Compositions made from crosslinked single casein

Abstract

Provided herein are compositions that are edible and suitable for use in food products, comprising recombinant crosslinked alpha casein, including for example, recombinant crosslinked alpha-s1-casein. Controlling and tailoring crosslinking approach leads to tailoring and improvement of particular product properties and quality attributes of the composition. For example, in dairy-like edible compositions containing only alpha-s1-casein as a dairy protein source, crosslinked recombinant alpha-s1-casein, in comparison with the same non-crosslinked recombinant alpha-s1-casein, enables a more desirable and higher quality product attribute, closer to animal-derived product attributes. Crosslinking has been found to surprisingly improve key analogue cheese properties, including stretch, melt onset, bite resistance, chewiness, and firmness. Surprisingly, crosslinking also enables the reduction of the amount of casein required to achieve desirable levels of key cheese properties.

Description

Attorney Docket No. 263942000740 COMPOSITIONS MADE FROM CROSSLINKED SINGLE CASEIN CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 685,449, filed August 21, 2024, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (263942000740seqlist.xml; Size: 24,464 bytes; and Date of Creation: August 19, 2025) is herein incorporated by reference in its entirety. FIELD

[0003] The present disclosure relates generally to casein compositions that are edible and suitable for use in food products, and more specifically to compositions made from crosslinked single casein, including crosslinked recombinant alpha casein. BACKGROUND

[0004] The clean food space is comprised of both plant-based and cell-based foods. Cell- based food is a large umbrella term that includes culturing muscle and fat cells to replace slaughtered meat and culturing bioengineered organisms to express recombinant animal proteins to replace other animal products such as dairy and eggs. The need to find an alternate source of animal protein comes from the inefficiencies and unsustainability of current animal food production.

[0005] Cheese is the third most unsustainable animal product globally (when measuring greenhouse gas emissions per kg of product), and the consumption of dairy cheese hasn’t been slowed down by plant-based alternatives introduced into the market in the last 10 years. On the contrary, mozzarella cheese consumption is growing year on year in the US and in developing markets. Current cheese alternatives do not match the functionality, nutrition and taste of dairy cheese due to their lack of caseins.

[0006] What is desired in the art are caseins that can be incorporated into cheese analogues that can improve their properties, and preferably also require less of the caseins to 1MOFO-359836952Attorney Docket No. 263942000740 achieve such improved properties. Achieving such properties is an important step to developing commercially viable cheese analogues. BRIEF SUMMARY

[0007] In some aspects, provided herein are compositions that are edible and suitable for use in food products, comprising recombinant crosslinked alpha casein. In some embodiments, the alpha casein is alpha-s1-casein. Controlling and tailoring crosslinking approach leads to tailoring and improvement of particular product properties and quality attributes of the composition. For example, in dairy-like edible compositions containing only alpha-s1-casein as a dairy protein source, crosslinked recombinant alpha-s1-casein, in comparison with the same non-crosslinked recombinant alpha-s1-casein, enables a more desirable and higher quality product attribute, closer to animal-derived product attributes. Crosslinking has been found to surprisingly improve key analogue cheese properties, including stretch, melt onset, bite resistance, chewiness, and firmness. Surprisingly, crosslinking also enables the reduction of the amount of casein required to achieve desirable levels of key cheese properties.

[0008] In some aspects, provided is a composition comprising crosslinked recombinant alpha casein, wherein the recombinant alpha casein is crosslinked to recombinant alpha casein. In some embodiments, the recombinant alpha casein is not crosslinked to beta casein or kappa casein. In some variations, beta casein is not present in the composition; and / or kappa casein is not present in the composition. In certain variations of the foregoing, the recombinant alpha casein is a single variant of an alpha casein. In one variation, the recombinant alpha casein is alpha-s1-casein or alpha-s2-casein.

[0009] In some aspects, provided is a coagulate comprising any one of the compositions comprising crosslinked recombinant alpha casein, as described herein.

[0010] In other aspects, provided is a cheese analogue comprising any one of the compositions comprising crosslinked recombinant alpha casein, as described herein. In some embodiments, the cheese analogue is a mozzarella cheese analogue. In some variations of the foregoing, one or more cheese-like features of the cheese analogue incorporating any one of the compositions comprising crosslinked recombinant alpha casein, as described herein, are improved as compared to a comparator cheese analogue. 2MOFO-359836952Attorney Docket No. 263942000740

[0011] In yet other aspects, provided is a method of producing a casein composition, comprising: crosslinking recombinant alpha casein protein in the absence of beta casein and kappa casein proteins. In some embodiments, the recombinant alpha casein is crosslinked by heat treatment, irradiation, or enzymatically. In some variations, the recombinant alpha casein is enzymatically crosslinked using one or more of transglutaminase, laccase, tyrosinase, peroxidase, sulfhydryl oxidase, and glucose oxidase. DESCRIPTION OF THE FIGURES

[0012] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0013] FIG. 1 depicts photographs from a melt test on pizza and stretch for NC1 and NC2 (Example 2).

[0014] FIG. 2 depicts an SDS-PAGE analysis (Example 3).

[0015] FIGS. 3A-3D depict photographs from a pizza melt test for cheese analogues NC3-NC6 (Example 3). FIG. 3A depicts NC3. FIG. 3B depicts NC4. FIG. 3C depicts NC5. FIG. 3D depicts NC6.

[0016] FIGS. 4A-4D depict photographs from a browning test for cheese analogues NC3- NC6 (Example 3). FIG. 4A depicts NC3. FIG. 4B depicts NC4. FIG. 4C depicts NC5. FIG. 4D depicts NC6.

[0017] FIGS. 5A-5L depict photographs from a pizza melt test for cheese analogues NC7-NC10 (Example 4). FIG. 5A depicts shreds of NC7. FIG. 5B depicts shreds of NC8. FIG. 5C depicts shreds of NC9. FIG. 5D depicts shreds of NC10. FIG. 5E depicts a pizza melt test for NC7. FIG. 5F depicts a pizza melt test for NC8. FIG. 5G depicts a pizza melt test for NC9. FIG. 5H depicts a pizza melt test for NC10. FIG. 5I depicts a stretch test of NC7. FIG. 5J depicts a stretch test of NC8. FIG. 5K depicts a stretch test of NC9. FIG. 5L depicts a stretch test of NC10.

[0018] FIG. 6 depicts the results of a sensory evaluation of cheese analogues NC7-NC10 (Example 4).

[0019] FIGS. 7A-7L depict photographs from a pizza melt test for cheese analogues NC11-NC14 (Example 5). FIG. 7A depicts shreds of NC11. FIG. 7B depicts shreds of NC12. 3MOFO-359836952Attorney Docket No. 263942000740 FIG. 7C depicts shreds of NC13. FIG. 7D depicts shreds of NC14. FIG. 7E depicts a pizza melt test for NC11. FIG. 7F depicts a pizza melt test for NC12. FIG. 7G depicts a pizza melt test for NC13. FIG. 7H depicts a pizza melt test for NC14. FIG. 7I depicts a stretch test of NC11. FIG. 7J depicts a stretch test of NC12. FIG. 7K depicts a stretch test of NC13. FIG. 7L depicts a stretch test of NC14.

[0020] FIG. 8 depicts the results of a sensory evaluation of cheese analogues NC11- NC14 (Example 5).

[0021] FIGS. 9A-9D depict photographs from a pizza melt test for cheese analogues NC15 and NC16 (Example 6). FIG. 9A depicts shreds of NC15. FIG. 9B depicts shreds of NC16. FIG. 9C depicts a pizza melt test for NC15. FIG. 9D depicts a pizza melt test for NC16. FIG. 9E depicts a stretch test of NC15. FIG. 9F depicts a stretch test of NC16.

[0022] FIG. 10 depicts the results of a sensory evaluation of cheese analogues NC15 and NC16 (Example 6). DETAILED DESCRIPTION

[0023] The following description sets forth exemplary compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0024] In some aspects, provided is a composition comprising crosslinked recombinant alpha casein, wherein the recombinant alpha casein is crosslinked to recombinant alpha casein. In some embodiments, the recombinant alpha casein is not crosslinked to beta casein or kappa casein. In certain embodiments, beta casein is not present in the composition; and / or kappa casein is not present in the composition.

[0025] In some variations of the foregoing, the composition comprising crosslinked recombinant alpha casein further comprises fat or oil. In certain variations, the fat or oil comprises a plant or plant-derived fat or oil. In some variations of the foregoing, e.g., where fat or oil is present, the composition comprising crosslinked recombinant alpha casein is an emulsion. 4MOFO-359836952Attorney Docket No. 263942000740

[0026] In some aspects, provided is a coagulate comprising any one of the compositions comprising crosslinked recombinant alpha casein, as described herein.

[0027] In other aspects, provided is a cheese analogue comprising any one of the compositions comprising crosslinked recombinant alpha casein, as described herein. In some embodiments, the cheese analogue is a mozzarella cheese analogue. In some variations of the foregoing, one or more cheese-like features of the cheese analogue incorporating any one of the compositions comprising crosslinked recombinant alpha casein, as described herein, are improved as compared to a comparator cheese analogue.

[0028] In yet other aspects, provided is a method of producing a casein composition, comprising: crosslinking recombinant alpha casein protein in the absence of beta casein and kappa casein proteins. In some embodiments, the recombinant alpha casein is crosslinked by heat treatment, irradiation, or enzymatically. In some variations, the recombinant alpha casein is enzymatically crosslinked using one or more of transglutaminase, laccase, tyrosinase, peroxidase, sulfhydryl oxidase, and glucose oxidase. Recombinant alpha casein

[0029] In certain variations of the foregoing compositions and methods, the recombinant alpha casein is a single variant of an alpha casein. In one variation, the recombinant alpha casein is alpha-s1-casein or alpha-s2-casein. In one variations, the recombinant alpha casein is alpha-s1-casein.

[0030] In some variations, the recombinant alpha casein is a microbially-produced recombinant alpha casein. In some such variations, the recombinant alpha casein is a bacterially-produced recombinant alpha casein. In some variations, the recombinant alpha casein is not present in a micellar form. In some variations, the recombinant alpha casein is in a liquid or emulsion form. In yet other variations, the recombinant alpha casein is in a dried or powder form.

[0031] In some embodiments, the alpha casein is not in micellar form and not derived from milk or derived from milk casein. In some aspects, the recombinant alpha casein is the only casein in the composition.

[0032] In some embodiments, the consumable compositions (and products made therefrom) do not include any animal-derived caseins. In some embodiments, the 5MOFO-359836952Attorney Docket No. 263942000740 compositions do not comprise any casein proteins isolated from any animal-derived products or micelles. In other embodiments, the consumable compositions have no detectable amount of any animal-derived caseins. The consumable compositions herein do not include any beta and / or kappa caseins. In other variations, the consumable compositions have no detectable amount of any beta and / or kappa caseins. In some embodiments, the consumable compositions herein (and products made therefrom) do not include any dairy proteins other than a single variant of the alpha casein. In some variations, the consumable compositions do not include any whey proteins or any milk-derived whey proteins. In some embodiments, compositions herein (and products made therefrom) do not include any animal-derived dairy proteins.

[0033] In some variations, the alpha casein is a modified casein protein relative to a native casein protein. The modifications in the alpha casein protein may comprise one or more amino acid insertions, deletions, or substitutions relative to a wild-type or native casein protein.

[0034] In some embodiments, the alpha casein in a consumable composition is only an alpha-S1 casein. In such compositions, the alpha-S1 casein may comprise a modified alpha- S1 casein, such as a modified alpha-S1 casein that lacks or has substantially reduced post- translational phosphorylation. In some variations, the alpha-S1 casein may be a full-length alpha-S1 casein. In some variations, the compositions comprising an alpha-S1 casein lacks any animal-derived proteins. In some variations of the foregoing, the consumable composition has no alpha-S2 casein. In other variations, the consumable composition has no detectable amount of alpha-S2 casein. In yet other variations, the consumable composition does not include a mixture of alpha-S1 and alpha-S2 casein.

[0035] The alpha casein may match the sequence (i.e., be identical in amino acid sequence) of that from a ruminant species. In some embodiments, an alpha casein may have substantial identity (such as at least 80%, 85%, 90%, 95% or 99%) of the amino acid sequence of an alpha casein from a ruminant species. Accordingly, in some variations, an alpha casein may be a bovine alpha casein. In other variations, the alpha casein may be a caprine alpha casein. In other variations, the alpha casein may be an ovine alpha casein. In other variations, the alpha casein may be an equine alpha casein. In other variations, the alpha casein may be a camel or camelid alpha casein. In other variations, the alpha casein may be a human alpha casein. 6MOFO-359836952Attorney Docket No. 263942000740

[0036] In some embodiments, the alpha casein may be a mature form of an alpha casein (lacking a signal sequence, such as exemplified in SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, or 20). The alpha casein may be a bovine alpha casein, for instance, casein protein with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 1-3 or 10-12. A single variant of an alpha casein may be an ovine alpha casein, for instance, casein protein with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 4-6. The alpha casein may be a caprine alpha casein, for instance, casein protein with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 7-9. The alpha casein may be an equine alpha casein, for instance, casein protein with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 13-15. The alpha casein may be a camel alpha casein, for instance, casein protein with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 16-18. The alpha casein may be a human alpha casein, for instance, casein protein with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 19-20.

[0037] In some variations, the recombinant alpha casein is present in the composition at an amount between 1% and 30%, between 1% and 18%, between 1% and 17%, between 1% and 16%, between 1% and 15%, between 1% and 14%, between 1% and 13%, between 1% and 12%, between 1% and 11%, between 1% and 10%, between 1% and 9%, between 1% and 8%, between 1% and 7%, between 1% and 6%, or between 1% and 5% wt / wt of the composition. In some variations, the recombinant alpha casein is present in the composition at an amount less than or about 5% wt / wt, such as between 1% and 2%, between 1% and 3%, between 1% and 4%, between 1% and 5%, or about 1%, 2%, 3%, 4% or 5% wt / wt of the composition. In some variations, the recombinant alpha casein is present in the composition at an amount greater than 5% wt / wt, such as between 5% and 18%, between 5% and 17%, between 5% and 16%, between 5% and 14%, between 5% and 13%, between 5% and 12%, between 5% and 11%, between 5% and 10%, between 5% and 11%, between 5% and 10%, between 5% and 9%, between 5% and 8%, between 5% and 7%, between 5% and 6%, or about 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% or 18% wt / wt of the composition. In other variations, the recombinant alpha casein is present in the composition at an amount greater than 1%, greater than 2%, greater 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, or greater than 10%. In other variations, the recombinant alpha casein is present in the composition at an amount less 7MOFO-359836952Attorney Docket No. 263942000740 than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4% or less than 3% wt / wt of the composition. In one variation, the recombinant casein is present in the composition at an amount greater than or about 5% and less than 30% wt / wt of the composition. In another variation, the recombinant casein is present in the composition at an amount greater than 5% and less than 18% wt / wt of the composition.

[0038] It should be understood that reference to “between” two values or parameters herein includes (and describes) aspects that include those two values or parameters per se. For example, description referring to “between x and y” includes the values of “x” and “y” per se. Crosslinking

[0039] In crosslinking the alpha casein, e.g., recombinant alpha casein, the alpha casein is crosslinked to other alpha casein, but not other caseins (e.g., not to beta casein or kappa casein) or other dairy proteins. This is a distinction from crosslinking typically used in the dairy industry where alpha casein is present with beta and kappa caseins, and these proteins are typically present in a micellar form, that is then crosslinked. The present invention is based, at least in part, on the discovery that alpha casein proteins crosslinked to other alpha casein proteins in the absence of any other caseins, while not able to form micellar structures, improve the quality of the key functional attributes of food products critical for commercially-relevant dairy compared to non-crosslinked alpha casein, which is also not able to form micellar structures.

[0040] In some embodiments, dry crosslinking is employed to produce an alpha casein ingredient. In some variations, casein may be crosslinked in the dry state, for example by heating an alpha casein powder or irradiating an alpha casein powder.

[0041] In some embodiments, alpha casein is crosslinked in solution (e.g., in a liquid or in an emulsion form). In some variations, alpha casein may be crossed by adding a suitable crosslinking enzyme. In certain variations, the crosslinking enzyme comprises transglutaminase, laccase, tyrosinase, peroxidase, sulfhydryl oxidase, or glucose oxidase. In one variation, any combination of suitable crosslinking enzymes may be used. In another variation, the crosslinking enzyme is transglutaminase. In some embodiments, the crosslinked alpha casein in solution is immediately used for making another composition (e.g., a 8MOFO-359836952Attorney Docket No. 263942000740 downstream composition), or may be dried, and optionally stored, for subsequent use. For instance, in some variations, the alpha casein is crosslinked, spray-dried, and subsequently used in cheese-making. In certain embodiments, the solution is at rest during the crosslinking process. In other embodiments, the solution is mixed during crosslinking.

[0042] In some embodiments, the alpha casein is crosslinked in an emulsion. In some variations, alpha casein is crosslinked when present in an emulsion, for example, by adding a suitable crosslinking enzyme. In certain embodiments, the emulsion is at rest during the crosslinking process. In other embodiments, the emulsion is mixed during crosslinking.

[0043] In other embodiments, alpha casein is crosslinked when present in a water-fat emulsion, for example by adding a suitable crosslinking enzyme. In certain embodiments, the water-fat emulsion is at rest during the crosslinking process. In other embodiments, the water-fat emulsion is mixed during crosslinking.

[0044] In some variations of the foregoing, the alpha casein is recombinant alpha casein. In one variation of the foregoing, the alpha casein is alpha-s1-casein or alpha-s2-casein. In another variation, the alpha casein is unphosphorylated alpha-s1-casein or alpha-s2-casein, such as recombinant alpha-s1-casein or alpha-s2-casein that lacks phosphorylation.

[0045] When crosslinking enzymes are employed, such enzymes may be deactivated, such as by heating, subsequent to a crosslinking step. The degree of crosslinking is related to the degree and conditions of exposure to the crosslinking enzyme.

[0046] In some embodiments, at least about 0.1% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer. In some embodiments, between about 0.1% and about 100% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer. In certain embodiments, between about 0.1% and about 1%, between about 0.1% and about 10%, between about 0.1% and about 20%, between about 0.1% and about 30%, between about 0.1% and about 40%, between about 0.1% and about 50%, between about 0.1% and about 60%, between about 0.1% and about 70%, between about 0.1% and about 80%, between about 0.1% and about 90%, between about 0.1% and about 95%, between about 0.1% and about 99%, between about 0.1% and about 100%, between about 1% and about 10%, between about 1% and about 20%, between about 1% and about 30%, between about 1% and about 40%, between about 1% and about 50%, between about 1% and about 9MOFO-359836952Attorney Docket No. 263942000740 60%, between about 1% and about 70%, between about 1% and about 80%, between about 1% and about 90%, between about 1% and about 95%, between about 1% and about 99%, between about 1% and about 100%, between about 10% and about 20%, between about 10% and about 30%, between about 10% and about 40%, between about 10% and about 50%, between about 10% and about 60%, between about 10% and about 70%, between about 10% and about 80%, between about 10% and about 90%, between about 10% and about 95%, between about 10% and about 99%, between about 10% and about 100%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 20% and about 60%, between about 20% and about 70%, between about 20% and about 80%, between about 20% and about 90%, between about 20% and about 95%, between about 20% and about 99%, between about 20% and about 100%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 30% and about 70%, between about 30% and about 80%, between about 30% and about 90%, between about 30% and about 95%, between about 30% and about 99%, between about 30% and about 100%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 40% and about 80%, between about 40% and about 90%, between about 40% and about 95%, between about 40% and about 99%, between about 40% and about 100%, between about 50% and about 60%, between about 50% and about 70%, between about 50% and about 80%, between about 50% and about 90%, between about 50% and about 95%, between about 50% and about 99%, between about 50% and about 100%, between about 60% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 60% and about 95%, between about 60% and about 99%, between about 60% and about 100%, between about 70% and about 80%, between about 70% and about 90%, between about 70% and about 95%, between about 70% and about 99%, between about 70% and about 100%, between about 80% and about 90%, between about 80% and about 95%, between about 80% and about 99%, between about 80% and about 100%, between about 90% and about 95%, between about 90% and about 99%, between about 90% and about 100%, between about 95% and about 99%, between about 95% and about 100%, or between about 99% and about 100% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer.

[0047] In some embodiments, at least about 1% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer. In 10MOFO-359836952Attorney Docket No. 263942000740 certain embodiments, at least about 1%, about 2%, about 5%, about 10%, about 15%, or about 20% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer. In some embodiments, between about 1% and about 30% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer. In certain embodiments, between about 1% and about 5%, between about 1% and about 10%, between about 1% and about 15%, between about 1% and about 20%, between about 1% and about 30%, between about 2% and about 5%, between about 2% and about 10%, between about 2% and about 15%, between about 2% and about 20%, between about 2% and about 30%, between about 5% and about 10%, between about 5% and about 15%, between about 5% and about 20%, between about 5% and about 30%, between about 10% and about 15%, between about 10% and about 20%, between about 10% and about 30%, or between about 20% and about 30% of the recombinant alpha casein protein monomers in the composition are crosslinked to at least one other casein protein monomer. In some variations, the percentage of crosslinked recombinant alpha casein protein monomers is determined by SDS-PAGE.

[0048] In some embodiments, no more than 99.9% of the recombinant alpha casein protein in the composition is in monomeric form. In some embodiments, between about 0.1% and about 99.9% of the recombinant alpha casein protein in the composition is in monomeric form. In certain embodiments, between about 0.1% and about 1%, between about 0.1% and about 10%, between about 0.1% and about 20%, between about 0.1% and about 30%, between about 0.1% and about 40%, between about 0.1% and about 50%, between about 0.1% and about 60%, between about 0.1% and about 70%, between about 0.1% and about 80%, between about 0.1% and about 90%, between about 0.1% and about 95%, between about 0.1% and about 99%, between about 0.1% and about 99.9%, between about 1% and about 10%, between about 1% and about 20%, between about 1% and about 30%, between about 1% and about 40%, between about 1% and about 50%, between about 1% and about 60%, between about 1% and about 70%, between about 1% and about 80%, between about 1% and about 90%, between about 1% and about 95%, between about 1% and about 99%, between about 1% and about 99.9%, between about 10% and about 20%, between about 10% and about 30%, between about 10% and about 40%, between about 10% and about 50%, between about 10% and about 60%, between about 10% and about 70%, between about 10% and about 80%, between about 10% and about 90%, between about 10% and about 95%, between about 10% and about 99%, between about 10% and about 99.9%, between about 11MOFO-359836952Attorney Docket No. 263942000740 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 20% and about 60%, between about 20% and about 70%, between about 20% and about 80%, between about 20% and about 90%, between about 20% and about 95%, between about 20% and about 99%, between about 20% and about 99.9%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 30% and about 70%, between about 30% and about 80%, between about 30% and about 90%, between about 30% and about 95%, between about 30% and about 99%, between about 30% and about 99.9%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 40% and about 80%, between about 40% and about 90%, between about 40% and about 95%, between about 40% and about 99%, between about 40% and about 99.9%, between about 50% and about 60%, between about 50% and about 70%, between about 50% and about 80%, between about 50% and about 90%, between about 50% and about 95%, between about 50% and about 99%, between about 50% and about 99.9%, between about 60% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 60% and about 95%, between about 60% and about 99%, between about 60% and about 99.9%, between about 70% and about 80%, between about 70% and about 90%, between about 70% and about 95%, between about 70% and about 99%, between about 70% and about 99.9%, between about 80% and about 90%, between about 80% and about 95%, between about 80% and about 99%, between about 80% and about 99.9%, between about 90% and about 95%, between about 90% and about 99%, between about 90% and about 99.9%, between about 95% and about 99%, between about 95% and about 99.9%, or between about 99% and about 99.9%, of the recombinant alpha casein protein in the composition is in monomeric form.

[0049] In some embodiments, no more than 99% of the recombinant alpha casein protein in the composition is in monomeric form. In certain embodiments, no more than 99%, 98%, 95%, 90%, 85%, 80%, or 70% of the recombinant alpha casein protein in the composition is in monomeric form. In some embodiments, between about 70% and about 99% of the recombinant alpha casein protein in the composition is in monomeric form. In certain embodiments, between about 70% and about 80%, between about 70% and about 85%, between about 70% and about 90%, between about 70% and about 95%, between about 70% and about 98%, between about 70% and about 99%, between about 80% and about 85%, between about 80% and about 90%, between about 80% and about 95%, between about 80% and about 98%, between about 80% and about 99%, between about 85% and about 90%, 12MOFO-359836952Attorney Docket No. 263942000740 between about 85% and about 95%, between about 85% and about 98%, between about 85% and about 99%, between about 90% and about 95%, between about 90% and about 98%, between about 90% and about 99%, between about 95% and about 98%, or between about 95% and about 99% of the recombinant alpha casein protein in the composition is in monomeric form. In some variations, the remainder of the recombinant alpha casein protein in the composition is crosslinked to other recombinant alpha casein protein. In some variations, the percentage of crosslinked recombinant alpha casein protein monomers is determined by SDS-PAGE. Cheese Analogue and Cheese Properties

[0050] In certain aspects, provided is a cheese analogue comprising any one of the compositions comprising crosslinked recombinant alpha casein (e.g., alpha-S1 casein), as described herein. In some embodiments, the cheese analogue is a mozzarella cheese analogue. In some variations of the foregoing, one or more cheese-like features of the cheese analogue incorporating any one of the compositions comprising crosslinked recombinant alpha casein, as described herein, are improved as compared to a comparator cheese analogue. In one variation, the comparator cheese analogue comprises non-crosslinked alpha casein, and does not comprise beta casein or kappa casein. In some variations, the cheese analogue and the comparator cheese analogue comprise recombinant alpha casein in the same amount ± 1%, 2%, 3%, 4%, or 5% w / w. In one variation, the comparator cheese analogue comprises essentially the same components as the cheese analogue except that it lacks crosslinked alpha casein protein.

[0051] In some embodiments, the cheese analogue comprises recombinant alpha casein protein, wherein at least about 1% of the recombinant alpha casein protein is crosslinked recombinant alpha casein protein. In certain embodiments, the recombinant alpha casein protein is a single variant of an alpha casein protein. In some variations, the single variant of the alpha casein protein is alpha-S1 casein. In other variations, the single variant of the alpha casein protein is alpha-S2 casein. In some variations, 100% of the recombinant alpha casein is alpha-S1 casein. In some variations, 100% of the recombinant alpha casein is alpha-S2 casein.

[0052] In some variations, the recombinant alpha casein is present in the cheese analogue at an amount between 1% and 30%, between 1% and 18%, between 1% and 17%, between 1% and 16%, between 1% and 15%, between 1% and 14%, between 1% and 13%, between 13MOFO-359836952Attorney Docket No. 263942000740 1% and 12%, between 1% and 11%, between 1% and 10%, between 1% and 9%, between 1% and 8%, between 1% and 7%, between 1% and 6%, or between 1% and 5% wt / wt of the cheese analogue. In some variations, the recombinant alpha casein is present in the cheese analogue at an amount less than or about 5% wt / wt, such as between 1% and 2%, between 1% and 3%, between 1% and 4%, between 1% and 5%, or about 1%, 2%, 3%, 4% or 5% wt / wt of the cheese analogue. In some variations, the recombinant alpha casein is present in the cheese analogue at an amount greater than 5% wt / wt, such as between 5% and 18%, between 5% and 17%, between 5% and 16%, between 5% and 14%, between 5% and 13%, between 5% and 12%, between 5% and 11%, between 5% and 10%, between 5% and 11%, between 5% and 10%, between 5% and 9%, between 5% and 8%, between 5% and 7%, between 5% and 6%, or about 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% or 18% wt / wt of the cheese analogue. In other variations, the recombinant alpha casein is present in the cheese analogue at an amount greater than 1%, greater than 2%, greater 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, or greater than 10%. In other variations, the recombinant alpha casein is present in the cheese analogue at an amount less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4% or less than 3% wt / wt of the cheese analogue. In one variation, the recombinant casein is present in the cheese analogue at an amount greater than or about 5% and less than 30% wt / wt of the cheese analogue. In another variation, the recombinant casein is present in the cheese analogue at an amount greater than 5% and less than 18% wt / wt of the cheese analogue.

[0053] In some variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount of between about 5% and about 18% wt / wt of the cheese analogue, wherein the alpha casein imparts stretch to the cheese analogue. In some variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount of between about 5% and about 6%, between about 5% and about 7%, between about 5% and about 8%, between about 5% and about 9%, between about 5% and about 10%, between about 5% and about 11%, between about 5% and about 12%, between about 5% and about 13%, between about 5% and about 14%, between about 5% and about 15%, between about 5% and about 16%, between about 5% and about 17%, between about 5% and about 18%, between about 6% and about 7%, between about 6% and about 8%, between about 6% and about 9%, between about 6% and about 10%, between about 6% and about 11%, between 14MOFO-359836952Attorney Docket No. 263942000740 about 6% and about 12%, between about 6% and about 13%, between about 6% and about 14%, between about 6% and about 15%, between about 6% and about 16%, between about 6% and about 17%, between about 6% and about 18%, between about 7% and about 8%, between about 7% and about 9%, between about 7% and about 10%, between about 7% and about 11%, between about 7% and about 12%, between about 7% and about 13%, between about 7% and about 14%, between about 7% and about 15%, between about 7% and about 16%, between about 7% and about 17%, between about 7% and about 18%, between about 8% and about 9%, between about 8% and about 10%, between about 8% and about 11%, between about 8% and about 12%, between about 8% and about 13%, between about 8% and about 14%, between about 8% and about 15%, between about 8% and about 16%, between about 8% and about 17%, between about 8% and about 18%, between about 9% and about 10%, between about 9% and about 11%, between about 9% and about 12%, between about 9% and about 13%, between about 9% and about 14%, between about 9% and about 15%, between about 9% and about 16%, between about 9% and about 17%, between about 9% and about 18%, between about 10% and about 11%, between about 10% and about 12%, between about 10% and about 13%, between about 10% and about 14%, between about 10% and about 15%, between about 10% and about 16%, between about 10% and about 17%, between about 10% and about 18%, between about 11% and about 12%, between about 11% and about 13%, between about 11% and about 14%, between about 11% and about 15%, between about 11% and about 16%, between about 11% and about 17%, between about 11% and about 18%, between about 12% and about 13%, between about 12% and about 14%, between about 12% and about 15%, between about 12% and about 16%, between about 12% and about 17%, between about 12% and about 18%, between about 13% and about 14%, between about 13% and about 15%, between about 13% and about 16%, between about 13% and about 17%, between about 13% and about 18%, between about 14% and about 15%, between about 14% and about 16%, between about 14% and about 17%, between about 14% and about 18%, between about 15% and about 16%, between about 15% and about 17%, between about 15% and about 18%, between about 16% and about 17%, between about 16% and about 18%, or between about 17% and about 18% wt / wt of the cheese analogue. In some variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, or about 18% wt / wt of the cheese analogue. 15MOFO-359836952Attorney Docket No. 263942000740

[0054] In some embodiments, the recombinant alpha casein is present in the cheese analogue in an amount of no more than about 18% wt / wt of the cheese analogue. In certain embodiments, the recombinant alpha casein is present in the cheese analogue in an amount of no more than about 15% wt / wt of the cheese analogue. In some embodiments, the recombinant alpha casein is present in the cheese analogue in an amount of no more than about 11% wt / wt of the cheese analogue. In certain embodiments, the recombinant alpha casein is present in the cheese analogue in an amount of no more than about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, or about 5% wt / wt of the cheese analogue.

[0055] In some embodiments, the cheese analogue may comprise one or more additional components including, for example, solvents (such as water), fats, salts, starch, sugars, flavors, acids, pH stabilizers, and carbohydrates.

[0056] In some variations, a cheese analogue may comprise 10% w / w to 30% w / w fats (w / w herein referring to weight of the component / total weight of the composition, such as the total weight of a cheese analogue composition). Examples of fats which can be added to a cheese analogue include coconut, canola, high-oleic sunflower, palm oils. Other examples are provided elsewhere herein. In some variations, a cheese analogue may comprise at least 10% w / w fats. In some variations, a cheese analogue may comprise at most 30% w / w fats. In some variations, a cheese analogue may comprise from 10% w / w to 30% w / w, 10% w / w to 15% w / w, 10% w / w to 20% w / w, 10% w / w to 25% w / w, 15% w / w to 20% w / w, 15% w / w to 25% w / w, 15% w / w to 30% w / w, 15% w / w to 40% w / w, 20% w / w to 25% w / w, 20% w / w to 30% w / w, 25% w / w to 30% w / w, 24% w / w to 30% w / w, 24% w / w to 26% w / w, 24% w / w to 28% w / w, 26% w / w to 30% w / w, 26% w / w to 28% w / w, or 28% w / w to 30% w / w fats. In some variations, a cheese analogue may comprise about 10% w / w, 15% w / w, 20% w / w, 24% w / w, 25% w / w, 26% w / w, 27% w / w 28% w / w, 29% w / w, or 30% w / w fats. In some variations, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein (such as a recombinant alpha-S1 casein), and from 10% w / w to 30% w / w fats. In some variations, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein, and from 28% w / w to 30% w / w fats. In some variations, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein, and from 24% w / w to 30% w / w fats. 16MOFO-359836952Attorney Docket No. 263942000740

[0057] In other variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount relative to fats / oils, such that the ratio of the recombinant alpha casein to fat / oil is between about 1:0.7 and 1:3.3. In some variations, the recombinant alpha casein is present in the cheese analogue at an amount between about 9% and about 11% alpha casein dry weight / total cheese analogue composition weight and fats / oils are present in the cheese analogue composition such that relative to fats / oils, the ratio of the recombinant alpha casein to fat / oil is between about 1:1.1 and 1:3.3. In some such variations, the ratio of the recombinant alpha casein to fat / oil is about 1:1.1, 1:2, 1:3, or 1:3.3. In some such variations, the ratio of the recombinant alpha casein to fat / oil is about 1:3, about 1:3.1, about 1:3.2, or about 1:3.3. In some such variations, the ratio of the recombinant alpha casein to fat / oil is at least 1:3. In some such variations, the ratio of the recombinant alpha casein to fat / oil is at least 1:1.1.

[0058] In some variations, a cheese analogue may comprise 0% w / w to 20% w / w starch. Examples of starches that can be added to a cheese analogue include modified potato starch and modified corn starch. Other examples are provided elsewhere herein. In some variations, a cheese analogue may comprise 0% w / w starch. In some variations, a cheese analogue may comprise at least 0.1% w / w starch. In some variations, a cheese analogue may comprise at most 10% w / w starch. In some variations, a cheese analogue may comprise at most 15% w / w starch. In some variations, a cheese analogue may comprise at most 20% w / w starch. In some variations, a cheese analogue may comprise starch in an amount of between 0% and about 2%, between about 0% and about 4%, between about 0% and about 6%, between about 0% and about 8%, between about 0% and about 10%, between about 0% and about 15%, between about 0% and about 20%, between about 2% and about 4%, between about 2% and about 6%, between about 2% and about 8%, between about 2% and about 10%, between about 2% and about 15%, between about 2% and about 20%, between about 4% and about 6%, between about 4% and about 8%, between about 4% and about 10%, between about 4% and about 15%, between about 4% and about 20%, between about 5% and about 7%, between about 6% and about 8%, between about 6% and about 10%, between about 6% and about 15%, between about 6% and about 20%, between about 8% and about 10%, between about 8% and about 15%, between about 8% and about 20%, between about 10% and about 15%, between about 10% and about 20%, or between about 15% and about 20% w / w. 17MOFO-359836952Attorney Docket No. 263942000740

[0059] In other variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount relative to starch of no more than 1:0.33, or no more than 1:0.02 or no more than 1:0.01. In some variations, no starch is present. In some variations, the ratio of recombinant alpha casein to starch is between 1:0.01 to 1:4. In some variations, the ratio of recombinant alpha casein to starch is between 1:0.02 to 1:1.2. In some variations, the ratio of recombinant alpha casein to starch is at least 1:4, at least 1:1.2, or at least 1:1.

[0060] In some variations, a cheese analogue may contain salts such as calcium salts, emulsifying salts, table salts, etc. Examples of such salts are also provided elsewhere herein. In some variations, a cheese analogue may comprise emulsifying salts such as disodium phosphate, trisodium citrate or other emulsifying salts. In some variations, a cheese analogue may comprise calcium ions. The calcium ions may be added to a cheese analogue in the form of a calcium-based salt, for instance, calcium chloride (e.g., calcium chloride dihydrate or calcium chloride anhydrous), calcium carbonate, calcium lactate, calcium gluconate, or any other calcium salts known in the art. In some variations, a cheese analogue may comprise 0% to 0.5% w / w calcium ions. In some variations, a cheese analogue may comprise at least 0.03% w / w calcium ions. In some variations, a cheese analogue may comprise at most 0.5% w / w calcium ions. In some variations, a cheese analogue may comprise 0.03% to 0.3%, 0.30% to 0.5%, 0.2% to 0.3%, 0.2% to 0.5% w / w calcium ions. In some variations, a cheese analogue may comprise about 0.03%, 0.07%, 0.1%, 0.13%, 0.17%, 0.2%, 0.23%, 0.27%, 0.3%, 0.33%, 0.37%, 0.4%, 0.43%, 0.47% or 0.5% w / w calcium ions.

[0061] In other variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount relative to calcium ions of 1:0.027, between 1:0.030 to 1:0.055, or between 1:0.030 to 1:0.015. In some variations, no calcium ions are present. In other variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount relative to calcium ions of 1:0.180 to 1:0.089 or 1:0.016 to 1:0.089.

[0062] In some variations, a cheese analogue may comprise 0% to 1.5% w / w calcium salts. In some variations, a cheese analogue may comprise at least 0.1% w / w calcium salts. In some variations, a cheese analogue may comprise at most 1.5% w / w calcium salts. In some variations, a cheese analogue may comprise 0.1% to 1%, 0.1% to 1.5%, 0.5% to 1%, 0.5% to 1.5% w / w calcium salts. In some variations, a cheese analogue may comprise about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5% w / w calcium salts. The calcium salt may be calcium chloride (e.g., calcium chloride 18MOFO-359836952Attorney Docket No. 263942000740 dihydrate or calcium chloride anhydrous), calcium carbonate, calcium lactate, calcium gluconate, or any other calcium salts known in the art.

[0063] In other variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount relative to calcium salts of 1:0.1, between 1:0.11 to 1:0.166 , or between 1:0.11 to 1:0.055. In some variations, no calcium salts are present. In other variations of the foregoing, the recombinant alpha casein is present in the cheese analogue in an amount relative to calcium salts of 1:0.666 to 1:0.33 or 1:0.06 to 1:0.033.

[0064] In some embodiments, the cheese analogue has a moisture content of between 30% and 60%. In certain embodiments, the cheese analogue has a moisture content of between 35% to 52%. In some variations, the moisture of the cheese analogue is 30-52%, 30-60%, 35%-52%, 40%-52%, 40%-50%, 40%-45%, 45%-52% or between 45-50% moisture w / w. In some variations, the moisture content is about 30%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% w / w. In some variations, the ratio of recombinant alpha casein to moisture is between 1:2.3 to 1:3.5, 1:3 to 1:3.5, or 1:3 to 1:3.3. In some such variations, the ratio of recombinant alpha casein to moisture is between 1:3.9 to 1:5.8, 1:5 to 1:5.7, or 1:5 to 1:5.6.

[0065] In some examples, the recombinant alpha casein may be combined with water, sodium chloride (salt), calcium chloride, emulsifying salts (disodium phosphate, trisodium citrate), starch, and in some variations also with natural vegan flavors, and acid. Optional ingredients such as plant-based or other animal-free protein, hydrocolloids, sugars such as mono-, di- and oligosaccharides, emulsifying agents such as mono- and diglycerides, lecithins, and / or polysorbates, natural flavor maskers, color additives, preservatives, anti- caking agents and micronutrients such as vitamins can be incorporated into a cheese analogue as well.

[0066] In some variations, the cheese analogue comprises: a. recombinant alpha casein in an amount of between about 5% and about 15% w / w; b. fat in an amount of between about 20% and about 25% w / w; c. emulsifying salt in an amount of between about 1% and about 2%; 19MOFO-359836952Attorney Docket No. 263942000740 d. transglutaminase in an amount of between 0.2% and about 1%, optionally wherein the transglutaminase has an activity of 100-120 units per gram; e. salt (e.g., sodium chloride) in an amount of between about 1% and about 2%; f. water in an amount of between about 45% and about 55%; g. sugar in an amount of between about 1% and about 3%; h. gums in an amount of between about 1% and about 4%; i. starch in an amount of between about 2% and about 8%; or j. any combination of (a)-(i).

[0067] In some embodiments, the cheese analogue comprises fat in an amount of between about 20% and about 25% w / w. In certain embodiments, the cheese analogue comprises a saturated fat in an amount of between about 15% and about 21% w / w. In some variations, the saturated fat is coconut oil. In other variations, the saturated fat is palm stearin. In certain embodiments, the cheese analogue comprises an unsaturated fat in an amount of between about 3.5% and about 7.5%. w / w. In some variations, the unsaturated fat comprises sunflower oil. In other variations, the unsaturated fat comprises canola oil.

[0068] In some embodiments, the cheese analogue comprises emulsifying salt in an amount of between about 1% and about 2%. In certain embodiments, the emulsifying salt comprises trisodium citrate. In other variations, the emulsifying salt comprises disodium phosphate.

[0069] In some embodiments, the following ingredients are pre-mixed: recombinantly produced alpha casein protein, water and salts, such as emulsifying salts, and calcium salts. In some embodiments, pH adjustment is performed at this stage to bring the composition to neutral pH, 6.8-7.2, using a pH adjuster such as sodium hydroxide (lye). Optional ingredients such as plant-based or other animal-free protein, starches, fats, sugars, hydrocolloids and emulsifying agents can be added at this step or at a later stage. Pre-mixing can occur at ambient or elevated temperatures (e.g., 15-65 -melted (e.g., 30-70 °C) (exemplary range 40- incorporation. The calcium chloride and emulsifying salts may be added at the pre-mixing stage or at a later stage. Alternatively, the calcium chloride and emulsifying salts may be added consecutively, in any order: calcium chloride may be added before or after emulsifying salts. For instance, calcium chloride and emulsifying salts may be added in 2 stages over the course of 4 minutes to 1 hour (exemplary range 10 to 20 minutes), with 2-minute to 30- 20MOFO-359836952Attorney Docket No. 263942000740 minute (exemplary range 5 to 10 min) incubation intervals at ambient or elevated temperature. Alternatively, a cheese analogue may be produced without calcium chloride or emulsifying salts. Calcium chloride can also be added at the end of the cheese analogue making process, before or after the acid addition.

[0070] The mixtures may be heated over a temperature ramp from pre-mixingtemperature (ambient or elevated) to 50- -period of 1 to 30 minutes (exemplary range 1 to 5 mins), while being mixed mechanically. Heated mixtures may then be held for 0 to 20 minutes (exemplary range 2-5 mins) at the final ramp temperature as ingredients are mechanically incorporated to form an emulsion. Mechanical incorporation (e.g., mixing) can be achieved using a variety of mixers, such as a vertical cutter mixer or a twin-screw mixer.

[0071] The crosslinking of the recombinant alpha casein protein may be carried out prior to or after mixing of the alpha casein powder with the other ingredients. In some embodiments, the recombinant alpha casein protein is heat treated prior to mixing to achieve the crosslinking. In some embodiments, the recombinant alpha casein protein is mixed (hydrated) with water and possibly salts and / or emulsifiers and subsequently transglutaminase (TG) is added to the mixture for cross-linking, prior to fat emulsification. In other embodiments, the recombinant alpha casein protein is mixed with fat(s), water, emulsifiers and / or emulsifying salts to create an emulsion, and subsequently transglutaminase (TG) is added to the mixture. The emulsion comprising the TG may then be incubated at a temperature appropriate for optimal TG activity, e.g., around 45°C, for a length of time to allow optimal crosslinking of the recombinant alpha casein protein, e.g., between 20 minutes and about 35 minutes. In yet other embodiments, the recombinant alpha casein protein is mixed with fat(s), water, emulsifiers and / or emulsifying salts to create an emulsion, that emulsion is heated at 75°C-90°C, then cooled to 45°C, then TG is added to the mixture for a length of time to allow optimal crosslinking of the recombinant alpha casein protein, e.g., between 20 minutes and about 35 minutes. In some variations, the emulsion may then be heated briefly (e.g., for about 2 min) to about 70°C to inactivate the TG enzyme. Additional ingredients, such as starch, sugar, salts, gums, flavoring agents, and the like may be added to the emulsion comprising the cross-linked recombinant alpha casein to produce the cheese analogue. Exemplary methods of making cheese analogues comprising crosslinked recombinant alpha casein protein are described in the Examples herein. 21MOFO-359836952Attorney Docket No. 263942000740

[0072] The acidity of the mixtures may be regulated by incorporating an acid, such as lactic or citric acid and continuing to mix briefly, to a final pH of about 5-6.5 (exemplary range 5.7-6.2). Acidity may also be regulated by using glucono-delta-lactone earlier in the mixing process. The resulting mixtures may then be set into moulds, other shaping containers, or vacuum seal packaging. The resulting product may be chilled to 4 °C immediately after portioning into moulds to create the cheese analogue. Such cheese analogues can then be used as food products, toppings and incorporated into other food products.

[0073] In some embodiments, the amount of salts or minerals in a cheese analogue may be altered to generate favorable qualities. For instance, in one example, an amount of calcium may be altered to improve melt, texture, stretch, etc. In one example, the amount of calcium in a cheese analogue may be reduced to improve the melt of the cheese analogue. In another example, the amount of calcium may be increased in a cheese analogue to improve the texture or stretch of the cheese analogue.

[0074] Compared to cheese analogue made with non-crosslinked protein, cheese analogue made with crosslinked protein as described herein can have longer and higher quality stretch. In some variations, a “higher quality” refers to a firmer stretch with an elastic snap and / or has multiple strands as opposed to having a single strand that can be goopy. Cheese analogue with a higher quality stretch will exhibit more similar stretch compared to animal-derived cheese (e.g. higher work to extend and longer distance to failure as measured on texture analyzer).

[0075] Compared to cheese analogue made with non-crosslinked protein, cheese analogue made with crosslinked protein as described herein has increased browning, bite resistance, shred quality and / or mouthfeel. In some variations, the cheese analogue has all of the foregoing properties, e.g., browning, bite resistance, shred quality and mouthfeel.

[0076] In some embodiments, the crosslinked cheese analogue can exhibit increased browning. Increased browning can include the appearance or increase in size of colored spots upon heating. For example, increased browning can be measured by an increase in the number and / or size of colored spots upon heating. Colored spots can be any color that is not the original color of the cheese upon heating, including yellow or yellowish, tan or tannish, 22MOFO-359836952Attorney Docket No. 263942000740 brown or brownish, or black or blackish spots upon exposure to heat. Exposure to heat can include exposure to direct heat such as direct exposure to a heating element or fire.

[0077] In some embodiments, the crosslinked cheese analogue can exhibit improved texture, such as increased bite resistance. Bite resistance can be measured using instruments, including texture analyzers, that measure various textural properties contributing to bite resistance, such as hardness, fracturability, cohesiveness and springiness responsive to compression. In some examples, bite resistance is measured using probes, such as blade probes or platen probes to measure tenderness, toughness, and / or firmness responsive to compression or shearing action upon simulation of chewing or shearing action of incisor or molar teeth. In some embodiments, rheometers can be used to measure properties such as viscosity, elasticity, and plasticity under certain conditions to evaluate bite resistance. In some embodiments, bite resistance is assessed using sensory information from a human panel. Such evaluations of bite resistance can include description of cohesiveness, disintegration rate upon chewing, extent of gooeyness, gumminess, sliminess, crunchiness, firmness, and / or rubberiness. In some embodiments instrumental and sensory panel measurements can be used and / or correlated to determine bite resistance.

[0078] In some embodiments, the crosslinked cheese analogue can exhibit improved texture, such as improved mouthfeel. Mouthfeel is typically assessed using sensory information from a human panel. Mouthfeel can include characteristics such as smoothness, chunkiness, graininess, mealiness, viscosity, creaminess, astringency, and brittleness.

[0079] In some embodiments, the crosslinked cheese analogue can exhibit improved texture, such as improved mouthcoating. Mouthcoating is typically assessed using sensory information from a human panel. Mouthcoating can include characteristics such as stickiness, oiliness, waxiness, tackiness, slickness, slipperiness, pastiness and level of tooth packing.

[0080] In some embodiments, the crosslinked cheese analogue can exhibit improved shred quality. Shred quality can also be referred to as shred integrity or shreddability. In some instances, shred quality is determined by factors such as shred length and / or thickness, evenness of shreds, production of fines, and adherence to the shredding blade. Shred quality can be measured manually, using sieves for fines or individual shred measurement, or automated methods, such as automated methods combined with image analysis and / or sensory and visual information by a human panel. Aspects measured in determining shred 23MOFO-359836952Attorney Docket No. 263942000740 quality can include length and uniformity of the shreds (longer, more consistent shreds are generally preferred); cohesiveness of the shreds (cohesive shreds are preferred); shred texture, such as pastiness or crumbliness; fines content: (e.g., excessive fines can indicate poor shred quality); adhesion to other shreds or the blade (increased adhesion is typically not desirable); firmness of the shreds (slightly to moderately firm are preferable); elasticity (moderate elasticity is preferable); visual assessment of qualities such as appearance, color, and presence of defects like ragged edges, pastiness, or crumbliness.

[0081] In other variations, one or more additional properties may be improved, including: chewiness, opaqueness, firmness, springiness, hardness, adhesiveness, resilience, and cohesiveness as measured by texture analyzer, smoothness, oiling off, and higher melting temperature.

[0082] In some embodiments, the onset of melting and / or melting rate of the cheese analogue is slower than the onset of melting and / or melting rate of the comparator cheese analogue. In other embodiments, the quality of the stretch (e.g., elasticity) of the cheese analogue is improved as compared to the quality of the stretch (e.g., elasticity) of the comparator cheese analogue. In certain embodiments, the bite-resistance of the cheese analogue is higher than the bite-resistance of the comparator cheese analogue. In certain embodiments, the bite-resistance of the cheese analogue is similar to the bite-resistance of the comparator cheese analogue, such as one with animal-derived dairy ingredients or to animal- derived cheese. In some embodiments, the mouthfeel of the cheese analogue is similar to the mouthfeel of the comparator cheese analogue, such as an animal-derived cheese or cheese analogue. In some embodiments, the mouthfeel of the cheese analogue is more preferable to the mouthfeel of the comparator cheese analogue. In some embodiments, the amount of cheese analogue browning upon cooking is increased as compared to the cheese analogue browning upon cooking of the comparator cheese analogue. In some embodiments, the shred quality, also referred to as shreddability or shred integrity, of the cheese analogue is improved compared to the shred quality, shreddability, or shred integrity of the comparator cheese analogue.

[0083] Any suitable methods known in the art may be employed to determine and / or measure the aforementioned properties. For instance, stretch may be measured as stretch length by doing a fork test and strand quality can be evaluated by a trained individual to assess the properties of the strand (e.g., elasticity, firmness, resilience) and number of strands. 24MOFO-359836952Attorney Docket No. 263942000740 Stretch can also be measured in a texture analyzer that yields work to extend (e.g., force needed to pull the cheese) and distance to failure (e.g., stretch length). In some embodiments, a cheese analogue is analyzed for stretch using a texture analyzer (e.g., TA.XTPlus Texture and measuring the extensibility on a texture analyzer as the distance to failure, which is generally understood to be the distance at which all of the strands of cheese break.

[0084] In some embodiments, the cheese analogue has a stretch length of at least 10 mm. In certain embodiments, the cheese analogues has a stretch length of at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, at least 150 mm, at least 200 mm, at least 250 mm, or at least 300 mm. In some embodiments, the cheese analog comprises the recombinant alpha casein protein in an amount of no more than about 5% w / w (e.g., in an amount of about 5% w / w) and has a stretch length of at least 10 mm, at least 20 mm, or at least 50 mm. In some embodiments, the cheese analog comprises the recombinant alpha casein protein in between about 5% and about 10% w / w (e.g. between about 6% and about 8% w / w) and has a stretch length of at least 50 mm, at least 100 mm, or at least 150 mm. In some embodiments, the cheese analog comprises the recombinant alpha casein protein in between about 10% and about 15% w / w (e.g. between about 11% and about 13% w / w) and has a stretch length of at least 150 mm, at least 200 mm, at least 200 mm, or at least 300 mm. In some variations, the stretch length is determined by a pizza fork stretch test as described in Example 2.

[0085] In some embodiments, the cheese analogue has a stretch length at least 10 mm greater than a comparator cheese analogue. In certain embodiments, the cheese analogues has a stretch length at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, at least 150 mm, at least 200 mm, at least 250 mm, or at least 300 mm greater than a comparator cheese analogue. In some embodiments, the cheese analogue has a stretch length at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 4 fold, or at least 5 fold greater than a comparator cheese analogue. In some variations, the cheese analogue and the comparator cheese analogue have the same amount of recombinant alpha casein protein ± 1%, 2%, 3%, 4%, or 5% w / w. In some variations, the stretch length is determined by a pizza fork stretch test as described in Example 2.

[0086] In some embodiments, the cheese analogue has a stretch distance to failure of at least 200 mm, 210 mm, 220 mm, or 230 mm as measured by a texture analyzer. In some embodiments, the cheese analogue has a work to extend of at least about 100, 125, 150, 175, 25MOFO-359836952Attorney Docket No. 263942000740 200, 225, or 250 g*s as measured by a texture analyzer. In certain embodiments, the cheese analogue has a work to extend of at least about 200 g*s as measured by a texture analyzer.

[0087] Melt may be measured by a modified Schreiber melt test to get melt rate, extent of melt and time of melt. Melt may also be assessed on a food product, such as pizza, to see the extent of melt and spread. Melt may also be measured on a rheometer to get the critical temperature at which the cheese melts and time needed to melt.

[0088] Cheese analogues as described herein may have a melting ability which is comparable to a similar type of cheese made using animal-derived dairy proteins, such as cheese made from animal milk. Cheese analogues as described herein may have a melting ability which is comparable to a similar type of cheese or cheese analogue made using micellar form of casein, such as cheese made from milk, or cheese analogue made from caseinate or rennet casein. Cheese analogues as described herein may have a melting ability which is improved when compared to a similar type of cheese or cheese analogue made using a plant-derived cheese analogue lacking dairy proteins (such as a cheese-like product made either with plant-derived protein such as pea, chickpea, nut and / or other vegetable protein as the sole / primary protein source, or with no protein (such as cheese-like products made primarily with starch)). Melting ability of a cheese analogue as described herein may be tested using a modified Schreiber melt test and computer imaging. Alternatively, melting ability of a cheese analogue as described herein may be tested in an application, such as on a pizza cooked in an oven and assessed visually by a trained expert individual or by a trained sensory panel. Exemplary assays that measure this melt property are provided in the examples section.

[0089] In some embodiments, a cheese analogue is analyzed for melt characteristics by heating. In some variations, a cheese analogue incorporated into or onto a food product is analyzed for melt characteristics, in which the food product is heated at suitable oven temperatures for a suitable duration of time to cook the food product. In certain variations, such oven temperatures may include, for example, at least about 400°F, at least about 450°F, at least about 500°F, at least about 550°F, at least about 600°F, at least about 650°F, at least about 700°F, or at least about 750°F; or between about 400°F and maximum oven temperature, or between about 400°F and about 900°F. Cheese analogue analyzed for melt characteristics by heating, such as on pizza and cooked in an oven, is then assessed visually by a train expert individual or by a trained sensory panel. 26MOFO-359836952Attorney Docket No. 263942000740

[0090] In other variations, the cheese analogue on its own is analyzed for melt characteristics. In certain variations, the cheese analogue is directedly heated, e.g., on a heat surface (such as a hot plate) in an enclosed area to a temperature between about 80oC and about 95oC for a predetermined time period (e.g., about 5-15 minutes), and the time it takes for the cheese analogue to melt is recorded. In one variation of the foregoing, the cheese analogue melts within 1 minute, within 2 minutes, within 5 minutes, within 10 minutes or within 15 minutes. In certain embodiments, melt 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, the cheese analogue as described herein has a melt value of 1 or greater than 1. In some embodiments, the cheese analogue as described herein has a melt value 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, the cheese analogue as described herein has a melt value 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, the cheese analogue as described herein has a melt value greater than 1 and the melted area retains an opaque appearance. In some embodiments, the cheese analogue as described herein has a melt value greater than the melt value of a plant-based cheese analogue, for example the melt value the cheese analogue comprising a recombinant single variant alpha casein is 1.5x, 2x, 2.5 x or greater than 2.5x the melt value of the plant- based cheese analogue.

[0091] In some embodiments, the cheese analogue has a hardness of at least 1,600 g as measured by a texture analyzer. In certain embodiments, the cheese analogue has a hardness of at least 1,900 g as measured by a texture analyzer. In certain embodiments, the cheese analogue has a hardness of at least 2,100 g as measured by a texture analyzer. In some embodiments, the cheese analogue has a hardness of no more than 4,000 g. In certain embodiments, the cheese analogue has a hardness of at least 1,600 g, 1,700 g, 1,800 g, 1,900 g, 2,000 g, 2,100 g, 2,200 g, 2,300 g, 2,400 g, 2,500 g, 2,600 g, 2,700 g, 2,800 g, 2,900 g, or 3,000 g as measured by a texture analyzer. In some embodiments, the cheese analogue has a hardness of between about 1,600 g and about 4,000 g as measured by a texture analyzer. In certain embodiments, the cheese analogue has a hardness of between about 1,900 g and about 3,000 g as measured by a texture analyzer. In certain embodiments, the cheese analogue has a hardness of between about 1,600 g and about 1,900 g, between about 1,600 g and about 2,100 g, between about 1,600 g and about 2,300 g, between about 1,600 g and about 2,500 g, between about 1,600 g and about 2,700 g, between about 1,600 g and about 2,900 g, between 27MOFO-359836952Attorney Docket No. 263942000740 about 1,600 g and about 3,100 g, between about 1,600 g and about 3,500 g, between about 1,600 g and about 4,000 g, between about 1,900 g and about 2,100 g, between about 1,900 g and about 2,300 g, between about 1,900 g and about 2,500 g, a between about 1,900 g and about 2,700 g, between about 1,900 g and about 2,900 g, between about 1,900 g and about 3,100 g, between about 1,900 g and about 3,500 g, between about 1,900 g and about 4,000 g, between about 2,100 g and about 2,300 g, between about 2,100 g and about 2,500 g, a between about 2,100 g and about 2,700 g, between about 2,100 g and about 2,900 g, between about 2,100 g and about 3,100 g, between about 2,100 g and about 3,500 g, between about 2,100 g and about 4,000 g, between about 2,300 g and about 2,500 g, a between about 2,300 g and about 2,700 g, between about 2,300 g and about 2,900 g, between about 2,300 g and about 3,100 g, between about 2,300 g and about 3,500 g, between about 2,300 g and about 4,000 g, between about 2,500 g and about 2,700 g, between about 2,500 g and about 2,900 g, between about 2,500 g and about 3,100 g, between about 2,500 g and about 3,500 g, between about 2,500 g and about 4,000 g, between about 2,700 g and about 2,900 g, between about 2,700 g and about 3,100 g, between about 2,700 g and about 3,500 g, between about 2,700 g and about 4,000 g, between about 2,900 g and about 3,100 g, between about 2,900 g and about 3,500 g, between about 2,900 g and about 4,000 g, between about 3,100 g and about 3,500 g, between about 3,100 g and about 4,000 g, or between about 3,500 g and about 4,000 g as measured by a texture analyzer.

[0092] In some embodiments, the cheese analogue has an adhesiveness of at least about - 150 g*sec, as measured by a texture analyzer. In certain embodiments, the cheese analogue has an adhesiveness of at least -100 g*sec as measured by a texture analyzer. In some embodiments, the cheese analogue has an adhesiveness of between about -150 g*sec and about -50 g*sec as measured by a texture analyzer. In certain embodiments, the cheese analogue has an adhesiveness of between about -150 g*sec and about -100 g*sec, between about -150 g*sec and about -50 g*sec, or between about -100 g*sec and about -50 g*sec as measured by a texture analyzer.

[0093] In some embodiments, the cheese analogue has a resilience of at least about 0.10% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a resilience of at least about 0.11% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a resilience of at least about 0.10%, 0.11%, 0.15%, 0.20%, or 0.25% as measured by a texture analyzer. In some embodiments, the cheese analogue has a 28MOFO-359836952Attorney Docket No. 263942000740 resilience of between about 0.10% and about 0.25% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a resilience of between about 0.10% and about 0.15%, between about 0.10% and about 0.20%, between about 0.10% and about 0.25%, between about 0.10% and about 0.30%, between about 0.15% and about 0.20%, between about 0.15% and about 0.25%, between about 0.15% and about 0.30%, between about 0.15% and about 0.25%, between about 0.15% and about 0.30%, or between about 0.25% and about 0.30% as measured by a texture analyzer.

[0094] In some embodiments, the cheese analogue has a cohesiveness of at least about 0.32% as measured by a texture analyzer. In certain embodiments, the cheese analogues has a cohesiveness of about 0.32%, 0.33%, 0.35%, 0.40%, 0.45%, or 0.50% as measured by a texture analyzer. In some embodiments, the cheese analogues has a cohesiveness of between about 0.32% and about 0.50% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a cohesiveness of between about 0.32% and 0.35%, between about 0.32% and 0.40%, between about 0.32% and 0.45%, between about 0.32% and 0.50%, between about 0.35% and 0.40%, between about 0.35% and 0.45%, between about 0.35% and 0.50%, between about 0.40% and 0.45%, between about 0.40% and 0.50%, or between about 0.45% and about 0.50% as measured by a texture analyzer.

[0095] In some embodiments, the cheese analogue has a springiness of at least about 0.5% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a springiness of at least about 0.50%, 0.55%, 0.60%, 0.65%, or 0.70% as measured by a texture analyzer. In some embodiments, the cheese analogue has a springiness of between about 0.50% and about 0.70% as measured by a texture analyzer. In certain embodiments, the cheese analogues has a springiness of between about 0.50% and about 0.55%, between about 0.50% and about 0.60%, between about 0.50% and about 0.65%, between about 0.50% and about 0.70%, between about 0.55% and about 0.60%, between about 0.55% and about 0.65%, between about 0.55% and about 0.70%, between about 0.60% and about 0.65%, between about 0.60% and about 0.70%, or between about 0.65% and about 0.70% as measured by a texture analyzer.

[0096] In some embodiments, the cheese analogue has a chewiness of at least about 300% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a chewiness of at least about 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000% as measured by a texture analyzer. In some embodiments, the cheese analogue has a chewiness 29MOFO-359836952Attorney Docket No. 263942000740 of between about 300% and about 1,000% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a chewiness of between about 300% and about 400%, between about 300% and about 500%, between about 300% and about 600%, between about 300% and about 700%, between about 300% and about 800%, between about 300% and about 900%, between about 300% and about 1,000%, between about 400% and about 500%, between about 400% and about 600%, between about 400% and about 700%, between about 400% and about 800%, between about 400% and about 900%, between about 400% and about 1,000%, between about 500% and about 600%, between about 500% and about 700%, between about 500% and about 800%, between about 500% and about 900%, between about 500% and about 1,000%, between about 600% and about 700%, between about 600% and about 800%, between about 600% and about 900%, between about 600% and about 1,000%, between about 700% and about 800%, between about 700% and about 900%, between about 700% and about 1,000%, between about 800% and about 900%, between about 800% and about 1,000%, or between about 900% and about 1,000% as measured by a texture analyzer.

[0097] In some embodiments, the cheese analogue has a gumminess of at least about 700% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a gumminess of at least about 700%, 800%, 900%, 1,000%, 1,100%, or 1,200% as measured by a texture analyzer. In some embodiments, the cheese analogue has a gumminess of between about 700% and about 1,200% as measured by a texture analyzer. In certain embodiments, the cheese analogue has a gumminess of between about 700% and about 800%, between about 700% and about 900%, between about 700% and about 1,000%, between about 700% and about 1,100%, between about 700% and about 1,200%, between about 800% and about 900%, between about 800% and about 1,000%, between about 800% and about 1,100%, between about 800% and about 1,200%, between about 900% and about 1,000%, between about 900% and about 1,100%, between about 900% and about 1,200%, between about 1,000% and about 1,100%, between about 1,000% and about 1,200%, or between about 1,100% and about 1,200% as measured by a texture analyzer.

[0098] Sensory attributes may be assessed by a trained sensory panel and by trained expert individuals.

[0099] Other properties, such as pH and moisture, may be measured using any suitable methods or techniques that are well-known in the art. 30MOFO-359836952Attorney Docket No. 263942000740 Casein Content

[0100] In some embodiments, the crosslinked alpha casein described herein is incorporated into a food product, such as cheese analogue. In some variations of the foregoing, when the crosslinked alpha casein described herein is used, a lower amount of crosslinked alpha casein is used to achieve a particular property, e.g., stretch, as compared to non-crosslinked casein. To illustrate this point, as an example, in situation where a stretch distance of x cm is achieved by using an amount y% of non-crosslinked alpha casein, an amount less than y% of the crosslinked protein (produced according to the methods herein) is required to achieve the same stretch distance of x cm.

[0101] Thus, in some variations, a crosslinked alpha casein as described herein is incorporated into a food product or a downstream composition, such as cheese or cheese analogue, at an amount (sometimes referred to as utilization rate or casein content) that does not exceed about 30% by weight (wt / wt) of the cheese analogue. In certain variations, the total casein content is less than 25%, 20%, 15%, 10%, or 5% by weight (wt / wt) of the cheese analogue. The ability of crosslinking alpha casein to improve any one or more of cheese properties of a cheese or cheese analogue containing reduced alpha casein content is valuable for commercial production. Production Methods

[0102] In other aspects, provided is a method of producing any one of the compositions comprising crosslinked recombinant alpha casein, as described herein. In some embodiments, the method comprises: exposing alpha casein, particularly recombinant alpha casein, to a crosslinking condition or agent to produce alpha casein crosslinked to other alpha casein.

[0103] In some embodiments, the recombinant alpha casein is crosslinked by heat treatment, irradiation, or enzymatically. In certain embodiments the recombinant alpha casein is enzymatically crosslinked using one or more of transglutaminase, laccase, tyrosinase, peroxidase, sulfhydryl oxidase, and glucose oxidase. Any combination of the crosslinking enzymes may also be employed in the methods herein. In one variation, the recombinant alpha casein is enzymatically crosslinked using transglutaminase.

[0104] In some embodiments, the recombinant alpha casein is crosslinked in aqueous solution, in an emulsion, or in dried powder form. 31MOFO-359836952Attorney Docket No. 263942000740

[0105] In some variations, the recombinant alpha casein is a microbially-produced protein. In some variations, the recombinant alpha casein is a bacterially-produced protein. In some variations, the recombinant alpha casein lacks one or more post-translational modifications found in a native (animal-derived) alpha casein. In some variations, the recombinant alpha casein is not phosphorylated.

[0106] In some embodiments, the method comprises: producing recombinant alpha casein in a microbial host cell, extracting and / or purifying the recombinant alpha casein, and exposing the recombinant alpha casein to a crosslinking agent or condition to produce crosslinked recombinant alpha casein. In some variations, the recombinant alpha casein produced in the microbial host lacks one or more post-translational modifications found in a native (animal-derived) alpha casein. In some variations, the recombinant alpha casein is not phosphorylated. In some variations, the microbial host cell is a bacterial cell. In some variations, the microbial host cell is E. coli. In some variations, the microbial host cell is Bacillus subtilis (B. subtilis). In some variations, the recombinant alpha casein is dried into a powder prior to the crosslinking step. In some variations, the recombinant alpha casein is comprised in a solution prior to the crosslinking step. In some variations, the recombinant alpha casein is comprised in an emulsion, such as an oil / water or fat / water emulsion, prior to the crosslinking step.

[0107] In some embodiments, the method further comprises: incorporating any of the crosslinked alpha casein described herein into a cheese analogue. In some variations, the recombinant alpha casein is present in the cheese analogue at an amount between 1% and 30%, between 1% and 18%, between 1% and 17%, between 1% and 16%, between 1% and 15%, between 1% and 14%, between 1% and 13%, between 1% and 12%, between 1% and 11%, between 1% and 10%, between 1% and 9%, between 1% and 8%, between 1% and 7%, between 1% and 6%, or between 1% and 5% wt / wt of the cheese analogue. In some variations, the recombinant alpha casein is present in the cheese analogue at an amount less than or about 5% wt / wt, such as between 1% and 2%, between 1% and 3%, between 1% and 4%, between 1% and 5%, or about 1%, 2%, 3%, 4% or 5% wt / wt of the cheese analogue. In some variations, the recombinant alpha casein is present in the cheese analogue at an amount greater than 5% wt / wt, such as between 5% and 18%, between 5% and 17%, between 5% and 16%, between 5% and 14%, between 5% and 13%, between 5% and 12%, between 5% and 11%, between 5% and 10%, between 5% and 11%, between 5% and 10%, between 5% and 32MOFO-359836952Attorney Docket No. 263942000740 9%, between 5% and 8%, between 5% and 7%, between 5% and 6%, or about 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% or 18% wt / wt of the cheese analogue. In other variations, the recombinant alpha casein is present in the cheese analogue at an amount greater than 1%, greater than 2%, greater 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, or greater than 10% wt / wt of the cheese analogue. In other variations, the recombinant alpha casein is present in the cheese analogue at an amount less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4% or less than 3% wt / wt of the cheese analogue. In one variation, the recombinant casein is present in the cheese analogue at an amount greater than or about 5% and less than 30% wt / wt of the cheese analogue. In another variation, the recombinant casein is present in the cheese analogue at an amount greater than 5% and less than 18% wt / wt of the cheese analogue. EXAMPLES

[0108] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation Example 1: Expression in E. coli

[0109] Bovine alpha-s1-casein protein coding sequence (without the native signal peptide) codon-optimized for Escherichia coli was cloned into a vector with an inducible promoter. The construct was transformed into E. coli cells and casein expression confirmed. The transformed E.coli was used to produce alpha-s1-casein by fed-batch fermentation. At the end of the fermentation process, cells were harvested. The recombinant alpha-s1-casein was extracted from the cells and purified by phase separation and centrifugation. The resulting slurry was spray-dried to a powder generating the finished ingredient. Purified protein powder (referred to as casein powder) was analyzed by HPLC, Dumas method and mass spectrometry. The casein protein powder containing recombinant alpha-s1-casein produced was used for the experiments herein. The percentage (%) of casein throughout the examples and application refers to the % of pure alpha-s1-casein, except when the percentage of “casein powder” is explicitly referenced, which refers instead to the amount of casein-containing powder used in a formulation. For example, for a casein powder with a 33MOFO-359836952Attorney Docket No. 263942000740 purity of 80%, a casein powder inclusion rate of 10% (wt / wt) in a composition is a pure casein inclusion rate of 8% (wt / wt) in that composition. Example 2: Properties of mozzarella cheese analogue from recombinant single variant alpha casein protein with an altered formulation containing transglutaminase (“TG”)

[0110] Recombinant unphosphorylated alpha-s1-casein, in the form of casein powder, was used to make non-micellar mozzarella cheese analogue termed as NC1 and NC2; wherein NC2 has an altered formulation containing transglutaminase (TG) compared to NC1. The casein powder used to make NC1 and NC2 contains about 85% (wt / wt) of pure recombinant alpha-s1-casein as measured by a combination of the Dumas method (total protein), mass spectrometry and HPLC; the rest of the casein powder is residual material from the recombinant protein production and purification (ash, moisture etc.). For making cheese analogues, all the ingredients were added at a concentration specified in Table 1. For NC1 cheese analogue, casein powder, trisodium citrate, dipotassium phosphate, water, lecithin, coconut oil, sunflower oil, starch, sodium chloride, sugar and flavoring agent were added to a can. The contents in the can were mixed for at least 5 mins in RVA 4500. To the mix, CaCl2 6.5 mins. Lactic acid was added, and the ingredients were mixed for an additional 1 minute. The resulting mixtures turned to a homogeneous non-micellar mass, transferred to standard molds, and allowed to sit in the fridge for 7 days before analysis. For making NC2 cheese analogue, casein powder, trisodium citrate, dipotassium phosphate, water, lecithin, fats were mixed in a can for at least 5 mins. CaCl2and TG were added and were mixed. The mix was addition and aging steps were the same as described above. 34MOFO-359836952Attorney Docket No. 263942000740 Table 1: Ingredient Composition and Concentration for Recombinant Single Casein Variant Cheese Analogue (NC1 and NC2)

[0111] The NC1 and NC2 cheese analogue samples were analyzed for qualitative and quantitative parameters such as pH, moisture, melt and stretch.

[0112] A pizza melt test was performed to determine the melt of the NC cheese analogue. For this, 9g of NC cheese analogue was shredded on a 4” pizza topped with 8g of tomato ine the melt of the NC cheese analogue on the pizza. The cheese stretch was also qualitatively analyzed on the pizza by pulling apart the pizza slice when the cheese temperature was

[0113] A pizza fork stretch test was performed to quantitate cheese extensibility by inserting a fork into the center of freshly baked pizza and lifted until all the cheese strands broke. For this, 6g of cheese was shredded on a 4” inch pizza topped with 4g of tomato sauce. 35 MOFO-359836952Attorney Docket No. 263942000740Samples were baked at ~ for 90 seconds. A ruler was used to measure the stretch andthe test was performed immediately after removing the pizza from the oven. The strand quality on the pizza was assessed by observing the number of strands formed when the pizza slices were separated post baking.

[0114] The NC1 and NC2 cheese analogues made from recombinant unphosphorylated alpha-s1-casein melted well on the pizza. There was more browning observed upon cooking in NC2 cheese analogue than the NC1 cheese analogue (FIG. 1). The pH of cheese analogues NC1 and NC2 was 5.7 and 5.9, and final moisture content was 48%, and 47%, respectively.

[0115] The NC2 cheese analogue, treated with TG, had a 15% longer stretch than the NC1 cheese analogue lacking TG. The NC2 cheese analogue stretched to a length of ~230 mm. In comparison, the NC1 cheese analogue stretched to a length of ~200 mm and formed a few strands. The NC1 cheese analogue formed 1-2 strands when the pizza slice was pulled apart. In comparison, the NC2 cheese analogue formed multiple strands suggesting a superior stretch quality (FIG.1).

[0116] The cheese analogues were evaluated blindly for bite-resistance and mouthfeel by a panel of 4 trained individuals, and all of the panel participants found the NC2 cheese analogue treated with TG had a higher bite-resistance and strand elasticity compared to the NC1 cheese analogue lacking TG. Example 3: Properties of mozzarella cheese analogue from recombinant single variant alpha casein protein with an additional pre-processing heating step

[0117] Recombinant unphosphorylated alpha-s1-casein, in the form of casein powder, make non-micellar mozzarella cheese analogue termed NC3, NC4, NC5, and NC6 respectively. The heat treatment resulted in crosslinking approximately <1%, 2%, 16%, and 19% of monomeric alpha-s1-casein when treated for 5 minutes, 20 minutes, 120 minutes, and 180 minutes, respectively. The crosslinking of monomeric alpha-s1-casein was evaluated by SDS-PAGE analysis and relative gel quantification (FIG. 2). The casein powder used to make NC3, NC4, NC5, and NC6 cheese analogues contains about 79 % (wt / wt) of pure recombinant alpha-s1-casein as measured by a combination of the Dumas method (total protein), mass spectrometry and HPLC; the rest of the casein powder is residual material from recombinant protein production and purification. For making cheese analogues, all the 36MOFO-359836952Attorney Docket No. 263942000740 ingredients were added at a concentration specified in Table 2. Heat treated casein powder, trisodium citrate, dipotassium phosphate, water, palm stearin, canola oil, starch, sodium chloride, sugar, lysine and flavoring agent were added to a can. The contents in the can were mixed for at least 5 mins in RVA 4500. To the mix, CaCl2was added and mixed. The were mixed for an additional 1 minute. The resulting mixtures turned to a homogeneous non- micellar mass, transferred to standard molds, and allowed to sit in the fridge for 7 days before analysis. Table 2: Ingredient Composition and Concentration for Recombinant Single Casein Variant Cheese Analogue (NC3 to NC6 cheese analogues)

[0118] The NC3, NC4, NC5 and NC6 cheese analogue samples were analyzed for qualitative and quantitative parameters such as pH, moisture, melt and stretch. A pizza melt test was performed as described in Example 2 above.

[0119] An extensibility test was performed on a texture analyzer to quantitate cheese stretch by inserting a 6-pronged hook into a 6 g sample of hand-cut cheese shreds that had been melted for 10 min at 90°C and lifted until all the cheese strands broke. Distance to failure (breakage of all strands) representing extent of cheese stretch and work to extend representing tensile strength needed to stretch the cheese were quantified. The tests were 37MOFO-359836952Attorney Docket No. 263942000740 performed on samples taken out of fridge and then kept at ambient temperatures for at least 30 mins.

[0120] The pH of NC3, NC4, NC5 and NC6 cheese analogues were 5.87, 5.78, 5.8, 5.78 respectively, and final moisture content was 43%, 42%, 45% and 46%, respectively. The NC3, NC4, NC5 and NC6 cheese analogues containing alpha-s1-casein heat-treated for 5 mins, 20 mins, 120 mins and 180 mins respectively melted well on pizza. When the stretch on pizza was compared, the NC3 cheese analogue stretched minimally. In comparison, the NC4 cheese analogue had moderately longer stretch and the NC5 and NC6 cheese analogues had significantly longer stretch as well as multiple strands demonstrating a superior strand quality with increased heat-treatment (and therefore increased crosslinking) (FIGS. 3A-3D).

[0121] Surprisingly, there were differences in the cheese analogue browning, demonstrating a trend. The NC3 cheese analogue had minimal brown spots (FIGS. 4A-4D). The extent of brown spots increased in NC4 cheese analogue compared to NC3 cheese analogue. The extent of browning was similar in NC5 and NC6 cheese analogues, however, both had more browning in comparison with the NC3 and NC4 cheese analogue (FIGS. 4A- 4D) suggesting the browning increases with increased crosslinking of alpha-s1-casein. Table 3: Stretch (extensibility) of New Culture Cheese Analogues

[0122] The extensibility test on texture analyzer showed maximum stretch for all the four cheese analogues. Interestingly, the work to extend varied among the cheese analogues, demonstrating a trend. The NC3 cheese analogue had work to extend of 65 g*sec. In comparison, the NC4, NC5 and NC6 cheese analogue had over 3x, 3.9x and 3.6x higher work to extend (Table 3). This suggests the cheese analogues elasticity, more specifically the force required to stretch the cheese to its maximum, increases as the extent of heat-treatment (and 38MOFO-359836952Attorney Docket No. 263942000740 therefore increased crosslinking) increases and the effect plateaus when the heat treatment is at ~120 mins. Example 4: Improved properties of mozzarella cheese analogue made from lower usage rate of recombinant single variant alpha casein (< =8 % wt / wt pure recombinant casein) with an altered formulation containing transglutaminase

[0123] Recombinant unphosphorylated alpha-s1-casein, in the form of casein powder, was used to make non-micellar mozzarella cheese analogues termed as NC7, NC8, NC9 and NC10. The casein powder used to make NC7-NC10 cheese analogues contains about 85% (wt / wt) of pure recombinant alpha-s1-casein as measured by the methods described in previous examples. For making cheese analogues, all the ingredients were added at a concentration specified in Table 4. The NC7 and NC8 cheese analogues contained 8% recombinant alpha-s1-casein (wt / wt) and 6% recombinant alpha-S1-casein (wt / wt) respectively, treated by TG during the cheesemaking. Casein powder, trisodium citrate, dipotassium phosphate, CaCl2and water was mixed for at least 5 mins in RVA 4500. To the mix, lecithin, sunflower oil and coconut oil were added and mixed further at high speed until a well emulsified mixture was formed. To this emulsified mixture, TG was added, mixed and for 30 mins to enable crosslinking. After the TG crosslinking, the contents were for an additional 1.5 minutes. The resulting mixtures turned into a homogeneous non-micellar mass, were transferred to standard molds, and allowed to sit in the fridge for 7 days before analysis. The NC9 and NC10 cheese analogues contained 8% recombinant alpha-S1-casein (wt / wt) and 6% recombinant alpha-S1-casein respectively (wt / wt), made similarly to theabove process but without the TG addition and lacking the 45 crosslinking treatment step.39MOFO-359836952Attorney Docket No. 263942000740 Table 4: Ingredient Composition and Concentration for Recombinant Single Casein Variant Cheese Analogue (NC7, NC8, NC9 and NC10)

[0124] The NC7, NC8, NC9 and NC10 cheese analogue samples were subjected to pH and moisture analysis, and melt and stretch assessment when cooked on pizza. A pizza melt test and a pizza fork stretch test, as described in Example 2, were performed to determine the melt and stretch of the NC cheese analogues.

[0125] The NC7, NC8, NC9 and NC10 cheese analogue samples were also blindly assessed for visual and sensory characteristics relevant to mozzarella cheese by a panel of 4 trained individuals, utilizing a 5-point acceptability scale. The scorecard ranged from a score of 1-5, with score 5 indicating high product acceptability and product quality comparable to animal-derived mozzarella control cheese and with score 3 being a threshold for acceptability 40 MOFO-359836952Attorney Docket No. 263942000740 relevant to commercially-viable product quality (score 1 – highly not acceptable, score 2 – not acceptable, score 3 – moderately acceptable, score 4 – acceptable, score 5 – highly acceptable). The average, median, minimal and total scores are shown in Table 5. Table 5 Attributes and Scores from the Visual and Sensory Evaluation Relevant to Mozzarella Cheese (5-point acceptability scale)

[0126] The NC7, NC8, NC9 and NC10 cheese analogues made from recombinant unphosphorylated alpha-s1-casein with reduced casein usage rates (<= 8% wt / wt pure recombinant casein) with and without crosslinking of alpha casein made cheeses that were shreddable, meltable, had a good oiling off and flavor, and were opaque (FIGS. 5A-5H, FIG. 6 & Table 5). The pH range of set product after 7 days of aging was 6.2 – 6.4 for all 4 cheese analogues, and moisture content range of set product after 7 days of aging was 49%-50% (wt / wt) for all 4 cheese analogues, demonstrating cheeses were controlled for with respect to pH and moisture effects.

[0127] The NC7 and NC8 cheese analogues, treated with TG, having 8% and 6% pure recombinant alpha-s1-casein respectively stretched well and had elastic strands when tested by a pizza fork test. The NC7 and NC8 cheese analogue stretched to a length of 180 mm and 100 mm respectively in that assay (FIGS. 5I and 5J). The strand length correlated with casein usage: strand length decreased for decreased casein usage. In contrast, NC9 and NC10 cheese 41MOFO-359836952Attorney Docket No. 263942000740 analogs, not treated with TG, having 8% and 6% pure recombinant alpha-s1-casein respectively stretched to a length of 10 mm and lacked elasticity (FIGS. 5K and 5L). This demonstrates that crosslinking of alpha-s1-casein by TG in the cheese analogs promotes stretch and maintains strand quality even at alpha-s1-casein usage rates of <=8% wt / wt.

[0128] In the visual and sensory evaluation relevant to mozzarella cheese, the NC7 and NC8 cheese analogues scored higher in mouthfeel, stretch length and stretch quality, bite resistance, and mouth coating compared to their comparator cheeses NC9 and NC10 (Table 5, FIG 6). Furthermore, the NC7 and NC8 cheese analogues, with different reduced usage rates of alpha-s1-casein, scored similarly in all attributes, indicating that crosslinking alpha- s1-casein maintains overall product quality inclusive of all critical mozzarella cheese attributes even with lower usage rates of 6% wt / wt pure recombinant alpha-s1-casein (Table 5). Additionally, both the minimal and median scores were 3 or higher for NC7 and NC8, indicating commercially-relevant acceptable product quality for mozzarella. The NC9 and NC10 cheese analogues had poor stretch, elasticity, bite resistance, and had inferior mouthfeel, with average scores of 2.5 or less for these attributes (Table 5). Interestingly, melt, flavor, oiling off and opaqueness were acceptable in all 4 cheese analogues, with similar scores, higher than 3 in the visual and sensory evaluation. This finding shows that while certain mozzarella-specific product attributes, such as stretch and bite-resistance, are lacking in low casein usage cheese analogues (6%-8% wt / wt pure recombinant casein usage per this example) without crosslinking treatment, these cheese analogues do demonstrate high quality product attributes critical for other types of cheeses, such as melt, flavor, oiling off and opaqueness which are critical for cheddar cheese or American cheese. Example 5: Improved properties of mozzarella cheese analogue made from reduced recombinant single variant alpha casein content (13% and 11%) and transglutaminase

[0129] Recombinant unphosphorylated alpha-S1-casein, in powder form, was used to make non-micellar mozzarella cheese analogues, designated NC11, NC12, NC13 and NC14, using ingredients at concentrations specified in Table 6. The casein powder used to make NC11-NC14 cheese analogues contained about 85% (wt / wt) pure recombinant alpha-S1- casein as measured by the methods described in previous examples. The NC11 and NC13 cheese analogues contained 13% total recombinant alpha-S1-casein (wt / wt) and 11% total recombinant alpha-S1-casein (wt / wt), respectively, and were treated with transglutaminase (TG) during the cheesemaking. Casein powder, trisodium citrate, dipotassium phosphate, 42MOFO-359836952Attorney Docket No. 263942000740 CaCl2and water were mixed for at least 5 mins in RVA 4500. Lecithin, sunflower oil and coconut oil were added to the mixture and mixed further at high speed until a well-emulsified mixture was formed. TG was added, and the TG and the emulsified mixtures were the mixed crosslinking. After the TG crosslinking, the for 7 mins. Lactic acid was added, and the ingredients were mixed at 75 resulting mixtures became a homogeneous non-micellar mass, they were transferred to standard molds, and were refrigerated for 7 days before analysis. The NC12 and NC14 cheese analogues contained 13% total recombinant alpha-S1-casein (wt / wt) and 11% total recombinant alpha-S1-casein (wt / wt), respectively, and were made similarly to the aboveprocess but without the TG addition and 45 crosslinking treatment step.Table 6: Ingredient Composition and Concentration for Recombinant Single Casein Variant Cheese Analogue (NC11, NC12, NC13 and NC14)

[0130] The NC11, NC12, NC13 and NC14 cheese analogue samples were subjected to pH and moisture analysis, texture profile of raw cheese block, melt, and stretch assessment when cooked on pizza.

[0131] A pizza melt and fork stretch test was performed to determine the melt of the NC cheese analogues. For this, 14g of NC cheese analogue was shredded on a 5” pizza topped 43MOFO-359836952Attorney Docket No. 263942000740 with 12g of tomato sauce. It was then baked in an oven preset at ~370 qualitatively determine the melt of the NC cheese analogue on the pizza. A fork stretch test was performed afterwards to quantitate cheese extensibility by inserting a fork into the center of freshly baked pizza when the cheese temperature reached ~ 60 - the cheese strands break. A ruler was used to measure the maximum stretch length of the cheese. The strand quality on the pizza was assessed by observing the number of strands formed either when the pizza slices were separated post baking or during the fork lift.

[0132] The texture profile was analyzed on TA-XTPlus texture analyzer using a TA-11 acrylic probe. 1.5-2.5g of cheese sample was prepared with dimensions 1.5 cm (L) x 1.5 cm (W) x 1 cm (H). The test was performed in triplicate on samples wrapped and stored at 4 ºC for at least 30 minutes, with the following instrument settings: 65% deformation, 2 mm / sec test speed, and 2.5g trigger force.

[0133] The NC11, NC12, NC13 and NC14 cheese analogue samples were also blindly assessed for visual and sensory characteristics relevant to mozzarella cheese by a panel of 4 trained individuals, utilizing a 5-point acceptability scale as described in Example 4. Table 7 Attributes and Mean Scores from the Visual and Sensory Evaluation Relevant to Mozzarella Cheese (5-point acceptability scale)44MOFO-359836952Attorney Docket No. 263942000740

[0134] The NC11, NC12, NC13 and NC14 cheese analogues made from recombinant unphosphorylated alpha-S1-casein with a low amounts of casein (13 % and 11% wt / wt), with and without crosslinking of alpha-S1-casein, made cheeses that were shreddable, meltable, had a good oiling off and flavor, and were opaque (FIGS. 7A-7H & Table 7). The NC11, NC12, NC13, and NC14 cheese analogues, had pH values of 6.4, 6.3, 6.2, and 6.1, respectively, with moisture contents of 47, 49, 51, and 49% by weight after 7 days of aging, respectively, which were all within controlled ranges, demonstrating effective regulation of key quality parameters.

[0135] The NC11 and NC13 cheese analogues, treated with TG, having 13% and 11% alpha-S1-casein respectively stretched well and had elastic strands when tested by a pizza fork test. Both NC11 and NC13 cheese analogues stretched to a length > 300 mm and showed superior strand quality (i.e., multiple thick strands) (FIGS. 7I and 7K). Despite the lower level of casein content (11%) in NC13 cheese analogue, the strand length and quality were similar to the NC11 cheese analogue containing 13% casein. In contrast, NC12 and NC14 cheese analogs, not treated with TG, having 13% and 11% alpha-S1-casein respectively stretched only to a length of 100 mm and 50 mm respectively, lacked elasticity, and exhibited poor strand quality (1-2 thin strands) (FIGS. 7J and 7L). These results demonstrate that crosslinking of alpha-S1-casein by TG in NC cheese analogues enhanced stretchability andstrand integrity relative to non- -casein, while enabling equivalentperformance at reduced casein concentrations.

[0136] In visual and sensory evaluations relevant to NC cheese analogues, the NC11 and NC13 analogues, incorporating crosslinking, demonstrated significantly superior performance relative to non-crosslinked comparatives. Specifically, improved shred quality was observed with cohesive shreds; strand quality was enhanced by the formation of multiple, thick, elastic strands that exhibited clean snap upon breakage; and sensory attributes showed increased bite resistance, acceptable mouthcoating, and favorable mouthfeel (Table -casein content (11% vs 13%), showed comparable quality across all attributes, indicating that 45MOFO-359836952Attorney Docket No. 263942000740 crosslinking preserves key mozzarella characteristics despite the lower percentages of casein (Table 7).

[0137] The NC12 and NC14 cheese analogues, lacking crosslinking, exhibited significantly reduced functional and organoleptic performance. Shred integrity was poor, with pasty shreds; strand quality was compromised, yielding short, thin strands with low elasticity; and sensory attributes were inferior, including mealy mouthfeel and oily, sticky mouthcoating. Average scores across these attributes were below 3 (Table 7).

[0138] These findings demonstrate that the addition of crosslinking in mozzarella cheese analogs with significantly lower casein content (13% and 11%), and where the only source of casein is recombinant alpha-S1-casein, has a strong positive affect on the structural properties (shred quality, strand quality, strand elasticity) and sensory characteristics (bite resistance, mouthfeel, mouthcoating), resulting in a highly desirable product at 13% and 11% alpha-S1- casein levels relative to the non-crosslinked (non-TG treated) NC analogues. Table 8 Texture Profile Analysis (TPA) of 11% and 13% alpha-S1-casein-containing crosslinked and non-crosslinked cheese analogues

[0139] Texture Profile Analysis (TPA) using a TA.XT Texture Analyzer was conducted to measure various attributes of the NC11 and NC13 crosslinked cheese analogs, N12 and N14 non-crosslinked cheese analogues, and a commercial mozzarella cheese analog. As shown in Table 8, enzymatically crosslinked alpha-S1-casein in NC11 and NC13 cheese analogues significantly enhanced viscoelastic properties compared to non-crosslinked controls at 11% and 13% alpha-S1 casein content. Crosslinking resulted in average increases in hardness (34%), springiness (46%), and cohesiveness (50%), with a reduction in adhesiveness (37%), and substantial gains in chewiness (170%) and gumminess (101%) (Table 8). These enhancements were preserved in the NC13 cheese analogue despite having 46MOFO-359836952Attorney Docket No. 263942000740 an even lower alpha-S1-casein content than the NC11 cheese analogue, underscoring the structural reinforcement imparted by crosslinking. In contrast, NC12 and NC14, lacking crosslinking, exhibited weaker mechanical strength and chewiness and gumminess, consistent with sensory panel assessments identifying a softer, pastier mouthfeel. These data establish crosslinked alpha-S1-casein networks as highly beneficial for achieving commercial-quality cheese analogue texture when using low levels of alpha-S1 casein (e.g., 11%-13% alpha-S1 casein content). Example 6: Improved properties of a cheese analogue made from 5% recombinant single variant alpha casein with an altered formulation containing transglutaminase

[0140] Recombinant unphosphorylated alpha-S1-casein, in the form of casein powder containing ~85% (wt / wt) pure alpha-S1-casein, was used to produce non-micellar cheese analogues NC15 and NC16. Both formulations contained 5% (wt / wt) total recombinant alpha-S1-casein, as specified in Table 9. NC15 was treated with transglutaminase (TG), while NC16 remained untreated. For NC15, casein powder, trisodium citrate, dipotassium sunflower oil, and coconut oil were then added and mixed at high speed to emulsify. TG was crosslinking. mixing for 2 minutes. The final homogeneous mass was molded and refrigerated for 7 days before analysis. NC16 was prepared identically, excluding the TG addition and incubation Table 9: Ingredient Composition and Concentration for Recombinant Single Casein Variant Cheese Analogue (NC15, NC16)47MOFO-359836952Attorney Docket No. 263942000740

[0141] The NC15 and NC16 cheese analogues were evaluated for pH, moisture content, raw block texture by texture analysis, melt and stretch performance on pizza, and visual and sensory attributes by human panel, following the methods outlined in Example 5.

[0142] The NC15 and NC16 cheese analogues formulated with 5% (wt / wt) recombinant unphosphorylated alpha-S1-casein, with and without transglutaminase crosslinking, respectively, produced shreddable and meltable non micellar cheeses. NC15 and NC16 exhibited pH values of 6.2 and 6.4, respectively, and moisture contents of approximately 50 percent by weight, demonstrating controlled and reproducible regulation of key quality parameters. Table 10 Attributes and Mean Scores from the Visual and Sensory Evaluation Relevant to Mozzarella Cheese (5-point acceptability scale)48MOFO-359836952Attorney Docket No. 263942000740

[0143] As shown in Table 10 and FIGS. 9A-9f, in comparative sensory and visual evaluations, the crosslinked NC15 cheese analogue exhibited superior performance relative to the non-crosslinked NC16 cheese analogue. NC15 demonstrated acceptable shreddability, including defined shred quality and length, as well as desirable opacity and controlled oiling- off (Table 10, FIGS. 9A and 9C). In contrast, NC16 generated butter-like, pasty shreds that adhered to the grater, rendering it non-shreddable. Upon cooking, non-crosslinked NC16 appeared translucent and exhibited excessive surface oiling, indicative of emulsion breakdown and phase separation, making it unsuitable for intended applications (FIGS. 9B and 9D). This suggests that crosslinking can rescue certain desirable cheese attributes, such as shreddability, opacity and oiling off, even with a reduced alpha-S1-casein content of 5% wt / wt.

[0144] Crosslinking conferred measurable improvements in stretch, mouthfeel, bite resistance, and mouthcoating. In a standardized pizza fork stretch test, NC15 (5% wt / wt alpha-S1-casein, transglutaminase- (Table 10, FIGS. 9E and 10). In contrast, NC16 (5% wt / wt alpha-S1-casein, untreated) displayed no stretch at all (FIG. 9F). These findings demonstrate that transglutaminase- induced crosslinking imparts some stretch to non-crosslinked formulations in which stretch is otherwise absent. During sensory evaluation, NC15 exhibited slight bite resistance during the first bite followed by cohesive breakdown. Mouthfeel was moderately oily and mildly mealy, with panel scores ranging from 1.75 to 2.0. NC16 lacked bite resistance, presented an excessively oily and sticky mouthfeel, and was distinctly mealy, resulting in scores of 1.25 or lower across these attributes (Table 10, FIG. 10). 49MOFO-359836952Attorney Docket No. 263942000740 Table 11 Cheese Properties based on Texture Profile Analysis

[0145] Texture Profile Analysis (TPA) using a TA.XT Texture Analyzer demonstrated that enzymatic crosslinking of the 5% (wt / wt) alpha-S1-casein cheese analogue, NC15, exhibited enhanced viscoelastic properties relative to the non-crosslinked control, NC16. As shown in Table 11, crosslinking led to increased hardness by 42%, springiness by 15%, cohesiveness by 40%, with significant gains in chewiness (125%) and gumminess (97%). Adhesiveness also decreased by approximately 200%, which was also reflected in visual and sensory assessments, particularly in shreddability and interaction with the grater (Tables 10 and 11). These textural improvements generated a raw cheese analogue (i.e., NC15) that meets baseline quality thresholds as confirmed by TPA and sensory evaluation. 50MOFO-359836952

Claims

1. Attorney Docket No. 263942000740 CLAIMS What is claimed is:

1. A composition comprising crosslinked recombinant alpha casein, wherein the recombinant alpha casein is crosslinked to recombinant alpha casein, and wherein the recombinant alpha casein is not crosslinked to beta casein or kappa casein.

2. The composition of claim 1, wherein beta casein is not present in the composition.

3. The composition of claim 1 or 2, wherein kappa casein is not present in the composition.

4. The composition of any one of claims 1 to 3, wherein the recombinant alpha casein is a single variant of an alpha casein.

5. The composition of claim 4, wherein the recombinant alpha casein is alpha-s1-casein or alpha-s2-casein.

6. The composition of any one of claims 1 to 5, wherein the recombinant alpha casein is a bacterially-produced recombinant alpha casein.

7. The composition of any one of claims 1 to 6, wherein the recombinant alpha casein is not present in a micellar form.

8. The composition of any one of claims 1 to 7, wherein the recombinant alpha casein is in a dried or powder form.

9. The composition of any one of claims 1 to 7, further comprising fat or oil.

10. The composition of claim 9, wherein the fat or oil comprises a plant or plant-derived fat or oil.

11. The composition of claim 9 or 10, wherein the composition is an emulsion.

12. The composition of any one of claims 1 to 11, wherein the recombinant alpha casein is present in the composition at an amount between 1% and 30% wt / wt of the composition. 51MOFO-359836952 Attorney Docket No. 263942000740 13. The composition of claim 12, wherein the recombinant alpha casein is present in the composition at an amount between 1% and 18% wt / wt of the composition.

14. The composition of claim 12, wherein the recombinant alpha casein is present in the composition at an amount greater than 5% and less than 18% wt / wt of the composition.

15. The composition of claim 12, wherein the recombinant alpha casein is present in the composition at an amount less than 5% wt / wt of the composition.

16. A coagulate comprising the composition of any one of claims 1 to 15.

17. A cheese analogue comprising the composition of any one of claims 1 to 15.

18. The cheese analogue of claim 17, wherein one or more cheese-like features are improved as compared to a comparator cheese analogue, wherein the comparator cheese analogue comprises non-crosslinked alpha casein and does not comprise beta casein or kappa casein.

19. The cheese analogue of claim 18, wherein the onset of melting and / or melting rate of the cheese analogue is slower than the onset of melting and / or melting rate of the comparator cheese analogue.

20. The cheese analogue of claim 18 or 19, wherein the quality of the stretch (elasticity) of the cheese analogue is improved as compared to the quality of the stretch (elasticity) of the comparator cheese analogue.

21. The cheese analogue of any one of claims 18 to 20, wherein stretch length of the cheese analogue is at least 2-fold greater or at least 50 mm greater than the stretch length of the comparator cheese analogue.

22. The cheese analogue of any one of claims 18 to 21, wherein the bite-resistance of the cheese analogue is improved as compared to the bite-resistance of the comparator cheese analogue.

23. The cheese analogue of any one of claims 18 to 22, wherein the mouthfeel of the cheese analogue is improved as compared to the mouthfeel of the comparator cheese analogue. 52MOFO-359836952 Attorney Docket No. 263942000740 24. The cheese analogue of any one of claims 18 to 23, wherein the amount of cheese analogue browning upon cooking is increased as compared to cheese analogue browning upon cooking of the comparator cheese analogue.

25. The cheese analogue of any one of claims 18 to 24, wherein the shred quality of the cheese analogue is improved as compared to the shred quality of the comparator cheese analogue 26. A method of producing a casein composition, comprising: crosslinking recombinant alpha casein protein in the absence of beta casein and / or kappa casein proteins.

27. The method of claim 26, wherein the recombinant alpha casein is crosslinked by heat treatment, irradiation, or enzymatically.

28. The method of claim 27, wherein the recombinant alpha casein is enzymatically crosslinked using one or more of transglutaminase, laccase, tyrosinase, peroxidase, sulfhydryl oxidase, and glucose oxidase.

29. The method of claim 26, wherein the recombinant alpha casein is crosslinked in aqueous solution, in an emulsion, or in dried powder form.

30. The method of any one of claims 26 to 29, wherein the recombinant alpha casein is a bacterially-produced protein.

31. The method of any one of claims 26 to 30, further comprising incorporating the crosslinked alpha casein into a cheese analogue.

32. The method of claim 31, wherein the recombinant alpha casein is present in the cheese analogue at an amount between 1% and 30% wt / wt of the cheese analogue.

33. The method of claim 31, wherein the recombinant alpha casein is present in the cheese analogue at an amount between 1% and 18% wt / wt of the cheese analogue.

34. The method of claim 31, wherein the recombinant alpha casein is present in the cheese analogue at an amount greater than 5% and less than 18% wt / wt of the cheese analogue. 53MOFO-359836952 Attorney Docket No. 263942000740 35. The method of claim 31, wherein the recombinant alpha casein is present in the cheese analogue at an amount less than 5% wt / wt of the cheese analogue. 54MOFO-359836952

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