, pandan, paprika, turmeric, curcuminoids, quinoline yellow, carmoisine, ponceau 4R, patent blue V, and green S.

[0168] Ingredients for adjusting pH include, but are not limited to, Tris buffer, potassium phosphate, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, sodium bicarbonate, and hydrochloric acid. F. End-User Products

[0169] The consumable compositions of the single variant casein protein described herein can be used as ingredients to make final products for end users.For example, the cheese products or cheese analogs described herein can be used by end users to make final products, such as pizza, Italian food toppings, Mexican food toppings, frozen foods, toppings for savory baked goods, soups, macaroni cheese, cheese sticks, etc. In some examples, the yogurt analogs can be used to make yogurt-like products or products that contain yogurt as an ingredient. In some examples, the consumable compositions can be used to make milk-like products or milk analogs or other beverages. Recombinant expression

[0170] The protein or proteins used in forming the cheese composition may be recombinantly produced. In some cases, the single variant casein protein (e.g., single variant alpha S1 or single variant alpha S2) is recombinantly produced. The single variant casein protein, e.g., single variant alpha S1 or single variant alpha S2 casein protein, may have an amino acid sequence from any species. For example, the recombinant alpha casein protein may have the amino acid sequence of cow, sheep, goat, buffalo, horse, human, deer or camel alpha casein. The nucleotide sequence encoding the casein protein may be codon-optimized for increased production efficiency. Exemplary alpha casein protein sequences are provided in Table 1 below for use in the recombinant production of single variant casein proteins. The recombinant single variant casein protein may be a non-naturally occurring variant of casein. Such variants may include one or more amino acid insertions, deletions or substitutions compared to the native casein sequence.

[0171] Such variants may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 1-56. In some cases, the variants may be truncated forms of alpha S1 casein protein, such as those having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 4-12, 16-24.

[0172] Recombinant single variant casein protein, e.g., single variant alpha S1 casein protein, is recombinantly expressed in a host cell. As used herein, "host" or "host cell" refers to any protein production host that is selected or genetically modified to produce a desired product. Exemplary hosts include bacteria, yeast, fungi, plants, insects and mammalian cells. In some cases, bacterial host cells, e.g., Lactococcus lactis, Bacillus subtilis or Escherichia coli, can be used to produce alpha casein protein and / or its truncated forms. Other host cells include bacterial hosts, including, but not limited to, Lactococci sp., Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, and Synechocystis sp. 6803.

[0173] In some embodiments, the full-length single variant alpha casein protein and / or its truncated forms are recombinantly produced in a host cell. For example, full-length and truncated single variant alpha S1 casein proteins can be produced in the same host cell, and such production can begin from the same open reading frame (i.e., the same expression cassette), with the truncated forms being produced, for example, by post-translational proteolytic cleavage or produced from separate open reading frames, such as using an expression cassette encoding the full-length alpha casein variant and one or more expression cassettes encoding the truncated open reading frames for the truncated forms of the alpha casein variant. Alternatively, full-length and truncated single variant alpha S1 casein proteins can be produced in different host cells. Expression of the target protein can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome, or other means. For example, a vector for expression can include (a) a promoter element, (b) a signal peptide, (c) a heterologous casein sequence, and (d) a terminator element.

[0174] Expression vectors that can be used for the expression of casein include expression vectors containing an expression cassette having elements (a), (b), (c) and (d). In some embodiments, the signal peptide (b) and / or the terminator element (d) may not necessarily be included in the vector. In some cases, the signal peptide may be part of the native signal sequence of the casein protein, for example, the protein may comprise the native signal sequence shown in bold in SEQ ID NO: 1, 13, 25, 28, 31, 34, 37, 39, 42, 45, 48, 51 or 54. In some cases, the vector may comprise the mature protein sequence exemplified in SEQ ID NO: 2, 3, 4-12, 14, 15, 16-24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56 together with a heterologous signal sequence. In some cases, the protein may not include a signal sequence, but instead may include an initiator methionine, as exemplified in SEQ ID NOs: 3, 5, 7, 9, 11, 12, 15, 17, 19, 21, 23, 24, 27, 30, 33, 36, 41, 44, 47, 50, 53, or 56. Generally, expression cassettes are designed to mediate transcription of a transgene when integrated into the genome of a cognate host microbial organism or when present on a plasmid or other replicating vector that is maintained in a host cell.

[0175] An origin of replication (e) may be contained in the vector to aid in the amplification of the vector prior to transformation into the host microorganism. A vector may also contain a selection marker (f) to aid in the selection of microorganisms stably transformed with the expression vector. An expression vector may also contain a restriction enzyme site (g) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate stable integration of the expression vector into the host genome. In some embodiments, an expression vector may contain any subset of elements (b), (e), (f) and (g), including none of elements (b), (e), (f) and (g). Other expression elements and vector elements known to those skilled in the art may be used in combination with or in place of the elements described herein.

[0176] Gram-positive bacteria (such as Lactococcus lactis and Bacillus subtilis) can be used to secrete the target protein into the medium, and Gram-negative bacteria (such as Escherichia coli) can be used to secrete the target protein into the periplasm or medium. In some embodiments, the protein expressed by the expressed bacteria may not have any post-translational modifications (PTMs), meaning that it may not be glycosylated and / or phosphorylated. Both Gram-positive and Gram-negative bacteria can be used to produce the protein intracellularly. In such instances, the cells can be lysed and the protein can be recovered.

[0177] Single variant casein proteins can be expressed and produced in L. lactis in both nisin-inducible expression systems (regulated by the PnisA promoter), lactate-inducible expression systems (regulated by the P170 promoter) or other similar inducible systems, as well as in constitutively expressed systems (regulated by the PsecA promoter), both in food-grade selection strains such as NZ3900 using vector pNZ8149 (lacF gene supplementation / rescue principle). Secretion of functional proteins can be enabled by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by L. lactis. For example, alpha S1 casein and its truncates can be co-expressed or individually expressed in L. lactis using synthetic operons.

[0178] B. subtilis has multiple intracellular and extracellular proteases that can interfere with protein expression. In some embodiments, B. subtilis strains are modified to reduce the types and amounts of intracellular and / or extracellular proteases, for example, strains deleted for 7 (KO7) and 8 (WB800N), respectively, can be used.

[0179] To drive recombinant protein secretion, the signal peptide of Clostridium thermocellum alpha-amylase, amyQ, can be used, or another bacterial signal peptide known in the art can be used. Moreover, native casein signal peptide sequences can be heterologously expressed in B. subtilis. Each casein protein has its own signal peptide sequence that can be used in the system. Signal proteins can cross-combine with casein proteins. The pHT01 vector can be used as a transformation and expression shuttle for inducible protein expression in B. subtilis. The vector contains a strong σ preceding the B. subtilis groES-groE operaon. A Based on a CpG-dependent promoter, this was converted into an efficiently controllable (IPTG-inducible) promoter by the addition of the lac operator. pHT01 is an E. coli / B. subtilis shuttle vector that provides ampicillin resistance to E. coli and chloramphenicol resistance to B. subtilis.

[0180] The single variant casein proteins can be produced in E. coli using safe laboratory strains, for example E. coli BL21 (exemplary strains BL21(DE3) or BL21 AI) or its derivatives, or wild type or its derivatives, such as K12 strains (exemplary strains MG1655 or W3110). Inducible (IPTG-inducible, lactose-inducible, arabinose-inducible, rhamnose-inducible, etc.), autoinducible (phosphate depletion-based, etc.) and constitutive promoters can be used to drive casein expression. The single variant casein proteins can be produced intracellularly or secreted into the periplasm and / or supernatant. Bacterial signal peptides of the Sec-dependent secretion pathway (OmpA, OmpC, OmpT, pelB, LamB, etc.), the SRP secretion pathway (TolA, DsbA, DsbC, TorT, etc.) and the TAT secretion pathway (TorA, SufI, etc.) can be used to drive recombinant protein secretion. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0181] Embodiment [Embodiment 1]: A consumable composition comprising a recombinant single variant of alpha-casein protein, wherein the single variant provides at least one dairy-like property selected from the group consisting of adhesion, extensibility, texture, mouthfeel, melting, browning, hardness, creaminess, taste, odor and softness, wherein the single variant is not an animal-derived casein and is not physically dissociated from the casein micelles, and wherein the composition is devoid of any added casein.

[0182] [Embodiment 2]: The consumable composition of embodiment 1, wherein the single variant of alpha-casein protein is not derived from caseinate.

[0183] [Embodiment 3]: A consumable composition according to embodiment 1 or embodiment 2, wherein the single variant of alpha casein protein is alpha S1 casein protein.

[0184] [Embodiment 4]: A consumable composition according to embodiment 1 or embodiment 2, wherein the single variant of alpha casein protein is alpha S2 casein protein.

[0185] [Embodiment 5]: A consumable composition according to any of embodiments 1 to 3, which does not contain any animal-produced protein.

[0186] [Embodiment 6]: The consumable composition of embodiment 4, lacking any other animal-derived dairy protein.

[0187] [Embodiment 7]: A consumable composition according to any one of embodiments 1 to 5, wherein at least one dairy-like property is improved compared to a cheese analogue derived from milk.

[0188] [Embodiment 8]: A consumable composition according to any one of embodiments 1 to 5, wherein at least one dairy-like property is improved compared to a cheese analogue derived from caseinate or improved compared to a cheese analogue derived from rennet casein.

[0189] [Embodiment 9]: A consumable composition according to any one of embodiments 1 to 5, wherein at least one dairy-like property is improved compared to a plant-derived cheese analog.

[0190] [Embodiment 10]: A consumable composition according to any of embodiments 1 to 9, wherein the single variant of alpha-casein protein comprises at least one non-native post-translational modification.

[0191] [Embodiment 11]: A consumable composition according to embodiment 10, wherein the single variant of alpha-casein protein further comprises at least one native post-translational modification.

[0192] [Embodiment 12]: A consumable composition described in any of embodiments 1 to 11, wherein the single variant of alpha-casein protein lacks one or more post-translational modifications of native alpha-casein protein.

[0193] [Embodiment 13]: A consumable composition according to embodiment 12, wherein the single variant of alpha-casein protein further comprises at least one non-native post-translational modification.

[0194] [Embodiment 14]: A consumable composition according to embodiment 13, wherein the single variant of alpha-casein protein is not post-translationally modified.

[0195] [Embodiment 15]: A consumable composition according to any one of embodiments 1 to 14, comprising full-length alpha-casein protein.

[0196] [Embodiment 16]: A consumable composition according to any one of embodiments 1 to 14, wherein the single variant of alpha casein protein comprises any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56.

[0197] [Embodiment 17]: The consumable composition of embodiment 16, further comprising one or more truncated alpha-casein proteins.

[0198] [Embodiment 18]: A consumable composition according to embodiment 17, wherein the truncated alpha-casein protein lacks one or more N-terminal amino acids of mature native alpha-casein protein.

[0199] [Embodiment 19]: The consumable composition of embodiment 18, wherein the truncated alpha-casein protein is selected from the group consisting of alpha-casein lacking between 1 and 23 N-terminal amino acids of native alpha-casein protein or alpha-casein lacking between 1 and 59 N-terminal amino acids of native alpha-casein protein, or a combination thereof.

[0200] [Embodiment 20]: The consumable composition of embodiment 19, wherein the truncated alpha-casein protein comprises any one of SEQ ID NOs: 4-12, 16-24.

[0201] [Embodiment 21]: A consumable composition according to any of embodiments 17 to 20, wherein the truncated alpha-casein protein lacks one or more C-terminal amino acids of native alpha-casein protein.

[0202] [Embodiment 22]: A consumable composition as described in embodiment 17, wherein between 0% and 20% wt / wt of the total recombinant alpha-casein protein of the composition is one or more truncated forms of alpha-casein protein.

[0203] [Embodiment 23]: A consumable composition according to embodiment 22, wherein the one or more truncated forms of alpha-casein protein constitute between 1% and 20% wt / wt of the total recombinant alpha-casein protein of the composition.

[0204] [Embodiment 24]: A consumable composition according to any of embodiments 1 to 23, wherein the recombinant alpha-casein protein comprises the amino acid sequence of bovine, caprine or ovine alpha-casein protein, or any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56, or a sequence having at least 70%, 80%, 85% or 90% identity to any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56.

[0205] [Embodiment 25]: A consumable composition described in any of embodiments 1 to 24, wherein the single variant of alpha-casein protein contains one or more non-native amino acids at the N-terminus.

[0206] [Embodiment 26]: A consumable composition described in embodiment 25, wherein the single variant of alpha-casein protein includes a non-native methionine at the N-terminal position.

[0207] [Embodiment 27]: A consumable composition described in any one of embodiments 1 to 26, wherein the single variant of alpha-casein protein is not derived from casein micelles.

[0208] [Embodiment 28]: A dairy product analog comprising any of the consumable compositions described in embodiments 1 to 27, the dairy product analog being selected from the group consisting of cheese analogs, yogurt analogs, cream analogs, and ice cream analogs.

[0209] [Embodiment 29]: The dairy analog of embodiment 28, further comprising a fat or oil derived from a non-animal source.

[0210] [Embodiment 30]: A dairy analogue as described in embodiment 28 or embodiment 29, which lacks any animal-derived dairy protein.

[0211] [Embodiment 31]: A dairy product analog described in any of embodiments 28 to 30, lacking any other casein protein.

[0212] [Embodiment 32]: A dairy product analog described in any of embodiments 28 to 31, wherein a single variant of alpha-casein protein is not contained in a micellar form within the dairy product analog.

[0213] [Embodiment 33]: A dairy product analog described in any one of embodiments 28 to 32, which is a cheese analog.

[0214] [Embodiment 34]: The dairy analog of embodiment 33, wherein the cheese analog is a mozzarella analog, a cheddar analog, or a parmesan analog.

[0215] [Embodiment 35]: A dairy product analog described in embodiment 33, wherein the cheese analog is a mozzarella analog and the single variant of alpha casein protein is alpha S1 casein.

[0216] [Embodiment 36]: A dairy analogue as described in embodiment 35, wherein the alpha S1 casein is bovine alpha S1 casein and the alpha S1 casein in the composition comprises between 0-20% of one or more truncated forms of alpha casein.

[0217] [Embodiment 37]: A dairy analogue as described in embodiment 35, wherein the alpha S1 casein in the composition comprises between 1 and 20% of one or more truncated forms of alpha S1 casein.

[0218] [Embodiment 38]: A dairy analogue according to embodiment 36 or embodiment 37, comprising an N-terminally truncated form of alpha S1 casein.

[0219] [Embodiment 39]: The dairy product analog of embodiment 38, wherein the N-terminal truncated form is selected from any one of SEQ ID NOs: 4-12, 16-24, or a combination thereof.

[0220] [Embodiment 40]: A dairy product analog described in any one of embodiments 28 to 39, further comprising one or more of: (a) oil derived from a plant; (b) starch derived from a plant; (c) sugar; and (d) salt. EXAMPLES

[0221] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way. Example 1 Expression of casein proteins in Lactococcus lactis using a nisin-inducible system (NICE) Construct design, cloning and transformation The bovine alpha S1 casein (variant C) protein coding sequence (without native signal peptide) was codon-optimized for expression in Lactococcus lactis and a synthetic operon was constructed for simultaneous expression and secretion of two proteins under a nisin-inducible promoter. A signal peptide sequence from the natively secreted lactococcal protein Usp45 was used to drive protein secretion. The synthetic operon was then cloned into an E. coli custom vector via restriction enzyme digestion compatible sites, verified by Sanger sequencing, and from there subcloned into the nisin-inducible pNZ8149 vector by restriction enzyme digestion and ligation.

[0222] The vector was transformed into the competent L. lactis strain NZ3900 by electroporation, and complete defined medium (CDM) supplemented with lactose was used for selection. Positive clones were confirmed by colony PCR, and three positive clones were subjected to protein expression induction and analysis. Protein expression and analysis

[0223] Individual colonies were grown in liquid culture at 30°C and protein production was induced with nisin for 2.5 hours (control samples were left uninduced). Cells were then harvested by centrifugation and TCA precipitated supernatants and lysed cell pellets were analyzed by Coomassie gel staining (SDS-PAGE) and chemiluminescence (western blot against alpha S1 casein, LSBio primary antibody). Example 2 Expression in L. lactis by a pH-inducible system

[0224] Similar to the constructs described above, the alpha-casein protein constructs were created by replacing the nisin promoter with the P170 promoter, a pH / lactate inducible promoter for L. lactis. Each of these constructs contained a secretion signal peptide.

[0225] Alpha S1 casein and its truncated forms were detected in L. lactis by secretion in Western blots. The unprocessed protein product accumulated intracellularly, whereas secretion of the mature protein and its truncated forms was detected. Example 3 Expression in B. subtilis Construct design, cloning and transformation

[0226] C-terminally His-tagged bovine alpha S1 casein (variant C) protein coding sequence (without native signal peptide) was codon-optimized for expression in Bacillus subtilis. Constructs were created with and without the codon-optimized signal peptide of amyQ, an alpha-amylase Bacillus amyloliquefaciens reported for efficient secretion of recombinant proteins. Transformation and Expression Constructs were cloned in E. coli by Gibson cloning into IPTG-inducible vector pHT01 and confirmed by Sanger sequencing. pHT01 is an E. coli / B. subtilis shuttle vector that provides ampicillin resistance to E. coli and chloramphenicol resistance to B. subtilis. Positive clones were further transformed into chemically competent B. subtilis WB800N. Positive clones were confirmed by colony PCR and three positive clones were subjected to protein expression induction and analysis. Protein expression and analysis

[0227] Individual colonies were grown in liquid culture at 37°C and protein production was induced with IPTG for 1, 2 and 6 hours (control samples were left uninduced). Cells were then harvested by centrifugation and TCA precipitated supernatants and lysed cell pellets were analyzed by Coomassie gel staining (SDS-PAGE) and chemiluminescence (Western blot against His-tag and alpha S1 casein).

[0228] Western blotting showed the expression of alpha S1 casein in B. subtilis. Example 4 Expression in E. coli Construct design, cloning and transformation

[0229] Bovine alpha S1 casein (variant C) or ovine alpha S1 casein protein coding sequence (without native signal peptide) codon-optimized for Escherichia coli was cloned into IPTG-inducible commercially available pET vectors. Cloning was performed by Gibson reaction of DNA fragments and vectors in such a way that only the protein coding sequence remained in the open reading frame. Gibson reactions were transformed into competent cells and verified by Sanger sequencing. The vectors were then transformed into chemically competent E. coli BL21(DE3) cells or its derivatives or wild type or its derivatives such as K12 strains and several single colonies were screened for expression. Protein expression, analysis and purification

[0230] Individual colonies were grown in liquid culture at 37C and protein production was induced with IPTG for 4 hours. Cells were then collected by centrifugation and lysed cell pellets were analyzed by Coomassie gel staining (SDS-PAGE) and chemiluminescence (Western blot against alpha S1 casein). Phase separation was used to purify the protein. The purified product was analyzed in a Coomassie stained gel as described above. Alpha S1 casein was expressed intracellularly in E. coli and successfully detected and purified in a Coomassie stained protein gel. An exemplary production of alpha casein is illustrated in Figure 1, where alpha S1 casein and two variants were also found: N-terminally truncated F24-199 bovine alpha S1 casein and N-terminally truncated M60-199 bovine alpha S1 casein. Example 5 Mozzarella cheese analogues derived from recombinant single variant alpha-casein proteins and their properties

[0231] Recombinant non-phosphorylated bovine alpha S1 casein was used to produce a non-micellar mozzarella cheese analog named NC mozzarella cheese. Casein, pre-warmed coconut oil, trisodium citrate, disodium phosphate, salt and glucose were added to a beaker at the concentrations specified in Table 2. To this, a mix of water, CaCl2 and modified potato starch was added at the concentrations specified in Table 2. The beaker was transferred to a water bath preset at a temperature of 85°C and the contents were mixed using a mixing propeller at a speed of 300 rpm for 9 minutes. Lactic acid was added and mixed for an additional minute. The resulting mixture became a homogenous non-micellar mass which was transferred to a standard mold and left in a refrigerator for 16-24 hours. After incubation, the NC mozzarella cheese analog was weighed to obtain a yield estimate. [Table 2-1] [Table 2-2]

[0232] The NC1 mozzarella cheese analogue samples were analyzed for qualitative and quantitative parameters, such as pH, extensibility and texture profile. These parameters were compared with commercial low moisture mozzarella cheese, commercial imitation mozzarella cheese and commercial plant-based vegan mozzarella style cheese. The low moisture mozzarella cheese is made from milk, where casein is micellar, resulting in its native high complexity of variants (alpha S1, alpha S2, beta, kappa; phosphorylation and glycosylation). The imitation mozzarella cheese is made from milk casein blend with plant-based fat, where micellar casein from milk is renneted and dried to milk protein concentrate. The plant-based vegan mozzarella style cheese does not have casein protein.

[0233] A pizza fork extensibility test was performed to quantify cheese extensibility by inserting a fork into the center of a freshly baked pizza and lifting until all of the cheese strings were broken. To do this, 6g of cheese was chopped into strips and placed on a 4" inch tortilla topped with 4g of tomato sauce. The samples were baked at 600°F for 90 seconds. A ruler was used to measure extensibility and the test was performed immediately after removing the pizza from the oven.

[0234] Texture profiles were analyzed on a TA-XT plus texture analyzer equipped with a TA-55 puncture probe. Cheese samples of 1.5-1.9 g were cut to dimensions of 1.5 cm l x 1.5 cm w x 1 cm h. Tests were performed on samples stored at 4°C and at ambient temperature for at least 30 minutes.

[0235] The NC1 Mozzarella cheese analog made from recombinant non-phosphorylated alpha S1 casein and its truncated forms as the sole protein component in the cheese showed animal-derived dairy-like melting, dairy-like stretchiness and dairy-like texture properties in the tests performed. The pH of the set cheese was 5.9.

[0236] Pizza fork extensibility testing showed that the NC1 mozzarella cheese analog stretched >12 inches (Table 3, Figure 2). In comparison, the low moisture mozzarella stretched >18 inches, while the imitation mozzarella stretched only <10 inches. The plant-based vegan mozzarella style cheese showed no extensibility, stretching <1 inch.

[0237] Texture profile analysis showed hardness, adhesion, resilience, cohesiveness, springiness and chewiness as shown in Table 3 (Table 3, Figure 3). The NC1 Mozzarella cheese analog showed almost the same cohesiveness and springiness (<10% deviation) and very similar hardness and chewiness profile (<30% deviation) when compared to real dairy low moisture mozzarella. It was consistently slightly harder, more resilient, more cohesive and chewier compared to both low moisture mozzarella and imitation mozzarella cheese. In comparison, the plant-based vegan mozzarella style cheese deviated from the dairy behavior and was extremely hard (approximately 4.6x harder), chewy (approximately 3.9x chewier) and adhesive (approximately 19.6x adhesive).

[0238] Interestingly, when compared to real dairy low moisture mozzarella, the NC1 mozzarella cheese analog shows different trends from the imitation mozzarella in terms of adhesion and elasticity. The imitation mozzarella is more adhesive and elastic than real dairy mozzarella, while the NC1 mozzarella cheese analog is less adhesive and elastic than real dairy mozzarella. Reduced adhesion is an advantageous cheese property, since adhesion represents the force required to remove the cheese from the probe (i.e., to remove it from the teeth). [Table 3-1] [Table 3-2] Example 6 Properties of Mozzarella Cheese Analogues Derived from Recombinant Single Variant Alpha-Casein Proteins with an Altered Formulation Lacking Calcium Chloride

[0239] Recombinant non-phosphorylated alpha S1 casein was used to make a non-micellar Mozzarella cheese analog named New Culture (NC2) Mozzarella cheese. Casein, pre-warmed coconut oil, trisodium citrate, disodium phosphate, sodium chloride and glucose were added to a beaker at the concentrations specified in Table 4. To this, water and a mix of modified potato starch were added at the concentrations specified in Table 4. The beaker was transferred to a water bath preset at a temperature of 85°C and the contents were mixed using a mixing propeller at a speed of 300 rpm for 9 minutes. Lactic acid was added and mixed for an additional minute. The resulting mixture became a homogenous non-micellar mass which was transferred to a standard mold and left in a refrigerator for 3 days. After refrigerator storage, the NC Mozzarella cheese analog was weighed to obtain a yield estimate. [Table 4]

[0240] The NC2 Mozzarella cheese analog samples were analyzed for qualitative and quantitative parameters, such as pH, moisture, melting, extensibility and texture profile, and were compared to a commercial low moisture Mozzarella cheese, a commercial imitation Mozzarella cheese and a commercial plant-based vegan Mozzarella style cheese (see Example 5 for further description of these cheeses).

[0241] Cheese melting was quantified by a modified Schreiber melting test using a custom imaging station mounted on a black magnetic hotbed. 0.5 g of cheese was melted on the magnetic hotbed at 95° C. for 15 min. A time lapse of melting was recorded and the increase in melted area was measured by measuring pixels. Melting values ​​were calculated by dividing the melted area by the unmelted area before melting. A melting value greater than 1 indicates melting.

[0242] Extensibility tests were performed on a texture analyzer to quantify cheese extensibility by inserting a six-pronged hook into a 6 g sample of hand-cut cheese strips placed in an extensibility fixture that had been molten at 90° C. in an oven for 10 minutes and lifting until all of the cheese strings had broken. The distance to break (breaking all strings), which represents the degree of cheese extensibility, and the work to stretch, which represents the tensile strength required to stretch the cheese, were quantified. Tests were performed on samples that had been removed from the refrigerator and then held at ambient temperature for at least 30 minutes. [Table 5]

[0243] NC2 Mozzarella cheese analog made from recombinant non-phosphorylated alpha S1 casein in a calcium-deficient formulation exhibited dairy-like melting, dairy-like extensibility and dairy-like spreadability properties. The moisture and pH of the solidified NC2 Mozzarella cheese analog were 45.7% and 5.7, respectively.

[0244] Surprisingly, the NC2 Mozzarella cheese analog made using the compositions described herein demonstrated superior melting to the formulations in Examples 5 and 7, reaching a melting value of 2.1× (Table 5, FIG. 4). In comparison, the low moisture Mozzarella and imitation Mozzarella reached melting values ​​of 2.5× and 2.0×, respectively. The plant-based vegan Mozzarella style cheese reached a melting value of 0.8×, suggesting a lack of melting (and even shrinkage).

[0245] The NC2 Mozzarella cheese analog melted at a slower rate than the low moisture mozzarella during the modified Schreiber melt test and reached its peak melt slightly later. The imitation and low moisture mozzarella required approximately 4-6 minutes for maximum melting, while the NC2 Mozzarella cheese analog was completely melted in approximately 6.5 minutes (Figure 4).

[0246] The NC2 mozzarella cheese analog showed nearly identical extensibility as the low moisture and imitation mozzarella, while the plant-based vegan mozzarella style cheese was not able to stretch at all (Table 5, FIG. 5). The tensile strength required to stretch the NC2 mozzarella cheese analog (as indicated by the work to stretch values, Table 5) was between the range required to stretch the low moisture and imitation mozzarella. As indicated by the distance to break values ​​in Table 5, the NC2 cheese analog stretched comparably to the low moisture mozzarella and significantly better than the imitation mozzarella. The NC2 mozzarella cheese analog stretched to a length of approximately 228 mm. In comparison, the low moisture and imitation mozzarella stretched to lengths of approximately 224 mm and 189 mm, respectively (Table 5). Example 7 Properties of Mozzarella Cheese Analogues Derived from Recombinant Single Variant Alpha-Casein Proteins with Different Fat Compositions

[0247] Recombinant non-phosphorylated alpha S1 casein was used to make a non-micellar Mozzarella cheese analog named New Culture (NC3) Mozzarella cheese. Casein, water, palm stearin, canola oil, trisodium citrate, disodium phosphate, modified potato starch and sodium chloride were added to a beaker at the concentrations specified in Table 6. To this, CaCl2 was added at the concentrations specified in Table 6. The beaker was transferred to a water bath preset at 85°C and the contents were mixed using a mixing propeller at a speed of 300 rpm for 9 minutes. Natural flavors and lactic acid were added and the ingredients were mixed for an additional minute. The resulting mixture became a homogenous non-micellar mass which was transferred to a standard mold and left in a refrigerator for 7 days. After incubation, the NC3 Mozzarella cheese analog was weighed to obtain a yield estimate. [Table 6-1] [Table 6-2]

[0248] The NC3 mozzarella cheese analog samples were analyzed for qualitative and quantitative parameters, such as pH, moisture, melting, extensibility and texture profile, as shown in Example 6. These parameters were compared to low moisture mozzarella cheese, imitation mozzarella cheese and a plant-based vegan mozzarella style cheese (see Example 5 for further description).

[0249] Cheese melting was quantified by a modified Schreiber melting test as described in Example 6. A spreadability test was performed in a TA.XTPlus texture analyzer to quantify cheese spreadability as described in Example 6. The test was performed on samples stored at 4°C. [Table 7]

[0250] Texture profiles were analyzed on a TA-XT plus texture analyzer equipped with a TA-55 puncture probe. Cheese samples of 1.5-1.9 g were cut to dimensions of 1.5 cm l x 1.5 cm w x 1 cm h. Tests were performed on samples stored at 4°C. [Table 8]

[0251] NC3 Mozzarella cheese analogs made from recombinant non-phosphorylated alpha S1 casein with different fat compositions exhibited dairy-like melting, dairy-like extensibility and dairy-like spreadability properties. The moisture and pH of the solidified NC3 Mozzarella cheese analogs were 44.8% and 5.7, respectively.

[0252] The NC3 Mozzarella cheese analog made using the compositions described herein reached a melting value of 1.6x (Table 7, Figure 6). In comparison, the low moisture Mozzarella and imitation Mozzarella reached melting values ​​of 2.5x and 2x, respectively. The plant-based vegan Mozzarella style cheese reached a melting value of 0.8x, suggesting a lack of melting (and even shrinkage).

[0253] Surprisingly, the NC3 Mozzarella cheese analog melted at a faster rate and reached its peak melt sooner than the low moisture mozzarella during the modified Schreiber melt test. The imitation and low moisture mozzarella required approximately 4-6 minutes for maximum melting, while the NC3 Mozzarella cheese analog was completely melted in approximately 2 minutes (Figure 6).

[0254] This is likely due to the replacement of coconut oil with a combination of palm stearin and canola oil.

[0255] The NC3 mozzarella cheese analog showed nearly identical extensibility as the low moisture and imitation mozzarella, while the plant-based vegan mozzarella style cheese was not able to stretch at all (Table 7, FIG. 7). The tensile strength required to stretch the NC1 mozzarella cheese analog (as indicated by the work to stretch values, Table 7) was similar to that required to stretch the low moisture mozzarella, while the imitation mozzarella required a significantly lower tensile strength. The NC3 cheese analog stretched to the same extent as the low moisture and imitation mozzarella, as indicated by the distance to break values ​​in Table 7. The NC3 mozzarella cheese analog stretched to a length of approximately 230 mm. In comparison, the low moisture and imitation mozzarella stretched to lengths of approximately 228 mm and approximately 225 mm, respectively (Table 7).

[0256] The texture profile analysis showed hardness, adhesion, resilience, cohesiveness, springiness and chewiness (as shown in Table 8, Figure 8). The NC3 Mozzarella cheese analog showed almost the same chewiness (<10% deviation) when compared to the imitation mozzarella. In comparison, the plant-based vegan mozzarella style cheese has a higher chewiness (>1.5x) compared to the low moisture mozzarella and imitation mozzarella. The NC3 Mozzarella cheese shows similar resilience (<10% deviation), higher hardness, springiness and lower adhesion and cohesiveness compared to other commercial cheeses.

[0257] Interestingly, when compared to animal-derived dairy low moisture mozzarella, the NC3 mozzarella cheese analogue shows a different trend in adhesion than the imitation mozzarella. The imitation mozzarella is more adhesive than the real dairy mozzarella, while the NC3 mozzarella cheese analogue is less adhesive. Reduced adhesion is a favorable cheese attribute, since adhesion represents the force required to remove the cheese from the probe, which corresponds to stickiness in the consumer's mouth (i.e., sticking to the teeth when biting). Example 8 Properties of Mozzarella Cheese Analogues Derived from Recombinant Single Variant Alpha-Casein Proteins with Modified Formulations and Altered Processing Parameters

[0258] Recombinant non-phosphorylated alpha S1 casein was used to make a non-micellar mozzarella cheese analog named New Culture (NC4) Mozzarella cheese. Casein, water, palm stearin, canola oil, modified potato starch, sodium chloride and CaCl2 were added to a beaker at the concentrations specified in Table 9. To this, trisodium citrate and dipotassium phosphate were added at the concentrations specified in Table 9. The beaker was transferred to a water bath preset at 85°C and the contents were mixed using a mixing propeller at a speed of 500 rpm for 4 minutes, followed by 300 rpm for 1 minute, at which point the natural flavors were also added. Lactic acid was added and the ingredients were mixed for an additional minute at 300 rpm. The resulting mixture became a homogenous non-micellar mass which was transferred to a standard mold and left in a refrigerator for 7 days. After incubation, the NC4 mozzarella cheese analog was weighed to obtain a yield estimate. [Table 9-1] [Table 9-2]

[0259] The NC4 Mozzarella cheese analog samples were analyzed for qualitative and quantitative parameters such as pH, moisture, melting, extensibility and texture profile, and were compared with low moisture Mozzarella cheese, imitation Mozzarella cheese and plant-based vegan Mozzarella style cheese (see Example 5 for further description).

[0260] Cheese melting was quantified by a modified Schreiber melting test as described in Example 6. A spreadability test was performed in a TA.XTPlus texture analyzer to quantify cheese spreadability as described in Example 6. The test was performed on samples stored at 4°C. [Table 10]

[0261] NC4 Mozzarella cheese analogs made from recombinant non-phosphorylated alpha S1 casein and its truncated forms as the sole protein component in the cheese using different formulations and different processing parameters compared to Examples 5, 6 and 7 also exhibited dairy-like melting, dairy-like extensibility and dairy-like spreadability properties. The moisture and pH of the set NC4 Mozzarella cheese analogs were 48.1% and 5.7, respectively.

[0262] The NC4 Mozzarella cheese analog using the compositions described herein reached a melting value of 1.5x (Table 10, Figure 9). In comparison, the low moisture Mozzarella and imitation Mozzarella reached melting values ​​of 2.5x and 2x, respectively. The plant-based vegan Mozzarella style cheese reached a melting value of 0.8x, suggesting a lack of melting (and even shrinkage).

[0263] More notably, the NC4 Mozzarella cheese analog melted at a faster rate and reached its peak melt sooner than the low moisture mozzarella during the modified Schreiber melt test: the imitation and low moisture mozzarella required approximately 4-6 minutes for maximum melting, while the NC4 Mozzarella cheese analog was completely melted in approximately 2 minutes (Figure 9).

[0264] The NC4 mozzarella cheese analog showed similar extensibility as the low moisture mozzarella and imitation mozzarella, while the plant-based vegan mozzarella style cheese was not able to stretch at all (Table 10, Figure 10). The tensile strength required to stretch the NC4 mozzarella cheese analog (as indicated by the work to stretch values, Table 10) was within the range of the tensile strengths of the low moisture mozzarella and imitation mozzarella. The NC4 cheese analog stretched better than the low moisture mozzarella and imitation mozzarella, as indicated by the distance to break values ​​in Table 10. The NC4 mozzarella cheese analog stretched to a length of approximately 241 mm. In comparison, the low moisture mozzarella and imitation mozzarella stretched to a length of approximately 228 mm and approximately 225 mm, respectively (Table 10, Figure 10). Example 9 Properties of Mozzarella Cheese Analogues Derived from Recombinant Single Variant Alpha-Casein Proteins from Different Species

[0265] Recombinant non-phosphorylated ovine alpha S1 casein was used to make a non-micellar Mozzarella cheese analog named New Culture (NC5) Mozzarella cheese. Casein, water, palm stearin, canola oil, trisodium citrate, disodium phosphate, modified potato starch and sodium chloride were added to a beaker at the concentrations specified in Table 11. The beaker was transferred to a water bath preset at 85°C and the contents were mixed using a mixing propeller at a speed of 300 rpm for 9 minutes. Natural flavors were added and the ingredients were mixed for an additional minute. The resulting mixture became a homogenous non-micellar mass which was transferred to a standard mold and left in a refrigerator for 5 days. After incubation, the NC5 Mozzarella cheese analog was weighed for yield estimation. [Table 11]

[0266] The NC5 mozzarella cheese analog samples were analyzed for qualitative and quantitative parameters, such as pH, moisture, melting, extensibility and texture profile, as described in Example 7. These parameters were compared to low moisture mozzarella, imitation mozzarella and a plant-based vegan mozzarella style cheese (see Example 7 for further description). [Table 12]

[0267] NC5 Mozzarella cheese analog made from recombinant non-phosphorylated ovine alpha S1 casein as the sole protein component in the cheese exhibited the dairy-like melting, dairy-like extensibility and dairy-like spreadability properties of Mozzarella cheese. The moisture and pH of the NC5 Mozzarella cheese analog were 45.6% and 6.85, respectively.

[0268] The NC5 Mozzarella cheese analog had higher melting values ​​than the plant-based mozzarella cheese, which exhibited a lack of melting (and even shrinkage) (Table 12, Figure 11). The melting rate of the NC5 Mozzarella cheese analog was slightly faster and reached its peak melt sooner compared to the melting of the low moisture and imitation mozzarella during the modified Schreiber melt test. The imitation and low moisture mozzarella required >10 minutes for maximum melting, while the NC5 Mozzarella cheese analog was completely melted in nearly 7 minutes (Figure 11).

[0269] The NC5 mozzarella cheese analog showed similar extensibility to the low moisture and imitation mozzarella (19.1 cm compared to 22.4 and 22.2 cm, respectively), while the plant-based mozzarella was unable to stretch at all (<2.5 cm) (Table 12, Figure 12). The extensibility and melting of the NC5 cheese analog was inferior to that of the NC1, 2 and 4 cheese analogs.

[0270] The texture profile analysis results for hardness, adhesion, resilience, cohesiveness, springiness and chewiness are shown in Table 13. The NC5 Mozzarella cheese analogue exhibited hardness and chewiness within the range of low moisture and imitation mozzarella. In comparison, the plant-based mozzarella cheese deviated from dairy behavior, exhibiting more chewiness (approximately 1.6x) and higher adhesion (approximately 1.5x) when compared to low moisture mozzarella.

[0271] Interestingly, the NC5 Mozzarella cheese analog showed improved adhesion that was not found in the imitation mozzarella. The imitation mozzarella is more adhesive than the low moisture mozzarella, while the NC5 Mozzarella cheese analog is less adhesive. Reduced adhesion is an advantageous cheese property. Adhesion represents the force required to remove the cheese from the probe and can be used as a proxy for whether the cheese will be adhesive to the teeth when eaten. [Table 13-1] [Table 13-2] Example 10 Properties of yogurt and yogurt drinks made from recombinant single variant alpha-casein

[0272] Yogurt analogs and yogurt drink analogs were made using recombinant non-phosphorylated bovine alpha S1 casein, native alpha casein (mix of alpha S1 and alpha S2 with native phosphorylation purified from cow's milk) and commercial micellar casein. For comparison, yogurt and yogurt drink were also made from commercial homogenized milk. Protein, trisodium citrate, disodium phosphate, calcium chloride and carbohydrates (sugars) were mixed in water at the concentrations specified in Table 14. To this solution, lecithin and pre-warmed fat were added at the concentrations listed in Table 14. The mixture was homogenized using an ultrasonic sonicator with a 16 mm probe at 90% power for 4 minutes. The mixture was pasteurized in a water bath at 90°C for 10 minutes and then cooled to 40C. 0.1 g of standard lactic acid bacteria cultures (Lactobacillus bulgaricus and Streptococcus thermophilus) were added. The milk-like colloid was fermented for 14 hours. The yogurt making process for whole milk consisted only of the pasteurization and fermentation steps described above. [Table 14]

[0273] The yogurt and yogurt simulants were analyzed for qualitative and quantitative parameters such as pH, odor, appearance, relative consistency and texture.

[0274] The recombinant alpha S1 casein yogurt analogs had a milk yogurt-like odor and appearance, fermented well, and displayed milk yogurt-like texture properties as shown in Table 15. The starting pH of the milk and milk-like solutions was in the range of 6.8-7.4, which dropped to 4-4.8 after 14 hours of fermentation (Table 15). All yogurt analogs except for the one without protein had the typical odor of fermented yogurt made from milk.

[0275] Yogurt analogs made from recombinant alpha S1 casein, micellar casein and native alpha casein became gel-like colloids with stable emulsions after fermentation (Figure 13). In comparison, the protein-free milk-like solution resulted in phase separation after fermentation and remained in liquid form (Figure 13). The yogurt analog formulations do not contain any added thickeners but instead rely on proteins to form a yogurt-like product. All yogurt analogs showed some level of syneresis.

[0276] Yogurt structure was analysed using a repeated test on a TA.XT Plus texture analyser equipped with a TA-18 ½” ball probe. This method disrupts the yogurt structure with a series of 10 consecutive sample insertions giving insight into the degree of deformation over time. Firmness (g) is quantified as the probe penetrates the sample, along with tackiness (g) and adhesion (g*sec). This test also makes it possible to infer the relative consistency by the work required to perform each cycle (insertion and removal of the probe from the sample). It can be hypothesized that the greater the work required to complete each cycle, the greater the consistency and robustness of the yogurt. Tests were performed on set, undisturbed and chilled (4° C.) yogurt or yogurt simulants. Texture analysis could not be performed on the protein-free samples as they remained liquid.

[0277] The recombinant alpha S1 yogurt analog had almost identical relative consistency to the milk yogurt (Table 15). The recombinant alpha S1 casein was only 15% less sticky than the milk yogurt. In comparison, the native alpha casein and micellar casein yogurt analogs were 26% and 75% less sticky than the milk yogurt (Table 15). [Table 15-1] [Table 15-2]

[0278] The stickiness of the native alpha casein yogurt analog was 12% higher than the milk-derived yogurt. In comparison, the micellar casein and recombinant alpha S1 casein yogurt analogs were less sticky than the milk-derived yogurt, being 36% and 68% less sticky, respectively. The micellar casein yogurt analog was less firm, while the native alpha casein and recombinant alpha S1 casein yogurt analogs were firmer compared to the milk yogurt (Table 15).

[0279] Yogurt drink analogs were made by mixing the yogurt or yogurt analog using a handheld homogenizer for 15 seconds. The drink analogs were analyzed for smoothness, texture, appearance and smell in comparison to a yogurt drink made from milk. The recombinant alpha S1 casein and native alpha casein yogurt drinks were similar in smoothness, texture, appearance and smell (aroma) to the milk yogurt drink and micellar casein yogurt drink analog (Figure 14). Example 11 Properties of Mozzarella Cheese Analogs Made from Alpha-Casein with Varying Phosphorylation Levels and Varying Calcium in the Formulation

[0280] Recombinant non-phosphorylated bovine alpha S1 casein, native alpha casein (a mix of alpha S1 and alpha S2 with native phosphorylation purified from bovine milk) and 70% dephosphorylated alpha casein (a mix of alpha S1 and alpha S2 caseins purified from milk and then enzymatically dephosphorylated to 70% supplied by Sigma Aldrich) were used to make non-micellar Mozzarella cheese analogs designated NC6-8 with varying amounts of CaCl2 as outlined in Table 16. Casein, water, palm stearin, canola oil, trisodium citrate, disodium phosphate, CaCl2, starch and sodium chloride were added to a beaker at the concentrations specified in Table 17. The formulations in Table 17 vary depending on the amount of calcium chloride added. The beaker was transferred to a water bath preset at 85°C and the contents were mixed using a mixing propeller at a speed of 300 rpm for 9 minutes. Natural flavors were added and the ingredients were mixed for an additional minute. The resulting mixture became a homogenous non-micellar mass, which was transferred into a standard mold and placed in a refrigerator for 5 days. After incubation, the NC6-8 Mozzarella cheese analogs were weighed for yield estimation. [Table 16] [Table 17]

[0281] The NC6-8 Mozzarella cheese analog samples were analyzed for qualitative and quantitative parameters such as pH, moisture, melting, extensibility and texture profile as described in Example 7. Additional qualitative properties such as cheese / cheese analog appearance after melting and cheese / cheese analog string quality are described as shown in Table 18. These parameters were compared between the tested cheese analogs, animal-derived low moisture mozzarella, animal-derived imitation mozzarella and plant-based vegan mozzarella style cheese. [Table 18]

[0282] The moisture and pH of the NC6–8 mozzarella cheese analogues were 47.2–49.4% and 5.7–6.7, respectively.

[0283] Cheese analogs made from native alpha casein showed melting response to varying amounts of added calcium, with the cheese analogs melting better and spreading better with reduced amounts of calcium; however, the cheese analogs with reduced calcium became clear / translucent liquids upon melting, suggesting the elimination of cheese-like emulsions. Surprisingly, cheese analogs made from recombinant non-phosphorylated alpha S1 casein showed similar trends of melting and spreading response to reduced calcium amounts while preserving cheese-like emulsion properties at lower calcium conditions (Table 18). In comparison, cheese analogs made from 70% dephosphorylated alpha casein showed no melting response to varying amounts of calcium, and the reduced calcium samples also formed translucent edges upon melting, indicating instability of the cheese-like emulsion.

[0284] All cheese analogs tested stretched comparable to dairy-like cheese in terms of stretch length. Table 18 shows a qualitatively assigned extensibility quality metric scale of 0-5, with 0 indicating the poorest quality and 5 indicating the highest quality extensibility. These values ​​are based on string toughness and thickness observed while stretching the cheese analogs using the TA.XT Texture Analyzer. Cheese analogs made from 70% dephosphorylated alpha casein and native alpha casein showed mild improvement in extensibility quality with calcium addition. Surprisingly, cheese analogs made from recombinant non-phosphorylated alpha S1 casein showed a significant improvement in extensibility quality with high calcium conditions producing extensibility quality comparable to animal-derived mozzarella, and low / medium calcium conditions producing extensibility quality comparable to milk-derived micellar imitation mozzarella. This strong dose response to calcium levels was specifically observed in cheese analogs made from recombinant non-phosphorylated alpha S1 casein.

[0285] The textural properties of the cheese analogs are outlined in Table 18. The cheese analogs made from recombinant alpha S1 casein showed a significant reduction in adhesiveness. In comparison, the cheese analogs made from native and 70% dephosphorylated alpha casein had only slightly reduced adhesiveness. The adhesiveness of the analog cheese also decreased with increasing amounts of calcium in the formulation.

[0286] The cheese analogs made from native alpha casein were as hard / firm as milk-derived low moisture mozzarella at any calcium dosage (approximately 5% softer). Surprisingly, the recombinant non-phosphorylated alpha S1 casein cheese analogs were also similarly hard (approximately 5% softer compared to low moisture mozzarella) for the no calcium and low / medium calcium conditions. In comparison, the analogs with 70% dephosphorylated alpha casein also showed increased firmness for the low / medium calcium conditions (>5% harder compared to low moisture mozzarella and >10% harder compared to cheese analogs made from native alpha casein or recombinant non-phosphorylated alpha S1 casein). Increased firmness is an undesirable attribute of cheese analogs, as typically described for plant-based cheese analogs.

[0287] Other textural properties such as chewiness, cohesiveness, springiness and resilience were also measured and showed no significant differences between the conditions tested.

[0288] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the present invention described herein can be used in the practice of the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. 1. A consumable composition comprising a recombinant single variant of alpha-casein protein, comprising: wherein the single variant provides the consumable composition with at least one dairy-like property selected from the group consisting of adhesiveness, extensibility, firmness, consistency, stickiness, chewiness, resilience, elasticity, mouthfeel, melting, hardness, creaminess, and softness; the recombinant single variant of the alpha-casein protein is not an animal-derived casein and is not physically dissociated from casein micelles; wherein the dairy-like properties are substantially provided by the recombinant single variant of the alpha-casein protein; A consumable composition, wherein said consumable composition is comparable to a dairy-derived consumable composition in at least one of said dairy-like properties.

2. 10. The consumable composition of claim 1, wherein the casein content of the consumable composition substantially comprises the recombinant single variant of the alpha-casein protein.

3. 10. The consumable composition of claim 1, lacking any additional casein other than said recombinant single variant of said alpha-casein protein.

4. The consumable composition of claim 1, (a) not containing any animal-produced protein, (b) lacking any animal-derived dairy protein, and / or (c) lacking any dairy protein other than the recombinant single variant of the alpha-casein protein.

5. A consumable composition described in any one of claims 1 to 4, wherein the recombinant single variant of alpha-casein protein is an alpha-casein protein that lacks one or more post-translational modifications of native alpha-casein protein.

6. 6. The consumable composition of claim 5, wherein the recombinant single variant of the alpha-casein protein is non-phosphorylated.

7. A consumable composition as described in claim 5, wherein the recombinant single variant of alpha-casein protein is at least 95% less phosphorylated compared to animal-derived alpha-casein protein.

8. A consumable composition as described in claim 5, wherein the recombinant single variant of the alpha-casein protein has substantially reduced phosphorylation or no phosphorylation compared to the animal-derived alpha-casein protein without chemical or enzymatic treatment of the recombinant single variant of the alpha-casein protein.

9. A consumable composition as described in claim 5, comprising 5% w / w to 30% w / w of the recombinant single variant of the alpha-casein protein.

10. 5. The consumable composition of any one of claims 1 to 4, wherein the recombinant single variant of alpha-casein protein is recombinant alpha-S1 casein protein.

11. 5. The consumable composition of any one of claims 1 to 4, wherein the recombinant single variant of alpha-casein protein comprises the amino acid sequence of bovine, caprine or ovine alpha-casein protein, or any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56, or a sequence with at least 80% identity to any one of SEQ ID NOs: 2, 3, 14, 15, 26, 27, 29, 30, 32, 33, 35, 36, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55 or 56.

12. 5. The consumable composition of any one of claims 1 to 4, wherein the recombinant single variant of the alpha-casein protein comprises one or more non-native amino acids at the N-terminus.

13. A consumable composition according to any one of claims 1 to 4 which is a cheese analogue.

14. The consumable composition of claim 13, wherein the recombinant single variant of alpha-casein protein comprises the amino acid sequence of ruminant alpha-sl casein protein.

15. A consumable composition as described in claim 13, wherein at least 5% of the cheese analog is the recombinant single variant wt / wt of the alpha-casein protein.

16. The consumable composition of claim 15, wherein the recombinant single variant of alpha-casein protein (a) lacks one or more post-translational modifications of native alpha-casein protein, (b) is not phosphorylated, or (c) is at least 95% less phosphorylated compared to animal-derived alpha-casein protein.

17. The consumable composition of claim 15, wherein the consumable composition is a cheese analog, and the cheese analog further comprises one or more of: (a) plant-derived oil, (b) plant-derived starch, (c) sugar, and (d) salt.

18. 18. The consumable composition of claim 17, wherein the cheese analog is a mozzarella analog, a cheddar analog, or a parmesan analog.

19. The cheese analogue of claim 19, wherein the cheese analogue is a mozzarella cheese analogue and comprises the recombinant single variant of the alpha-casein protein, a vegetable-derived oil, and a moisture level of between 45-52%. the recombinant single variant of the alpha-casein protein has substantially reduced or no phosphorylation; the mozzarella cheese analog lacks any added casein or animal-derived protein; and the recombinant single variant of the alpha-casein protein provides stretchability properties to the mozzarella cheese analog; 14. The consumable composition of claim 13.

20. The consumable composition is a dairy analog; (i) the dairy analog contains: (a) at most 25 mg calcium per gram of casein; (b) about 15% to about 30% fat w / w; (c) about 0.5% to about 4% starch w / w, or at most 10% starch w / w; and / or (ii) the dairy analogue comprises a ratio of the recombinant single variant of alpha-casein to emulsifying salt of between 12:1 and 6:1; and / or (iii) the dairy analog does not contain any emulsifiers other than the emulsifying salt; The consumable composition of any one of claims 1 to 4.

21. The consumable composition of any one of claims 1 to 4, which is a yogurt analog.

22. 22. The consumable composition of claim 21, wherein the recombinant single variant of alpha casein is bovine alpha S1 casein.

23. 22. The consumable composition of claim 21, wherein the emulsification, firmness, adhesion or viscosity of the yogurt analog is comparable to or improved compared to a dairy-based yogurt or a dairy-based yogurt analog, and / or the adhesiveness of the yogurt analog is reduced compared to a dairy-based yogurt or a dairy-based yogurt analog.

24. 24. The consumable composition of claim 23, wherein the dairy-derived yogurt or dairy-derived yogurt analog comprises micellar casein.

25. 22. The consumable composition of claim 21, wherein the yogurt analog comprises (a) from about 1% to about 4% of the recombinant single variant of alpha-casein w / w, (b) from about 2% to about 6% fat w / w, and / or (c) from about 4% to about 8% carbohydrates w / w.

26. The consumable composition of claim 21, wherein the recombinant single variant of alpha-casein protein (a) lacks one or more post-translational modifications of native alpha-casein protein, (b) is not phosphorylated, or (c) is at least 95% less phosphorylated compared to animal-derived alpha-casein protein.

27. A consumable composition according to any one of claims 1 to 4, which is a dairy analogue beverage.

28. 28. The consumable composition of claim 27, wherein the smoothness of the beverage is comparable to or improved compared to the smoothness, texture and / or emulsification of dairy-based beverages.

29. 28. The consumable composition of claim 27, wherein the beverage comprises from about 0.5% to about 10% of the recombinant single variant of alpha-casein w / w, and / or from about 0.1% to about 6% fat w / w.

30. The consumable composition of claim 27, wherein the recombinant single variant of alpha-casein protein (a) lacks one or more post-translational modifications of native alpha-casein protein, (b) is not phosphorylated, or (c) is at least 95% less phosphorylated compared to animal-derived alpha-casein protein.

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