Cheese analogue compositions having certain stretch and other properties

Abstract

Provided herein are consumable compositions, such as cheese analogues, and the methods of making the same using certain amounts of a recombinant single alpha casein variant, which imparts certain desirable properties, such as stretch, to the compositions.

Description

CHEESE ANALOGUE COMPOSITIONS HAVING CERTAIN STRETCH AND OTHER PROPERTIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 486,402 filed on February 22, 2023, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (263943000600SEQLIST.xml; Size: 24,226 bytes; and Date of Creation: February 21, 2023) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates generally to plant-based and cell-based foods, and more specifically to cheese analogue compositions comprising recombinant alpha casein to impart certain stretch, melt and other properties to the cheese analogue compositions.BACKGROUND

[0004] The clean food space is comprised of both plant-based and cell-based foods. Cellbased 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 generally do not match the functionality, nutrition and taste of dairy cheese due to their lack of casein proteins. Thus, there is an unmet need in the art.BRIEF SUMMARY

[0006] In some aspects, provided is a cheese analogue composition comprising a recombinant alpha casein. In some embodiments, the recombinant alpha casein is the only casein in the composition. In some embodiments, the recombinant alpha casein is at a concentration between about 9% and about 15% alpha casein dry weight casein / total composition weight. In some embodiments, the alpha casein imparts stretch to the cheese analogue composition. In certain embodiments, the composition further comprises fat, starch and salt.

[0007] In certain variations of the foregoing, the recombinant alpha casein comprises only alphaS 1 casein. In certain variations, the composition has no animal -derived caseins. In one variation, the composition has no alphaS2 casein. In some variations, the recombinant alpha casein is devoid of phosphorylation. In some variations, the recombinant alpha casein is substantially reduced in phosphorylation. In certain variations, the recombinant alpha casein comprises an amino acid sequence of a bovine, ovine, or caprine alpha casein.

[0008] In certain embodiments, the composition has a distance to failure of at least 150 mm, for example, when measured on a texture analyzer and the composition is melted for 10 min at 90°C prior to the distance to failure measurement. In certain embodiments, the composition melts when heated to a suitable temperature for a suitable duration.

[0009] In certain embodiments, the composition further comprises a mozzarella, cheddar or parmesan flavoring.

[0010] In another aspect, provided is a food product comprising any of the cheese analogue compositions described herein. In some embodiments, the food product is a pizza, a frozen food product, or a shredded cheese-like product.DESCRIPTION OF THE FIGURES

[0011] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0012] Fig. 1 shows the expression of recombinant alphaS 1 protein from a microbial system.

[0013] Fig. 2 is a graph comparing aspects of stretch ability (extensibility) quality between three exemplary mozzarella cheese analogues NCI, NC2, and NC3 as described herein. The lighter bars represent the measured level of work to extend (in g*sec), and the darker bars represent the measured distance to failure, at which all cheese strands break, in mm.

[0014] Figs. 3 A-3C illustrate the melt properties on a pizza of exemplary mozzarella cheese analogues NCI, NC2, and NC3, respectively, as described herein. Left and right pictures of each figure represent uncooked and cooked mozzarella cheese analogue respectively.

[0015] Fig. 4 is a graph comparing aspects of stretch ability (extensibility) quality between three exemplary cheese analogues NC4, NC5, and NC6 as described herein. The lighter bars represent the measured level of work to extend (in g*sec), and the darker bars represent the measured distance to failure, at which all cheese strands break, in mm.

[0016] Figs. 5A-5C illustrate the melt properties on a pizza of exemplary mozzarella cheese analogues NC4, NC5, and NC6, respectively, as described herein. Left and right pictures represent uncooked and cooked mozzarella cheese analogue respectively.DETAILED DESCRIPTION

[0017] 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.

[0018] Although the dairy industry is worth $330 billion, research needs to be performed for a clean dairy solution using recombinant dairy proteins. Because dairy cheese is an inefficient dairy product, in terms of resources needed per gram as well as being the hardest dairy product to accurately reproduce from just plant-based ingredients, presented herein are methods and compositions of dairy-like products made using recombinant proteins.

[0019] A component that gives dairy cheese its unique characteristics is the casein proteins. When milk or milk-derived ingredients are used in dairy products, the caseins are found in micelles. Micelles are protein colloids, and typically in cow milk, the micelles arecomprised of four casein proteins (alphaS 1 casein, alphaS2 casein, beta casein, and kappa casein) that interact with insoluble calcium phosphate at the colloid center. It is the micelles in milk that attract each other once chymosin is added to milk. This forms the curd, which is then used to make 99% of all cheeses. A recombinant non-naturally occurring single variant of casein can be used to generate dairy or dairy-like products without the presence of other caseins and without the formation of a micelle.

[0020] The current disclosure is based on the surprising discovery made by the inventors that the amount of a single recombinantly produced alpha casein is critical to achieving certain desired properties of a cheese analogue. In fact, the inventors surprisingly discovered that there is a threshold effect that requires a minimal amount of such single recombinantly produced alpha casein in order to achieve certain desired stretch properties of the cheese analogue.

[0021] In certain variations, the consumable compositions described herein are formed from a recombinant single alpha casein variant. Recombinant casein protein may be expressed in a microbial organism, for example, bacteria such as gram-positive bacteria Lactococcus lactis and Bacillus sublilis. as well as a gram-negative model organism E. coli. as well as other host organisms such as yeasts, fungi, and plants. These recombinant casein proteins may be combined with other components (e.g., minerals, fats, sugars, and vitamins) to make dairy -like products, for example, cheese that behaves, smells, tastes, looks and feels like animal-derived dairy cheese. Such dairy-like products may have no: i) lactose, ii) cholesterol, iii) animal-derived saturated fats, iv) milk-derived whey proteins; and / or v) milk- derived casein proteins. In certain variations, the single variant of alpha casein is produced in a bacterial host cell, such that such proteins are secreted from the cell into the surrounding media. In some examples, the single variant of an alpha casein is produced in a bacterial host cell, such that such proteins are secreted from the cell into the surrounding media. In some embodiments, the recombinant protein is produced in a bacterial host cell, such that such proteins are intracellular. Recombinant protein can then be isolated, purified or partially purified and used in methods for making compositions which can be used as a dairy ingredient, or emulsified with plant-based fats and other nutrients to form a cheese analogue product. In certain variations, the single casein may be a variant selected from a variety of different species. For example, the single alpha casein variant may be selected from a variety of different species such as human, Bovinae (cattle, bison, buffalo), Caprinae (sheep andgoat), Equine (horses, zebra) and Camelus (camels). Compositions described herein are produced without beta and kappa casein. In some variations, the recombinant caseins can be isolated, purified or partially purified from genetically modified microorganisms or their cultivation broth.

[0022] In some embodiments, the term “about” as used herein can mean within 1 or 2 standard deviations. Alternatively, in other embodiments, “about” can mean a range of up to 5%, or up to 1% of a given value. For example, about can mean up to ±5%, ±4%, ±3%, ±2%, or ±1% of a given value.

[0023] The term “dairy protein” as used herein means a protein that has an amino acid sequence derived from a protein found in milk (including variants thereof).

[0024] The term “animal -derived dairy protein” as used herein means a protein derived from milk, such as a protein obtained and / or isolated from milk of a milk-producing organism, including but not limited to cow, sheep, goat, human, bison, buffalo, camel and horse. “Animal-derived casein protein” means casein protein obtained and / or isolated from milk of a milk-producing organism. Exemplary animal -derived dairy protein includes caseinate, and caseins purified from caseinate.

[0025] The term “recombinant dairy protein” as used herein means a protein that is expressed in a heterologous or recombinant organism that has an amino acid sequence derived from a protein found in milk (including variants thereof). “Recombinant casein protein” means a casein produced by a recombinant organism or in a heterologous host cell.

[0026] The term “single variant of casein” (also referred to as “single variant of a casein protein”) as used herein may describe a composition comprising or created from one variant of a casein protein amino acid sequence. For instance, a composition comprising a single variant of casein comprises only alpha casein. In some variations, the term “single variant of a casein protein” may describe a composition wherein a single casein protein provides one or more dairy -like properties to the composition irrespective of the presence of other caseins. A composition comprising a single variant of casein may be created from only one casein protein amino acid sequence but may comprise truncated forms of the protein sequence in place of or in addition to the full-length version of the protein.

[0027] The term “single variant of alpha casein” as used herein may describe a composition comprising or created from one variant of an alpha casein amino acid sequence. For instance, a composition comprising a single variant of alpha casein comprises only one of alphaS 1 or alphaS2 casein. A composition comprising a single variant of alpha casein may be created from only one alpha casein amino acid sequence but may comprise truncated forms of the protein sequence in place of or in addition to the full-length version of the protein. For instance, a composition comprising a single variant of alpha casein may comprise a mixture of full length alphaS 1 casein and truncated forms thereof. A composition comprising a single variant of alpha casein may comprise only the full-length alphaS 1 casein or only a truncated form of alphaS 1 casein, or only a mixture of truncated forms of the alphaS 1 protein.

[0028] A percentage of “sequence identity” as used herein in the context of polynucleotide or polypeptide (amino acid) sequences refers to the percentage of residues in two sequences that are the same when the sequences are aligned for maximum correspondence. There are a number of different algorithms known in the art that can be used to measure polynucleotide or polypeptide sequence identity. For instance, sequences can be compared using FASTA (e.g., using its default parameters as provided in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, WI), Gap (e.g., using its default parameters as provided in the Wisconsin Package Version 10.0, GCG, Madison, WI), Bestfit, ClustalW (e.g., using default paramaters of Version 1.83), or BLAST (e.g., using reciprocal BLAST, PSLBLAST, BLASTP, BLASTN) (see, for example, Pearson. 1990. Methods Enzymol. 183:63; Altschul et al. 1990. J. Mol. Biol. 215:403).Compositions Comprising Only Recombinant Alpha CaseinA. ALPHA CASEIN

[0029] Traditionally, cheese begins with animal -derived milk. The process of animal- derived cheese production includes precipitating micellar forms from milk wherein the micellar forms are in most cases complex protein mixtures (comprising multiple types of casein proteins such as alpha, beta and kappa proteins). Milk is acidified, the micelles shrink and dissociate slightly, then milk is renneted and made into curd, and the curd made into cheese. Cheese analogues may be created from animal milk by first precipitating casein micelles from milk using one of the following methods: 1) a sodium salt to make sodium caseinate, 2) an acid to make acid casein, 3) enzymatic coagulation with rennet to makerennet casein. Casein precipitated in this way from milk is then further processed with fats to create the cheese analogue.

[0030] Provided herein are consumable compositions with a single variant of alpha casein, produced recombinantly, that is not in micellar form and not derived from milk or derived from milk casein. In some aspects, provided are consumable compositions comprising a recombinant alpha casein, wherein the recombinant alpha casein is the only casein in the cheese analogue composition.

[0031] In some embodiments, the consumable compositions (and products made therefrom) do not include any animal-derived caseins. The compositions herein 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.

[0032] The compositions described herein comprise single variants of alpha casein that are made through recombinant production. In some variations, the single variant casein in a consumable composition may be a modified casein protein relative to a native casein protein. The modifications in the single variant of a casein protein may comprise one or more amino acid insertions, deletions, or substitutions relative to a wild-type or native casein protein.

[0033] In some embodiments, the single variant of an alpha casein in a consumable composition is only an alphaS 1 casein. In such compositions, the alphaS 1 casein may comprise a modified alphaS 1 casein, such as a modified alphaS 1 casein which lacks or has substantially reduced post-translational phosphorylation. In some variations, the alphaS 1 casein may be a full-length alphaS 1 casein. In some variations, the compositions comprising an alphaS 1 casein lacks any animal -derived proteins. In some variations of the foregoing, the consumable composition has no alphaS2 casein. In other variations, the consumablecomposition has no detectable amount of alphaS2 casein. In yet other variations, the consumable composition does not include a mixture of alphaS 1 and alphaS2 casein.

[0034] A single variant of an alpha casein may match the sequence (i.e., be identical in amino acid sequence) of that from a ruminant species. In some embodiments, a single variant of 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, a single variant of an alpha casein may be a bovine alpha casein. In other variations, a single variant of an alpha casein may be a caprine alpha casein. In other variations, a single variant of an alpha casein may be an ovine alpha casein. In other variations, a single variant of an alpha casein may be an equine alpha casein. In other variations, a single variant of an alpha casein may be a camel or camelid alpha casein. In other variations, a single variant of an alpha casein may be a human alpha casein.

[0035] In some embodiments, a single variant of an 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) A single variant of an 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. A single variant of an 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. A single variant of an 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. A single variant of an 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. A single variant of an 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.B. POST-TRANSLATIONAL MODIFICATIONS

[0036] Depending on the host organism used to express the casein, the single variant of the alpha caseins may have a post-translational modification (or “PTM” as used herein)different from animal-derived casein proteins. In some variations, the single variant of the alpha casein has no post translational modifications (PTMs). In some variations, the single variant of the alpha casein comprises substantially reduced PTMs. As used herein, substantially reduced PTMs means at least 50% reduction of one or more types of PTMs as compared to the amount of PTMs in an animal-derived casein protein. For instance, the single variant of alpha casein may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less post-translationally modified as compared to animal-derived alpha casein. In some variations, the post-translationally modified caseins may be lacking one or more sites of post translational modifications found in animal -derived casein proteins.

[0037] In some variations, the single variant of the alpha casein comprises a reduced or modified phosphorylation that differs from an animal -derived alpha casein. In some variations, the single variant of alpha casein is not phosphorylated. In some variations, no phosphorylation of the single variant of alpha casein is detectable.

[0038] Alternatively, in some variations, the single variant of the alpha casein may comprise PTMs comparable to animal -derived casein PTMs. In some variations, the single variant of the alpha casein comprises substantially increased PTMs. As used herein, substantially increased PTMs means at least 5% increase in one or more types of PTMs as compared to the amount of PTMs in an animal-derived casein protein. For instance, the single variant of alpha caseins may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% more post-translationally modified as compared to animal-derived alpha casein.

[0039] The PTMs in the alpha casein may be modified chemically or enzymatically. In some variations, the single variant of the alpha casein comprises substantially reduced or no PTMs without chemical or enzymatic treatment. Compositions may be generated using single variant of the alpha casein with reduced or no PTMs, wherein the lack of PTMs is not due to chemical or enzymatic treatments of the protein, such as producing a single variant of alpha casein through recombinant production where the recombinant protein lacks PTMs.

[0040] In some embodiments, the single variant of alpha casein is a recombinantly produced protein and comprises substantially reduced or no phosphorylation. For instance, single variant of alpha caseins may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% less phosphorylated as compared to animal-derived alpha casein. Insome variations, the recombinant single variant of alpha casein has no phosphorylation or no detectable phosphorylation.

[0041] In some variations, compositions may comprise single variants of alpha casein that have a mix of alpha caseins with no PTMs, reduced PTMs, and / or nature comparable (or native-like) PTMs. In some variations, compositions may comprise single variants of the alpha casein with native PTMs, casein proteins lacking one or more types of PTMs, and / or no PTMs. Alternatively, compositions may comprise a single variant of the alpha casein with uniform PTMs. The PTM structures in such cases may include reduced PTMs, PTMs lacking one or more types of PTM. For instance, in one variation, the consumable composition may comprise only a single variant of alpha casein with reduced phosphorylation.Consumable CompositionsA. CHEESE ANALOGUES

[0042] A recombinant alpha casein, in which the recombinant alpha casein is the only casein in the composition, may be used to produce cheese analogues. Thus, in one aspect, provided is a cheese analogue that comprises a recombinant alpha casein, wherein the recombinant alpha casein is the only casein in the cheese analogue. In some variations of the foregoing, the recombinant alpha casein is at a concentration between about 9% and about 15% alpha casein (dry weight casein / total cheese analogue weight) and wherein the alpha casein imparts stretch to the cheese analogue.

[0043] In some embodiments, the cheese analogue comprises a recombinant alpha casein, wherein the recombinant alpha casein is the only casein in the cheese analogue. In some variations of the foregoing, the recombinant alpha casein is at a concentration between about 9% and about 15% alpha casein (dry weight casein / total cheese analogue weight) and wherein the alpha casein imparts stretch to the cheese analogue. In some variations of the foregoing, the recombinant alpha casein is at a concentration between about 9% and about 11%, about 9% and about 10%, about 9% and about 12%, about 9% and about 13%, about 9% and about 14% or about 9% and about 15% alpha casein (dry weight casein / total cheese analogue weight) and wherein the alpha casein imparts stretch to the cheese analogue. In some variations of the foregoing, the recombinant alpha casein is at a concentration of about9%, about 10% or about 11%, alpha casein (dry weight casein / total cheese analogue weight) and wherein the alpha casein imparts stretch to the cheese analogue.

[0044] In some embodiments, the recombinant alpha casein is at a concentration no more than about 15% alpha casein (dry weight casein / total cheese analogue weight) . In some embodiments, the recombinant alpha casein is at a concentration no more than about 11% alpha casein (dry weight casein / total cheese analogue weight). In some embodiments, the recombinant alpha casein is at a concentration no more than about 14%, about 13%, or about 12% alpha casein (dry weight casein / total cheese analogue weight). In some embodiments, the recombinant alpha casein is at a concentration at least about 9% alpha casein (dry weight casein / total cheese analogue weight). In some embodiments, the recombinant alpha casein is at a concentration at least about 10% alpha casein (dry weight casein / total cheese analogue weight).

[0045] 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.

[0046] 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 alphaS 1 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.

[0047] 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.

[0048] In some variations, a cheese analogue may comprise 0% w / w to 50% w / w starch. Examples of starches which can be added to a cheese analogue include modified potato, com. Other examples are provided elsewhere herein. In some variations, a cheese analogue may comprise at least 0% 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 from 0% w / w to 15% w / w, 0% w / w to 10% w / w, 0% w / w to 3% w / w, 0% w / w to 5% w / w, 0% w / w to 7% w / w, 5% w / w to 10% w / w, or 5% w / w to 7% w / w starch. In some variations, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein (such as a recombinant alphaS 1 casein) and from 0% to 15%, 5% to 10%, or 5% to 7% w / w starch.

[0049] In other variations of the foregoing, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein (such as a recombinant alphaS 1 casein) and the recombinant alpha casein is present in the cheese analogue in an amount relative to starch of no more than 1 : 1.7, or no more than 1 : 1 : 1.1 or no more than 1 :0.8. In some variations, no starch is present. In some variations, the ratio of recombinant alpha casein to starch is between 1 :0.6 to 1 : 1.1. In some variations, no starch is present. In some variations,the ratio of recombinant alpha casein to starch is between 1 :0.6 to 1 :0.8. In some variations, the ratio of recombinant alpha casein to starch is at least 1 :0.6, at least 1 :0.8, or at least 1 : 1.1. In other variations of the foregoing, a cheese analogue may comprise at least 11% alpha casein or greater than 11% alpha casein, and the recombinant alpha casein is present in the cheese analogue in an amount relative to starch of no more than 1 :0.7, or no more than 1 :0.33 or no more than 1 :0.2.

[0050] 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 salts such as calcium chloride. 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. 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.

[0051] In other variations of the foregoing, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein (such as a recombinant alphaS 1 casein) and 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, a cheese analogue may comprise at least 11% alpha casein or greater than 11% alpha casein, and 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.

[0052] In some variations, a cheese analogue contains moisture, such as between 35% to 52% moisture. In some variations, the moisture is between 35%-52%, 40%-52%, 40%-50%, 40%-45%, 45%-52% or between 45-50% moisture w / w. In some variations, the moisture is 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51% or 52% 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 variations of the foregoing, a cheese analogue may comprise at least 9% alpha casein or between 9% and 11% alpha casein (such as a recombinant alphaSl casein) and the moisture is about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% w / w. 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.

[0053] In some examples, a single variant of an alpha casein recombinantly produced 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, natural flavor maskers, color additives, preservatives, anti -caking agents and micronutrients such as vitamins can be incorporated into a cheese analogue as well.

[0054] In some embodiments, the following ingredients are pre-mixed: a recombinantly produced single variant of an alpha casein, fat(s), water, starch, salt(s) such as sodium chloride. 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, 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., 15-50 °C). In some variations, fats are pre-melted (e.g., 30-70 °C) (exemplary range 40-50 °C) and held at their melting temperature prior to 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- 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.

[0055] The mixtures may be heated over a temperature ramp from pre-mixing temperature (ambient or elevated) to 50-95 °C (exemplary range 75-90 °C), over a ramp 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.

[0056] 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.

[0057] 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.

[0058] In some embodiments, the cheese analogue comprises a single variant of recombinant alpha casein, such as alphaS 1 casein, present in a certain amount within the composition or relative to certain specific components in the composition imparts one or more specific properties that are desirable, such as stretch and / or melt, to the cheese analogue.Stretch

[0059] The stretching ability of a cheese analogue made with a recombinant single variant alpha casein such as by methods described herein, may be comparable to the stretching ability of a similar type of cheese made using animal-derived dairy proteins, suchas cheese made from animal milk. Stretching ability of a cheese analogue as described herein may be comparable to the stretching ability of a 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. Stretching ability of a cheese analogue as described herein may be improved / more desirable when compared to the stretching ability of a 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, or when compared to 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)). Stretching ability of a cheese analogue as described herein may be tested using a trained panel of human subjects or using a machine such as a texture analyzer. Stretch of a cheese analogue as described herein may be greater than 2.5 cm when measured on a texture analyzer post cooking. Exemplary assays that measure this stretch property are provided in the examples section.

[0060] In some embodiments, the cheese analogue as described herein exhibits a distance to failure of at least 150 mm. In some embodiments, a cheese analogue is analyzed for stretch using a texture analyzer (e.g., TA.XTPlus Texture Analyzer), such as by heating the cheese in an oven at 90°C for 10 min in an extensibility rig 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. In some embodiments, the cheese analogue as described herein has distance to failure of at least 150 mm. In some embodiments, the cheese analogue as described herein has distance to failure of at least at least 160mm, at least 170 mm, at least 180 mm, at least 190 mm, at least 200 mm, at least 210 mm, at least 220 mm or at least 230 mm. In some embodiments, the cheese analogue has a recombinant alpha casein (such as alphaS 1 casein) concentration of between about 9% and about 12% or between about 9% and about 11%, as described herein and has a distance to failure comparable to the distance to failure of a cheese analogue with a higher concentration of alpha casein, such as a concentration of at least 13%, at least 14%, or at least 15% dry weight / total weight. In some embodiments, such lower casein cheese analogue as described herein has a distance to failure that is within 10-20%, 15-25%, 20-40% or 10-50% of the distance to failure of a cheeseanalogue with a higher concentration of alpha casein, such as a concentration of at least 13%, at least 14%, or at least 15% dry weight / total weight.

[0061] In some variations, the cheese analogue is analyzed for stretch using a texture analyzer, such as by heating the cheese in an oven at 90°C for 10 min in an extensibility rig and measuring the amount of work required to extend the cheese analogue (in g*sec). In some embodiments, the cheese analogue as described herein has a work to extend greater than 60 g*sec, greater than 50 g*sec, or greater than 40 g*sec. In some embodiments, the cheese analogue described herein has a recombinant alpha casein (such as alphaS 1 casein) concentration of between 9% to about 12% or about 9% to about 11% as described herein and has a work to extend comparable to the work to extend of a cheese analogue with a higher concentration of alpha casein, such as a concentration of at least 13%, 14%, 15% or more than 15% dry weight / total weight. In some variations, the cheese analogue as described herein has a higher ratio of starch to alpha casein and has a work to extend of at least 10 g*sec or at least 15 g*sec.Melt

[0062] 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. Exemplary assays that measure this melt property are provided in the examples section.

[0063] 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 oventemperatures 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.

[0064] 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 80 °C and about 95 °C 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 plantbased cheese analogue.Texture

[0065] The texture of a cheese analogue as described herein may be comparable to the texture of a similar type of cheese made using animal -derived dairy proteins, such as cheese made from animal milk. Texture of a cheese analogue as described herein may be comparable to the texture of a 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. Texture of a cheese analogue as described herein may be improved / more desirable when compared tothe texture of a 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, or when compared to 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)). Texture of a cheese analogue as described herein may be tested using a trained panel of human subjects or using a machine such as a texture analyzer.Browning Ability

[0066] Cheese analogues as described herein may have a browning 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 browning 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 browning 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)). Browning ability of a cheese analogue as described herein may be tested using an oven and computer imaging.Mouthfeel

[0067] The mouthfeel of a cheese analogue as described herein may be comparable to the mouthfeel of a similar type of cheese made using animal -derived dairy proteins, such as cheese made from animal milk. Mouthfeel of a cheese analogue as described herein may be comparable to the mouthfeel of a 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. Mouthfeel of a cheese analogue as described herein may be improved when compared to the mouthfeel of a 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)).B. OTHER COMPONENTS

[0068] The compositions described herein may be used as ingredients in generating consumable compositions such as food products. The food products may include cheeseanalogues and other suitable food products described elsewhere herein. In some embodiments, the cheese analogue is incorporated into a food product. In some embodiments, the food products may include a shredded cheese-like product. In some embodiments, the food products may include a cheese-like topping or a cheese-like filling. In some embodiments, the food products may include a cheese analogue incorporated into a pizza, a frozen food or prepared meal item. For instance, a cheese product or a cheese analogue described herein may be used by end-user to make a final product such as pizza, Italian food toppings, Mexican food toppings, frozen meals, toppings for savory baked goods, soups, macaroni cheese, cheese sticks, etc. Such consumable compositions may comprise one or more ingredients in addition to the single variant casein protein present in a certain amount in the compositions. The ingredients may include but are not limited to solvents, salts, sugar, fats, flavorings, colorants, etc.

[0069] The consumable compositions may further comprise salts such as calcium, phosphorous, citrate, potassium, sodium and / or chloride salts. The calcium salt may be selected from calcium chloride, calcium carbonate, calcium citrate, calcium glubionate, calcium lactate, calcium gluconate, calcium acetate, equivalents thereof and / or combinations thereof. The phosphate salt may be selected from orthophosphates such as monosodium (dihydrogen) phosphate, di sodium phosphate, trisodium phosphate, monopotassium (dihydrogen) phosphate, dipotassium phosphate, tripotassium phosphate; pyrophosphates such as disodium or dipotassium pyrophosphate, trisodium or tripotassium pyrophosphate, tetrasodium or tetrapotassium pyrophosphate; polyphosphates such as pent sodium or potassium tripolyphosphate, sodium or potassium tetrapolyphosphate, sodium or potassium hexametaphosphate. The citrate salt may be selected from calcium citrate, potassium citrate, sodium citrate, trisodium citrate, tripotassium citrate or equivalents thereof. The consumable composition may comprise a combination of salts. In some embodiments, the consumable composition comprises calcium, phosphate and citrate salts. In some embodiments, the consumable composition comprises calcium and phosphate salts. In some embodiments, the consumable composition comprises calcium and citrate salts. In some embodiments, the consumable composition comprises phosphate and citrate salts.

[0070] In some embodiments, fat is added to the consumable composition. In some variations, fats may be essentially free of animal-derived fats. Fats used herein may include plant-based fats such as canola oil, sunflower oil, coconut oil, palm oil, or combinations thereof. Fats used herein may include microbially-made recombinant animal or plant fats. Fats used herein may include mammalian cell-cultured recombinant animal or plant fats.

[0071] Consumable composition as described herein may further comprise sugars. Sugars used herein may include plant-based monosaccharides, disaccharides and / or oligosaccharides. Examples of sugars include sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allulose, tagatose, xylose, and arabinose.

[0072] The consumable compositions as described herein and the methods of making such compositions may including adding or mixing with one or more ingredients. For example, food additives may be added in or mixed with the compositions. Food additives can add volume and / or mass to a composition. A food additive may improve functional performance and / or physical characteristics. For example, a food additive may prevent gelation or increased viscosity due to the lipid portion of the lipoproteins in the freeze-thaw cycle. An anticaking agent (cellulose, potato starch, com starch, starch blends) may be added to make a free-flowing composition. Carbohydrates can be added to increase resistance to heat damage, e.g., less protein denaturation during drying and improve stability and flowability of dried compositions. Food additives include, but are not limited to, starch (e.g., potato, modified potato, com, rice), food coloring, pH adjuster (e.g. glucono-delta-lactone, sodium hydroxide), natural flavouring (e.g., mozzarella, parmesan, butter, cream, colby, provolone, asiago, etc.), artificial flavoring, flavor enhancer, flavour maskers, batch marker, food acid (e.g., lactic acid, citric acid), filler, anticaking agent (e.g., sodium silicoaluminate), antigreening agent (e.g., citric acid), food stabilizer, foam stabilizer or binding agent, antioxidant, acidity regulatory, bulking agent, color retention agent, whipping agent (e.g., ester-type whipping agent, triethyl citrate, sodium lauryl sulfate), emulsifier (e.g., lecithin, monoglycerides, diglycerides), humectant, thickener, pharmaceutical excipient, solid diluent, nutrient, sweetener, glazing agent, preservative (e.g., sorbic acid, nisin), vitamins (e.g. vitamin B, vitamin D, vitamin A), dietary elements, carbohydrates, polyol, gums, starches, flour, oil, and bran. In some variations, flavoring may comprise a mozzarella flavoring, cheddar flavoring, parmesan flavoring or other similar cheese flavorings.

[0073] Food coloring includes, but is not limited to, FD&C Yellow #5, FD&C Yellow #6, FD&C Red #40, FD&C Red #3, FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 3, carotenoids (e.g., saffron, P-carotene), annatto, betanin, butterfly pea, caramel coloring, chlorophyllin, elderberry juice, lycopene, carmine, pandan, paprika, turmeric, curcuminoids, quinoline yellow, carmoisine, Ponceau 4R, Patent Blue V, and Green S.

[0074] Ingredients for pH adjustment include, but are not limited to, Tris buffer, potassium phosphate, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, sodium bicarbonate, and hydrochloric acid.RECOMBINANT EXPRESSION

[0075] One or more proteins used in the formation of cheese compositions may be produced recombinantly. In some variations, a single variant alpha casein (including specifically a single variant of alphaS 1 casein) is produced recombinantly. The single variant alpha casein, e.g., a single variant alphaS 1 casein can have an amino acid sequence from any milk-producing species. For example, a recombinant alpha casein may have an amino acid sequence of cow, sheep, goat, buffalo, horse, human, deer or camel alpha casein. The nucleotide sequence encoding the casein protein may be codon-optimized for increased efficiency of production. Exemplary alpha casein sequences are provided in Table 1 below for use in recombinant production of a single variant casein protein. A recombinant single variant casein protein can be a non-naturally occurring variant of a casein. Such variant can comprise one or more amino acid insertions, deletions, or substitutions relative to a native casein sequence.

[0076] Such a variant can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1-20.

[0077] A recombinant single variant casein protein, such as a single variant alphaS 1 casein, is recombinantly expressed in a host cell. As used herein, a “host” or “host cell” denotes any protein production host selected or genetically modified to produce a desired product. Exemplary hosts include bacteria, yeast, fungi, plants, insects and mammalian cells. In some variations, a bacterial host cell such as Lactococcus laclis. Bacillus subtilis or Escherichia coli may be used to produce alpha caseins and / or its truncated forms. Other host cells include bacterial host such as, but not limited to, Lactococci sp., Bacillusamyloliquefaciens, Bacillus licheniformis and Bacillus megalerium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glulamicum. Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum and Synechocystis sp. 6803.

[0078] In some embodiments, a full-length single variant alpha casein and / or truncated forms thereof are produced recombinantly in a host cell. For example, full-length and truncated single variant alphaS 1 caseins may be produced in the same host cell and such production can originate from the same open reading frame (or the same expression cassette) and truncated forms generated for example, by post-translational proteolytic cleavage. Expression of a target protein can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome or other means. For example, a vector for expression can include: (a) a promoter element, (b) a signal peptide, (c) a heterologous casein sequence, and (d) a terminator element.

[0079] Expression vectors that can be used for expression of casein include those containing an expression cassette with elements (a), (b), (c) and (d). In some embodiments, the signal peptide (b) and / or terminator element (d) need not be included in the vector. In some variations, a signal peptide may be part of the native signal sequence of the casein protein, for instance, the protein may comprise a native signal sequence as bolded in SEQ ID NOs: 1, 4, 7, 10, 13, 16 or 19. In some variations, the vector may comprise a mature protein sequence, as exemplified in SEQ ID NOs: 2, 5, 8, 11, 14, 17, or 20, to which a heterologous signal sequence is added. In some variations, the protein may comprise no signal sequence but instead an initiator methionine, as exemplified in SEQ ID NOs: 3, 6, 9, 12, 15, or 18. In general, the expression cassette is designed to mediate the transcription of the transgene when integrated into the genome of a cognate host microorganism or when present on a plasmid or other replicating vector maintained in a host cell.

[0080] To aid in the amplification of the vector prior to transformation into the host microorganism, a replication origin (e) may be contained in the vector. To aid in the selection of microorganisms stably transformed with the expression vector, the vector may also include a selection marker (f). The expression vector may also contain a restriction enzyme site (g) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate the expression vector’s stable integration into thehost genome. In some embodiments the expression vector may contain any subset of the elements (b), (e), (f), and (g), including none of elements (b), (e), (f), and (g). Other expression elements and vector elements known to one of skill in the art can be used in combination or substituted for the elements described herein.

[0081] Gram-positive bacteria (such as Lactococcus lactis and Bacillus subtilis) may be used to secrete target proteins into the media, and gram-negative bacteria (such as Escherichia coif) may be used to secrete target proteins into periplasm or into the media. In some embodiments, the bacterially-expressed proteins expressed may not have any post- translational modifications (PTMs), which means they are not glycosylated and / or may not be phosphorylated. Both gram positive and gram-negative bacteria may be used to produce proteins intracellularly. In such examples, the cells may be lysed to recover the protein.

[0082] Single variant casein proteins may be expressed and produced in L. lactis both in a nisin-inducible expression system (regulated by PnisA promoter), lactate-inducible expression system (regulated by Pl 70 promoter) or other similar inducible systems, as well as a constitutively expressed system (regulated by P secA promoter), wherein both are in a foodgrade selection strain, such as NZ3900 using vector pNZ8149 (lacF gene supplementation / rescue principle). The secretion of functional proteins may be enabled by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by L. lactis.For example, alphaS 1 casein and truncates thereof may be co-expressed or individually expressed in L. lactis using a synthetic operon.

[0083] B. subtilis has multiple intracellular and extracellular proteases, which may interfere with protein expression. In some embodiments, B. subtilis strains are modified to reduce the type and amount of intracellular and / or extracellular proteases, for example strains which may have deletions for 7 (KO7) and 8 (WB800N) proteases, respectively, may be used.

[0084] In order to drive the recombinant protein secretion, the signal peptide of amyQ, alpha-amylase of Clostridium thermocellum may be used or another bacterial signal peptide known in the art. Additionally, native casein signal peptide sequences may be expressed heterologously in B. subtilis. Each casein protein has its own signal peptide sequence and may be used in the system. The signal proteins may be cross-combined with the casein proteins. The pHTOl vector may be used as a transformation and expression shuttle forinducible protein expression in / A subtilis. The vector is based on the strong oA-dependent promoter preceding the groES-groELoperon of B. subtilis, which has been converted into an efficiently controllable (IPTG-inducible) promoter by addition of the lac operator. pHTOl is an E. coli / B. subtilis shuttle vector that provides ampicillin resistance to E.coli and chloramphenicol resistance to / / . subtilis.

[0085] Single variant casein proteins may be produced in E. coli using safe laboratory strains such as E. coli BL21 (exemplary strains BL21 (DE3) or BL21 Al) or their derivatives, or a wild-type like K12 strains (exemplary strains MG1655 or W3110) or their derivatives.Inducible (such as IPTG-inducible, lactose-inducible, arabinose-inducible, rhamnose- inducible), auto-inducible (such as phosphate depletion based) and constitutive promoters may be used to drive the casein expression. Single variant casein proteins may be produced intracellularly, or may be secreted into the periplasm and / or supernatant. In order to drive the recombinant protein secretion, bacterial signal peptides of Sec-dependent secretion pathway (such as OmpA, OmpC, OmpT, pelB, LamB), SRP secretion pathway (such as TolA, DsbA, DsbC, TorT) and TAT secretion pathway (such as TorA, Sufi) can be used.TABLE 1: SEQUENCESEXAMPLES

[0086] 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: Exemplary methods for recombinant expressionExpression in Lactococcus lactis

[0087] Bovine alphaS 1 casein (variant C) protein coding sequence (without the native signal peptide) was codon-optimized for expression in Lactococcus lactis and a synthetic operon was constructed for co-expression and secretion of the two proteins under a nisin- inducible promoter. Signal peptide sequence from natively secreting lactococcal protein Usp45 was used to drive protein secretion. A synthetic operon was then cloned into an E. coli custom vector via restriction digest compatible sites and confirmed via Sanger sequencing, from which it was subcloned into nisin-inducible pNZ8149 vector via restriction digestion and ligation.

[0088] The vector was transformed into compatible L. lactis strain NZ3900 via electroporation and completely defined media (CDM) supplemented with lactose was used for selection. Positive clones were confirmed via colony PCR and 3 positive clones were taken forward for the protein expression induction and analysis.

[0089] Individual colonies were grown at 30 °C in liquid culture and protein production was induced with nisin for 2.5 hours (control samples left uninduced). Cells were then harvested by centrifugation and TCA-precipitated supernatants and lysed cell pellets were analysed by Coomassie gel staining (SDS-PAGE) and chemiluminescence (Western Blot against alphaS 1 casein, LSBio primary antibodies).

[0090] Similar to the constructions above, alpha casein constructions were created replacing the nisin promoter with the Pl 70 promoter, a pH / lactate inducible promoter for L. lactis. Each of these constructs contained a secretion signal peptide.

[0091] AlphaS 1 casein and its truncated forms were detected in L. lactis upon secretion on western blot. Unprocessed protein product accumulated intracellularly but secretion was detected for the mature protein and its truncated forms.Expression in B. subtilis

[0092] Bovine alphaS 1 casein (variant C) protein coding sequence (without the native signal peptide) His-tagged C-terminally was codon-optimized for expression in Bacillus subtilis. Constructs were created with and without the codon-optimized signal peptide of amyQ, alpha-amylase Bacillus amyloliquefaciens which has been reported for the efficient secretion of recombinant proteins. Constructs were cloned through E. coli via Gibson cloning into transformation and expression IPTG-inducible vector pHTOl and confirmed via Sanger sequencing. pHTOl is an E. coli / B. subtilis shuttle vector that provides ampicillin resistance to E. coli and chloramphenicol resistance to B. subtilis. Positive clones were further transformed into chemically competent B. subtilis WB800N. Positive clones were confirmed via colony PCR and 3 positive clones were taken forward for the protein expression induction and analysis.Protein expression and analysis

[0093] Individual colonies were grown at 37°C in liquid culture and protein production was induced with IPTG for 1 hour, 2 hours and 6 hours (control samples were left uninduced). Cells were then harvested by centrifugation, and TCA-precipitated supernatants and lysed cell pellets were analyzed by Coomassie gel staining (SDS-PAGE) and chemiluminescence (Western Blot against His tag and alphaS 1 casein). Western blotting showed expression of the alphaS 1 casein in B. subtilis.Expression in E. Coli

[0094] The bovine alphaS 1 casein (variant C) coding sequence (without the native signal peptide) was codon-optimized for Escherichia coli and cloned into a T7-driven commercially available pET vector (pET28). Cloning was performed via a Gibson reaction of DNAfragments and the vector in such a way that only the alphaS 1 casein coding sequence was left within the open reading frame (no N or C-terminal tags). The vector was then transformed into chemically competent E. coli BL21(DE3) cells, and expression after induction with IPTG was confirmed from an isolated single colony.

[0095] Individual colonies were grown at 37 C in liquid culture, and protein production was induced with IPTG for 4 hours. Cells were then harvested by centrifugation, resuspended in a buffered solution, and lysed. The lysate was clarified by centrifugation, and the clarified lysate was treated with ammonium sulfate at room temperature, and the precipitate was collected by centrifugation. The pellet was resuspended in 8M urea, followed by dialysis against disodium phosphate. The insoluble proteins were removed by centrifugation, and the remaining contaminants were removed by precipitation with ethanol (60% v / v) and ammonium acetate (150mM), followed by centrifugation. The resulting alphaS 1 casein solution was concentrated using a centrifugal filtration unit and then dialyzed against disodium phosphate. The purified product was analyzed on a coomassie-stained SDS-PAGE gel where the alphaS 1 casein appeared as the predominant protein band (Fig. 1).Example 2: Mozzarella Cheese analogue made from the reduced amount of recombinant single variant alpha casein

[0096] Recombinant unphosphorylated alphaS 1 casein from Example 1 was used to make non-micellar mozzarella cheese analogue. A casein prep was generated from the recombinant host expressing alphaS 1 casein, by purifying the expressed alphaS 1 casein as described generally in Example 1 to a final purity between 60-75%, as measured by protein dumas and label-free quantification by LC-MS / MS, with the remainder of the prep composed of moisture, fat, ash, and host proteins.

[0097] The casein prep containing alphaS 1 casein was combined with ingredients shown in Table 2 to create 3 exemplary mozzarella cheese analogues, referred to as NCI, NC2, and NC3, containing a final amount of 6%, 9%, and 11.1% alphaS 1 casein, respectively. Water, palm stearin, canola oil, modified potato starch, sodium chloride, and calcium chloride were added in a beaker at concentrations specified in Table 2. To this, trisodium citrate and dipotassium phosphate were added at the concentration specified in Table 2. The beaker was moved to a water bath at a preset temperature, and the contents were mixed at a first speed using a mixing propeller, followed by mixing for additional time at a lower speed, at whichpoint natural flavors were also added. Lactic acid was added, and the ingredients were mixed for additional time at the lower speed. The resulting mixture turned to a homogeneous non- micellar mass, transferred to standard molds, and allowed to sit in the fridge for multiple days. After incubation, the mozzarella cheese analogues were weighed to get yield estimation.Table 2: Ingredient Composition and Concentration for Cheese Analogues

[0098] The mozzarella cheese analogue samples were analyzed for qualitative and quantitative parameters such as pH, moisture, melt and stretch. The pH of all three mozzarella cheese analogues was 6, and final moisture content was 44%, 42%, and 40%, for NCI, NC2 and NC3, respectively.

[0099] A pizza melt test was performed to determine the melt of the cheese analogues. For this, 9g of each mozzarella cheese analogue was shredded on a 4” pizza topped with 8g of tomato sauce. It was then baked in an oven preset at 750°F for 1 minute to qualitatively determine the melt on the pizza. The mozzarella cheese analogues showed good spread and melt on the pizza (Figs. 3A-3C).

[0100] Despite the cheese analogues showing similar melting properties, reducing the amount of alpha casein had a surprising effect on the ability of the cheese analogue to stretch. Stretch was assessed by an extensibility test. The test was performed on a TA.XTPlus Texture Analyzer to quantitate cheese extensibility by inserting a 6-pronged hook into a 6 g sample of hand-cut cheese analogue shreds that had been melted for 10 min at 90°C and lifted until all the cheese analogue strands broke. Distance to failure (breakage of all strands) representing the extent of cheese stretch and work to extend representing tensile strength needed to stretch the cheese was quantified. The tests were performed on samples taken out of the fridge and then kept at ambient temperatures for at least 30 mins.

[0101] As shown in Fig. 2, the extensibility of the NC2 mozzarella cheese analogue containing 9% alphaS 1 casein was comparable to NC3 mozzarella cheese analogue containing 11.1% alphaS 1 casein (-224 mm, and -226 mm, respectively). However, surprisingly, the NCI mozzarella cheese analogue with only 6 % alphaS 1 casein only stretched to a length of 96.2 mm, less than half of the stretch exhibited by cheese analogues containing the higher amounts of alphaS 1 casein, suggesting a threshold effect of alphaS 1 casein required to achieve stretch.Example 3: Mozzarella Cheese analogue with reduced amount of recombinant single variant alpha casein and increased fat content

[0102] Recombinant unphosphorylated alphaS 1 casein as produced in Example 1 was used to make non-micellar mozzarella cheese analogue termed NC4, NC5, and NC6 having different alphaS 1 casein amounts of 7%, 8%, and 9% (wt / wt), respectively. The recombinant unphosphorylated alphaS 1 casein was produced according to the general methods described in Example 1 and quantified as described in Example 2. The casein-prep contained 75% (wt / wt) alphaS 1 casein. The three mozzarella cheese analogues were made using the method described in Example 2 using the ingredients shown in Table 3.Table 3: Ingredient Composition for NC4, NC5, and NC6, mozzarella cheese analogues

[0103] The pH of the three mozzarella cheese analogues was 6.3, 6.3, and 6.2, for NC4, NC5 and NC6 respectively, and the final moisture content was 47%, 47%, and 42%, for NC4, NC5 and NC6 respectively.

[0104] The melt test was performed as described in Example 2. The NC4, NC5, and NC6 mozzarella cheese analogues with 7%, 8%, and 9% alphaS 1 casein each melted on the pizza comparably (Figs. 5A-5C).

[0105] The extensibility test was performed on a TA.XTPlus Texture Analyzer as described in Example 2. The extensibility was severely affected when the mozzarella cheese analogues contained less than 9% alphaS 1 casein. NC4 and NC5 mozzarella cheese analogues containing 7% and 8% alphaS 1 casein had a stretch less than 47mm. In comparison, NC3 mozzarella cheese analogue containing 9% alphaS 1 casein stretched to a length of 163mm. The stretch results are shown in Fig. 4. These results demonstrate a threshold effect of alphaS 1 casein, that below 9% the ability to provide stretch to the cheese analogue is significantly impacted.

Claims

CLAIMSWhat is claimed is:

1. A cheese analogue composition comprising a recombinant alpha casein, wherein the recombinant alpha casein is the only casein in the composition, and the recombinant alpha casein is at a concentration between about 9% and about 15% alpha casein dry weight casein / total composition weight, and wherein the alpha casein imparts stretch to the cheese analogue composition.

2. The composition of claim 1, wherein the recombinant alpha casein is at a concentration between about 9% and about 12% alpha casein, or between about 9% and about 11% alpha casein dry weight casein / total composition weight.

3. The composition of any one of claims 1 to 3, further comprising fat at a concentration between about 10% and about 30% weight / total composition weight.

4. The composition of any one of claims 1 to 3, further comprising fat at a concentration between about 20% and about 30%, between about 24% and about 30%, or between about 28% and about 30% weight / total composition weight.

5. The composition of any one of claims 1 to 4, further comprising starch.

6. The composition of claim 5, wherein the starch is at a concentration no more than 15% weight / total composition weight.

7. The composition of claim 5, wherein the starch is at a concentration between about 5% and about 10%, or between about 5% and about 7% weight / total composition weight.

8. The composition of any one of claims 1 to 7, wherein the composition has a distance to failure of at least 150 mm.

9. The composition of claim 8, wherein the distance to failure is measured on a texture analyzer and the composition is melted for 10 min at 90°C prior to the distance to failure measurement.

10. The composition of any one of claims 1 to 9, wherein the recombinant alpha casein is at a concentration no more than about 11% alpha casein dry weight / total composition weight.

11. The composition of any one of claims 1 to 10, wherein the recombinant alpha casein comprises only alphaS 1 casein.

12. The composition of any one of claims 1 to 11, wherein the composition has no animal-derived caseins.

13. The composition of any one of claims 1 to 12, wherein the composition has no alphaS2 casein.

14. The composition of any one of claims 1 to 13, wherein the recombinant alpha casein is devoid of phosphorylation.

15. The composition of any one of claims 1 to 13, wherein the recombinant alpha casein has substantially reduced phosphorylation.

16. The composition of any one of claims 1 to 15, wherein the recombinant alpha casein comprises an amino acid sequence of a bovine, ovine, or caprine alpha casein.

17. The composition of any one of claims 1 to 16, wherein the recombinant alpha casein comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID Nos: 1 - 20.

18. The composition of any one of claims 3 to 17, wherein the fat comprises a plant- derived fat or oil.

19. The composition of any one of claims 1 to 18, wherein the composition has a moisture content less than about 52%.

20. The composition of claim 19, wherein the moisture content is between about 35% and about 50%.

21. The composition of any one of claims 1 to 20, further comprising salt.

22. The composition of claim 21, wherein the salt is calcium salt, phosphorous salt, citrate salt, potassium salt, sodium salt, or chloride salt, or any combination thereof.

23. The composition of claim 21, wherein the salt is a calcium salt present at a concentration between 0.5-1.5% dry weight / total composition weight.

24. The composition of any one of claims 1 to 23, wherein the composition further comprises a mozzarella, cheddar or parmesan flavoring.

25. A food product comprising the cheese analogue composition of any one of claims 1 to 24.

26. The food product of claim 25, wherein the food product is a pizza.

27. The food product of claim 25, wherein the food product is a frozen food product.

28. The food product of claim 25, wherein the food product is a shredded cheese-like product.

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