CN117813318A

CN117813318A - Dairy-like compositions and related methods

Info

Publication number: CN117813318A
Application number: CN202280056407.9A
Authority: CN
Inventors: 因加·拉德曼; 迪拉杰考尔·潘费尔; 陈梦圆
Original assignee: New Training Co
Current assignee: New Training Co
Priority date: 2021-08-17
Filing date: 2022-08-17
Publication date: 2024-04-02

Abstract

Provided herein are dairy-like analog compositions and methods of making the compositions using recombinant single variants of casein, including, for example, recombinant single variants of alpha casein.

Description

Dairy-like compositions and related methods

Cross reference

The present application claims the benefit of U.S. provisional application No. 63/234,193, filed on 8 months 17 of 2021, and U.S. patent application No. 17/829,951, filed on 1 of 6 months 2022, both of which are incorporated herein by reference in their entireties.

Background

The clean food area (clean food space) includes both plant-based foods and cell-based foods. Cell-based food products are a large covered term that includes culturing muscle and fat cells to replace slaughter meat, and culturing bioengineered organisms to express recombinant animal proteins to replace other animal products such as dairy products and eggs. The need to find alternative sources of animal proteins comes from the inefficiency and sustainability of current animal food production.

Cheese is the third least sustainable animal product worldwide (when measured as greenhouse gas emissions per kg of product) and the consumption of dairy cheese is not slowed by plant-based substitutes introduced into the market over the last 10 years. In contrast, in the united states and developing markets, mozzarella (mozzarella) cheese consumption has grown year by year. Current cheese substitutes do not match the function, nutrition and taste of dairy cheese due to their lack of casein.

SUMMARY

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, 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 are to be regarded as illustrative in nature and not as restrictive.

In some aspects, provided herein are edible compositions. In some embodiments, the edible composition may comprise a recombinant single variant of alpha casein. In some embodiments, the single variant provides the edible composition with at least one dairy-like property selected from the group consisting of: adhesion, stretchability, texture, mouthfeel, melting, browning, hardness, creaminess, taste, smell and flexibility. In some embodiments, the recombinant single variant may not be casein of animal origin and is not physically dissociated from the casein micelles. In some embodiments, the dairy-like properties may be provided substantially by a recombinant single variant casein; and wherein the edible composition may be comparable in at least one dairy-like property to the dairy-derived edible composition.

In some embodiments, the edible composition may comprise one or more dairy-like properties selected from the group consisting of: adhesion, stretchability, firmness, viscosity, tackiness, chewiness, resilience, elasticity, mouthfeel, melting, hardness, creaminess and flexibility.

In some embodiments, the casein content of the edible composition may substantially comprise a recombinant single variant of alpha casein.

In some embodiments, the casein content of the edible composition may comprise only a recombinant single variant of alpha casein.

In some embodiments, a recombinant single variant of alpha casein may comprise at least 95% or at least 97% of the casein content of the edible composition.

In some embodiments, a recombinant single variant of alpha casein may comprise at least 99% of the casein content of the edible composition.

In some embodiments, the edible composition lacks any additional casein other than a recombinant single variant of alpha casein.

In some embodiments, a single variant of alpha casein may not be derived from caseinate (caseinate).

In some embodiments, the composition may be free of any animal-produced proteins.

In some embodiments, the composition lacks any other dairy protein of animal origin.

In some embodiments, the dairy-like properties may be comparable to or improved over a dairy-derived edible composition; wherein the milk-derived edible composition may be identical in all ingredients to the edible composition except that the milk-derived edible composition may comprise milk, one or more milk-derived proteins, or milk-derived ingredients in addition to or in place of the recombinant single variant of alpha casein.

In some embodiments, the dairy-like properties may be comparable to or improved over a micellar casein composition, wherein the micellar casein composition may be identical to the edible composition in all ingredients, except that the micellar casein composition may comprise a recombinant single variant of alternative alpha casein or micellar casein isolated from milk in addition to the recombinant single variant of alpha casein.

In some embodiments, the recombinant single variant may be an alpha casein that may comprise at least one unnatural post-translational modification.

In some embodiments, a recombinant single variant of alpha casein may also comprise at least one natural post-translational modification.

In some embodiments, the recombinant single variant may be an alpha casein lacking post-translational modification of one or more native alpha casein.

In some embodiments, a single variant of alpha casein may also comprise at least one non-natural post-translational modification.

In some embodiments, a single variant of alpha casein may not be post-translationally modified.

In some embodiments, a single variant of alpha casein may not be phosphorylated.

In some embodiments, the single variant of alpha casein may be alpha-s 1 casein. In some embodiments, the single variant of alpha casein may be alpha-s 2 casein. In some embodiments, the composition may comprise full-length alpha casein.

In some embodiments, a single variant of alpha casein may comprise any 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.

In some embodiments, a single variant of alpha casein may comprise the amino acid sequence of bovine, caprine, or ovine alpha casein, or any 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 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.

In some embodiments, a single variant of alpha casein may comprise one or more unnatural amino acids at the N-terminus.

In some embodiments, a single variant of alpha casein may comprise a non-natural methionine at the N-terminal position.

In some embodiments, a single variant of alpha casein may not be derived from casein micelles.

In some aspects, described herein may be a dairy analog. The dairy analogue may comprise any edible composition provided herein, wherein the analogue may be selected from the group consisting of: cheese analogues, yoghurt analogues, cream analogues and ice cream analogues.

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

In some embodiments, the dairy analog may also comprise fats or oils from non-animal sources. In some embodiments, the dairy analog lacks any dairy protein of animal origin. In some embodiments, the dairy analogue lacks any other casein. In some embodiments, a recombinant single variant of alpha casein may not be contained within a dairy analog in micellar form.

In some embodiments, the dairy analogue may comprise one or more of the following: (a) an oil of vegetable origin; (b) starch of vegetable origin; (c) a sugar; and (d) a salt.

In some embodiments, the dairy analogue may be a cheese analogue. In some embodiments, the cheese analogue may be a mozzarella analogue, a cheddar analogue or a parma cheese (parresan) analogue. In some embodiments, the cheese analogue may be a low moisture cheese analogue. In some embodiments, the cheese analogue may be a soft cheese analogue. In some embodiments, the cheese analogue may be a hard cheese analogue.

In some embodiments, the cheese analogue may be a mozzarella cheese analogue, and wherein the single variant of casein may be alpha casein. In some embodiments, the alpha casein may be alpha S1 casein. In some embodiments, the αs1 casein may be bovine αs1 casein. In some embodiments, the αs1 casein may be full length casein. In some embodiments, the alpha casein may be alpha S2 casein. In some embodiments, the αs2 casein may be bovine αs2 casein. In some embodiments, the αs2 casein may be full length casein.

In some embodiments, the stretchability of the cheese analogue may be comparable to or improved relative to the stretchability of the dairy-derived cheese or dairy-derived cheese analogue.

In some embodiments, the cheese analogue has a melting area/time that is comparable to or greater than the melting area/time of the dairy-derived cheese or dairy-derived cheese analogue.

In some embodiments, the malleability of the cheese analogue may be comparable to or improved relative to the malleability of the dairy-derived cheese or dairy-derived cheese analogue. In some embodiments, the texture of the cheese analogue may be comparable to or improved relative to the texture of the dairy-derived cheese or dairy-derived cheese analogue. In some embodiments, the adhesiveness of the cheese analogue may be reduced relative to the adhesiveness of the dairy-derived cheese or dairy-derived cheese analogue.

In some embodiments, the dairy-derived cheese or dairy-derived cheese analogue may comprise micellar casein. In some embodiments, the stretchability of the cheese analogue may be improved relative to the stretchability of a plant-derived cheese analogue. In some embodiments, melting of the cheese analogue may be improved relative to melting of a cheese analogue of plant origin. In some embodiments, the texture of the cheese analogue may be improved relative to the texture of a cheese analogue of plant origin. In some embodiments, the adhesiveness of the cheese analogue may be reduced relative to the adhesiveness of a plant-derived cheese analogue.

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

In some embodiments, the cheese analogue may comprise at most 25mg of calcium per gram of casein. In some embodiments, the cheese analogue may comprise from 0mg to 25mg of calcium per gram of casein. In some embodiments, the cheese analogue may comprise from 0mg to 20mg of calcium per gram of casein. In some embodiments, the cheese analogue may comprise from 0mg to 10mg of calcium per gram of casein. In some embodiments, the cheese analogue may comprise from 5mg to 15mg of calcium per gram of casein. In some embodiments, the cheese analogue may comprise about 0mg of calcium per gram of casein. In some embodiments, the cheese analogue may comprise about 10mg of calcium per gram of casein.

In some embodiments, the cheese analogue may comprise from about 15% w/w to about 30% w/w fat. In some embodiments, the cheese analogue may comprise from about 18% w/w to about 28% w/w fat. In some embodiments, the cheese analogue may comprise from about 20% w/w to about 25% w/w fat.

In some embodiments, the cheese analogue may comprise from about 0.5% w/w to about 4% w/w starch. In some embodiments, the cheese analogue may comprise from about 1% w/w to about 3% w/w starch. In some embodiments, the cheese analogue may comprise from about 2% w/w to about 3% w/w starch. In some embodiments, the cheese analogue may comprise up to 10% w/w starch. In some embodiments, the cheese analogue may comprise up to 5% w/w starch.

In some embodiments, the ratio of recombinant single variant alpha casein to emulsifying salt may be from 12:1 to 6:1. In some embodiments, the cheese analogue does not contain an emulsifier other than an emulsifying salt.

In some embodiments, the dairy analog may be a yogurt analog. In some embodiments, the single variant of casein may be alpha casein. In some embodiments, the alpha casein may be alpha S1 casein. In some embodiments, the αs1 casein may be bovine αs1 casein. In some embodiments, the αs1 casein may be full length casein. In some embodiments, the alpha casein may be alpha S2 casein. In some embodiments, the αs2 casein may be bovine αs2 casein. In some embodiments, the αs2 casein may be full length casein.

In some embodiments, emulsification of the yogurt analog may be comparable to or improved relative to emulsification of the dairy-derived yogurt or dairy-derived yogurt analog. In some embodiments, the firmness, viscosity, or viscosity of the yogurt analog may be comparable to or improved relative to a dairy-derived yogurt or dairy-derived yogurt analog. In some embodiments, the adhesiveness of the yogurt analog may be reduced relative to a dairy-derived yogurt or dairy-derived yogurt analog. In some embodiments, the dairy-derived yogurt or dairy-derived yogurt analogue may comprise micellar casein.

In some embodiments, the dairy-derived yogurt or dairy-derived yogurt analogue may be identical in all ingredients to the dairy analogue, except that the dairy-derived yogurt or dairy-derived yogurt analogue may comprise micellar casein that replaces a recombinant single variant of alpha casein. In some embodiments, emulsification of the yogurt analog may be comparable to or improved relative to emulsification of a plant-derived yogurt analog. In some embodiments, the firmness, viscosity, or viscosity of the yogurt analog may be comparable to or improved relative to a plant-derived yogurt analog. In some embodiments, the adhesiveness of the yogurt analog may be reduced relative to a plant-derived yogurt analog.

In some embodiments, the plant-derived yogurt analog lacks any dairy protein.

In some embodiments, the yogurt analog may comprise from about 1% w/w to about 4% w/w of a single variant of casein. In some embodiments, the yogurt analog may comprise from about 2% w/w to about 4% w/w of a single variant of casein. In some embodiments, the yogurt analog may comprise from about 2% w/w to about 6% w/w fat. In some embodiments, the yogurt analog may comprise from about 4% w/w to about 8% w/w carbohydrate.

In some embodiments, the dairy analog may be a beverage. In some embodiments, the single variant of casein may be alpha casein. In some embodiments, the smoothness of the beverage may be comparable to or improved relative to the smoothness of the dairy-derived beverage. In some embodiments, the texture of the beverage may be comparable to or improved relative to the texture of a dairy-derived beverage. In some embodiments, emulsification of the beverage may be comparable to or improved relative to a beverage of dairy origin. In some embodiments, the beverage may comprise from about 0.5% w/w to about 10% w/w of a single variant of casein. In some embodiments, the beverage may comprise from about 0.1% w/w to about 6% w/w fat.

Incorporated by reference

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.

Brief Description of Drawings

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 and drawings that set forth an illustrative embodiment in which the principles of the invention are utilized.

Figure 1 illustrates recombinantly produced full length αs1 casein and two truncated forms of αs1 casein present.

Fig. 2 illustrates a comparison of tensile capacity (ductility) between imitation cheese (analog cheese) prepared using the methods described herein and several commercially available cheeses and cheese analogs (cheese analogs).

Fig. 3 illustrates a comparison of texture (on a logarithmic scale) between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 4 illustrates a comparison of melting profiles between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 5 illustrates a comparison of tensile capacity (ductility) between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 6 illustrates a comparison of melting between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 7 illustrates a comparison of tensile capacity (ductility) between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 8 illustrates a comparison of texture (on a logarithmic scale) between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 9 illustrates a comparison of melting profiles between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 10 illustrates a comparison of tensile capacity (ductility) between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 11 illustrates a comparison of melting profiles between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 12 illustrates a comparison of ductility profiles between imitation cheese prepared using the methods described herein and several commercially available cheeses and cheese analogues.

Fig. 13 illustrates yogurt and milk-derived yogurt prepared using the methods described herein.

Fig. 14 illustrates a yogurt beverage and a milk-derived yogurt beverage prepared using the methods described herein.

Detailed Description

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

Despite the 3300 billion dollars worth of the dairy industry, research into clean dairy solutions using recombinant dairy proteins is still needed. Since dairy cheese is an inefficient dairy product in terms of resources required per gram and is the most difficult dairy product to reproduce accurately from plant-based ingredients alone, methods and compositions for dairy-like products prepared using recombinant proteins are presented herein.

The component that imparts a unique characteristic to dairy cheese is casein. When milk or milk-derived ingredients are used in dairy products, casein is present as micelles. Micelles are protein colloids and typically in cow milk, micelles contain four kinds of casein (αs1 casein, αs2 casein, βcasein and κcasein) that interact with insoluble calcium phosphate in the colloid center. After chymosin is added to milk, micelles in the milk attract each other. This forms a clot which is then used to make up 99% of all cheeses. In the case of yoghurt, acidification of micelles comprising liquid colloids may be performed using starting cultures of bacteria known for yoghurt production. The present disclosure is based on the finding that a single variant of recombinant non-naturally occurring casein can be used to produce a dairy product or dairy-like product in the absence of other casein and without micelle formation. Those skilled in the art have attempted to separate different casein proteins from milk or milk micelles for use in the production of dairy-like products, but the inventors of the present application have for the first time found that a single variant casein protein in recombinant form, in particular in some embodiments lacking or having a post-translational modification (PTM) different from that of native casein, may provide dairy-like characteristics without being present in micellar form and without being associated with or otherwise present in other casein or other dairy proteins.

Those skilled in the art will know that micelles are complex structures containing multiple proteins, and thus will not be able to be expected to form dairy-like products such as cheese, cheese analogues, yoghurt and other dairy products using a single casein. The present disclosure is based on the surprising discovery made by the inventors that a single recombinantly produced casein (a single variant of casein, such as alpha casein) is capable of forming consumables (concumables) without forming micelles or incorporating into micelles. The present disclosure also describes recombinantly produced dairy products, such as cheese analogs and other dairy analog products, as well as powders using the compositions formed by the methods described herein.

Those skilled in the art will know that micelles are complex structures containing a variety of proteins carrying a variety of post-translational modifications, and thus will not be expected to be able to form dairy-like products using a single recombinant casein, such as cheeses, cheese analogues, yoghurt and other dairy products with comparable or improved dairy-like properties. The present disclosure is based on the surprising discovery made by the inventors that a single recombinant casein (a single variant of casein, such as alpha casein) is capable of forming a consumable with comparable or improved dairy-like properties despite differences in post-translational modifications and despite not being incorporated into micelles.

The present disclosure also describes edible compositions incorporating truncated forms of a single variant of recombinantly produced alpha casein. The compositions described herein may comprise truncated forms of different casein.

The edible compositions described herein are formed from recombinant single casein variants such as alpha casein variants. Recombinant casein may be expressed in microbial organisms, for example bacteria such as the gram positive bacteria lactococcus lactis (Lactococcus lactis) and bacillus subtilis (Bacillus subtilis), as well as the gram negative mode organisms escherichia coli (e.coli), as well as other host organisms such as yeasts, fungi and plants. These recombinant caseins can be combined with other components (e.g., minerals, fats, sugars, and vitamins) to produce dairy-like products such as cheese that behaves, smells, tastes, looks, and feels like dairy cheese of animal origin. Such dairy-like products may be free of: i) Lactose, ii) cholesterol, iii) saturated fat of animal origin, iv) whey protein of milk origin; and/or v) casein of milk origin.

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

In some embodiments, the method comprises using a single casein producing composition from a plurality of different species. In some examples, the method comprises producing the composition using a single alpha casein variant from a plurality of different species. The casein may be from humans, bovine subfamilies (cattle, bison, buffalo), ovine subfamilies (sheep and goats), equine (horses, zebras) and camels (camels). A single casein variant may be modified compared to native alpha casein, for example a truncated form of native casein. In some examples, the compositions described herein may be produced in the absence of beta casein or kappa casein. ,

in some embodiments, the recombinant casein may be isolated, purified or partially purified from the genetically modified microorganism or its culture broth.

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

The term "dairy protein" as used herein means a protein having an amino acid sequence derived from proteins found in milk (including variants thereof).

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

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 found in milk (including variants thereof). "recombinant casein" means casein produced by a recombinant organism or in a heterologous host cell.

The term "single variant of casein (single variant of casein)" (also referred to as "single variant of casein (single variant of a casein protein)") as used herein may describe a variant comprising or a composition produced by the amino acid sequence of casein. For example, a composition comprising a single variant of casein comprises only alpha casein. In some cases, the term "single variant of casein" may describe a composition in which a single casein provides one or more dairy-like properties to the composition, irrespective of the presence of other casein.

Compositions comprising a single variant of casein may be produced from only one casein amino acid sequence, but may comprise a truncated form of the protein sequence instead of or in addition to the full length form of the protein.

The term "single variant of alpha casein" (also referred to as "single variant alpha casein") as used herein may describe a variant comprising or a composition produced from an amino acid sequence of alpha casein. For example, a composition comprising a single variant of αcasein comprises only one of αs1 casein or αs2 casein. Compositions comprising a single variant of alpha casein may be produced from only one alpha casein amino acid sequence, but may comprise a truncated form of the protein sequence that replaces or is outside of the full length form of the protein. For example, a composition comprising a single variant of αcasein may comprise a mixture of full-length αs1 casein and truncated forms thereof. A composition comprising a single variant of αcasein may comprise only full length αs1 casein, or only truncated forms of αs1 casein, or a mixture of truncated forms of αs1 protein.

The percentage of "sequence identity" as used herein in the context of polynucleotide or polypeptide (amino acid) sequences refers to the percentage of residues in the two sequences that are identical when the sequences are aligned for maximum correspondence. Many different algorithms are known in the art and can be used to measure polynucleotide or polypeptide sequence identity. For example, sequences may be compared using the following: FASTA (e.g., using its default parameters as provided in Wisconsin Package version 10.0,Genetics Computer Group (GCG), madison, WI), gap (e.g., using its default parameters as provided in Wisconsin Package version 10.0, GCG, madison, WI), bestfit, clustalW (e.g., using default parameters of version 1.83), or BLAST (e.g., using bi-direction BLAST (reciprocal BLAST), PSI-BLAST, BLASTP, BLASTN) (see, e.g., pearson 1990.methods enzymol 183:63; altschul et al 1990.j. Mol. Biol 215:403).

Composition comprising casein

A. Casein protein

Traditionally, cheese has been started from animal derived milk. Cheese production processes of animal origin involve precipitation of micellar forms from milk, wherein the micellar forms are in most cases complex protein mixtures (comprising various types of casein, such as alpha protein, beta protein and kappa protein). The milk is acidified, the micelles shrink and dissociate slightly, then the milk is treated with rennet (renneted) and made into a clot, and the clot is made into cheese. Cheese analogues can be produced from animal milk by first precipitating casein micelles from the milk using one of the following methods: 1) sodium salt is used to prepare sodium caseinate (sodium caseinate), 2) acid is used to prepare acid casein (acid casein), 3) rennet enzyme is used to coagulate to prepare rennet casein. Casein precipitated from milk in this way is then further treated with fat to produce cheese analogues.

Provided herein are edible compositions containing a single variant of recombinantly produced casein that is not in micellar form and is not derived from milk or from milk casein. In some aspects, the edible compositions of the present disclosure are not only prepared from a single variant of casein, but are also capable of providing dairy-like properties to food and beverage products without being in a micellar structure or derived from starting materials having a micellar structure. In some aspects, the edible compositions of the present disclosure are not only prepared using a single variant of casein, but the single variant of casein is capable of providing desired or improved dairy-like properties in the absence of other casein and without the complexity of the milk-derived casein structure. In some aspects, the edible compositions of the present disclosure comprise a single variant of alpha casein. In some alternative examples, the edible compositions of the present disclosure comprise a single variant of β casein or κ casein.

In some embodiments, the compositions herein (and products made therefrom) do not comprise any dairy protein other than a single variant of casein. In some cases, the single variant of casein is a single variant of alpha casein. In some cases, the compositions herein (and products made therefrom) do not comprise any whey protein or any whey protein of milk origin. In some embodiments, the compositions herein (and products made therefrom) do not comprise any dairy proteins of animal origin. The compositions herein do not comprise any casein isolated from any animal derived products or micelles.

The compositions described herein comprise a single variant of casein prepared by recombinant production. In some cases, the single variant casein in the edible composition may be modified casein relative to native casein. Modifications in a single variant of casein may include one or more amino acid insertions, deletions or substitutions relative to wild-type or native casein. In some cases, the single variant casein is a single variant of alpha casein. In some examples, the casein may be beta casein or kappa casein or gamma casein.

The single variant of alpha casein may be a recombinant protein, which is a truncated alpha casein relative to wild-type or native alpha casein. Truncations may be similar to truncations found in nature (e.g., having a common number of amino acids). The truncations may be non-naturally occurring truncations of alpha casein. A single variant of alpha casein may have an N-terminal truncation relative to wild-type or native alpha casein. A single variant of alpha casein may have a C-terminal truncation relative to wild-type or native alpha casein. A single variant of alpha casein may have an N-terminal truncation and a C-terminal truncation relative to wild-type or native alpha casein.

In some embodiments, the single variant of αcasein in the edible composition is αs1 casein. In such compositions, the αs1 casein may comprise modified αs1 casein, such as αs1 casein in which the type of post-translational modification (phosphorylation, glycosylation, the location of such modification, or the amount of such modification) is altered. In some cases, the αs1 casein may be full length αs1 casein. In some cases, the composition comprising αs1 casein lacks any protein of animal origin.

In some embodiments, the single variant of αcasein in the edible composition is αs2 casein. In such compositions, the αs2 casein may comprise modified αs2 casein, such as αs2 casein in which the type of post-translational modification (phosphorylation, glycosylation, location of such modification, or amount of such modification) is altered. In some cases, the αs2 casein may be full length αs2 casein. In some cases, the composition comprising αs2 casein lacks any protein of animal origin.

The compositions herein comprising a single variant of alpha casein are recombinant proteins and do not comprise alpha casein isolated from casein micelles or alpha casein isolated from any naturally occurring micellar form or product comprising micelles or micellar forms.

In some embodiments, the edible compositions described herein comprise a single variant of casein, such as alpha casein. In some cases, the casein content of the edible composition comprises substantially a single variant of casein. In some cases, the casein content of the edible composition comprises only a single variant of casein. In some cases, a single variant of casein comprises at least 95% or at least 97% of the casein content of the edible composition. In some cases, a single variant of casein comprises at least 99% of the casein content of the edible composition. In some cases, the edible composition lacks any additional casein other than a single variant of casein. In a preferred embodiment, the single variant of casein is alpha casein.

A single variant of alpha casein may be from a ruminant species. The single variant of alpha casein may be bovine alpha casein. The single variant of alpha casein may be goat alpha casein. The single variant of alpha casein may be sheep alpha casein. The single variant of alpha casein may be equine alpha casein. The single variant of alpha casein may be camelid or camelid alpha casein. The single variant of alpha casein may be human alpha casein.

A single variant of alpha casein 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 (as exemplified by SEQ ID NOs: 4-12, 16-24). A single variant of alpha casein may be bovine alpha casein, e.g. casein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO 1-3, 28-30, 39-41 or 48-50 or a truncated form thereof. A single variant of alpha casein may be sheep alpha casein, for example casein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO 13-15 or 42-44 or a truncated form thereof. A single variant of alpha casein may be goat alpha casein, e.g. casein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NOs 25-27 or 45-47 or truncated forms thereof. A single variant of alpha casein may be equine alpha casein, for example casein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO 31-33 or 51-53 or truncated forms thereof. A single variant of alpha casein may be camelid alpha casein, e.g. casein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO 34-36 or 54-56 or truncated forms thereof. A single variant of alpha casein may be human alpha casein, e.g. casein having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO 37-38 or a truncated form thereof.

The single variant of alpha casein in the edible composition may be alpha S1 casein. The αs1 casein may be full length αs1 casein. In some cases, the αs1 protein is a truncated αs1 protein relative to wild-type or native αs1 casein. In some cases, the αs1 casein has an N-terminal truncation relative to wild-type or native αs1 casein. In some cases, the αs1 casein has a C-terminal truncation relative to wild-type or native αs1 casein. In some cases, the αs1 casein may have an N-terminal truncation and a C-terminal truncation relative to wild-type or native αs1 casein. In some cases, the αs1 protein lacks between 1 and 59N-terminal amino acids. In some cases, the αs1 protein lacks between 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 50, 1 to 59N-terminal amino acids. In some cases, bovine αs1 protein having SEQ ID No. 2 lacks between 1 and 59N-terminal amino acids. In some cases, bovine αS1 protein having SEQ ID NO. 2 lacks 22, 23, 24, or 25N-terminal amino acids (see examples such as SEQ ID NO. 4-12). In some cases, sheep αS1 protein having SEQ ID NO. 14 lacks between 1 and 59N-terminal amino acids (see examples such as SEQ ID NO. 16-24). In some cases, the ovine αs1 protein having SEQ ID No. 14 lacks 22, 23, 24, or 25N-terminal amino acids.

In some cases, the αs1 casein is a mixture of full-length αs1 casein and one or more truncated forms of αs1 casein (such as any one or more of the truncated forms described herein).

In some cases, the compositions prepared herein from a single variant of alpha casein comprise a mixture of full length forms and one or more truncated forms of alpha casein, and in such compositions, the total alpha casein may comprise up to 20% wt/wt of the truncated form of one or more alpha S1 casein. The truncated form may be any truncated form of the full-length single variant αs1 casein, examples of which are provided elsewhere herein. In some cases, the compositions herein prepared from a single variant of αcasein comprise only truncated forms of αs1 casein.

In some cases, a single variant of alpha casein in a composition (such as a cheese analogue) comprises more than 0% of truncated forms, such as 0.1% wt/wt, 0.2% wt/wt, 0.5% wt/wt, 0.8% wt/wt of the single variant of alpha casein is a truncated form of alpha S1 protein. In some embodiments, a single variant of αs1 casein comprises at least 1% wt/wt of one or more truncated forms of αs1 casein. In some cases, a single variant of αs1 casein comprises up to 20% wt/wt of one or more truncated forms of αs1 casein. In some of the cases where the number of the cases, single variants of αS1 casein comprise 1% wt/wt to 3% wt/wt, 1% wt/wt to 5% wt/wt, 1% wt/wt to 7% wt/wt, 1% wt/wt to 10% wt/wt, 1% wt/wt to 12% wt/wt, 1% wt/wt to 15% wt/wt, 1% wt/wt to 20% wt/wt, 3% wt/wt to 5% wt/wt, 3% wt/wt to 7% wt/wt, 3% wt/wt to 10% wt/wt, 3% wt/wt to 12% wt/wt, 3% wt/wt to 15% wt/wt, 3% wt/wt to 20% wt/wt, 5% wt/wt to 7% wt/wt, 5% wt/wt to 10% wt/wt from 5% wt/wt to 12% wt/wt, from 5% wt/wt to 15% wt/wt, from 5% wt/wt to 20% wt/wt, from 7% wt/wt to 10% wt/wt, from 7% wt/wt to 12% wt/wt, from 7% wt/wt to 15% wt/wt, from 7% wt/wt to 20% wt/wt, from 10% wt/wt to 12% wt/wt, from 10% wt/wt to 15% wt/wt, from 10% wt/wt to 20% wt/wt, from 12% wt/wt to 15% wt/wt, from 12% wt/wt to 20% wt/wt, or from 15% wt/wt to 20% wt/wt of one or more truncated forms of αs1 casein. In some cases, a single variant of αs1 casein comprises about 1%, 3%, 5%, 7%, 10%, 12%, 15%, or 20% of truncated forms of one or more αs1 casein. In some cases, a single variant of αs1 casein comprises at least 1%wt/wt, 3%wt/wt, 5%wt/wt, 7%wt/wt, 10%wt/wt, 12%wt/wt, or 15%wt/wt of one or more truncated forms of αs1 casein. In some cases, a single variant of αs1 casein comprises up to 3%, 5%, 7%, 10%, 12%, 15%, or 20% of truncated forms of αs1 casein. In such compositions, the remaining percentage of αs1 casein in a single variant of αcasein is the full length form of αs1 casein.

B. Post-translational modification

Depending on the host organism used to express the casein, a single variant of casein, such as alpha casein, may have a different glycosylation or phosphorylation pattern (post-translational modification) than animal derived casein. In some cases, a single variant of casein, such as alpha casein, does not comprise post-translational modification (PTM). In some cases, a single variant of casein, such as alpha casein, comprises significantly reduced PTM. As used herein, significantly reduced PTM means at least a 50% reduction in one or more types of PTM as compared to the amount of PTM in casein of animal origin. For example, a single variant of alpha casein may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% fewer post-translational modifications than an animal-derived alpha casein. In some cases, the post-translationally modified casein may lack one or more post-translational modification sites found in animal-derived casein.

In some cases, a single variant of casein, such as alpha casein, comprises one or more PTMs that are different from animal-derived casein, e.g., a modification in the single variant of alpha casein at an amino acid that is not modified in animal-derived alpha casein, or a chemically different modification, such as a different phosphorylated structure, than animal-derived alpha casein.

Alternatively, a single variant of casein, such as alpha casein, may comprise PTM comparable to animal derived casein PTM. In some cases, a single variant of casein, such as alpha casein, comprises a significantly increased PTM. As used herein, significantly increased PTM means at least a 5% increase in one or more types of PTM as compared to the amount of PTM in casein of animal origin. For example, a single variant of alpha casein may have 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% more post-translational modifications than an animal-derived alpha casein.

The PTM in casein, such as alpha casein, may be chemically or enzymatically modified. In some cases, a single variant of casein, such as alpha casein, comprises significantly reduced or no PTM without chemical or enzymatic treatment. Single variants of casein, such as alpha casein with reduced or no PTM, may be used to produce the composition, wherein the lack of PTM is not due to chemical or enzymatic treatment of the protein, such as by recombinant production, to produce single variants of alpha casein, wherein the recombinant protein lacks PTM.

Single variants of casein, such as phosphorylation in alpha casein, may be chemically or enzymatically modified. In some cases, a single variant of casein comprises significantly reduced or no phosphorylation without chemical or enzymatic treatment. For example, a single variant of alpha casein may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less phosphorylated than an alpha casein of animal origin. Single variants of casein such as alpha casein with reduced or no phosphorylation may be used to produce the composition, wherein the lack of phosphorylation is not due to chemical or enzymatic treatment, such as where recombinant production provides single variants of casein with reduced or no phosphorylation.

The composition may be produced using a single variant of casein, such as alpha casein, which is a mixture of casein having different PTMs. For example, the composition may comprise a single variant of alpha casein, may comprise a mixture of alpha casein without PTM, with reduced PTM, with naturally equivalent (or naturally-like) PTM, and/or with increased PTM. In some cases, the composition may comprise a single variant of casein, such as alpha casein with natural (natural equivalent or animal origin) PTM, casein lacking one or more types of PTM, and/or no PTM. Alternatively, the composition may comprise a single variant of casein, such as alpha casein with a consistent PTM. In such a case, the PTM structure may include reduced PTM, PTM lacking one or more types of PTM. For example, the edible composition may comprise only a single variant of alpha casein having reduced phosphorylation.

Edible composition

A. Cheese and cheese-like analogues

The compositions of single variant casein described in this application can be used to produce cheese analogues. Cheese analogues may comprise components other than a single variant of recombinantly produced casein. Cheese analogues may comprise components other than a single variant of recombinantly produced alpha casein. In some cases, cheese analogues may include solvents such as water, fat, salt, starch, sugar, flavoring agents, acids, pH stabilizers, carbohydrates, and the like. Cheese analogues may comprise proteins other than a single variant of casein. For example, other proteins may include proteins found in dairy products of animal origin (in addition to casein). Alternatively, cheese analogues may comprise proteins not found in dairy products of animal origin, examples of which may include, but are not limited to, vegetable proteins and/or microbial proteins.

The cheese analogues described herein may comprise from 5% w/w to about 30% w/w of a recombinant single variant of casein. In some cases, a cheese analogue may comprise at least 5% w/w of a recombinant single variant of casein, such as a single variant of any of the casein described herein. In some cases, the cheese analogue may comprise up to 30% w/w of a recombinant single variant of casein. In some cases, the cheese analogue may comprise from 5% to 7% w/w, 5% to 10% w/w, 5% to 15% w/w, 5% to 20% w/w, 5% to 25% w/w, 5% to 30% w/w, 7% to 10% w/w, 7% to 15% w/w, 7% to 20% w/w, 7% to 25% w/w, 7% to 30% w/w, 10% to 15% w/w, 10% to 20% w/w, 10% to 25% w/w, 10% to 30% w/w, 15% to 25% w/w, 15% to 30% w/w, 20% to 25% w/w, 15% to 30% w/w, 25% to 25% w/w, or a single recombinant protein. In some cases, the cheese analogue may comprise about 5% w/w, 7% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w or 30% w/w of a recombinant single variant of casein.

The cheese analogues described herein may comprise from 5% w/w to about 30% w/w of a recombinant single variant of alpha casein. In some cases, a cheese analogue may comprise at least 5% w/w of a recombinant single variant of alpha casein, such as a single variant of any of the alpha casein described herein. In some cases, the cheese analogue may comprise up to 30% w/w of a recombinant single variant of alpha casein. In some cases, the cheese analogue may comprise from 5% to 7% w/w, 5% to 10% w/w, 5% to 15% w/w, 5% to 20% w/w, 5% to 25% w/w, 5% to 30% w/w, 7% to 10% w/w, 7% to 15% w/w, 7% to 20% w/w, 7% to 25% w/w, 7% to 30% w/w, 10% to 15% w/w, 10% to 20% w/w, 10% to 25% w/w, 10% to 30% w/w, 15% to 25% w/w, 15% to 30% w/w, 20% to 25% w/w, 15% to 30% w/w, 25% to 25% w/w, or a single recombinant protein. In some cases, the cheese analogue may comprise about 5% w/w, 7% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w or 30% w/w of a recombinant single variant of alpha casein. In some preferred cases, the cheese analogue may comprise a recombinant single variant of alpha casein from 10% w/w to 25% w/w, 15% w/w to 25% w/w, 18% w/w to 25% w/w, 20% w/w to 25% w/w, 10% w/w to 20% w/w, 15% w/w to 20% w/w, 18% w/w to 20% w/w.

In some cases, the cheese analogue may comprise 5% w/w to 40% w/w fat. Examples of fats that may be added to the cheese analogue include coconut oil, canola oil, high oleic sunflower oil, palm oil. Other examples are provided elsewhere herein. In some cases, the cheese analogue may comprise at least 5% w/w fat. In some cases, cheese analogues may comprise up to 40% w/w fat. In some cases, the cheese analogue may comprise from 5% to 10% w/w, 5% to 15% w/w, 5% to 20% w/w, 5% to 25% w/w, 5% to 30% w/w, 5% to 40% w/w, 10% to 15% w/w, 10% to 20% w/w, 10% to 25% w/w, 10% to 30% w/w, 10% to 40% w/w, 15% to 20% w/w, 15% to 25% w/w, 15% to 30% w/w, 15% to 40% w/w, 20% to 30% w/w, 20% to 40% w/w, 25% to 40% w/w, or 40% to 40% w/w. In some cases, the cheese analogue may comprise about 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w, or 40% w/w fat.

In some cases, the cheese analogue may comprise 0% w/w to 50% w/w starch. Examples of starches that may be added to cheese analogues include modified potato starch, corn starch. Other examples are provided elsewhere herein. In some cases, the cheese analogue may comprise at least 0% w/w starch. In some cases, cheese analogues may comprise up to 50% w/w starch. In some cases, the cheese analogue may comprise from 0% to 10% w/w, 0% to 20% w/w, 0% to 30% w/w, 0% to 40% w/w, 0% to 50% w/w, 10% to 20% w/w, 10% to 30% w/w, 10% to 40% w/w, 10% to 50% w/w, 20% to 30% w/w, 20% to 40% w/w, 20% to 50% w/w, 30% to 40% w/w, 30% to 50% w/w, or 40% to 50% w/w of starch. In some cases, the cheese analogue may comprise 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 analogue may comprise from 0.5% w/w to 1% w/w, 0.5% w/w to 2% w/w, 0.5% w/w to 3% w/w, 0.5% w/w to 4% w/w, 1% w/w to 2% w/w, 1% w/w to 3% w/w, 1% w/w to 4% w/w, 2% w/w to 3% w/w, 2% w/w to 4% w/w, 3% w/w to 4% w/w starch.

Preferably, the cheese analogue may comprise up to 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% to 5% w/w, 0% to 10% w/w, 0% to 15% w/w, 0% to 20% w/w, 0% to 25% w/w, 0% to 30% w/w, 5% to 10% w/w, 5% to 15% w/w, 5% to 20% w/w, 5% to 25% w/w, 5% to 30% w/w, 10% to 15% w/w, 10% to 20% w/w, 10% to 25% w/w, 10% to 30% w/w, 15% to 25% w/w, 15% to 30% w/w, 20% to 25% w/w, or 15% to 30% w/w. In some cases, the cheese analogue may comprise 0% w/w, 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w or 30% w/w starch. In some cases, the cheese analogue may comprise up to 1%, 5%, 10%, 15%, 20%, 25% or 30% w/w starch.

In some cases, cheese analogues may comprise 0% w/w to 16% w/w of salts, such as calcium salts, emulsifying salts, common salts, and the like. Examples of such salts are also provided elsewhere herein.

In some cases, the cheese analogue may comprise a calcium salt, such as calcium chloride. In some cases, the cheese analogue may comprise 0.1% w/w to 6% w/w calcium salt. In some cases, the cheese analogue may comprise at least 0.1% w/w calcium salt. In some cases, the cheese analogue may comprise up to 6% w/w calcium salt. In some cases, the cheese analogue may comprise 0.1% to 1% w/w, 0.1% to 2% w/w, 0.1% to 3% w/w, 0.1% to 4% w/w, 0.1% to 5% w/w, 0.1% to 6% w/w, 1% to 2% w/w, 1% to 3% w/w, 1% to 4% w/w, 1% to 5% w/w, 1% to 6% w/w, 2% to 3% w/w, 2% to 4% w/w, 2% to 5% w/w, 3% to 4% w/w, 3% to 5% w/w, 6% to 6% w/w, or 4% to 6% w/w of the calcium salt. In some cases, the cheese analogue may comprise about 0.1% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w or 6% w/w calcium salt. In some cases, the cheese analogue may comprise less than 0.1% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w or 6% w/w calcium salt. In some cases, the cheese analogue may comprise more than 0.1% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w or 5% w/w calcium salt. In some cases, cheese analogues may contain calcium ions. The calcium ions may be added to the cheese analogue in the form of a calcium-based salt, such as calcium chloride.

In some cases, the cheese analogue may comprise 0% w/w to 0.6% w/w calcium ions. In some cases, the cheese analogue may comprise at least 0% w/w calcium ions. In some cases, cheese analogues may contain up to 0.6% w/w calcium ions. In some cases, the cheese analogue may comprise 0% w/w to 0.1% w/w, 0% w/w to 0.2% w/w, 0% w/w to 0.3% w/w, 0% w/w to 0.4% w/w, 0% w/w to 0.5% w/w, 0% w/w to 0.6% w/w, 0.1% w/w to 0.2% w/w, 0.1% w/w to 0.3% w/w, 0.1% w/w to 0.4% w/w, 0.1% w/w to 0.5% w/w, 0.1% w/w to 0.6% w/w, 0.2% w/w to 0.3% w/w, 0.2% w/w to 0.5% w/w, 0.2% w/w to 0.6% w/w, 0.2% w/w to 0.3% w/w, 0.1% w/w to 0.4% w/w, 0.6% w/w to 0.5% w/w, 0.1% w to 0.4% w/w, 0.6% w/w to 0.5% w/w. In some cases, the cheese analogue may comprise about 0% w/w, 0.1% w/w, 0.2% w/w, 0.3% w/w, 0.4% w/w, 0.5% w/w, or 0.6% w/w calcium ions. In some cases, the cheese analogue may comprise at least 0.1% w/w, 0.2% w/w, 0.3% w/w, 0.4% w/w, 0.5% w/w or 0.6% w/w calcium ions. In some cases, the cheese analogue may contain up to 0.1% w/w, 0.2% w/w, 0.3% w/w, 0.4% w/w or 0.5% w/w calcium ions.

In some cases, the cheese analogue may contain 0mg to 30mg of calcium ions per gram of casein. In some cases, the cheese analogue may contain at least 0mg of calcium ions per gram of casein. In some cases, the cheese analogue may contain at most 30mg of calcium ions per gram of casein. In some cases, the cheese analogue may contain 0 to 5mg, 0 to 10mg, 0 to 15mg, 0 to 20mg, 0 to 25mg, 0 to 30mg, 5 to 10mg, 5 to 15mg, 5 to 20mg, 5 to 25mg, 5 to 30mg, 10 to 15mg, 10 to 20mg, 10 to 25mg, 10 to 30mg, 15 to 20mg, 15 to 25mg, 15 to 30mg, 20 to 25mg, 20 to 30mg, or 25 to 30mg of calcium ions per gram of casein. In some cases, the cheese analogue may contain 0mg, 5mg, 10mg, 15mg, 20mg, 25mg or 30mg of calcium ions per gram of casein. In some cases, the cheese analogue may comprise at least 1mg, 5mg, 10mg, 15mg, 20mg, 25mg or 30mg of calcium ions per gram of casein. In some cases, the cheese analogue may contain at most 1mg, 5mg, 10mg, 15mg, 20mg, 25mg or 30mg of calcium ions per gram of casein.

In some cases, the cheese analogue may include an emulsifying salt, such as disodium phosphate, trisodium citrate, or other emulsifying salts. In some cases, the cheese analogue may comprise 0.1% w/w to 6% w/w of the emulsifying salt. In some cases, the cheese analogue may comprise at least 0.1% w/w of the emulsifying salt. In some cases, cheese analogues may contain up to 6% w/w of emulsified salt. In some cases, the cheese analogue may comprise 0.1% to 1% w/w, 0.1% to 2% w/w, 0.1% to 3% w/w, 0.1% to 4% w/w, 0.1% to 5% w/w, 0.1% to 6% w/w, 1% to 2% w/w, 1% to 3% w/w, 1% to 4% w/w, 1% to 5% w/w, 1% to 6% w/w, 2% to 3% w/w, 2% to 4% w/w, 2% to 5% w/w, 3% to 4% w/w, 3% to 5% w/w, 6% to 6% w/w, or 4% to 6% w/w. In some cases, the cheese analogue may comprise about 0.1% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w, or 6% w/w of the emulsifying salt. In some cases, the cheese analogue may contain less than 0.1% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w, or 6% w/w of the emulsifying salt. In some cases, cheese analogues may comprise more than 0.1% w/w, 1% w/w, 2% w/w, 3% w/w, 4% w/w, or 5% w/w of emulsifying salts. Alternatively, in some cases, the cheese analogue does not contain any emulsifying salts. In some cases, the cheese analogue does not contain any emulsifying agent other than an emulsifying salt.

In some embodiments, the cheese analogue may comprise a ratio of casein (e.g., recombinant single variant casein) to emulsifying salt. In some cases, the cheese analogue may 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 analogue may 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 analogue may 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.

In some cases, the cheese analogue may have a ratio (weight/weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of between about 3:1 and 6:1. In some cases, the cheese analogue may have a ratio (weight/weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of between about 6:1 and 9:1. In some cases, the cheese analogue may have a ratio (weight/weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of between about 8:1 and 10:1. In some cases, the cheese analogue may have a ratio (weight/weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of between about 8:1 and 12:1. In some cases, the cheese analogue may have a ratio (weight/weight) of casein (e.g., recombinant single variant casein) to emulsifying salt of between about 8:1 and 15:1.

In some cases, the cheese analogue may comprise a common salt such as sodium chloride salt. In some cases, the cheese analogue may comprise 0.1% w/w to 4% w/w sodium chloride. In some cases, the cheese analogue may comprise at least 0.1% w/w sodium chloride. In some cases, the cheese analogue may comprise up to 4% w/w sodium chloride. In some cases, the cheese analogue may comprise sodium chloride from 0.1% w/w to 1% w/w, from 0.1% w/w to 2% w/w, from 0.1% w/w to 3% w/w, from 0.1% w/w to 4% w/w, from 1% w/w to 2% w/w, from 1% w/w to 3% w/w, from 1% w/w to 4% w/w, from 2% w/w to 3% w/w, from 2% w/w to 4% w/w, or from 3% w/w to 4% w/w. In some cases, the cheese analogue may comprise about 0.1% w/w, 1% w/w, 2% w/w, 3% w/w or 4% w/w sodium chloride. In some cases, the cheese analogue may comprise less than 0.1% w/w, 1% w/w, 2% w/w, 3% w/w or 4% w/w sodium chloride. In some cases, the cheese analogue may comprise more than 0.1% w/w, 1% w/w, 2% w/w or 3% w/w sodium chloride.

In some examples, a single variant of casein, such as recombinantly produced alpha casein (5% w/w-30% w/w of cheese analogue) (exemplary optimal range 10% w/w-20% w/w) may be combined with: water (30% w/w-65% w/w) (exemplary optimal range 45% w/w-55% w/w), fat (5% w/w-40% w/w) (exemplary optimal range 20% w/w-25% w/w), sodium chloride (salt) (exemplary optimal range 0% w/w-1.5% w/w), calcium chloride (exemplary optimal range 0% w/w-6% w/w) (exemplary optimal range 0% w/w-1.5% w/w), emulsifying salt (disodium phosphate, trisodium citrate) (0% w/w-6% w/w) (exemplary optimal range 0% w/w-3% w/w), starch (0% w/w-50% w/w) (exemplary optimal range 0% w/w-8% w/w), natural pure flavoring agent (0% w/w-5% w/w) (exemplary optimal range 0.5% w/w-1.5% w/w), and acid (exemplary optimal range 0% w/w-1.5% w/w). Optional ingredients such as plant-based or other animal-free (animal-free) proteins (0% -30%) (exemplary optimal range 0% w/w-8% w/w), hydrocolloids (0% -5%) (exemplary optimal range 0% -2%), sugars such as mono-, di-and oligosaccharides (0% w/w-5% w/w) (exemplary optimal range 0% w/w-2% w/w), emulsifiers such as mono-and diglycerides (0% w/w-2% w/w) (exemplary optimal range 0% w/w-0.5% w/w), natural flavor masking agents, color additives (0% w/w-5% w/w), preservatives (0% w/w-1% w/w), anti-caking agents (0% w/w-2% w/w) and micronutrients such as vitamins (0% -1%) may also be incorporated into the cheese analogue.

In some embodiments, the following ingredients are premixed: a single variant of recombinantly produced alpha casein, fat, water, starch, salt such as sodium chloride. In some embodiments, a pH adjuster such as sodium hydroxide (lye) is used at this stage to adjust the pH of the composition to a neutral pH of 6.8-7.2. Optional ingredients such as plant-based proteins or other animal-component-free proteins, sugars, hydrocolloids, and emulsifiers may be added at this stage or later. Premixing may occur at ambient or elevated temperatures (15 ℃ C. -50 ℃ C.). In some cases, the fats are pre-melted [30 ℃ -70 ℃ ] (exemplary optimal range 40 ℃ -50 ℃), and maintained at their melting temperature prior to incorporation. The calcium chloride and emulsifying salt may be added during the pre-mixing stage or later. Alternatively, the calcium chloride and emulsifying salt may be added continuously in any order: the calcium chloride may be added before or after emulsifying the salt. For example, calcium chloride and emulsifying salts may be added in 2 stages over the course of 4 minutes to 1 hour (exemplary optimal range 10 minutes to 20 minutes), with an incubation interval of 2 minutes to 30 minutes (exemplary optimal range 5 minutes to 10 minutes) at ambient or elevated temperature. Alternatively, cheese analogues may be produced in the absence of calcium chloride or emulsifying salts. Calcium chloride may also be added at the end of the cheese analogue preparation process, either before or after the acid addition.

While mechanically mixing, the mixture may be heated for a ramp period of time ranging from a pre-mix temperature (ambient or high) to 50 ℃ -95 ℃ (exemplary optimal range 75 ℃ -90 ℃), 1 minute to 30 minutes (exemplary optimal range 1min to 5 min). The heated mixture may then be held at the final ramp temperature for 0 minutes to 20 minutes (an exemplary optimum range of 2min-5 min) at which time the ingredients are mechanically incorporated to form an emulsion. Mechanical incorporation (mixing) can be achieved using a variety of mixers, such as a vertical cutter mixer or a twin screw mixer.

The acidity of the mixture can be adjusted by incorporating an acid such as lactic acid or citric acid and continuing to mix briefly until the final pH is about 5-6.5 (an exemplary optimum range is 5.7-6.2). Acidity can also be regulated by using glucono-delta-lactone early in the mixing process. The resulting mixture may then be placed into a mold, other shaped container, or vacuum sealed package. The resulting product can be cooled to 4 ℃ immediately after dispensing into a mold to produce a cheese analogue. Such cheese analogues can then be used as food, ingredients and incorporated into other food products.

In some embodiments, the amount of salt or minerals in the cheese may be varied to produce advantageous qualities. For example, in one example, the amount of calcium may be varied to improve melting, texture, stretchability, and the like. In one example, the amount of calcium in the cheese analogue may be reduced to improve the melting of the cheese analogue. In another example, the amount of calcium in the cheese analogue may be increased to improve the texture or stretchability of the cheese analogue.

In some embodiments, the cheese analogue comprises a single variant casein, and the single variant casein, such as alpha casein, provides one or more properties of the cheese or cheese analogue to the composition.

The texture of cheese analogues prepared with a single variant of casein, e.g. alpha casein such as by the methods described herein, may be comparable to the texture of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The texture of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, described herein may be comparable to the texture of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The texture of cheese analogues prepared using compositions having a single variant of casein described herein, such as alpha casein, may be improved/more desirable when compared to the texture of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein, or when compared to cheese analogues of plant origin lacking dairy proteins, i.e. cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as sole/primary protein sources, or cheese-like products prepared without proteins, such as cheese-like products prepared primarily with starch. The texture of cheese analogues can be tested using a trained group of human subjects or using a machine such as a texture analyzer.

The taste of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, may be comparable to the taste of similar types of cheese prepared using dairy proteins of animal origin, such as cheese prepared from animal milk. The taste of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be comparable to the taste of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The taste of cheese analogues prepared using compositions described herein having a single variant of casein, such as alpha casein, may be improved when compared to the taste of cheese or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins, i.e. cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as the sole/primary protein source, or cheese-like products prepared without proteins, such as cheese-like products prepared mainly with starch. The taste of cheese can be tested using a trained group of human subjects.

Cheese analogue compositions described herein having a single variant of casein, such as alpha casein, may have browning capabilities comparable to similar types of cheese prepared using dairy proteins of animal origin, such as cheese prepared from animal milk. A cheese analogue composition with a single variant of casein, such as alpha casein, described herein may have browning capabilities comparable to similar types of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. A cheese analogue composition with a single variant of casein, such as alpha casein, described herein may have improved browning capabilities when compared to similar types of cheeses or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins (i.e., cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as the sole/primary protein source, or cheese-like products prepared without proteins (such as cheese-like products prepared primarily with starch)). The browning ability of cheese analogues can be tested using oven and computer imaging.

Cheese analogue compositions described herein having a single variant of casein, such as alpha casein, may have melting capacities comparable to similar types of cheese prepared using dairy proteins of animal origin, such as cheese prepared from animal milk. A cheese analogue composition with a single variant of casein, such as alpha casein, described herein may have a melting capacity comparable to similar types of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The cheese analogue compositions described herein with a single variant of casein, such as alpha casein, may have improved melting ability when compared to similar types of cheeses or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins (i.e., cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as the sole/primary protein source, or cheese-like products prepared without proteins (such as cheese-like products prepared primarily with starch)). The meltability of cheese analogues can be tested using a modified Schreiber meltdown test and computer imaging. Exemplary assays for measuring such melting properties are provided in the examples section.

In some embodiments, the melting characteristics of cheese or cheese analogue are analyzed by heating, such as by heating at 95 ℃ for 15 minutes on a hotplate, and the melting is assessed by the ratio of melted area to unmelted area, where melting is defined as a ratio of greater than or equal to 1. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a melting value of 1 or greater than 1. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a melting value of greater than 1, such as 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 than 2.6. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a melting value of greater than 1, such as 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, cheese analogues comprising recombinant single variant alpha casein have a melting value of greater than 1, and the melted regions remain opaque in appearance. In some embodiments, the cheese analogue comprising the recombinant single variant alpha casein has a melting value that is greater than the melting value of the plant-based cheese analogue, e.g., the melting value of the cheese analogue comprising the recombinant single variant alpha casein is 1.5×, 2×, 2.5×, or greater than 2.5× the melting value of the plant-based cheese analogue.

The stretching ability of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, may be comparable to the stretching ability of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The stretching ability of cheese prepared using a composition having a single variant of casein, such as alpha casein, described herein may be comparable to the stretching ability of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The stretching ability of cheeses prepared using a composition having a single variant of casein described herein, such as alpha casein, may be improved/more desirable when compared to the stretching ability of cheeses or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet, or when compared to cheese analogues of plant origin lacking dairy proteins, i.e. cheese-like products prepared with plant origin proteins such as peas, chickpeas, nuts and/or other plant proteins as sole/primary protein sources, or cheese-like products prepared without proteins, such as cheese-like products prepared mainly with starch. The stretching ability of cheese can be tested using a trained group of human subjects or using a machine such as a texture analyzer. The cheese analogue prepared using the methods described herein may have a stretchability of greater than 2.5cm when measured on a texture analyzer after cooking. Exemplary assays for measuring such tensile properties are provided in the examples section.

In some embodiments, the stretchability of the cheese or cheese analogue is analyzed using a texture analyzer, such as by heating the cheese in an oven at 90 ℃ for 10min in a malleability device, and the malleability is measured as the distance to failure (i.e., the distance that all the cheese filaments break) on the texture analyzer. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a stretch value of about 200mm (20 cm). In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a stretchability of at least 40mm, at least 50mm, at least 60mm, at least 80mm, at least 100mm, at least 120mm, at least 140mm, at least 160mm, at least 180mm, at least 190mm, at least 200mm, at least 210mm, at least 220mm, at least 230mm, at least 240mm, at least 250mm, or greater than 250 mm. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a stretchability of between 40mm-70mm, 70mm-100mm, 100mm-125mm, 125mm-150mm, 150mm-180mm, 180mm-200mm, 200mm-225mm, 225mm-250mm or 250mm-300 mm. In some embodiments, the cheese analogue comprising the recombinant single variant alpha casein has a stretchability that is greater than the stretchability of the plant-based cheese analogue, e.g., the stretchability of the cheese analogue comprising the recombinant single variant alpha casein is at least 2×, at least 3×, at least 4×, at least 5×, at least 7×, at least 10× the stretchability of the plant-based cheese analogue. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a stretchability comparable to that of low moisture milk-derived cheeses, such as low moisture mozzarella cheese, or caseinate-containing cheeses, such as mozzarella imitation cheese (imitation mozzarella cheese). In some embodiments, cheese analogues comprising recombinant single variant alpha casein have a stretchability within 10% -20%, 15% -25%, 20% -40% or 10% -150% of the stretchability of low moisture milk-derived cheese (such as low moisture mozzarella cheese) or caseinate-containing cheese (such as mozzarella imitation cheese).

The firmness of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, may be comparable to the firmness of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The firmness of a cheese analogue prepared using a composition having a single variant of casein, such as alpha casein, described herein may be comparable to the firmness of a cheese or cheese analogue prepared using a micellar form of casein, such as a cheese prepared from milk or a cheese analogue prepared from caseinate or rennet casein. The firmness of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be improved when compared to the firmness of cheese or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins, i.e. cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as sole/primary protein source, or cheese-like products prepared without proteins, such as cheese-like products prepared mainly with starch. The hardness of cheese analogues can be tested using a trained group of human subjects or using a machine such as a texture analyzer.

The adhesiveness of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, may be comparable to the adhesiveness of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The adhesiveness of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be comparable to the adhesiveness of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The adhesiveness of cheese analogues prepared by the methods described herein with a single variant of casein, such as alpha casein, may be reduced when compared to the adhesiveness of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The adhesiveness of cheese analogues prepared by the methods described herein with a single variant of casein, such as alpha casein, may be reduced when compared to the adhesiveness of cheese analogues of plant origin lacking dairy proteins (i.e. cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as sole/primary protein source, or cheese-like products prepared without proteins, such as cheese-like products prepared mainly with starch). The adhesion of cheese analogues can be tested using a trained group of human subjects or using a machine such as a texture analyzer. In texture analyzer testing, the adhesion of cheese analogues prepared using the methods described herein may be less than 2 (g x sec). Exemplary assays for measuring such adhesive properties are provided in the examples section.

The adhesion of cheese analogues can be tested using a texture analyser, e.g. equipped with TA-18 ¹ / ₂ "TA.XT Plus texture analyser of spherical probe, such as where the adhesion represents the force required to remove cheese from the probe. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have an adhesion between 0.1g x sec and 3.0g x sec. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have an adhesion of less than 3.0g x sec, less than 2.5g x sec, less than 2.0g x sec, less than 1.5g x sec, less than 1.0g x sec, or less than 0.5g x sec. In some embodimentsCheese analogues comprising recombinant single variant alpha casein have an adhesion such as between 0.1g sec-2.5g sec, 0.1g sec-2.0g sec, 0.1g sec-1.5g sec, 0.1g sec-1.0g sec, 0.1g sec-0.5g sec, 0.5g sec-2.5g sec, 0.5g sec-2.0g sec, 0.5g sec-1.5g sec, 0.5g sec-1.0g sec, or 0.5g sec-1.5g sec. In some embodiments, cheese analogues comprising recombinant single variant alpha casein have reduced adhesion compared to low moisture milk derived cheeses (such as low moisture mozzarella cheese) or caseinate containing cheeses (such as mozzarella imitation cheese). In some embodiments, the cheese analogue comprising the recombinant single variant α0 casein has a reduced adhesion of at least 2α1, at least 3α2, at least 5α3, at least 10α4, at least 20α5, or a reduced adhesion of greater than 20α6 when compared to the adhesion of a low moisture milk-derived cheese (such as low moisture mozzarella cheese) or a caseinate-containing cheese (such as mozzarella-like cheese). In some embodiments, cheese analogues comprising recombinant single variant alpha casein have reduced adhesion compared to plant-based cheese analogues. In some embodiments, the adhesiveness of a cheese analogue comprising a recombinant single variant alpha casein is reduced by at least 2 alpha 7, at least 3×, at least 5×, at least 10×, at least 15×, at least 20×, or by more than 20×, when compared to the adhesiveness of a plant-based cheese analogue.

The creaminess of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, can be comparable to the creaminess of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The creaminess of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be comparable to the creaminess of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The creaminess of cheese analogues prepared using compositions described herein having a single variant of casein, such as alpha casein, can be improved when compared to the creaminess of cheeses or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins (i.e., cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as the sole/primary protein source, or cheese-like products prepared without proteins (such as cheese-like products prepared with primarily starch)).

The mouthfeel of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, can be comparable to the mouthfeel of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The mouthfeel of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be comparable to the mouthfeel of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein. The mouthfeel of cheese analogues prepared using compositions described herein having a single variant of casein, such as alpha casein, can be improved when compared to the mouthfeel of cheeses or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins (i.e., cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as the sole/primary protein source, or cheese-like products prepared without proteins (such as cheese-like products prepared primarily with starch)).

When heated (e.g., melted), the opacity of cheese analogues prepared with a single variant of casein, such as alpha casein, such as by the methods described herein, can be comparable to the opacity of similar types of cheeses prepared using dairy proteins of animal origin, such as cheeses prepared from animal milk. The opacity of cheese analogues prepared using a composition having a single variant of casein, such as alpha casein, described herein may be comparable to the opacity of cheese or cheese analogues prepared using micellar forms of casein, such as cheese prepared from milk or cheese analogues prepared from caseinate or rennet casein, when heated. When heated, the opacity of cheese analogues prepared using compositions described herein having a single variant of casein, such as alpha casein, can be improved compared to the opacity of cheese or cheese analogues prepared using plant-derived cheese analogues lacking dairy proteins (i.e., cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as the sole/primary protein source, or cheese-like products prepared without proteins (such as cheese-like products prepared primarily with starch)).

The cheese analogue or similar composition described herein may be a low moisture cheese analogue. For example, the low moisture cheese analogue may contain from 45% w/w to 52% w/w moisture. The low moisture cheese analogue may contain less than 52% w/w moisture.

B. Yoghurt or yoghurt analogue

Single variant casein compositions, such as the compositions described herein comprising alpha casein, may be used to produce edible compositions such as yogurt analogues or yogurt-like compositions. For example, a single variant alpha casein composition may be used to form a yogurt analog product. Yoghurt analogue products may be formed without the formation of micelles or micelle-like compositions. Yoghurt analogue products prepared using the compositions described herein may provide similar or equivalent characteristics (such as texture, creaminess, firmness, viscosity, tackiness, viscosity, smell and taste) to dairy yoghurt of animal origin or to dairy yoghurt analogues prepared using micellar forms of casein (such as dairy yoghurt analogues prepared from caseinates, rennet casein or micellar casein). Yoghurt-like products prepared using the compositions described herein may provide improvements in one or more characteristics such as texture, creaminess, firmness, viscosity, adhesion, viscosity, smell and taste when compared to yoghurt-like products of plant origin (i.e. yoghurt-like products prepared without using protein) that lack any dairy proteins, or yoghurt-like products prepared using plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as sole protein sources, meaning that they do not comprise any casein.

The yogurt analogues described herein may comprise recombinant single variants of casein from 1% w/w to about 20% w/w. In some cases, the yogurt analog may comprise at least 1% w/w of a recombinant single variant of casein, such as a single variant of any of the casein described herein. In some cases, the yogurt analogue may comprise up to 20% w/w of a recombinant single variant of casein, such as a single variant of any of the casein described herein. In some of the cases where the number of the cases, the yogurt analog may comprise 1% w/w to 2% w/w, 1% w/w to 4% w/w, 1% w/w to 6% w/w, 1% w/w to 8% w/w, 1% w/w to 10% w/w, 1% w/w to 15% w/w, 1% w/w to 20% w/w, 2% w/w to 4% w/w, 2% w/w to 6% w/w, 2% w/w to 8% w/w, 2% w/w to 10% w/w, 2% w/w to 15% w/w, 2% w/w to 20% w/w, 4% w/w to 6% w/w 4% w/w to 8% w/w, 4% w/w to 10% w/w, 4% w/w to 15% w/w, 4% w/w to 20% w/w, 6% w/w to 8% w/w, 6% w/w to 10% w/w, 6% w/w to 15% w/w, 6% w/w to 20% w/w, 8% w/w to 10% w/w, 8% w/w to 15% w/w, 8% w/w to 20% w/w, 10% w/w to 15% w/w, 10% w/w to 20% w/w, or 15% w/w to 20% w/w of a recombinant single variant of casein, such as a single variant of any casein described herein. In some cases, the yogurt analog may comprise 1% w/w, 2% w/w, 4% w/w, 6% w/w, 8% w/w, 10% w/w, 15% w/w, or 20% w/w of a recombinant single variant of casein, such as a single variant of any casein described herein. In some preferred embodiments, the yogurt analogue may comprise from 2% w/w to 5% w/w of a recombinant single variant of casein.

The yogurt analogues described herein may comprise recombinant single variants of alpha casein from 1% w/w to about 20% w/w. In some cases, the yogurt analog may comprise at least 1% w/w of a recombinant single variant of alpha casein, such as a single variant of any of the alpha casein described herein. In some cases, the yogurt analog may comprise up to 20% w/w of a recombinant single variant of alpha casein, such as a single variant of any of the alpha casein described herein. In some of the cases where the number of the cases, the yogurt analog may comprise 1% to 2% w/w, 1% to 4% w/w, 1% to 6% w/w, 1% to 8% w/w, 1% to 10% w/w, 1% to 15% w/w, 1% to 20% w/w, 2% to 4% w/w, 2% to 6% w/w, 2% to 15% w/w, 2% to 20% w/w, 4% to 10% w/w, 4% to 6% w/w, 4% to 8% w/w, 4% to 10% w/w, 4% to 15% w/w, 4% to 10% w/w, 15% to 15% w/w, and 15% to 15% w/w, respectively, and the recombinant proteins may be present in a composition comprising the composition of the yogurt analog. Such as a single variant of any of the alpha casein described herein. In some cases, the yogurt analog may comprise 1% w/w, 2% w/w, 4% w/w, 6% w/w, 8% w/w, 10% w/w, 15% w/w, or 20% w/w of a recombinant single variant of alpha casein, such as a single variant of any of the alpha casein described herein. In some preferred embodiments, the yogurt analog may comprise from 2% w/w to 5% w/w of a recombinant single variant of alpha casein.

The yoghurt analogue described herein may comprise 0.5% w/w to 20% w/w fat. The yoghurt analogue described herein may comprise at least 0.5% w/w fat. The yoghurt analogue described herein may comprise up to 20% w/w fat. The yogurt analogs described herein may comprise 0.5% to 1% w/w, 0.5% to 4% w/w, 0.5% to 6% w/w, 0.5% to 8% w/w, 0.5% to 10% w/w, 0.5% to 12% w/w, 0.5% to 15% w/w, 0.5% to 18% w/w, 0.5% to 20% w/w, 1% to 4% w/w, 1% to 6% w/w, 1% to 8% w/w, 1% to 10% w/w, 1% to 12% w/w, 1% to 15% w/w, 1% to 10% w/w 1% to 18% w/w, 1% to 20% w/w, 4% to 6% w/w, 4% to 8% w/w, 4% to 10% w/w, 4% to 12% w/w, 4% to 15% w/w, 4% to 18% w/w, 4% to 20% w/w, 6% to 8% w/w, 6% to 10% w/w, 6% to 12% w/w, 6% to 15% w/w, 6% to 18% w/w, 6% to 20% w/w, 8% to 10% w/w, 8% to 12% w/w, 8% w/w to 15% w/w, 8% w/w to 18% w/w, 8% w/w to 20% w/w, 10% w/w to 12% w/w, 10% w/w to 15% w/w, 10% w/w to 18% w/w, 10% w/w to 20% w/w, 12% w/w to 15% w/w, 12% w/w to 18% w/w, 12% w/w to 20% w/w, 15% w/w to 18% w/w, 15% w/w to 20% w/w, or 18% w/w to 20% w/w of fat. The yogurt analogues described herein may comprise 0.5% w/w, 1% w/w, 4% w/w, 6% w/w, 8% w/w, 10% w/w, 12% w/w, 15% w/w, 18% w/w or 20% w/w fat. The yoghurt analogue described herein may comprise up to 0.5% w/w, 1% w/w, 4% w/w, 6% w/w, 8% w/w, 10% w/w, 12% w/w, 15% w/w or 18% w/w fat. The yogurt analogues described herein may comprise at least 0.5% w/w, 1% w/w, 4% w/w, 6% w/w, 8% w/w, 10% w/w, 12% w/w, 15% w/w or 18% w/w fat. Alternatively, the yogurt analogue may not comprise any fat.

The yoghurt analogue described herein may comprise from 0% w/w to 10% w/w starch. The yogurt analog described herein may comprise at least 0.5% w/w starch. The yoghurt analogue described herein may comprise up to 10% w/w starch. The yogurt analogues described herein may comprise 0.5% to 1% w/w, 0.5% to 2% w/w, 0.5% to 4% w/w, 0.5% to 6% w/w, 0.5% to 8% w/w, 0.5% to 10% w/w, 1% to 2% w/w, 1% to 4% w/w, 1% to 6% w/w, 1% to 8% w/w, 1% to 10% w/w, 2% to 4% w/w, 2% to 6% w/w, 2% to 8% w/w, 4% to 6% w/w, 4% to 8% w/w, 10% to 10% w/w of starch. The yoghurt analogue described herein may comprise 0.5% w/w, 1% w/w, 2% w/w, 4% w/w, 6% w/w, 8% w/w or 10% w/w starch. The yoghurt analogue described herein may comprise up to 0.5% w/w, 1% w/w, 2% w/w, 4% w/w, 6% w/w, 8% w/w or 10% w/w starch. The yogurt analogues described herein may comprise at least 0.5% w/w, 1% w/w, 2% w/w, 4% w/w, 6% w/w, 8% w/w or 10% w/w starch.

The yogurt analogues described herein may comprise proteins other than a single variant of casein. For example, other proteins may include proteins found in dairy products of animal origin (in addition to casein). Alternatively, yogurt analogs may comprise proteins not found in dairy products of animal origin, examples of which may include, but are not limited to, vegetable proteins and/or microbial proteins.

In some embodiments, the yogurt analog comprises a single variant casein, and the single variant casein, such as alpha casein, provides one or more properties of the yogurt analog to the composition.

The texture of a yoghurt analogue prepared with a single variant of casein, e.g. alpha casein such as by the method described herein, may be comparable to the texture of a similar type of yoghurt prepared using dairy proteins of animal origin, such as yoghurt prepared from animal milk. The texture of a yogurt analogue prepared using a composition having a single variant of casein described herein, such as alpha casein, may be comparable to the texture of a yogurt or yogurt analogue prepared using a micellar form of casein (such as a yogurt prepared from milk or a yogurt analogue prepared from caseinate, micellar casein or rennet casein). The texture of a yogurt analogue prepared using a composition having a single variant of casein described herein, such as alpha casein, may be improved/more desirable when compared to the texture of a yogurt or yogurt analogue prepared using micelles, such as yogurt prepared from milk or yogurt analogue prepared from caseinates, micellar casein or rennet casein, or when compared to a cheese analogue of plant origin lacking dairy proteins (i.e. cheese-like products prepared with plant-derived proteins such as peas, chickpeas, nuts and/or other plant proteins as sole/primary protein sources, or cheese-like products prepared without proteins, such as cheese-like products prepared primarily with starch). The texture of the yogurt analog may be tested using a trained group of human subjects or using a machine such as a texture analyzer. Texture properties may include, for example, one or more of firmness, tack, cohesiveness, and viscosity of yogurt or yogurt analogs.

The smoothness or creaminess of a yoghurt analogue prepared with a single variant of casein, e.g. alpha casein such as by the method described herein, may be comparable to the smoothness of a similar type of yoghurt prepared using milk product proteins of animal origin, such as yoghurt prepared from animal milk. The smoothness of a yogurt analogue prepared using a composition having a single variant of casein described herein, such as alpha casein, may be comparable to the smoothness of a yogurt or yogurt analogue prepared using a micellar form of casein (such as a yogurt prepared from milk or a yogurt analogue prepared from caseinate, micellar casein or rennet casein). The smoothness of a yogurt analogue prepared using a composition having a single variant of casein described herein, such as alpha casein, may be improved/more desirable when compared to the smoothness of a yogurt or yogurt analogue prepared using a micellar form of casein, such as yogurt prepared from milk or yogurt analogue prepared from caseinate, micellar casein or rennet casein, or when compared to a cheese analogue of plant origin lacking dairy proteins, i.e. a cheese-like product prepared with plant derived proteins such as peas, chickpeas, nuts and/or other plant proteins as sole/primary protein sources, or a cheese-like product prepared without proteins, such as cheese-like products prepared primarily with starch.

The odor of a yogurt analogue prepared with a single variant of casein, e.g. alpha casein, such as by the method described herein, may be comparable to the odor of a similar type of yogurt prepared using dairy proteins of animal origin, such as yogurt prepared from animal milk. The smell of a yoghurt analogue prepared using a composition with a single variant of casein, such as alpha casein, as described herein may be comparable to the smell of a yoghurt or yoghurt analogue prepared using a micellar form of casein, such as a yoghurt prepared from milk or a yoghurt analogue prepared from caseinate, micellar casein or rennet casein. The odor of a yogurt analogue prepared using a composition having a single variant of casein, such as alpha casein, described herein may be improved/more desirable when compared to the odor of a yogurt or yogurt analogue prepared using a micellar form of casein, such as yogurt prepared from milk or yogurt analogue prepared from caseinate, micellar casein or rennet casein, or when compared to a cheese analogue of plant origin lacking dairy proteins (i.e. cheese-like products prepared with plant-derived proteins, such as peas, chickpea, nuts and/or other plant proteins, as sole/primary protein sources, or cheese-like products prepared without proteins, such as cheese-like products prepared mainly with starch).

The taste of a yoghurt analogue prepared with a single variant of casein, e.g. alpha casein, such as by the method described herein, may be comparable to the taste of a similar type of yoghurt prepared using milk product proteins of animal origin, such as yoghurt prepared from animal milk. The taste of a yoghurt analogue prepared using a composition with a single variant of casein, such as alpha casein, as described herein may be comparable to the taste of a yoghurt or yoghurt analogue prepared using a micellar form of casein, such as a yoghurt prepared from milk or a yoghurt analogue prepared from caseinate, micellar casein or rennet casein. The taste of a yogurt analogue prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be improved/more desirable when compared to the taste of a yogurt or yogurt analogue prepared using a micellar form of casein, such as yogurt prepared from milk or yogurt analogue prepared from caseinate, micellar casein or rennet casein, or when compared to a cheese analogue of plant origin lacking dairy proteins (i.e. cheese-like products prepared with plant-derived proteins, such as peas, chickpea, nuts and/or other plant proteins, as sole/primary protein sources, or cheese-like products prepared without proteins, such as cheese-like products prepared mainly with starch). The taste of yogurt analogs can be tested using a trained group of human subjects.

C. Beverage

Single variant casein compositions, such as the compositions described herein comprising alpha casein, may be used to produce edible compositions such as beverages. For example, a single variant alpha casein composition may be used to form a milk-like beverage or a yoghurt-like beverage. The beverage product may be formed without forming micelles or micelle-like compositions. Beverage products prepared using the compositions described herein may provide similar or equivalent characteristics (such as texture, creaminess, and taste) to dairy beverages of animal origin or dairy beverages prepared using micellar forms of casein (such as dairy beverages prepared from caseinates or micellar casein). Beverage products prepared using the compositions described herein may provide improvements in one or more characteristics, such as texture, creaminess, and taste, when compared to beverages prepared with plant-derived proteins.

In some cases, the beverage may be selected from the group consisting of: fruit juice products, broths, soups, soda, soft drinks, nutritional beverages, energy beverages, sports drinks, recovery beverages, heated beverages, coffee-based beverages, tea-based beverages, milk-based beverages, yogurt-like beverages, milkshakes, non-dairy plant based mild beverages, infant formulas, meal replacement beverages. In some embodiments, the beverage comprises a carbonated beverage.

The compositions described herein may be used to produce beverage compositions, such as milk or milk-like compositions. For example, a single variant of alpha casein may be used to form a milk-like analogue product without forming micelles or micelle-like compositions. A milk-like analogue product prepared using the compositions described herein may provide similar or equivalent characteristics (such as texture, creaminess and taste) compared to animal milk or a milk-like analogue product prepared using micelles (such as a milk-like analogue product prepared from caseinate or rennet casein). Milk-like analogue products prepared using the compositions described herein may provide improvements in one or more characteristics (such as texture, creaminess and taste) compared to plant-derived milk analogues.

The beverages described herein can comprise from 0.5% w/w to about 10% w/w of a recombinant single variant of casein (such as alpha casein). In some cases, the beverage may comprise at least 0.5% w/w of a recombinant single variant of casein, such as a single variant of any of the casein described herein. In some cases, the beverage may comprise up to 10% w/w of a recombinant single variant of casein, such as a single variant of any casein described herein (such as alpha casein). In some of the cases where the number of the cases, the beverages described herein can comprise 0.5% to 1% w/w, 0.5% to 2% w/w, 0.5% to 4% w/w, 0.5% to 6% w/w, 0.5% to 8% w/w, 0.5% to 10% w/w, 1% to 2% w/w, 1% to 4% w/w, 1% to 6% w/w, 1% to 8% w/w, 1% to 10% w/w, 2% to 4% w/w, 2% to 6% w/w, 2% to 8% w/w, 4% to 6% w/w, 4% to 8% w/w, 4% to 10% to 6% w/w, 10% to 10% w/w, or a single variant of the recombinant protein. The beverages described herein can comprise 0.5% w/w, 1% w/w, 2% w/w, 4% w/w, 6% w/w, 8% w/w, or 10% w/w of a recombinant single variant of casein, such as a single variant of any casein described herein. In some preferred embodiments, the beverage may comprise from 0.5% w/w to 5% w/w of the recombinant single variant of casein.

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

In some embodiments, the beverage comprises a single variant casein, and the single variant casein, such as alpha casein, provides one or more properties of the beverage to the composition.

The texture of a beverage prepared with a single variant of casein (e.g., alpha casein), such as by the methods described herein, may be comparable to the texture of a similar type of beverage prepared using dairy proteins of animal origin, such as a yogurt beverage prepared from animal milk. The texture of a beverage prepared using a composition having a single variant of casein, such as alpha casein, described herein may be comparable to the texture of a beverage prepared using a micellar form of casein, such as milk, yogurt or a beverage prepared from caseinate or micellar casein. The texture of a beverage prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be improved/more desirable when compared to the texture of a beverage prepared using a micellar form of casein, such as milk, yogurt or beverage prepared from caseinate, micellar casein, or when compared to a beverage prepared with a protein of vegetable origin. The texture of the beverage can be tested using a trained group of human subjects and using a machine such as a viscometer for viscosity measurements. Texture characteristics may include, for example, viscosity, smoothness, mouthfeel, graininess, and creaminess.

The taste of a beverage prepared with a single variant of casein (e.g., alpha casein), such as by the methods described herein, may be comparable to the taste of a similar type of beverage prepared using dairy proteins of animal origin, such as a yogurt beverage prepared from animal milk. The taste of a beverage prepared using a composition having a single variant of casein, such as alpha casein, described herein may be comparable to the taste of a beverage prepared using a micellar form of casein, such as milk, yoghurt or a beverage prepared from caseinate, micellar casein or rennet casein. The taste of a beverage prepared using a composition having a single variant of casein, such as alpha casein, as described herein may be improved/more desirable when compared to the taste of a beverage prepared using a micellar form of casein, such as milk, yoghurt or a beverage prepared from caseinate, micellar casein, rennet casein, or when compared to a beverage prepared with a protein of vegetable origin. The taste of the beverage may be tested using a trained group of human subjects.

D. Other edible compositions

In some embodiments, the compositions described herein comprising a single variant of alpha casein may be used to produce a food product, a dairy product, or an analog of a dairy-like product. Dairy or dairy-like analog products that may be prepared using the compositions described herein may include the following analogs: milk, cream, milkshake, creamer (liquid and powder forms), ice cream, condensed milk, yoghurt or cheese. Cheese analogues or cheese-like products that are not derived from a real clot or are not prepared via coagulation of a hydrocolloid may also be prepared using a composition comprising a single variant of alpha casein, the composition comprising the full-length single variant of alpha casein described herein and optionally truncated forms thereof.

The single variant alpha-casein compositions described herein may be used to produce an edible composition such as a dairy cream analogue or a cream-like composition. For example, a single variant alpha casein composition may be used to form a dairy cream analogue product. The dairy creamer analogue product may be formed without forming micelles or micelle-like compositions. Cream analogue products prepared using the compositions described herein may provide similar or equivalent characteristics (such as texture, creaminess and taste) to dairy cream of animal origin or dairy cream analogues prepared using micelles (such as dairy cream analogues prepared from caseinates or rennet casein). Cream analogue products prepared using the compositions described herein may provide improvements in one or more characteristics such as texture, creaminess and taste when compared to cream analogues of vegetable origin.

The compositions described herein may be used to produce edible compositions such as ice cream analogue compositions. For example, a single variant alpha casein composition may be used to form an ice cream analogue product. The ice cream analogue product may be formed without the formation of micelles or micelle-like compositions. Ice cream analogue products prepared using the compositions described herein may provide similar or equivalent characteristics to dairy ice cream analogues of animal origin or ice cream analogues prepared using micelles (such as ice cream analogues prepared from caseinate or rennet casein). Ice cream analogue products prepared using the compositions described herein may provide improvements in one or more characteristics such as texture, creaminess and taste when compared to plant derived ice cream analogues.

The compositions described herein may be used to produce a variety of edible compositions including, but not limited to, beverages (such as nutritional beverages, dairy related beverages, and the like), salad dressing (salad dressing), baking ingredients (baking ingredient), cooking ingredients (cooking ingredient), and the like. For example, the single variant alpha casein compositions described herein may be used to produce yogurt drinks, rangeland seasoning (rangeland), and the like. As a further example, the single variant alpha-casein compositions described herein may be used to produce ingredients for baking and cooking.

E. Other components

The compositions described herein may be used as ingredients to produce edible compositions such as foods. The food product may include cheese analogues, yogurt analogue products and other food products described elsewhere herein. Such edible compositions may comprise one or more ingredients in addition to the single variant casein. Ingredients may include, but are not limited to, solvents, salts, sugars, fats, flavoring agents, coloring agents, and the like.

Edible compositions comprising a single variant casein may comprise salts, such as calcium, phosphorus, citrate, potassium, sodium and/or chloride salts. The calcium salt may be selected from calcium chloride, calcium carbonate, calcium citrate, calcium glucuronate (calcium glubionate), calcium lactate, calcium gluconate, calcium acetate, equivalents thereof, and/or combinations thereof. The phosphate may be selected from orthophosphates such as monosodium phosphate (dihydrogen), disodium phosphate, trisodium phosphate, monopotassium phosphate (dihydrogen), dipotassium phosphate, tripotassium phosphate; pyrophosphates, such as disodium dihydrogen pyrophosphate or dipotassium dihydrogen pyrophosphate, trisodium hydrogen pyrophosphate or tripotassium hydrogen pyrophosphate, tetrasodium pyrophosphate or tetrapotassium pyrophosphate; polyphosphates, such as pentasodium or pentapotassium tripolyphosphate, sodium or potassium tetrapolyphosphate, sodium or potassium hexametaphosphate. The citrate salt may be selected from calcium citrate, potassium citrate, sodium citrate, trisodium citrate, tripotassium citrate or equivalents thereof. The edible composition may comprise a combination of salts. In some embodiments, the edible composition comprises a calcium salt, a phosphate salt, and a citrate salt. In some embodiments, the edible composition comprises a calcium salt and a phosphate salt. In some embodiments, the edible composition comprises a calcium salt and a citrate salt. In some embodiments, the edible composition comprises phosphate and citrate.

In some embodiments, fat is added to the edible composition. In some cases, the fat may be substantially free of fat of animal origin. Fats as used herein may include vegetable-based fats such as canola oil, sunflower oil, coconut oil, palm oil, or combinations thereof. Fats for use herein may include recombinant animal or vegetable fats prepared by microorganisms. Fats as used herein may include recombinant animal or vegetable fats in mammalian cell culture.

The edible composition as described herein may further comprise a sugar. Sugar as used herein may include plant-based monosaccharides, disaccharides, and/or oligosaccharides. Examples of sugars include sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allose, tagatose, xylose, and arabinose.

The edible food compositions prepared from a single variant casein and methods of preparing such compositions described herein may include adding or mixing one or more ingredients. For example, food additives may be added to or mixed with the composition. The food additive may increase the volume and/or mass of the composition. The food additives may improve functional performance and/or physical characteristics. For example, the food additive may prevent gelation or viscosity increase due to the lipid fraction of lipoproteins in the freeze-thaw cycle. Anti-caking agents (cellulose, potato starch, corn starch, starch blends) can be added to prepare free-flowing compositions. Carbohydrates may be added to increase resistance to thermal damage, e.g., less protein denaturation during drying, and improve stability and flowability of the dried composition. Food additives include, but are not limited to, starches (e.g., potato starch, modified potato starch, corn starch, rice starch), food coloring, pH adjusting agents (e.g., glucono-delta-lactone, sodium hydroxide), natural flavoring agents (e.g., marsura cheese, pama cheese, butter, cream, colby cheese (colby), scindapsus Fu Luo cheese (pro volone), azimutag cheese (asigo), etc.), artificial flavoring agents, flavor enhancers, flavor masking agents, batch markers, food acids (food acids) (e.g., lactic acid, citric acid), fillers, anti-caking agents (e.g., sodium aluminosilicate), anti-greening agents (antigreening agent) (e.g., citric acid), food stabilizers, foam stabilizers or binders (binding agents), antioxidants, acidity regulators, fillers, color retention agents, whipping agents (e.g., ester whipping agents, triethyl citrate, sodium lauryl sulfate), emulsifiers (e.g., lecithin, monoglycerides, diglycerides), humectants, thickeners, pharmaceutical excipients, solid diluents, nutrients, sweeteners, glazing agents (glazings agents), preservatives (e.g., sorbic acid, nisin)), vitamins (e.g., vitamin B, vitamin D, vitamin a), dietary elements (diety elements), carbohydrates, polyols, gums, starches, flours, oils, and bran. In some cases, the flavoring may include a mozzarella flavoring, a cheddar flavoring, a parma flavoring, or other similar flavoring.

Food colors include, but are not limited to, FD & C yellow #5, FD & C yellow #6, FD & C red #40, FD & C red #3, FD & C blue # 1, FD & C blue # 2, FD & C Green #3, carotenoids (e.g., saffron (saffron), beta-carotene), carmine (annatto), betanin (betanin), pterocarpus flowers, caramel colorants, chlorophyll, elderberry juice (elderberry juice), lycopene, carmine (carmine), panan (parian), paprika (turmeric), curcuminoids (curcumin), quinoline yellow, azorubine (carmine), carmine 4R (poau 4R), patent blue V (Patent Blue V), and Green S (Green S).

Ingredients for pH adjustment 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 product

The edible compositions of single variant casein described herein may be used as an ingredient for the end user preparation of the end product. For example, the cheese products or cheese analogues described herein may be used by end users to prepare end products such as pizzas, pasta ingredients, mexico ingredients, frozen meals, savoury baked food ingredients, soups, macaroni cheese, cheese bars, and the like. In some examples, the yogurt analog may be used to prepare a yogurt-like product or a product comprising yogurt as an ingredient. In some examples, the edible composition may be used to prepare a dairy-like product or dairy analog or other beverage.

Recombinant expression

One or more proteins used to form the cheese composition may be recombinantly produced. In some cases, a single variant casein (e.g., a single variant of αs1 or a single variant of αs2) is recombinantly produced. A single variant casein, e.g. a single variant αs1 casein or a single variant αs2 casein, may have an amino acid sequence from any species. For example, the recombinant alpha casein may have the amino acid sequence of cow, sheep, goat, buffalo, horse, human, deer or camel alpha casein. The nucleotide sequence encoding casein may be codon optimized to increase production efficiency. Exemplary alpha casein sequences for recombinant production of single variant casein are provided in table 1 below. The recombinant single variant casein may be a non-naturally occurring variant of casein. Such variants may comprise one or more amino acid insertions, deletions or substitutions relative to the native casein sequence.

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 variant may be a truncated form of αS1 casein, such as a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 4-12, 16-24.

Recombinant single variant casein, such as single variant αs1casein, is expressed recombinantly in host cells. 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 bacterial, yeast, fungal, plant, insect, and mammalian cells. In some cases, bacterial host cells such as lactococcus lactis, bacillus subtilis, or Escherichia coli (Escherichia coli) may be used to produce alpha casein and/or truncated forms thereof. Other host cells include bacterial hosts such as, but not limited to, lactococcus species (Lactococci sp.), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus licheniformis (Bacillus licheniformis) and Bacillus megaterium (Bacillus megaterium), brevibacillus chondri (Brevibacillus choshinensis), mycobacterium smegmatis (Mycobacterium smegmatis), rhodococcus erythropolis (Rhodococcus erythropolis) and Corynebacterium glutamicum (Corynebacterium glutamicum), lactobacillus species (Lactobacillus sp.), lactobacillus fermentum (Lactobacillus fermentum), lactobacillus casei (Lactobacillus casei), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus plantarum (Lactobacillus plantarum) and Chlorella species 6803 (Synechocystis sp.6803).

In some embodiments, the full-length single variant alpha casein and/or truncated forms thereof are recombinantly produced in host cells. For example, full-length and truncated single variant αs1 casein may be produced in the same host cell, and such production may be derived from the same open reading frame (i.e., the same expression cassette) as well as truncated forms produced, for example, by post-translational proteolytic cleavage or from separate open reading frames, such as with an expression cassette encoding the full-length αcasein variant and one or more expression cassettes encoding truncated forms of the αcasein variant. Alternatively, the full length and truncated single variant αs1 casein may be produced in different host cells. Expression of the target protein may be provided by expression vectors, plasmids, nucleic acids integrated into the host genome, or other means. For example, a vector for expression may comprise: (a) a promoter element, (b) a signal peptide, (c) a heterologous casein sequence, and (d) a terminator element.

Expression vectors that can be used for expression of casein include expression vectors comprising expression cassettes having elements (a), (b), (c) and (d). In some embodiments, the signal peptide (b) and/or terminator element (d) need not be contained in a vector. In some cases, the signal peptide may be part of the native signal sequence of casein, e.g., the protein may comprise the native signal sequence as bolded 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 and heterologous signal sequence as set forth 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. In some cases, the protein may not comprise a signal sequence, but rather 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. In general, expression cassettes are designed to mediate transcription of a transgene when integrated into the genome of a homologous host microorganism or when present on a plasmid or other replicative vector maintained in the host cell.

To aid in the amplification of the vector prior to transformation into the host microorganism, an origin of replication (e) may be included in the vector. To assist in selection of microorganisms stably transformed with the expression vector, the vector may also include a selectable marker (f). The expression vector may also contain a restriction enzyme site (g) that allows 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, the expression vector may comprise 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 of skill in the art may be used in combination or in place of the elements described herein.

Gram-positive bacteria (such as lactococcus lactis and bacillus subtilis) can be used to secrete the target protein into the culture medium, and gram-negative bacteria (such as escherichia coli) can be used to secrete the target protein into the periplasm or the culture medium. In some embodiments, the expressed proteins expressed by bacteria may not have any post-translational modification (PTM), meaning that they are not glycosylated and/or may not be phosphorylated. Both gram-positive and gram-negative bacteria can be used to produce proteins within cells. In such an example, the cells may be lysed to recover the protein.

Single variant casein may be expressed and produced in lactococcus lactis (l.lactis) with both nisin inducible expression systems (regulated by the PnisA promoter), lactic acid inducible expression systems (regulated by the P170 promoter) or other similar inducible systems, as well as constitutive expression systems (regulated by the P secA promoter), both of which are carried out in food-grade selection strains, such as NZ3900 using the vector pNZ8149 (lacF gene supplementation/rescue principle). Secretion of functional proteins can be achieved by the signal peptide of Usp45 (SP (Usp 45)), usp45 being the major Sec-dependent protein secreted by lactococcus lactis. For example, αs1 casein and truncates thereof (truncates) may be co-expressed in lactococcus lactis or expressed separately using synthetic operons.

Bacillus subtilis (b. Subilis) has a variety of intracellular and extracellular proteases that may interfere with protein expression. In some embodiments, the bacillus subtilis strain is modified to reduce the type and amount of intracellular and/or extracellular proteases, e.g., strains with 7 (KO 7) and 8 (WB 800N) protease deletions, respectively, may be used.

To drive recombinant proteins Secretion, may use the signal peptide of amyQ (alpha-amylase of clostridium thermocellum (Clostridium thermocellum)) or another bacterial signal peptide known in the art. In addition, the native casein signal peptide sequence may be expressed heterologous in bacillus subtilis. Each casein has its own signal peptide sequence and can be used in the system. The signal protein may be cross-combined with casein. pHT01 vectors can be used as transformation and expression shuttle vectors for inducible protein expression in Bacillus subtilis. The vector is based on a strong sigma preceding the groES-groEL operon of Bacillus subtilis ^A A dependent promoter, which has been converted to an effectively controllable (IPTG inducible) promoter by addition of the lac operator. pHT01 is an E.coli/B.subtilis shuttle vector, providing ampicillin resistance to E.coli and chloramphenicol resistance to B.subtilis.

A safe laboratory strain, such as e.coli BL21 (exemplary strain BL21 (DE 3) or BL21 AI) or a derivative thereof, or a wild-type, such as K12 strain (exemplary strain MG1655 or W3110) or a derivative thereof, may be used to produce a single variant casein in e.coli. Inducible (such as IPTG-inducible, lactose-inducible, arabinose-inducible, rhamnose-inducible), self-inducible (such as based on phosphate depletion) and constitutive promoters can be used to drive casein expression. Single variant casein may be produced intracellularly or may be secreted into the periplasm and/or supernatant. To drive recombinant protein secretion, bacterial signal peptides of the Sec-dependent secretion pathway (such as OmpA, ompC, ompT, pelB, lamB), the SRP secretion pathway (such as TolA, dsbA, dsbC, torT), and the TAT secretion pathway (such as Torr, sufI) can be used.

Table 1: sequence(s)

Description of the embodiments

Embodiment 1: an edible composition comprising a recombinant single variant of alpha casein, wherein the single variant provides at least one dairy-like property selected from the group consisting of: adhesion, stretchability, texture, mouthfeel, melting, browning, hardness, creaminess, taste, smell and flexibility, wherein the single variant is not animal-derived casein and has not been physically dissociated from casein micelles, and wherein the composition lacks any additional casein.

Embodiment 2: the edible composition according to embodiment 1, wherein the single variant of alpha casein is not derived from caseinate.

Embodiment 3: the edible composition according to embodiment 1 or embodiment 2, wherein the single variant of αcasein is αs1 casein.

Embodiment 4: the edible composition according to embodiment 1 or embodiment 2, wherein the single variant of αcasein is αs2 casein.

Embodiment 5: the edible composition according to any one of embodiments 1-3, wherein the composition is free of any animal-produced proteins.

Embodiment 6: the edible composition according to embodiment 4, wherein the composition lacks any other dairy protein of animal origin.

Embodiment 7: the edible composition according to any one of embodiments 1-5, wherein at least one dairy-like property is improved compared to a dairy-derived cheese analogue.

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

Embodiment 9: the edible composition according to any one of embodiments 1-5, wherein at least one dairy-like property is improved compared to a cheese analogue of vegetable origin.

Embodiment 10: the edible composition according to any one of embodiments 1-9, wherein a single variant of alpha casein comprises at least one non-natural post-translational modification.

Embodiment 11: the edible composition according to embodiment 10, wherein the single variant of alpha casein further comprises at least one natural post-translational modification.

Embodiment 12: the edible composition according to any one of embodiments 1-11, wherein a single variant of alpha casein lacks one or more post-translational modifications of native alpha casein.

Embodiment 13: the edible composition according to embodiment 12, wherein the single variant of alpha casein further comprises at least one non-natural post-translational modification.

Embodiment 14: the edible composition according to embodiment 13, wherein the single variant of alpha casein is not post-translationally modified.

Embodiment 15: the edible composition according to any one of embodiments 1-14, wherein the composition comprises full-length alpha casein.

Embodiment 16: the edible composition according to any one of embodiments 1-14, wherein a single variant of alpha casein 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.

Embodiment 17: the edible composition according to embodiment 16, wherein the composition further comprises one or more truncated alpha caseins.

Embodiment 18: the edible composition according to embodiment 17, wherein the truncated alpha casein lacks 1 or more N-terminal amino acids of mature native alpha casein.

Embodiment 19: the edible composition according to embodiment 18, wherein the truncated alpha casein is selected from the group consisting of: alpha casein lacking between 1 and 23N-terminal amino acids of native alpha casein or alpha casein lacking between 1 and 59N-terminal amino acids of native alpha casein, or a combination thereof.

Embodiment 20: the edible composition according to embodiment 19, wherein the truncated alpha casein comprises any of SEQ ID nos. 4-12, 16-24.

Embodiment 21: the edible composition according to any one of embodiments 17-20, wherein the truncated alpha casein lacks 1 or more C-terminal amino acids of native alpha casein.

Embodiment 22: the edible composition according to embodiment 17, wherein between 0% wt/wt and 20% wt/wt of the total weight of the composition of alpha casein is a truncated form of one or more alpha casein.

Embodiment 23: the edible composition according to embodiment 22, wherein the truncated form of one or more alpha caseins comprises between 1% wt/wt and 20% wt/wt of the total weight of the composition alpha casein.

Embodiment 24: the edible composition according to any one of embodiments 1-23, wherein the recombinant alpha casein comprises the amino acid sequence of bovine, caprine, or ovine alpha casein, or any one of SEQ ID No. 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 No. 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.

Embodiment 25: the edible composition according to any one of embodiments 1-24, wherein a single variant of alpha casein comprises one or more unnatural amino acids at the N-terminus.

Embodiment 26: the edible composition according to embodiment 25, wherein the single variant of alpha casein comprises a non-natural methionine at the N-terminal position.

Embodiment 27: the edible composition according to any one of embodiments 1-26, wherein the single variant of alpha casein is not derived from casein micelles.

Embodiment 28: a dairy analogue comprising the edible composition of any one of embodiments 1-27, wherein the analogue is selected from the group consisting of: cheese analogues, yoghurt analogues, cream analogues and ice cream analogues.

Embodiment 29: the dairy analog according to embodiment 28, further comprising a fat or oil from a non-animal source.

Embodiment 30: the dairy analogue according to embodiment 28 or embodiment 29, wherein the dairy analogue lacks any dairy protein of animal origin.

Embodiment 31: the dairy analogue according to any one of embodiments 28-30, wherein the dairy analogue lacks any other casein.

Embodiment 32: the dairy analogue according to any one of embodiments 28-31, wherein a single variant of alpha casein is not comprised in the dairy analogue in micellar form.

Embodiment 33: the dairy analogue according to any one of embodiments 28-32, wherein the dairy analogue is a cheese analogue.

Embodiment 34: the dairy analogue according to embodiment 33, wherein the cheese analogue is a mozzarella analogue, a cheddar analogue or a parma analogue.

Embodiment 35: the dairy analogue according to embodiment 33, wherein the cheese analogue is a mozzarella cheese analogue, and wherein the single variant of alpha casein is alpha S1 casein.

Embodiment 36: the dairy analogue according to embodiment 35, wherein the αs1 casein is bovine αs1 casein and wherein the αs1 casein in the composition comprises between 0% -20% of the truncated form of one or more αcasein.

Embodiment 37: the dairy analogue according to embodiment 35, wherein the αs1 casein in the composition comprises between 1% -20% of one or more truncated forms of αs1 casein.

Embodiment 38: the dairy analog according to embodiment 36 or embodiment 37, comprising an N-terminal truncated form of αs1 casein.

Embodiment 39: the dairy analogue according to embodiment 38, wherein the N-terminal truncated form is selected from any one of the following: SEQ ID NOS 4-12, 6-24 or combinations thereof.

Embodiment 40: the dairy analogue according to any one of embodiments 28-39, further comprising one or more of the following: (a) an oil of vegetable origin; (b) starch of vegetable origin; (c) a sugar; and (d) a salt.

Examples

The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

Example 1: expression of casein in lactococcus lactis via nisin inducible System (NICE)

Construct design, cloning and transformation

Bovine αs1 casein (variant C) protein coding sequence (without natural signal peptide) was codon optimized for expression in lactococcus lactis, and synthetic operons were constructed for co-expression and secretion of both proteins under the nisin inducible promoter. The signal peptide sequence from the naturally secreted lactococcus protein Usp45 was used to drive protein secretion. The synthetic operon was then cloned into an E.coli custom vector via restriction digest compatible sites and subcloned into a nisin-inducible pNZ8149 vector via restriction digest and ligation as confirmed by Sanger sequencing.

Vectors were transformed into a compatible lactococcus lactis strain NZ3900 via electroporation and selected using fully defined medium supplemented with lactose (completely defined media, CDM). Positive clones were confirmed by colony PCR, and 3 positive clones were removed for protein expression induction and analysis.

Protein expression and analysis

Individual colonies were grown in liquid culture at 30 ℃ and protein production was induced with nisin for 2.5 hours (control samples were not induced). Cells were then harvested by centrifugation and the supernatant of TCA pellet and lysed cell pellet were analyzed by coomassie gel staining (SDS-PAGE) and chemiluminescence (western blot against αs1 casein, LSBio primary antibody).

Example 2: expressed in lactococcus lactis via a pH inducible system.

Similar to the above constructs, the nisin promoter was replaced with the P170 promoter (pH/lactic acid inducible promoter for lactococcus lactis) to produce an alpha casein construct. Each of these constructs contains a secretion signal peptide.

After secretion, αs1 casein and its truncated forms were detected in lactococcus lactis on western blots. Unprocessed protein products accumulate in the cell, but secretion of the mature protein and its truncated forms is detected.

Example 3: expression in Bacillus subtilis

Construct design, cloning and transformation

The C-terminally His-tagged bovine αs1 casein (variant C) protein coding sequence (without the native signal peptide) was codon optimized for expression in bacillus subtilis. Constructs were produced of signal peptide (which have been reported for efficient secretion of recombinant proteins) of amyQ (a-amylase of bacillus amyloliquefaciens) with and without codon optimization. The construct was cloned into the transformation and expression IPTG inducible vector pHT01 by escherichia coli via Gibson cloning and confirmed via Sanger sequencing. pHT01 is an E.coli/B.subtilis shuttle vector, providing ampicillin resistance to E.coli and chloramphenicol resistance to B.subtilis. Positive clones were further transformed into chemically competent bacillus subtilis WB 800N. Positive clones were confirmed by colony PCR, and 3 positive clones were removed for protein expression induction and analysis.

Protein expression and analysis

Individual colonies were grown in liquid culture at 37 ℃ and protein production was induced with IPTG for 1 hour, 2 hours and 6 hours (control samples were not induced). Cells were then harvested by centrifugation and the supernatant of TCA pellet and lysed cell pellet were analyzed by coomassie gel staining (SDS-PAGE) and chemiluminescence (western blot for His tag and αs1 casein).

Western blot shows expression of αs1 casein in bacillus subtilis.

Example 4: expression in E.coli

Construct design, cloning and transformation

Bovine αs1 casein (variant C) or ovine αs1 casein coding sequence optimized for e.coli codons (without native signal peptide) was cloned into IPTG-inducible commercially available pET vectors. Cloning is performed via the Gibson reaction of the DNA fragment and vector in such a way that only the protein coding sequence remains in open reading frame. The Gibson reaction was transformed into competent cells and confirmed by Sanger sequencing. The vector is then transformed into chemically competent E.coli BL21 (DE 3) cells or derivatives thereof or wild-type e.g.K12 strain or derivatives thereof and several single colonies are screened for expression.

Protein expression, analysis and purification

Individual colonies were grown in liquid culture at 37 ℃ and protein production was induced with IPTG for 4 hours. Cells were then harvested by centrifugation and lysed cell pellet analyzed by coomassie gel staining (SDS-PAGE) and chemiluminescence (western blot for αs1 casein). Phase separation was used to purify the protein. The purified product was analyzed on coomassie stained gel, similar to that described above. The αs1 casein was expressed intracellularly in e.coli, successfully detected and purified on coomassie stained protein gels. An exemplary production of αcasein is illustrated in fig. 1, wherein αs1 casein and 2 variants are found: n-terminally truncated F24-199 bovine αS1 casein and N-terminally truncated M60-199 bovine αS1 casein.

Example 5: marsella cheese analogues from recombinant single variant alpha casein and properties thereof

Recombinant unphosphorylated bovine αs1 casein was used to prepare non-micellar mozzarella cheese analogues, known as NC mozzarella cheese. Casein, preheated coconut oil, trisodium citrate, disodium phosphate, salt, and glucose were added to the beaker at the concentrations specified in table 2. To which water was added at the concentrations specified in Table 2, caCl ₂ And modified potato starch. The beaker was moved to a water bath preset at a temperature of 85 ℃ and the contents were mixed using a mixing propeller at a speed of 300rpm for 9min. Lactic acid was added and mixing continued for an additional 1min. The resulting mixture was turned into a homogeneous non-micellar mass, transferred to a standard mold, and allowed to stand in a refrigerator for 16-24 hours. After incubation, NC mozzarella cheese analogues were weighed to obtain yield estimates.

Table 2: composition and concentration of components of recombinant single casein variant cheese analogue (NC 1 mozzarella analogue)

Qualitative and quantitative parameters, such as pH, stretchability, and texture profile, were analyzed on NC1 mozzarella cheese analogue samples. These parameters were compared to commercially available low moisture mozzarella cheese, commercially available imitation mozzarella cheese, and commercially available plant-based plain mozzarella cheese. Low moisture mozzarella cheese is prepared from milk in which casein is micellar and has the natural high complexity of its variants (αs1, αs2, β, κ; phosphorylated and glycosylated). Mozzarella imitation cheese is prepared from a blend of milk casein and vegetable based fat, wherein micellar casein from milk is curdled and dried into a milk protein concentrate. Vegetable-based plain mozzarella cheese does not have casein.

A pizza fork tensile test was performed to quantify cheese extensibility by inserting a fork into the center of freshly baked pizza and lifting until all cheese filaments break. To this end, 6g of cheese was shredded onto a 4 "inch tortilla, which was topped with 4g of tomato sauce. The samples were baked at 600°f for 90 seconds. A ruler was used to measure stretchability and the test was performed immediately after the pizza was removed from the oven.

The texture profile was analyzed on a TA-XTplus texture analyzer with a TA-55 piercing probe. A sample of cheese, 1.5g-1.9g, was cut to dimensions 1.5cm long by 1.5cm wide by 1cm high. The test is performed on samples stored at 4 ℃ and at ambient temperature for at least 30 minutes.

NC1 mozzarella cheese analogues prepared from recombinant unphosphorylated αs1 casein and its truncated forms as the sole protein component in cheese showed animal derived dairy-like melting, dairy-like stretching and dairy-like texture properties in the tests performed. The pH of the coagulated cheese was 5.9.

Pizza fork tensile testing showed that NC1 mozzarella cheese analogues were stretched >12 inches (table 3, fig. 2). In contrast, low moisture mozzarella cheese stretched >18 inches, but imitation mozzarella cheese stretched only <10 inches. Vegetable-based plain mozzarella cheese did not show stretchability, it stretched <1 inch.

Texture profile shows hardness, adhesion, restoring force, cohesiveness, elasticity, and chewiness as shown in table 3 (table 3, fig. 3). NC1 mozzarella cheese analogues show almost the same cohesiveness and elasticity (< 10% deviation), and very similar hardness and chewiness profile (< 30% deviation) when compared to real dairy low moisture mozzarella cheese. It is always somewhat harder, more resilient, more cohesive and more chewy than both low moisture mozzarella cheese and mozzarella imitation cheese. In contrast, plant-based plain mozzarella cheese deviates from dairy behavior in that it is very hard (hard-4.6×), chewy (chewy-3.9×) and sticky (sticky-19.6×).

Interestingly, NC1 mozzarella cheese analogues show a different trend in terms of adhesion and elasticity than imitation mozzarella cheese when compared to real dairy low moisture mozzarella cheese. Although mozzarella imitation cheese is more adhesive and elastic than real dairy mozzarella, NC1 mozzarella analogues are less adhesive and elastic than real dairy mozzarella. Reduced adhesion is an advantageous property of cheese because it represents the force required to remove cheese from the probe (i.e., remove cheese adhering to the teeth).

Table 3: cheese Properties

Example 6: properties of mozzarella cheese analogues from recombinant single variant alpha casein with altered formulation lacking calcium chloride.

Recombinant unphosphorylated αs1 casein was used to prepare non-micellar mozzarella cheese analogues, known as New Culture (NC 2) mozzarella cheese. Casein, preheated coconut oil, trisodium citrate, disodium phosphate, sodium chloride, and glucose were added to the beaker at the concentrations specified in table 4. To this was added a mixture of water and modified potato starch at the concentrations specified in table 4. The beaker was moved to a water bath preset at a temperature of 85 ℃ and the contents were mixed using a mixing propeller at a speed of 300rpm for 9min. Lactic acid was added and mixing continued for an additional 1min. The resulting mixture was turned into a homogeneous non-micellar mass, transferred to a standard mold, and allowed to stand in a refrigerator for 3 days. After refrigerator storage, NC mozzarella cheese analogues were weighed to obtain yield estimates.

Table 4: composition and concentration of components of recombinant single casein variant cheese analogues (NC 2 mozzarella analogues).

Composition of the components	Concentration (%, wt/wt)
		Casein protein	18.5
Coconut oil	24.2
		Citric acid trisodium salt	1
Phosphoric acid disodium salt	2
		Sodium chloride	1.49
Water and its preparation method	49.37
		Modified potato starch	2.54
Glucose	0.4
		Lactic acid crystal	0.5

Qualitative and quantitative parameters such as pH, moisture, melting, stretchability, and texture profile were analyzed on NC2 mozzarella cheese analogue samples. These parameters were compared to commercially available low moisture mozzarella cheese, commercially available imitation mozzarella cheese, and commercially available plant-based pure mozzarella cheese (see example 5 for further description of these cheeses).

Cheese melting was quantified by a modified Schreiber melting test using a custom imaging station mounted on a black magnetic hotbed. 0.5g of cheese was melted on a magnetic hotbed at 95℃for 15min. The time lapse of melting was recorded to measure the increase in the melted area by measuring the pixels. The melt value is calculated by dividing the melt area by the unmelted area prior to melting. A melt value greater than 1 indicates melting.

A malleability test was performed on a texture analyzer to quantify cheese malleability by inserting a 6-fork hook into a 6g hand-cut cheese sliver sample placed in a malleability device (this sample had been melted in an oven at 90 ℃ for 10min and lifted until all cheese filaments had broken). The distance of failure (break of all filaments) representing the degree of cheese stretching and the work of extension representing the required tensile strength of the stretched cheese were quantified. The test was performed on samples taken from the refrigerator and then kept at ambient temperature for at least 30 min.

Table 5: melting and stretchability (malleability) of cheese

NC2 mozzarella cheese analogues prepared from recombinant unphosphorylated αs1 casein with calcium-deficient formulations show dairy-like melting, dairy-like stretchability and dairy-like extensibility properties. The moisture and pH of the coagulated NC2 mozzarella cheese analogue were 45.7% and 5.7, respectively.

Surprisingly, NC2 mozzarella cheese analogues prepared using the compositions described herein showed better melting than the formulations in example 5 and example 7 and reached a melting value of 2.1 x (table 5, fig. 4). In contrast, low moisture mozzarella cheese and mozzarella imitation cheese reached melting values of 2.5 x and 2.0 x, respectively. Plant-based plain mozzarella cheese achieved a melting value of 0.8 x, indicating lack of melting (and even shrinkage).

NC2 mozzarella cheese analogues melt at a slower rate than low moisture mozzarella cheese, reaching their peak melting later during the modified Schreiber melting test. Imitation mozzarella cheese and low moisture mozzarella cheese require-4-6 minutes to reach maximum melting, while NC2 mozzarella cheese analogues melt completely within-6.5 minutes (fig. 4).

NC2 mozzarella cheese analogues showed almost the same malleability as low moisture mozzarella cheese and mozzarella imitation cheese, whereas plant-based plain mozzarella cheese was not fully stretched (table 5, fig. 5). The tensile strength (indicated by the work of extension value, table 5) required to stretch NC2 mozzarella cheese analogues is between the range required to stretch low moisture mozzarella cheese and mozzarella imitation cheese. As indicated by the distance to failure values in table 5, NC2 cheese analogues were stretched comparable to low moisture mozzarella cheese and significantly better than mozzarella-like cheese. NC2 mozzarella cheese analogues were stretched to a length of-228 mm. In contrast, low moisture mozzarella cheese and mozzarella imitation cheese were stretched to lengths of 224mm and 189mm, respectively (Table 5).

Example 7: properties of mozzarella cheese analogues from recombinant single variant alpha-casein with different fat compositions

Recombinant unphosphorylated αs1 casein was usedIn preparing non-micellar mozzarella cheese analogues, they are known as New Culture (NC 3) mozzarella cheese. Casein, water, palm stearin, canola oil, trisodium citrate, disodium phosphate, modified potato starch and sodium chloride were added to the beaker at the concentrations specified in table 6. CaCl was added thereto at the concentrations specified in Table 6 ₂ . The beaker was moved to a water bath preset at 85 ℃ and the contents were mixed using a mixing propeller at a speed of 300rpm for 9 minutes. Natural flavoring and lactic acid were added and the ingredients were mixed for an additional 1 minute. The resulting mixture was turned into a homogeneous non-micellar mass, transferred to a standard mold, and allowed to stand in a refrigerator for 7 days. After incubation, NC3 mozzarella cheese analogues were weighed to obtain yield estimates.

Table 6: composition and concentration of components of recombinant single casein variant cheese analogue (NC 3 mozzarella analogue)

Composition of the components	Concentration (%, wt/wt)
		Casein protein	18.5
Palm stearin	17
		Canola oil	7
Citric acid trisodium salt	0.7
		Phosphoric acid disodium salt	1.3
Sodium chloride	1.5
		Water and its preparation method	49.7
CaCl ₂	0.5
		Modified potato starch	2.5
Natural flavoring agent	1.1
		88% lactic acid solution	0.2

Qualitative and quantitative parameters of NC3 mozzarella cheese analogue samples, such as pH, moisture, melting, stretchability and texture profile, were analyzed as set forth in example 6. These parameters were compared to low moisture mozzarella cheese, mozzarella imitation cheese, and plant-based plain mozzarella cheese (see example 5 for further description).

Cheese melting was quantified by a modified Schreiber melting test as described in example 6. As described in example 6, a ductility test was performed on a ta.xtplus texture analyzer to quantify cheese ductility. The test was performed on samples stored at 4 ℃.

Table 7: melting and stretchability (malleability) of cheese

The texture profile was analyzed on a TA-XTplus texture analyzer with a TA-55 piercing probe. A sample of cheese, 1.5g-1.9g, was cut to dimensions 1.5cm long by 1.5cm wide by 1cm high. The test was performed on samples stored at 4 ℃.

Table 8: cheese Properties

NC3 mozzarella cheese analogues prepared from recombinant unphosphorylated αs1 casein having different fat compositions show dairy-like melting, dairy-like stretching and dairy-like malleability properties. The moisture and pH of the coagulated NC3 mozzarella cheese analogue were 44.8% and 5.7, respectively.

NC3 mozzarella cheese analogues prepared using the compositions described herein reached a melting value of 1.6 x (table 7, fig. 6). In contrast, low moisture mozzarella cheese and mozzarella imitation cheese reached melting values of 2.5 x and 2 x, respectively. Plant-based plain mozzarella cheese achieved a melting value of 0.8 x, indicating lack of melting (and even shrinkage).

Surprisingly, NC3 mozzarella cheese analogues melt at a faster rate than low moisture mozzarella cheese, reaching their peak melting faster during the modified Schreiber melting test. Imitation mozzarella cheese and low moisture mozzarella cheese require-4-6 minutes to reach maximum melting, while NC3 mozzarella cheese analogues melt completely within-2 minutes (fig. 6).

This may be due to the replacement of coconut oil with a combination of palm stearin and canola oil.

NC3 mozzarella cheese analogues showed almost the same malleability as low moisture mozzarella cheese and mozzarella imitation cheese, whereas plant-based plain mozzarella cheese was not fully stretched (table 7, fig. 7). The tensile strength required to stretch NC1 mozzarella cheese analogues (indicated by the work of extension value, table 7) is similar to that required to stretch low moisture mozzarella cheese, whereas mozzarella imitation cheese requires significantly lower tensile strength. NC3 cheese analogues were stretched to the same extent as low moisture mozzarella cheese and mozzarella imitation cheese, as indicated by the distance to failure values in table 7. NC3 mozzarella cheese analogues were stretched to a length of-230 mm. In contrast, low moisture mozzarella cheese and mozzarella imitation cheese were stretched to lengths of 228mm and 225mm, respectively (Table 7).

Texture profile shows hardness, adhesiveness, restoring force, cohesiveness, elasticity, and chewiness (as shown in table 8, fig. 8). NC3 mozzarella analogues show almost the same chewiness (< 10% deviation) when compared to mozzarella imitation cheese. In contrast, plant-based, plain mozzarella cheese has a higher chewiness (> 1.5×) than low moisture mozzarella cheese and mozzarella-imitation cheese. NC3 mozzarella cheese shows similar restoring force (< 10% deviation), higher hardness, elasticity, and lower adhesion and cohesiveness compared to other commercial cheeses.

Interestingly, NC3 mozzarella cheese analogues show a different trend in terms of adhesion than imitation mozzarella cheese when compared to dairy low moisture mozzarella cheese of animal origin. Although mozzarella imitation cheese is more adhesive than real dairy mozzarella, NC3 mozzarella analogues are less adhesive. Reduced tackiness is an advantageous property of cheese because it represents the force required to remove cheese from the probe, which corresponds to tackiness in the mouth of the consumer (i.e., sticking to the teeth when chewing).

Example 8: properties of mozzarella cheese analogues from recombinant single variant alpha-casein with altered recipe and altered processing parameters

Recombinant unphosphorylated αs1 casein was used to prepare non-micellar mozzarella cheese analogues, known as New Culture (NC 4) mozzarella cheese. Casein, water, palm stearin, canola oil, modified potato starch, sodium chloride and CaCl ₂ Added to the beaker at the concentrations specified in table 9. To this was added trisodium citrate and dipotassium phosphate at the concentrations specified in table 9. The beaker was moved to a water bath preset at 85 ℃ and the contents were mixed using a mixing propeller at 500rpm for 4 minutes followed by 300rpm for 1 minute, at which time the natural flavoring was also added. Lactic acid was added and the ingredients were mixed at 300rpm for an additional 1 minute. The resulting mixture was turned into a homogeneous non-micellar mass, transferred to a standard mold, and allowed to stand in a refrigerator for 7 days. After incubation, NC4 mozzarella cheese analogues were weighed to obtain yield estimates.

Table 9: composition and concentration of components of recombinant single casein variant cheese analogue (NC 4 mozzarella cheese).

Qualitative and quantitative parameters such as pH, moisture, melting, stretchability, and texture profile were analyzed on NC4 mozzarella cheese analogue samples. These parameters were compared to low moisture mozzarella cheese, mozzarella imitation cheese, and plant-based plain mozzarella cheese (see example 5 for further description).

Table 10: melting and stretchability (malleability) of cheese

NC4 mozzarella cheese analogues prepared from recombinant unphosphorylated αs1 casein and its truncated forms as the sole protein component in cheese using different formulations and different processing parameters compared to examples 5, 6 and 7 also show dairy-like melting, dairy-like stretching and dairy-like malleability properties. The moisture and pH of the coagulated NC4 mozzarella cheese analogue were 48.1% and 5.7, respectively.

NC4 mozzarella cheese analogues using the compositions described herein reached a melting value of 1.5 x (table 10, fig. 9). In contrast, low moisture mozzarella cheese and mozzarella imitation cheese reached melting values of 2.5 x and 2 x, respectively. Plant-based plain mozzarella cheese achieved a melting value of 0.8 x, indicating lack of melting (and even shrinkage).

More notably, NC4 mozzarella analogues melt at a faster rate than low moisture mozzarella, reaching their peak melting faster during the modified Schreiber melting test. Imitation mozzarella cheese and low moisture mozzarella cheese require-4-6 minutes to reach maximum melting, while NC4 mozzarella cheese analogues melt completely within-2 minutes (fig. 9).

NC4 mozzarella cheese analogues showed similar malleability to low moisture mozzarella cheese and mozzarella imitation cheese, whereas plant-based plain mozzarella cheese was not fully stretched (table 10, fig. 10). The tensile strength (indicated by the work-of-extension values, table 10) required to stretch NC4 mozzarella cheese analogues is in the range of low moisture mozzarella cheese and mozzarella-imitation tensile strengths. NC4 cheese analogues stretched better than low moisture mozzarella cheese and mozzarella-imitation cheese as indicated by the distance to failure values in table 10. NC4 mozzarella cheese analogue was stretched to a length of 241 mm. In contrast, low moisture mozzarella cheese and mozzarella imitation cheese were stretched to lengths of 228mm and 225mm, respectively (table 10, fig. 10).

Example 9: properties of the analog of Marylala cheese of recombinant single variant alpha Casein from different species

Recombinant unphosphorylated sheep αs1 casein was used to prepare non-micellar mozzarella cheese analogues, known as New Culture (NC 5) mozzarella cheese. Casein, water, palm stearin, canola oil, trisodium citrate, disodium phosphate, modified potato starch and sodium chloride were added to the beaker at the concentrations specified in table 11. The beaker was moved to a water bath preset at 85 ℃ and the contents were mixed using a mixing propeller at a speed of 300rpm for 9 minutes. Natural flavoring is added and the ingredients are mixed for an additional 1 minute. The resulting mixture was turned into a homogeneous non-micellar mass, transferred to a standard mold, and allowed to stand in a refrigerator for 5 days. After incubation, NC5 mozzarella cheese analogues were weighed for yield estimation.

Table 11: composition and concentration of components of recombinant single casein variant cheese analogue (NC 5 mozzarella cheese).

Composition of the components	Final concentration (%)
		Casein protein	22.1
Palm stearin	16.9
		Canola oil	7.2
Citric acid trisodium salt	0.7
		Phosphoric acid disodium salt	1.3
Sodium chloride	1.5
		Water and its preparation method	47.25
Modified potato starch	2.5
		Natural flavoring agent	0.55

Qualitative and quantitative parameters of NC5 mozzarella cheese analogue samples, such as pH, moisture, melting, stretchability and texture profile were analyzed as described in example 7. These parameters were compared to low moisture mozzarella cheese, mozzarella imitation cheese, and plant-based plain mozzarella cheese (see example 7 for further description).

Table 12: melting and stretchability (malleability) of cheese

NC5 mozzarella cheese analogues prepared from recombinant non-phosphorylated sheep αs1 casein as the sole protein component in cheese showed dairy-like melting, dairy-like stretching and dairy-like malleability properties of mozzarella cheese. The moisture and pH of NC5 mozzarella cheese analogues were 45.6% and 6.85, respectively.

NC5 mozzarella cheese analogues have higher melting values than plant-based mozzarella cheeses, which exhibit lack of melting (and even shrinkage) (table 12, fig. 11). NC5 mozzarella analogues melt slightly faster than low moisture mozzarella and mozzarella-like cheese, reaching their peak melting faster during the modified Schreiber melting test. Imitation mozzarella cheese and low moisture mozzarella cheese require >10 minutes to reach maximum melting, while NC5 mozzarella cheese analogues completely melted within-7 minutes (fig. 11).

NC5 mozzarella cheese analogues showed similar malleability to low moisture mozzarella cheese and mozzarella imitation cheese (19.1 cm compared to 22.4cm and 22.2cm, respectively), whereas plant-based mozzarella cheese was not fully stretched (< 2.5 cm) (table 12, fig. 12). The stretchability and melting of NC5 cheese analogues is lower than that of NC1, NC2 and NC4 cheese analogues.

The results of texture profile analysis for hardness, adhesion, restoring force, cohesiveness, elasticity, and chewiness are shown in table 13. NC5 mozzarella cheese analogues show hardness and chewiness in the range of low moisture mozzarella cheese and mozzarella imitation cheese. In contrast, plant-based mozzarella cheese deviates from dairy behavior and it exhibits more chewiness (-1.6×) and higher adhesiveness (-1.5×) when compared to low-moisture mozzarella cheese.

Interestingly, NC5 mozzarella cheese analogues showed an improvement in adhesion, which was not found in mozzarella-like cheeses. Although mozzarella imitation cheese is more adhesive than low moisture mozzarella, NC5 mozzarella analogues are less adhesive. Reduced tackiness is an advantageous cheese property. The adhesion represents the force required to remove the cheese from the probe and can be used as an indicator of whether the cheese will adhere to the teeth when consumed.

Table 13: cheese Properties

Example 10: yogurt and yogurt beverage Properties prepared from recombinant Single variant alpha Casein

Recombinant unphosphorylated bovine αs1 casein, natural αcasein (a mixture of αs1 and αs2, with natural phosphorylation, purified from milk) and commercially available micellar casein are used for the preparation of yogurt analogs and yogurt beverage analogs. For comparison, yogurt and yogurt drinks were also prepared from commercially available homogeneous milk. Protein, trisodium citrate, disodium phosphate, calcium chloride, and carbohydrates (sugar) were mixed in water at the concentrations specified in table 14. Lecithin and preheated fat were added to the solution at the concentrations listed in table 14. The mixture was homogenized using an ultrasonic apparatus with a 16mm probe at 90% power for 4min. The mixture was pasteurized in a water bath at 90 ℃ for 10min and then cooled to 40 ℃. 0.1g of standard lactobacillus culture (lactobacillus bulgaricus (Lactobacillus bulgaricus) and streptococcus thermophilus (Streptococcus thermophilus)) was added. The milk-like colloid was fermented for 14 hours. The yoghurt preparation process of whole milk consists only of the pasteurization and fermentation steps described above.

Table 14: yogurt and yogurt analogue formulations

* The components in milk are derived from nutrition fact column

Qualitative and quantitative parameters such as pH, odor, appearance, relative viscosity, and texture were analyzed for yogurt and yogurt analogues.

The recombinant αs1 casein yogurt analogues have a milk yogurt-like odor and appearance, ferment well, and show 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, and after 14 hours of fermentation was reduced to 4-4.8 (Table 15). All yogurt analogues have the typical odor of fermented yogurt prepared from milk, except for yogurt analogues that do not have protein.

Yogurt analogues prepared from recombinant αs1 casein, micellar casein and native αcasein became gel-like colloids with stable emulsions after fermentation (fig. 13). In contrast, protein-free milk-like solutions caused phase separation upon fermentation and remained in liquid form (fig. 13). Yogurt analogue formulations do not include any added thickeners, but rely on proteins to form yogurt-like products. All yogurt analogs showed some level of syneresis.

Yogurt Structure use is equipped with TA-18 ¹ / ₂ "circular test on a TA.XT Plus texture analyser of spherical probes. This method breaks down the yogurt structure through a series of 10 consecutive sample insertions, thus providing insight into the extent of deformation over time. As the probe penetrated the sample, the firmness (g) and viscosity (g) and adhesion (g x sec) were quantified. The test can also infer the relative viscosity by performing the work required for each cycle (insertion and removal of probes from the sample). It can be assumed that the more work is required to complete each cycle, the more viscous and firm the yoghurt. The test was performed on set, undisturbed and cooled (4 ℃) yoghurt or yoghurt analogue. Since the protein-free sample remains in a liquid state, the texture analysis of the sample is not possible.

The recombinant αs1 yogurt analog has almost the same relative viscosity as the lactic acid milk (table 15). The viscosity of recombinant αs1 casein was only 15% lower than that of lactic acid milk. In contrast, native alpha casein and micellar casein yoghurt analogues were 26% and 75% less viscous than lactic acid milk (table 15).

Table 15: yogurt and yogurt analog properties prepared from different proteins.

Natural alpha casein yoghurt analogues have a viscosity of 12% higher than milk-derived yoghurt. In contrast, micellar casein and recombinant αs1 casein yogurt analogues have lower viscosity than milk-derived yogurt, 36% and 68% lower viscosity, respectively. Although micellar casein yogurt analogues were less firm, natural αcasein and recombinant αs1 casein yogurt analogues were stronger than lactic acid milk (table 15).

Yoghurt drink analogues were prepared by mixing yoghurt or yoghurt analogue for 15sec using a hand-held homogenizer. The beverage analogs were analyzed for smoothness, texture, appearance, and odor compared to yogurt beverages prepared from milk. The recombinant αs1 casein and natural αcasein yogurt drinks were similar in smoothness, texture, appearance and odor (aroma) to the lactic acid milk drinks and micellar casein yogurt drink analogs (fig. 14).

Example 11: properties of mozzarella cheese analogues prepared from alpha-casein having different levels of phosphorylation and different calcium in the formulation

Recombinant unphosphorylated bovine αs1 casein, native αcasein (mixture of αs1 and αs2, with native phosphorylation, purified from milk) and 70% dephosphorylated αcasein (mixture of αs1 casein and αs2 casein, enzymatically 70% dephosphorylated after purification from milk, derived from Sigma Aldrich) were used to prepare non-micellar analog of marsulla cheese, designated NC6-8, with varying amounts of CaCl as outlined in table 16 ₂ . Casein, water, palm stearin, canola oil, trisodium citrate, disodium phosphate, caCl ₂ Starch and sodium chloride were added to the beaker at the concentrations specified in table 17. The formulation in table 17 varies depending on the amount of calcium chloride added. The beaker was moved to a water bath preset at 85 ℃ and the contents were mixed using a mixing propeller at a speed of 300rpm for 9 minutes. Natural flavoring is added and the ingredients are mixed for an additional 1 minute. The resulting mixture was turned into a homogeneous non-micellar mass, transferred to a standard mold, and allowed to stand in a refrigerator for 5 days. After incubation, NC6-8 mozzarella cheese analogues were weighed for yield estimation.

Table 16: list of New Culture alpha casein cheese analogues

Table 17: NC6-8 composition of mozzarella cheese analogues.

Qualitative and quantitative parameters of NC6-8 mozzarella cheese analogue samples, such as pH, moisture, melting, stretchability and texture profile were analyzed as described in example 7. As shown in table 18, additional qualitative properties were recorded, such as appearance of the cheese/cheese analogue after melting and silk quality of the cheese/cheese analogue. These parameters were compared between the cheese analogues tested, low moisture mozzarella cheese of animal origin, imitation mozzarella cheese of animal origin and vegetable-based plain mozzarella cheese.

Table 18: cheese and cheese analogue properties

*0.5％CaCl ₂ And 1% CaCl ₂ Corresponding to the use of 10mg and 20mg of calcium ion per gram of casein, respectively.

NC6-8 mozzarella cheese analogues have a moisture and pH of 47.2% -49.4% and 5.7-6.7, respectively.

Cheese analogues prepared from natural alpha casein show melting reactions for varying amounts of added calcium, with reduced amounts of calcium they melt better and diffuse more; however, the reduced calcium cheese analogue became a transparent/translucent liquid upon melting, indicating the elimination of the cheese-like emulsion. Surprisingly, cheese analogues prepared from recombinant unphosphorylated αs1 casein showed a similar trend of melting and diffusion responsiveness to reduced amounts of calcium while maintaining cheese-like emulsion properties under low calcium conditions (table 18). In contrast, cheese analogues prepared from 70% dephosphorylated alpha casein showed no melting reaction for different amounts of calcium, and upon melting, the calcium-reduced samples also formed translucent edges, indicating instability of the cheese-like emulsion.

All cheese analogues tested were comparable to dairy-like cheeses in terms of tensile length. Table 18 shows the qualitatively assigned stretchability quality metrics from 0-5, where 0 indicates the worst quality and 5 indicates the highest quality stretchability. These values are based on the observed silk firmness and thickness when cheese analogues were stretched using a ta.xt texture analyser. Cheese analogues prepared from 70% dephosphorylated alpha casein and natural alpha casein showed a slight improvement in tensile quality after calcium addition. Surprisingly, cheese analogues prepared from recombinant unphosphorylated αs1 casein showed a significant improvement in tensile quality, high calcium conditions resulted in tensile quality comparable to animal-derived mozzarella cheese, and even low/medium calcium conditions resulted in tensile quality comparable to milk-derived micellar mozzarella imitation cheese. This strong dose response to calcium amounts was uniquely observed in cheese analogues prepared from recombinant unphosphorylated αs1 casein.

The texture properties of the cheese analogues are summarized in table 18. Cheese analogues prepared from recombinant αs1 casein showed a significant decrease in adhesion. In contrast, cheese analogues prepared from natural and 70% dephosphorylated alpha casein had only slightly reduced adhesion. As the amount of calcium in the formulation increases, the adhesion of the imitation cheese also decreases.

Regardless of the calcium dose, cheese analogues prepared from natural alpha casein are similarly hard/firm (soft-5%) as low moisture mozzarella cheese from milk. Surprisingly, recombinant unphosphorylated αs1 casein cheese analogues were similarly hard (soft-5% compared to low moisture mozzarella cheese) also under calcium-free and low/medium calcium conditions. In contrast, analogs containing 70% dephosphorylated αcasein showed increased hardness (hard >5% compared to low moisture mozzarella cheese and >10% compared to cheese analogs prepared from either natural αcasein or recombinant unphosphorylated αs1 casein) even under low/medium calcium conditions. Increased hardness, as generally noted for plant-based cheese analogues, is an undesirable property of cheese analogues.

Other texture properties such as chewiness, cohesiveness, elasticity and resilience were also measured, and these properties did not show significant differences in the conditions tested.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many alterations, modifications and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims

1. An edible composition comprising a recombinant single variant of alpha casein, wherein the single variant provides the edible composition with at least one dairy-like property selected from the group consisting of: adhesion, stretchability, texture, mouthfeel, melting, browning, hardness, creaminess, taste, smell and flexibility, wherein the reconstituted single variant is not casein of animal origin and has not been physically dissociated from casein micelles; wherein the dairy-like properties are substantially provided by the recombinant single variant casein; and wherein the edible composition is comparable to a dairy-derived edible composition in at least one of the dairy-like properties.

2. The edible composition of claim 1, wherein the edible composition comprises one or more dairy-like properties selected from the group consisting of: adhesion, stretchability, firmness, viscosity, tackiness, chewiness, resilience, elasticity, mouthfeel, melting, hardness, creaminess and flexibility.

3. The edible composition of any one of claims 1-2, wherein the casein content of the edible composition substantially comprises a recombinant single variant of alpha casein.

4. The edible composition of any one of claims 1-3, wherein the casein content of the edible composition comprises only recombinant single variants of alpha casein.

5. The edible composition of any one of claims 1-4, wherein the recombinant single variant of alpha casein comprises at least 95% or at least 97% of the casein content in the edible composition.

6. The edible composition of any one of claims 1-5, wherein the recombinant single variant of alpha casein comprises at least 99% of the casein content in the edible composition.

7. The edible composition of any one of claims 1-6, wherein the edible composition lacks any additional casein other than the recombinant single variant of alpha casein.

8. The edible composition of any one of claims 1-7, wherein the single variant of alpha casein is not derived from caseinate.

9. The edible composition of any one of claims 1-8, wherein the composition is free of any animal-produced proteins.

10. The edible composition according to any one of claims 1-9, wherein the composition lacks any other dairy protein of animal origin.

11. The edible composition according to any one of claims 1-10, wherein at least one dairy-like property is comparable to or improved compared to a dairy-derived edible composition; wherein the milk-derived edible composition is identical to the edible composition in all ingredients except that the milk-derived edible composition comprises milk, one or more milk-derived proteins, or milk-derived ingredients in addition to or in place of the recombinant single variant of alpha casein.

12. The edible composition of any one of claims 1-11, wherein at least one dairy-like property is comparable to or improved compared to a micellar casein composition, wherein the micellar casein composition is identical to the edible composition in all ingredients, except that the micellar casein composition comprises a recombinant single variant of alternative alpha casein or micellar casein isolated from milk other than a recombinant single variant of alpha casein.

13. The edible composition of any one of claims 1-12, wherein the recombinant single variant is an alpha casein comprising at least one non-natural post-translational modification.

14. The edible composition of claim 13, wherein the recombinant single variant of alpha casein further comprises at least one natural post-translational modification.

15. The edible composition of any one of claims 1-14, wherein the recombinant single variant is an alpha casein lacking one or more post-translational modifications of natural alpha casein.

16. The edible composition of claim 15, wherein the single variant of alpha casein further comprises at least one non-natural post-translational modification.

17. The edible composition of claim 16, wherein the single variant of alpha casein is not post-translationally modified.

18. The edible composition of claim 17, wherein the single variant of alpha casein is not phosphorylated.

19. The edible composition of any one of claims 1-18, wherein the single variant of alpha casein is alpha-s 1 casein.

20. The edible composition of any one of claims 1-19, wherein the single variant of alpha casein is alpha-s 2 casein.

21. The edible composition of any one of claims 1-20, wherein the composition comprises full-length alpha casein.

22. The edible composition of any one of claims 1-21, wherein the single variant of alpha casein 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.

23. The edible composition of any one of claims 1-22, wherein the single variant of alpha casein comprises the amino acid sequence of bovine, caprine, or ovine alpha casein, 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.

24. The edible composition of any one of claims 1-23, wherein the single variant of alpha casein comprises one or more unnatural amino acids at the N-terminus.

25. The edible composition of claim 24, wherein the single variant of alpha casein comprises a non-natural methionine at the N-terminal position.

26. The edible composition of any one of claims 1-25, wherein the single variant of alpha casein is not derived from casein micelles.

27. A dairy analogue comprising the edible composition of any one of claims 1-26, wherein the analogue is selected from the group consisting of: cheese analogues, yoghurt analogues, cream analogues and ice cream analogues.

28. The dairy analogue of claim 27, wherein the edible composition comprises the recombinant single variant alpha casein.

29. The dairy analogue according to claim 27 or 28, further comprising a fat or oil from a non-animal source.

30. The dairy analogue according to any one of claims 27-29, wherein the dairy analogue lacks any dairy protein of animal origin.

31. The dairy analogue according to any one of claims 27-30, wherein the dairy analogue lacks any other casein.

32. The dairy analogue according to any one of claims 27-31, wherein the recombinant single variant of alpha casein is not comprised within the dairy analogue in micellar form.

33. The dairy analogue according to any one of claims 27-32, further comprising one or more of the following: (a) an oil of vegetable origin; (b) starch of vegetable origin; (c) a sugar; and (d) a salt.

34. The dairy analogue according to any one of claims 27-33, wherein the dairy analogue is a cheese analogue.

35. The dairy analogue of claim 34, wherein the cheese analogue is a mozzarella analogue, a cheddar analogue or a parma analogue.

36. The dairy analogue according to claim 34 or claim 35, wherein the cheese analogue is a low moisture cheese analogue.

37. The dairy analogue according to claim 34 or claim 35, wherein the cheese analogue is a soft cheese analogue.

38. The dairy analogue according to claim 34 or claim 35, wherein the cheese analogue is a hard cheese analogue.

39. The dairy analogue according to any one of claims 34-38, wherein the cheese analogue is a mozzarella cheese analogue, and wherein the single variant of casein is alpha casein.

40. The dairy analogue according to any one of claims 34-39, wherein the alpha casein is alpha S1 casein.

41. The dairy analog of claim 40, wherein the αs1 casein is bovine αs1 casein.

42. The dairy analogue according to claim 40 or 41, wherein the αs1 casein is full length casein.

43. The dairy analogue according to any one of claims 34-39, wherein the alpha casein is alpha S2 casein.

44. The dairy analog of claim 43, wherein the αs2 casein is bovine αs2 casein.

45. The dairy analog of claim 44, wherein the αs2 casein is full-length casein.

46. The dairy analogue according to any one of claims 34-45, wherein the stretchability of the cheese analogue is comparable to or improved relative to the stretchability of a dairy-derived cheese or dairy-derived cheese analogue.

47. The dairy analogue according to any one of claims 34-46, wherein the cheese analogue has a melting area/time comparable to or greater than the melting area/time of dairy-derived cheese or dairy-derived cheese analogue.

48. The dairy analogue according to any one of claims 34-47, wherein the malleability of the cheese analogue is comparable to or improved relative to the malleability of dairy-derived cheese or dairy-derived cheese analogue.

49. The dairy analogue according to any one of claims 34-48, wherein the texture of the cheese analogue is comparable to or improved relative to the texture of dairy-derived cheese or dairy-derived cheese analogue.

50. The dairy analogue according to any one of claims 34-49, wherein the adhesiveness of the cheese analogue is reduced relative to the adhesiveness of a dairy-derived cheese or dairy-derived cheese analogue.

51. The dairy analogue according to any one of claims 34-50, wherein the dairy-derived cheese or dairy-derived cheese analogue comprises micellar casein.

52. The dairy analogue according to any one of claims 34-51, wherein the stretchability of the cheese analogue is improved relative to the stretchability of a plant-derived cheese analogue.

53. The dairy analogue according to any one of claims 34-52, wherein the melting of the cheese analogue is improved relative to the melting of a plant-derived cheese analogue.

54. The dairy analogue according to any one of claims 34-53, wherein the texture of the cheese analogue is improved relative to the texture of a plant-derived cheese analogue.

55. The dairy analogue according to any one of claims 34-54, wherein the adhesiveness of the cheese analogue is reduced relative to the adhesiveness of a plant-derived cheese analogue.

56. The dairy analogue according to any one of claims 34-55, wherein the cheese analogue comprises at least 5% w/w of a single variant of the casein.

57. The dairy analogue of any one of claims 34-56, wherein the cheese analogue comprises from about 5% w/w to about 30% w/w of the single variant of casein.

58. The dairy analogue of any one of claims 34-57, wherein the cheese analogue comprises from about 10% w/w to about 25% w/w of the single variant of casein.

59. The dairy analogue of any one of claims 34-58, wherein the cheese analogue comprises from about 15% w/w to about 25% w/w of the single variant of casein.

60. The dairy analogue according to any one of claims 34-59, wherein the cheese analogue comprises at most 25mg of calcium per gram of casein.

61. The dairy analogue according to any one of claims 34-60, wherein the cheese analogue comprises from 0 to 25mg of calcium per gram of casein.

62. The dairy analogue according to any one of claims 34-61, wherein the cheese analogue comprises from 0 to 20mg of calcium per gram of casein.

63. The dairy analogue according to any one of claims 34-62, wherein the cheese analogue comprises from 0 to 10mg of calcium per gram of casein.

64. The dairy analogue according to any one of claims 34-62, wherein the cheese analogue comprises from 5 to 15mg of calcium per gram of casein.

65. The dairy analogue of any one of claims 34-64, wherein the cheese analogue comprises about 0mg of calcium per gram of casein.

66. The dairy analogue of any one of claims 34-64, wherein the cheese analogue comprises about 10mg of calcium per gram of casein.

67. The dairy analogue of any one of claims 34-66, wherein the cheese analogue comprises from about 15% w/w to about 30% w/w fat.

68. The dairy analogue of any one of claims 34-67, wherein the cheese analogue comprises from about 18% w/w to about 28% w/w fat.

69. The dairy analogue of any one of claims 34-68, wherein the cheese analogue comprises from about 20% w/w to about 25% w/w fat.

70. The dairy analogue of any one of claims 34-69 wherein the cheese analogue comprises from about 0.5% w/w to about 4% w/w starch.

71. The dairy analogue of any one of claims 34-70 wherein the cheese analogue comprises from about 1% w/w to about 3% w/w starch.

72. The dairy analogue of any one of claims 34-71, wherein the cheese analogue comprises from about 2% w/w to about 3% w/w starch.

73. The dairy analogue of any one of claims 34-72, wherein the cheese analogue comprises at most 10% w/w starch.

74. The dairy analogue according to any one of claims 34-73, wherein the cheese analogue comprises at most 5% w/w starch.

75. The dairy analogue of any one of claims 34-74, wherein the ratio of recombinant single variant alpha casein to emulsifying salt is from 12:1 to 6:1.

76. The dairy analogue according to any one of claims 34-75, wherein the cheese analogue does not comprise an emulsifier other than an emulsifying salt.

77. The dairy analogue according to any one of claims 27-33, wherein the dairy analogue is a yoghurt analogue.

78. The dairy analogue of claim 77, wherein the single variant of casein is alpha casein.

79. The dairy analogue of claim 78, wherein the alpha casein is alpha S1 casein.

80. The dairy analogue of claim 79, wherein the αs1 casein is bovine αs1 casein.

81. The dairy analogue of any one of claims 77-80, wherein the αs1 casein is full length casein.

82. The dairy analogue of claim 78, wherein the alpha casein is alpha S2 casein.

83. The dairy analogue of claim 82, wherein the αs2 casein is bovine αs2 casein.

84. The dairy analogue of any one of claims 82-83, wherein the αs2 casein is full length casein.

85. The dairy analogue according to any one of claims 77-84, wherein the emulsification of the yoghurt analogue is comparable to or improved relative to the emulsification of a dairy-derived yoghurt or a dairy-derived yoghurt analogue.

86. The dairy analogue according to any one of claims 77-85, wherein the firmness, viscosity or viscosity of the yoghurt analogue is comparable to or improved relative to dairy-derived yoghurt or dairy-derived yoghurt analogue.

87. The dairy analogue according to any one of claims 77-86, wherein the adhesiveness of the yoghurt analogue is reduced relative to a dairy-derived yoghurt or dairy-derived yoghurt analogue.

88. The dairy analogue according to any one of claims 85-87, wherein the dairy-derived yoghurt or dairy-derived yoghurt analogue comprises micellar casein.

89. The dairy analogue of any one of claims 85-87, wherein the dairy-derived yoghurt or dairy-derived yoghurt analogue is identical to the dairy analogue in all ingredients except that the dairy-derived yoghurt or dairy-derived yoghurt analogue comprises micellar casein in place of a recombinant single variant of alpha casein.

90. The dairy analogue according to any one of claims 77-89, wherein the emulsification of the yoghurt analogue is comparable to or improved relative to the emulsification of a yoghurt analogue of plant origin.

91. The dairy analogue according to any one of claims 77-90, wherein the firmness, viscosity or viscosity of the yoghurt analogue is comparable to or improved relative to a yoghurt analogue of vegetable origin.

92. The dairy analogue according to any one of claims 77-91, wherein the adhesiveness of the yoghurt analogue is reduced relative to a yoghurt analogue of plant origin.

93. The dairy analogue according to any one of claims 90-92, wherein the plant derived yoghurt analogue lacks any dairy protein.

94. The dairy analogue of any one of claims 77-93, wherein the yoghurt analogue comprises from about 1% w/w to about 4% w/w of a single variant of the casein.

95. The dairy analogue of any one of claims 77-94, wherein the yoghurt analogue comprises from about 2% w/w to about 4% w/w of a single variant of the casein.

96. The dairy analogue of any one of claims 77-95, wherein the yoghurt analogue comprises from about 2% w/w to about 6% w/w fat.

97. The dairy analogue of any one of claims 77-96, wherein the yoghurt analogue comprises from about 4% w/w to about 8% w/w carbohydrate.

98. The dairy analogue according to any one of claims 27-33, wherein the dairy analogue is a beverage.

99. The dairy analogue of claim 98, wherein the single variant of casein is alpha casein.

100. The dairy analogue according to any one of claims 98-99, wherein the smoothness of the beverage is comparable to or improved relative to the smoothness of a dairy derived beverage.

101. The dairy analogue according to any one of claims 98-100, wherein the texture of the beverage is comparable to or improved relative to the texture of a dairy derived beverage.

102. The dairy analogue according to any one of claims 98-101, wherein the emulsification of the beverage is comparable to or improved relative to a beverage of dairy origin.

103. The dairy analogue of any one of claims 98-102, wherein the beverage comprises from about 0.5% w/w to about 10% w/w of the single variant of casein.

104. The dairy analogue of any one of claims 98-104, wherein the beverage comprises from about 0.1% w/w to about 6% w/w fat.