Hybrid micelle

Abstract

A hybrid micelle is provided comprising k-casein or a functionally equivalent variant thereof and a plant protein, wherein the plant protein is soluble under basic conditions and insoluble under neutral conditions, and forms a cross-linked micellar network with kappa-casein. The hybrid micelle can be used to prepare a curd and is useful to prepare animal-free food products such as dairy products. A method of preparing the hybrid micelles involves combining the plant protein and the k-casein at a pH that results in solubilization of the proteins, and adjusting the pH of the protein solution to result in dispersion of the proteins into the hybrid micelles.

Description

HYBRID MICELLEField of the Invention

[0001] The present invention generally relates to micelles, and in particular, to micelles comprising casein.Background

[0002] In recent years, the food industry has been in a constant state of change as innovative food products are required to cater to the increasingly complex wants and needs of the consumer. Animal-free dairy and cheese products are examples of such products that are being developed to satisfy a rapidly growing and evolving market.

[0003] Milk comprises casein proteins that are primarily responsible for the unique texture (i.e.; meltability and stretchability) of cheese. There are four different types of caseins in milk: alpha si, alpha s2, beta, and kappa. These four caseins form micelles that are held together by calcium phosphate molecules in a three-dimensional submicellar cross-linked network. The network encapsulates and emulsifies fat and minerals found in milk as well as provides structure to cheese at room temperature. When heat is applied, the calcium bonds that crosslink the network are broken and reformed, giving cheese its ability to melt and stretch when cooked.

[0004] In order to replicate the natural submicellar network found in animal-based cheese, at least two of the four main casein components are believed to be required, specifically alpha si casein and kappa casein. Alpha si casein forms the bulk and core of the micelle, but due to the hydrophobic nature of alpha si, kappa casein, an amphiphilic protein, is required to solubilize the network in water. Kappa casein functions as a surfactant. The hydrophobic region of kappa casein binds and envelopes the alpha si casein, while its hydrophilic region interacts with water, forming a micelle.

[0005] This need for two caseins poses significant technical and economic challenges for the cost-effective production of animal-free dairy products that are functionally equivalent to conventional animal-based products. Manufacturing and processing of two recombinant caseinproteins substantially increases the complexity of production, resulting in cost-prohibitive products.

[0006] Accordingly, it would be desirable to provide a novel, animal-free dairy product that overcomes one or more of the aforementioned disadvantages.Summary

[0007] A novel hybrid micelle has now been developed which is useful in the production of an animal-free product having properties of conventional dairy and cheese products. The micelle comprises kappa casein in combination with a plant protein and yields a submicellar network similar to that of the natural casein micellar network.

[0008] Thus, in one aspect of the invention, a hybrid micelle is provided comprising k- casein and a plant protein, wherein the plant protein is soluble under basic conditions and insoluble under neutral conditions, and the k-casein and plant protein form a cross-linked micellar network

[0009] In another aspect of the invention, a food product is provided comprising a curd prepared from hybrid micelles comprising k-casein and a plant protein in combination with one or more of a fat, a structuring agent, and a plasticising agent.

[0010] In a further aspect, a method of preparing a hybrid micelle is provided comprising: i) combining a plant protein and k-casein at a pH that results in solubilization of both the plant protein and k-casein to form a protein solution; ii) adjusting the pH of the protein solution to result in dispersion of the proteins into kappa casein-plant protein hybrid micelles; and iii) crosslinking and stabilizing the kappa casein-plant protein hybrid micelle.

[0011] These and other aspects of the invention are described in detail herein by reference to the following Figures.Brief Description of the Figures

[0012] Figure 1 graphically illustrates the stretchability of a cheese analogue containing different compositions of hybrid micelles according to embodiments of the invention as compared to bovine micellar casein;

[0013] Figure 1 illustrates the effects of gluten, k-casein, and k-casein: gluten ratio in a hybrid micelle on stretchability of a cheese analogue;

[0014] Figure 3 illustrates A) the amino acid, and B) transcript sequences of bovine k- casein; and

[0015] Figure 4 illustrates the amino acid sequence of human k-casein.Detailed Description

[0016] A hybrid micelle is provided comprising kappa-casein and a plant protein. The plant protein exhibits a kappa-casein-like pH-solubility profile in which it is soluble under basic conditions and insoluble under neutral conditions, and thus, is suitable to form a cross-linked micellar network with kappa-casein.

[0017] Kappa-casein, or k-casein or K-casein, are each used herein to refer to mammalian k-casein, which may be an endogenous protein or a functionally equivalent variant thereof. The term "functionally equivalent variant" as used with respect to k-casein is meant to encompass both naturally and non-naturally occurring variants of an endogenous casein that retains the biological activity of an endogenous casein, e.g. to form casein micelles, and to emulsify milk and milk products. The variant need not exhibit identical activity to a selected endogenous milk protein, but will exhibit sufficient activity for it to function effectively as an emulsifier in milk or milk products, e.g. it will retain at least about 25% of the biological activity of the endogenous milk protein, and preferably at least about 50% or greater of the biological activity of the endogenous milk protein.

[0018] In one embodiment, the k-casein is bovine k-casein such as alpha, beta or kappa casein, or a functionally equivalent variant thereof. Bovine k-casein is a 190 amino acid protein and is encoded by the CSN3 gene. The amino acid sequence of bovine k-casein is provided herein in Fig. 3 A. Nucleic acid-encoding sequences are available, for example, on the NCBI database. A transcript sequence for bovine k-casein is provided under NCBI accession no. NM_174294.2, and is shown in Fig. 3B.

[0019] In another embodiment, the k-casein protein is a human k-casein, or a functionally equivalent variant thereof. The amino acid sequence of human k-casein is provided in Fig. 4.Nucleic acid casein-encoding sequences are available, for example, on the NCBI database. A transcript sequence for human k-casein is provided under NCBI accession no. NM_005212.

[0020] Functionally equivalent variants of a k-casein protein may result naturally from alternative splicing during transcription or from genetic coding differences and may retain significant sequence homology with an endogenous k-casein, e.g. at least about 70% sequence homology, preferably at least about 80% sequence homology, and more preferably at least about 90% or greater sequence homology. Such variants can readily be identified using established cloning techniques employing primers derived from a k-casein. Additionally, such modifications may result from non-naturally occurring synthetic alterations made to a k-casein to yield functionally equivalent variants which may have more desirable characteristics for its use herein, for example, increased activity or stability. Non-naturally occurring variants of a k-casein include analogues, fragments and derivatives thereof.

[0021] A functionally equivalent analogue of a k-casein in accordance with the present invention may incorporate one or more amino acid substitutions, additions or deletions. Amino acid additions or deletions include both terminal and internal additions or deletions to yield a functionally equivalent peptide. Examples of suitable amino acid additions or deletions include those incurred at positions within the protein that are not closely linked to activity. Amino acid substitutions within a k-casein, particularly conservative amino acid substitutions, may also generate functionally equivalent analogues thereof. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine with another non-polar (hydrophobic) residue; the substitution of a polar (hydrophilic) residue with another such as between arginine and lysine, between glutamine and asparagine, between glutamine and glutamic acid, between asparagine and aspartic acid, and between glycine and serine; the substitution of a basic residue such as lysine, arginine or histidine with another basic residue; or the substitution of an acidic residue, such as aspartic acid or glutamic acid with another acidic residue.

[0022] A functionally equivalent fragment in accordance with the present invention comprises a portion of a k-casein sequence which maintains the function of intact k-casein, e.g. with respect to inducing micelle formation. Such biologically fragments of a k-casein can readily be identified using assays useful to evaluate the activity of selected k-casein fragments.

[0023] A functionally equivalent derivative of a k-casein in accordance with the present invention is a k-casein, or an analogue or fragment thereof, in which one or more of the amino acid residues therein is chemically derivatized. The amino acids may be derivatized at the amino or carboxy groups, or alternatively, at the side “R” groups thereof. Derivatization of amino acids within the peptide may render a peptide having more desirable characteristics such as increased stability or activity. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form, for example, amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form, for example, O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids, for example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Terminal derivatization of the protein to protect against chemical or enzymatic degradation is also encompassed including acetylation at the N- terminus and amidation at the C-terminus of the peptide.

[0024] Kappa-casein for use in the present micelles may be a naturally-occurring protein or may be a recombinantly produced protein, both of which are commercially available.

[0025] The present hybrid micelle comprises k-casein cross-linked with a plant protein having a similar pH-solubility profile to k-casein. A suitable plant protein is soluble under basic conditions, i.e. at a pH of 10 or higher, for example, a pH of 10-12, such as 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11,3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, and insoluble under neutral or nearneutral conditions, for example, a pH of from about 5.5 to about 7.5, such as 5.8 to 7.2, or a pH of about 7. A plant protein is soluble at a selected pH when it exhibits a solubility of at least 0.1 g per 100 mL of solvent. A plant protein is insoluble at a selected pH when it exhibits a solubility of less than 0.1 g per 100 mL of solvent and, thus, forms nanoparticles which are preferably stable, for example, 90% of the particles exhibit a particle size distribution of about 1 micron or less for 24 hours following their formation.

[0026] The term “about” is used herein to denote variation from a recited value that would not be expected to have a material effect on functionality, for example, a variation of 10%, either a variation of up to 10% less than the recited value or a variation of by up to 10% more than the recited value.

[0027] The plant protein comprises an amino acid content that promotes cross-linking with casein, for example, through disulfide linkages. In one embodiment, the plant protein comprises a proline amino acid content of at least about 10%, such as 15%, 20%, 25%, 30% or more.

[0028] Suitable plant proteins for use to make hybrid micelles in accordance with the invention include structural proteins and homologous seed storage proteins such as prolamins, for example, in cereal grains such as wheat (including durum, spelt, khorasan, emmer and einkorn), barley, rye, com, sorghum, oat, rice and the like, including cross hybrids such as triticale. Examples of such proteins include gluten, glutenins, gliadins, zein, hordein, secalin, kafirin, avenin, rice protein, legume protein (such as bean, lentil, lupin, peanut, chickpea), soy protein, pea protein, functionally equivalent variants thereof, and / or combinations thereof. In one embodiment, the plant protein is gluten.

[0029] As one of skill in the art will appreciate, the selected protein, including a functionally equivalent variant, may be a natural protein or a synthetically produced protein. As described herein, functionally equivalent variants of the selected plant protein may result naturally from alternative splicing during transcription or from genetic coding differences and may retain significant sequence homology with an endogenous plant protein, e.g. at least about 70% sequence homology, preferably at least about 80% sequence homology, and more preferably at least about 90% or greater sequence homology. Such variants can readily be identified using established cloning techniques employing primers derived from an endogenous protein. Additionally, such modifications may result from non-naturally occurring synthetic alterations made to the plant protein to render functionally equivalent variants which may have more desirable characteristics for its use in the hybrid micelle, for example, increased activity or stability. Non-naturally occurring variants include analogues, fragments and derivatives thereof as above described.

[0030] Hybrid micelles in accordance with the present invention comprise amounts of k- casein and plant protein sufficient to form stable crosslinks. Amounts of plant protein to k-caseinat a ratio in the range of 1 :20 to 1 :2 yield hybrid micelles. In an embodiment, an amount of plant protein to k-casein at a ratio in the range of 1 : 10 to 1 :5 is used to form the present micelles. In an an embodiment, the present hybrid micelle comprises an amount of plant protein ranging from 0.1 to 1% w / v of the micelle solution, and an amount of k-casein ranging from 1 to 2% w / v of the micelle solution.

[0031] The hybrid micelles may be formed by exposure of the k-casein and plant protein solution to conditions that promote the cross-linking thereof. Micelles of kappa casein and plant protein may be formed using conventional methods of micelle formation, by dissolving the k- casein and plant protein in water and combining the solution with a crosslinking solution to form a micellar mixture. As will be appreciated by one of skill in the art, preferred crosslinkers are those suitable for use in edible cheese products.

[0032] In one embodiment, the present hybrid micelles may be formed using enzymatic crosslinking solutions including, for example, an enzymatic crosslinker such as transglutaminase, a laccase, a peroxidase or a tyrosinase.

[0033] In another embodiment, the present hybrid micelles may be formed using chemical crosslinking agents may be used such as glutaraldehyde or tannic acid.

[0034] In another embodiment, the hybrid micelles are formed on addition of divalent cations to promote cross-linking. Suitable cations include calcium (Ca+2) and magnesium (Mg+2) cations. In an embodiment, the cation is calcium. The cation may be added in the form of a solution such as calcium chloride or magnesium chloride. An amount of divalent cation suitable to result in the formation of micelles is combined with the k-casein and plant protein. In an embodiment, the amount of divalent cation is 0.5 to 2.5% w / v of the micelle solution.

[0035] The present hybrid micelles may also be formed via pH-driven micelle induction. In this method, the plant protein and k-casein are combined at a pH that results in solubilization of both the plant protein and k-casein to form a protein solution. The pH may be a basic pH in the range of 10-12. The pH of the protein solution is then adjusted by the addition of acid with stirring to result in dispersion of the proteins into nanoparticles. This is achieved at a pH at or slightly below the isoelectric points of kappa casein and the plant protein. The isoelectric point (pl) of theproteins is the pH at which the protein carries no net electrical charge or is electrically neutral. For example, for hybrid micelles comprising kappa casein and gluten, micelle formation is induced at pH of 5.5-6.0, such as 5.8. Divalent cation solution is added to the dispersion to promote crosslinking and stabilization of the kappa casein-plant protein hybrid micelle.

[0036] An emulsifying agent may be added to the micellar mixture to increase stability of the micelles. Suitable emulsifiers for this purposed include those which are conventionally used for emulsification in micelle preparation such as, but not limited to, mono- or di-glyceride, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, soybean lecithin or salts such as potassium citrate.

[0037] The hybrid micelles may be formed into curds for use in food products using methods known in the art. In particular, curd formation may be induced by adding rennet extract, a suitable microbial (including fungal or bacterial) culture as a substitute for rennet comprising rennet enzymes including chymosin, pepsin and / or lipase, or an acidic solution or substance such as vinegar or lemon juice. Alternatively, the curds may be formed by heating the micellar mixture followed by drying for a sufficient period of time to yield dried curds.

[0038] The present hybrid micelles are useful to form food products, for example, food products that comprise casein micelles such as animal-free dairy products. Examples include, but are not limited to milk-like beverages, yogurt, cheese, sour cream, cottage cheese, and the like.

[0039] To make such food products, the hybrid micelles are formed into curds which may be combined with one or more additional ingredients to provide a product having desirable properties in terms of structure, texture, rheology, composition, flavour and nutrition. Such additional ingredients may include one or more of a structural component, a fat component and / or a plasticizer.

[0040] Alternatively, the micelles may be concentrated and dried for future use using methods known in the art, including heating, evaporating and spray-drying. The powder may then be reconstituted for future use.

[0041] A structural component such as a thickening and / or gelling agent may be combined with curds prepared from the present hybrid micelles to provide structure to the food product. Examples of structural agents include, but are not limited to, starch, microbial and vegetable gums such as alginin, guar gum, locust bean gum, gellan gum, tara gum, Arabic gum, Konjac and xanthan gum; proteins such as collagen, egg white and gelatin; polysaccharides such as agar, carboxymethyl cellulose, pectin and carrageen; and mixtures thereof. The amount of structural agent within the food product will vary with the food product and the properties of the food product. Greater amounts of structuring agent is added to a product requiring more structure, such as cheese, while less structuring agent is required for products that lack structure such as yogurts and beverages. The amount of structuring agent, thus, may be in the range of about 0.1 to 1% by weight of the product.

[0042] The food product may also include a fat component. The fat component may comprise saturated fats, unsaturated fats (either monounsaturated or polyunsaturated) or a mixture thereof. The fat component may be a vegetable fat or oil. Examples of suitable fats or oils include, but are not limited to sunflower oil, canola oil, safflower oil, soybean oil, avocado oil, olive oil, com oil, flaxseed oil, almond oil, coconut oil, peanut oil, pecan oil, cottonseed oil, algal oil, palm oil, palm stearin, palm olein, palm kernel oil, rice bran oil, sesame oil, butteroil, cocoa butter, grape seed oil, hazelnut oil, brazil nut oil, linseed oil, acai palm oil, passion fruit oil, walnut oil, shea butter, shea stearin, shea olein, palm kernel stearin, palm kernel olein, and mixtures thereof. As one of skill in the art will appreciate, the vegetable oils used may vary with respect to their triglyceride content, for example, to provide enhanced oxidative stability. Accordingly, the oil used may be high oleic acid-containing oil such as high-oleic sunflower, high-oleic & high-stearic sunflower oil, high-oleic soybean, high-oleic canola, high-oleic safflower oil, and mixtures thereof. The term “high-oleic acid” refers to an oil containing an increased amount of oleic acid as compared to the typical oleic acid content of the oil. The fat / oil component may comprise about 0.1-25% by weight of the food product.

[0043] The food product may optionally include a plasticizer to increase plasticity and / or decrease viscosity of the product. Suitable plasticizers include food grade plasticizers such as, but not limited to, food grade acids such as levulinic acid, palmitic acid, stearic acid, and oleic acid, food grade carboxylic acids, such as citric, malic, lactic, acetic, oxalic and tartaric acid, glycerol,polyethylene glycol, triethylene glycol, ethylene glycol, sorbitol, sugars such as fructose, galactose and glucose, and mixtures thereof. The plasticizer or mixture of plasticizers may comprise about 0.1-5% by weight of the product.

[0044] The food product may include additional ingredients including, but not limited to, flavorants, colorants, preservatives, anti-oxidants, nutrients, fillers, etc.

[0045] Flavorants may include salt, sugar, spices, herbs, fruit flavourings (e.g. berry, lemon, lime, banana, orange, melon, etc.), vegetable flavourings (e.g. garlic, onion, tomato, cucumber), and savoury flavourings such as beef, chicken or cheese flavouring. Flavorants may also include amino acids that provide a salty, sour, bitter or sweet taste, and free fatty acids such as butyric, lactic and capric acids which provide characteristic cheese flavor.

[0046] Examples of preservatives that may be used include, but are not limited to, sodium benzoate, sodium and calcium propionate, sorbic acid, ethyl formate, and sulfur dioxide.

[0047] Examples of anti-oxidants that may be used include, but are not limited to, ascorbic acid, tocopherols, butylated hydroxyanisole and propyl gallate.

[0048] Nutrients that may be included in the present cheese include vitamins (e.g. vitamin A, C, E, K, D, thiamin (vitamin Bl), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, folic acid (vitamin B9) and / or vitamin B12, and mixtures thereof), minerals (e.g. calcium, phosphorus, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper and mixtures thereof), and protein isolates such as pea protein, soy protein, fava protein, yeast protein and other organisms, corn protein, wheat protein, rice protein, canola protein, peanut protein, bean protein, lentil protein, pumpkin seed, rice, brown rice, peanut, almond, chia seed, flax seed and combinations thereof.

[0049] Such additional ingredients may each be included in the present cheese product in an amount in the range of about 0.01% by weight to about 1% by weight, for example.

[0050] The food product may also include a filler to provide volume / bulk to the product. Examples of suitable fillers include, but are not limited to, consumable inert components such as microcrystalline cellulose, maltodextrin, dextrin, pea protein, soy protein, inulin, sugars and mixtures thereof.

[0051] The balance of the food product is water. The food product comprises at least about30% by wt water, such as at least about 40% by weight, e.g. at least about 50%, 60%, 70% or more. As one of skill in the art will appreciate, water content will vary with the desired characteristics of the end product. For example, beverages will comprise a higher water content (e.g. 80% by weight or more), while creams and cheeses will comprise less water (e.g. less than 80% by weight, such as 40-70% by weight).

[0052] In an embodiment, the hybrid micelles are used to prepare a cheese product. To prepare a cheese analogue, the hybrid micelles are coagulated to form curds. Coagulation may be induced using rennet; however, to produce an animal-free product, non-rennet coagulation may be used employing recombinant chymosin or an acid such as citric acid. The resulting curds are then combined with additional selected ingredients such as a structuring agent, a fat, flavouring and water to yield a desired cheese product. The combined ingredients are heated to form a homogeneous product. The amount of each ingredient used will depend on the properties of the desired end product. For example, for a soft creamy cheese product, less structuring agent(s), more fat and / or more water may be used to provide a less structured creamy cheese product such as brie or cream cheese, while an increased amount of structuring agent and less water may be used to prepare a harder more structured cheese product such as a cheddar or parmesan cheese product. The flavouring ingredients are altered accordingly to result in a combination that results in an appropriate flavour profile for the desired cheese product.

[0053] The content of the micelles also has an impact on the properties of the resulting cheese product. For example, varying the plant protein, or combination of plant proteins, in the present hybrid micelles used to make curd results in a cheese product having different properties, such as more or less stretch. In one embodiment, the present hybrid micelles are made with gluten combined with k-casein to result in curds useful to make a stretchy cheese product, while the use of other plant proteins combined with k-casein, such as lentil, soy or pea protein, result in micelles that form a curd for use in a product with little or no stretch, e.g. less than 3 cm of stretch, such as less than 2 cm or less than 1 cm. Stretch of a cheese product is determined by pulling the final product in a single direction, or 2 opposing directions to determine the length the product will expand or stretch before breaking.

[0054] Micelles formed having higher plant protein content, e.g. gluten content, result in a cheese product with greater stretch, e.g. greater than 3 cm, for example, greater than 5 cm, 10 cm, 15 cm, 20 cm, or greater. The amounts of plant protein to k-casein in micelles for use to prepare a cheese product may be in the range of 1 :2 to 1 :200, for example, 1 :5 to 1 : 150. In one embodiment, a cheese product was prepared with micelles comprising 0.1-2% w / w gluten and 10- 15% w / w k-casein.

[0055] The method of making the micelles also has an impact on the properties of the product made using the hybrid micelles. For example, hybrid micelles prepared by a pH-driven method of micelle formation result in curds that yield a product with greater stretch and therefore useful in a cheese product that mimics a cheese such as mozzarella or cheddar cheese. On the other hand, micelles made using a conventional method of micelle formation result in curds that yield a product with little or no stretch, and thus, may be used to make a product that mimics a non-stretching cheese such as parmesan, brie or asiago.

[0056] Embodiments of the invention are described in the following specific examples which are not to be construed as limiting.Example 1

[0057] Conventional and novel casein micelles were prepared using the following methods.

[0058] Conventional micelle induction: Alpha-casein and k-casein were dissolved in water at a final mixture volume of 1.84% w / v and 0.46% w / v, respectively. The following stock solutions were sequentially mixed into the casein dispersion: 32% w / v CaCh, 20% w / v K2HPO4, and 20% w / v K3 citrate stock solutions to result in a final concentration of 0.21% w / v CaCh, 0.22% w / v K2HPO4, and 0.20% w / v K3 citrate in the final micellar mixture. The micellar composition is summarized in Table 1. The pH of the mixture was adjusted to 5.5 using 6.65% w / v citric acid solution. Rennet solution (0.15%) (microbial rennet derived from Rhizomucor miehei mold) was added at 1.36% v / v of mixture volume and gently mixed. The mixture was left undisturbed for 30 minutes until a curd formed.Table 1. Composition of a-casein and k-casein micelle solutionComponents Concentration (%w / v) FunctionCaCl20.21 crosslinkerMgCl20.05 crosslinkerK3 citrate 0.20 chelator, emulsifying saltK2HPO4 0.22 buffer k-casein 0.46 a-casein 1 84 water 97.03 solvent

[0059] Hybrid micelle production using conventional micelle induction: Hybrid micelles were prepared with various combinations of k-casein and different plant proteins (as shown in Table 3) using conventional micelle induction as described above. Table 3. Curd formation / stretchability Curd formation and strength were qualitatively evaluated to compare against curds made from rennetted casein derived from cow milk. Curds were dehydrated for 7 hours at 70 deg C and stored at ambient temperature until further processing into cheese analogue or other dairy products.

[0060] Cheese analogue preparation: Dried curds (28-30%w / w) were mixed with a slurry of palm shortening, starch, citric acid, K3 citrate, sodium chloride, and sodium alginate at concentrations as set out in Table 2 to form a cheese analogue. The cheese analogue was cooked at 85 deg C until soft, homogenous, and stretchy. The cheese analogue was rested at 4 deg C for 24 hours, and then heated at 200 deg C until melted.Table 2. Composition of cheese analogue slurry to be mixed with curds

[0061] Stretchability of the cheese analogue was evaluated to compare its performance to a cheese analogue containing casein derived from cow milk by melting one gram of cheese analogue shaped into a ball at 300 deg C and measuring the length of the intact / contiguous cheese strand pulled with a small flat spatula when the sample temperature was at 80 - 90 deg C. Results are shown in Table 3. No stretching was observed in cheese analogue made with curd of hybrid micelles prepared by conventional methods. Cheese analogue samples made from micellar casein showed stretchability of 18 - 22 cm.Table 3. Curd formation / stretchability of rennetted k-casein and plant protein mixtures after melting

[0062] Screening of plant proteins for use in pH-driven micelle induction: The solubility of each plant protein at high pH was determined by dissolving the protein in water having a pH of 11.5 (200 mg per 10 mL of water). Once solubilized, each plant protein solution was titrated to pH 5.8 with 3% w / v citric acid to induce nanoparticle formation of the plant protein. Stability of the nanoparticles was evaluated based on the lack of sedimentation or precipitation after standing for 24 hours. The results are shown in Table 4.Table 4. Plant protein solubility at pH 11.5 and stability of their nanoparticle dispersions at pH 5.8Plant protein Solubility at pH 11.5 Stability at pH 5.81(%w / v) brown rice protein o.5 no pea protein isolate o.5 no zein >1.0 yes faba protein isolate i.o novital wheat gluten 1 0 no soy protein >2.0 yes lentil protein >2.0 no

[0063] pH-driven hybrid micelle induction: Plant proteins exhibiting solubility at pH 11.5, including zein, soy protein, lentil protein and wheat gluten, were then used to prepare hybrid micelles. Plant protein and k-casein solutions were dissolved in water at pH 11.5 at concentrations according to Table 5 and mixed at 1000 rpm. The pH of the solution was gradually titrated to pH 5.6 with 3% w / v citric acid with stirring at 1000 rpm. Calcium chloride stock solution (32% w / v) was added to the solution while stirring at 1000 rpm for 5 minutes to reach a final concentration as set out in Table 5. The mixture was heated for 1 hour at 85 deg C with stirring. The mixture was dehydrated for 6 hours at 70 deg C to form dried curds and was stored at ambient temperature until further processing into cheese analogue or other dairy products.

[0064] Cheese analogues containing dried curds of hybrid micelles of k-casein and plant proteins (prepared by pH-driven micelle induction) were prepared by combination with a cheese analogue slurry as described above (Table 2). Stretchability of the resulting cheese analogue after heating at 200 deg C until melted were evaluated to determine performance against cheese analogue containing casein derived from cow milk as shown in Fig. 1.Table 5. Stretchability of cheese analogues containing hybrid micelles from pH-driven micelle induction

[0065] As shown, each of the proteins resulted in a cheese analogue that exhibited some stretch. Surprisingly, wheat gluten exhibited greater stretch despite exhibiting somewhat less solubility at pH 11.5.

[0066] Cheese analogues containing hybrid micelles of k-casein and gluten: Cheese analogue compositions predicted to result in composition with stretch were validated and theresults of their stretching performance determined to identify various compositions that yield acceptable results. Results are tabulated in Tables 6 - 9 below and summarized in Fig. 2.Table 6. Compositions (%w / w) of cheese analogues containing hybrid micelle of k-casein and gluten composition gluten k-casein calcium fat starch acid citrate sodium alginate water stretch (cm)5.54% 14.38% 14.18% 21.37% 5.44% 0.26% 1.01% 0.64% 0.07% 37.11%2 3.11% 12.83% 15.04% 18.44% 7.02% 0.26% 0.76% 0.62% 0.02% 41.90%3 4.19% 13.28% 11.85% 21.66% 6.74% 0.25% 1.35% 0.78% 0.06% 39.84%4 5.46% 15.41% 12.63% 21.83% 4.07% 0.21% 1.50% 0.54% 0.03% 38.32%5 5.28% 13.37% 15.31% 17.26% 4.31% 0.20% 0.99% 0.69% 0.08% 42.49%6 3.76% 15.68% 12.89% 21.48% 6.44% 0.31% 0.90% 0.47% 0.06% 38.01%7 3.49% 14.61% 13.98% 22.02% 4.45% 0.31% 1.19% 0.76% 0.02% 39.17%8 5.73% 14.59% 13.13% 17.51% 6.67% 0.24% 1.13% 0.77% 0.00% 40.23%9 2.61% 13.13% 14.38% 23.77% 6.05% 0.18% 1.58% 0.56% 0.01% 37.73%10 4.48% 12.08% 12.70% 18.32% 7.70% 0.16% 1.21% 0.62% 0.04% 42.69%11 4.16% 14.81% 10.21% 19.30% 6.96% 0.24% 1.39% 0.43% 0.07% 42.42%12 3.00% 16.46% 11.37% 17.12% 5.49% 0.21% 1.58% 0.61% 0.05% 44.11%13 2.16% 15.31% 12.16% 22.39% 5.94% 0.35% 1.55% 0.53% 0.08% 39.53%14 2.73% 12.66% 10.34% 23.63% 4.85% 0.29% 1.35% 0.44% 0.05% 43.67%15 3.51% 13.16% 9.68% 24.09% 8.48% 0.31% 1.07% 0.65% 0.00% 39.04%16 2.44% 16.32% 11.25% 18.24% 8.16% 0.25% 1.44% 0.82% 0.06% 41.03%17 1.59% 13.60% 11.36% 21.48% 6.44% 0.19% 1.05% 0.57% 0.05% 43.67%18 1.59% 15.36% 8.63% 20.77% 7.79% 0.26% 1.08% 0.56% 0.01% 43.93%19 1.87% 14.15% 10.39% 19.02% 5.57% 0.26% 1.72% 0.81% 0.04% 46.17%20 3.63% 16.78% 11.13% 20.76% 4.86% 0.19% 0.88% 0.44% 0.02% 41.30%Table 7. Compositions (%w / w) of cheese analogues containing hybrid micelle of k-casein and gluten composition gluten k-casein calcium fat starch acid citrate sodium alginate water stretch (cm)21 0.60% 14.37% 11.93% 24.94% 4.58% 0.21% 0.69% 0.72% 0.07% 41.88% 1222 2.21% 14.52% 10.73% 21.12% 8.47% 0.20% 0.87% 0.69% 0.07% 41.10% 423 3.51% 13.71% 7.54% 23.47% 7.45% 0.20% 0.77% 0.72% 0.03% 42.61% 224 2.73% 12.45% 12.74% 19.77% 5.46% 0.18% 0.71% 0.57% 0.06% 45.33% 625 2.01% 12.65% 14.01% 21.17% 6.87% 0.18% 0.71% 0.59% 0.05% 41.77% 1426 3.82% 14.47% 5.83% 23.39% 7.15% 0.21% 0.85% 0.56% 0.08% 43.63% 027 1.31% 13.69% 9.14% 23.16% 8.85% 0.20% 0.79% 0.52% 0.04% 42.29% 428 2.69% 13.76% 9.51% 22.76% 6.68% 0.20% 0.80% 0.61% 0.06% 42.94% 229 1.47% 13.03% 13.21% 17.21% 7.87% 0.19% 0.78% 0.56% 0.02% 45.67% 1030 4.32% 14.17% 9.31% 21.28% 5.81% 0.21% 0.94% 0.67% 0.08% 43.21% 2Table 8. Compositions (%w / w) of cheese analogues containing hybrid micelle of k-casein and gluten composition gluten k-casein calcium fat starch acid citrate sodium alginate water stretch (cm)31 1.51% 12.65% 13.88% 20.98% 6.66% 0.17% 0.62% 0.61% 0.05% 42.86% 1032 1.14% 12.63% 14.53% 21.55% 6.93% 0.18% 0.54% 0.64% 0.07% 41.79% 19±333 1.38% 13.85% 13.76% 22.95% 6.26% 0.17% 0.66% 0.62% 0.05% 40.28% 634 1.60% 13.02% 14.97% 22.83% 3.41% 0.19% 0.65% 0.65% 0.08% 42.60% 1235 1.64% 12.47% 14.20% 22.28% 6.46% 0.18% 0.67% 0.62% 0.06% 41.42% 636 1.38% 12.55% 14.53% 22.55% 4.92% 0.19% 0.67% 0.67% 0.06% 42.48% 1037 1.85% 14.08% 14.98% 21.92% 3.66% 0.12% 0.61% 0.64% 0.05% 42.10% 1038 1.84% 14.04% 14.58% 23.02% 3.92% 0.28% 0.57% 0.59% 0.09% 41.08% 839 1.80% 13.41% 13.95% 20.63% 6.05% 0.18% 0.55% 0.57% 0.08% 42.77% 1040 1.87% 13.03% 14.65% 23.55% 3.79% 0.19% 0.66% 0.53% 0.08% 41.65% 6Table 9. Compositions (%w / w) of cheese analogues containing hybrid micelle of k-casein and gluten composition gluten k-casein calcium fat starch acid citrate sodium alginate water stretch (cm)41 0.11% 11.89% 15.74% 21.05% 6.29% 0.22% 0.38% 0.74% 0.07% 43.50% 18±242 0.12% 12.18% 15.45% 20.14% 7.39% 0.12% 0.42% 0.71% 0.06% 43.43% 16±443 0.16% 12.90% 15.53% 20.87% 8.80% 0.17% 0.54% 0.74% 0.06% 40.24% 18±2Micellar casein 20±2

[0067] It was determined that stretch of a cheese analogue may be varied based on the relative amounts of gluten (plant protein) and k-casein used to form the micelles. In embodiments, amounts of gluten up to about 6%w / w combined with amounts of k-casein in the range of 10- 20%w / w resulted in products with varying amounts of stretch, with gluten of less than 3%w / w resulting in products with greater stretch.Example 2

[0068] Rennetted ternary mixtures of k-casein and various combinations of two plant proteins were prepared from micelles formed using conventional micelle induction and then evaluated for curd formation and stretchability at 80 deg C and after melting. Combinations of plant proteins were mixed at 1 : 1 ratio (total concentration of 0.92% w / v for each plant protein). Note: BR - Brown Rice, F - Faba Bean, P - Pea Protein Isolate, PB - Peanut Butter, PS - Pumpkin Seed, and Z - Zein. Results are shown on Table 10. These combinations resulted in hybridmicelles that formed curds, with little or no stretch. Some combinations exhibited weak stretch at 80 deg C, but no stretch following melting.Table 10.2-combination ofCurd Formation Stretch at 80 deg C Stretch after Melt proteins

[0069] Rennetted quaternary mixtures of k-casein, zein, faba bean protein, and various gums (structural agent in an amount of 5% by wt) were evaluated for curd formation and stretchability at 80 deg C and after melting as shown in Table 11. These combinations resulted in hybrid micelles that formed curds, with little or no stretch.Table 11.Gum Curd Formation Stretch at 80 deg C Stretch after Melt

[0070] Cheese analogue compositions of k-casein, lentil, and faba bean hybrid micelle formed using conventional micelle induction were prepared according to Table 3 and their ability to stretch was determined. Results are shown in Table 12.Table 12. composition CaC12 MgC12 K3 citrate K2HPO4 k-casein lentil faba water stretch length (cm)1 0.75% 1.25% 1.25% 1.25% 1.35% 2.50% 2.50% 89.15% 0.702 0.75% 0.25% 0.25% 0.25% 0.27% 1.25% 0.00% 96.98% 0.813 1.25% 0.75% 0.25% 0.25% 1.35% 1.25% 2.50% 92.40% 0.754 0.25% 0.75% 1.25% 1.25% 0.27% 2.50% 0.00% 93.73% 0.005 1.25% 1.25% 0.75% 0.25% 0.27% 2.50% 0.00% 93.73% 1.006 0.25% 0.25% 0.75% 1.25% 1.35% 1.25% 2.50% 92.40% 0.007 1.25% 1.25% 1.25% 0.75% 0.27% 1.25% 2.50% 91.48% 1.158 0.25% 0.25% 0.25% 0.75% 1.35% 2.50% 0.00% 94.65% 0.629 1.25% 0.25% 1.25% 1.25% 0.81% 1.25% 0.00% 93.94% 1.1610 0.25% 1.25% 0.25% 0.25% 0.81% 2.50% 2.50% 92.19% 0.6811 1.25% 1.25% 0.25% 1.25% 1.35% 1.88% 0.00% 92.78% 1.1512 0.25% 0.25% 1.25% 0.25% 0.27% 1.88% 2.50% 93.36% 0.6113 1.25% 0.25% 1.25% 0.25% 1.35% 2.50% 1.25% 91.90% 0.0014 0.25% 1.25% 0.25% 1.25% 0.27% 1.25% 1.25% 94.23% 0.9015 1.25% 0.25% 0.25% 1.25% 0.27% 2.50% 2.50% 91.73% 0.5516 0.25% 1.25% 1.25% 0.25% 1.35% 1.25% 0.00% 94.40% 0.0017 0.75% 0.75% 0.75% 0.75% 0.81% 1.88% 1.25% 93.07% 1.2118 0.75% 0.75% 0.75% 0.75% 0.81% 1.88% 1.25% 93.07% 1.2719 0.75% 0.75% 0.75% 0.75% 0.81% 1.88% 1.25% 93.07% 1.0820 0.75% 0.75% 0.75% 0.75% 0.81% 1.88% 1.25% 93.07% 1.2421 0.50% 1.25% 0.25% 0.75% 2.00% 1.70% 0.00% 93.55% 1.0022 0.50% 1.25% 0.25% 0.75% 4.00% 1.70% 0.00% 91.55% 0.0023 2.00% 1.25% 0.25% 0.75% 2.00% 1.70% 0.00% 92.05% 0.0024 2.00% 1.25% 0.25% 0.75% 4.00% 1.70% 0.00% 90.05% 0.0025 1.25% 1.25% 0.25% 0.75% 2.00% 1.70% 0.00% 92.80% 1.8526 1.25% 1.25% 0.25% 0.75% 2.00% 1.70% 0.00% 92.80% 0.861.25% 1.25% 0.25% 0.75% 4.00% 1.70% 0.00% 90.80% 0.0028 1.25% 1.25% 0.25% 0.75% 4.00% 1.70% 0.00% 90.80% 0.0029 0.50% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 92.55% 1.2230 2.00% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 91.05% 0.0031 0.50% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 92.55% 0.0032 2.00% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 91.05% 0.0033 1.25% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 91.80% 0.9634 1.25% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 91.80% 0.931.25% 1.25% 0.25% 0.75% 3.00% 1.70% 0.00% 91.80% 0.00

[0071] Cheese analogue compositions comprising k-casein, pumpkin seed, and faba bean hybrid micelles, prepared using conventional induction, were prepared according to Table 3 and their ability to stretch determined as set out in Table 13.Table 10 compositio CaCl stretch length n 2 MgC12 K3Citrate K2HPO4 kcasein pumpkin faba water (cm)1 0.11% 0.00% 0.00% 0.22% 0.16% 0.22% 0.36% 98.93% o2 0.22% 0.22% 0.02% 0.16% 0.13% 0.36% 0.51% 98.38% 03 0.07% 0.25% 0.09% 0.24% 0.24% 0.18% 0.18% 98.75% 04 0.13% 0.20% 0.20% 0.09% 0.07% 0.55% 0.47% 98.29% 05 0.20% 0.05% 0.04% 0.04% 0.02% 0.29% 0.22% 99.15% 06 0.15% 0.16% 0.13% 0.02% 0.20% 0.00% 0.33% 99.02% 07 0.05% 0.02% 0.18% 0.05% 0.15% 0.04% 0.55% 98.96% 08 0.00% 0.18% 0.05% 0.15% 0.00% 0.15% 0.44% 99.04% 09 0.25% 0.27% 0.07% 0.11% 0.11% 0.33% 0.00% 98.85% 010 0.04% 0.11% 0.22% 0.07% 0.04% 0.25% 0.04% 99.24% 011 0.24% 0.07% 0.16% 0.27% 0.09% 0.47% 0.15% 98.55% 012 0.18% 0.04% 0.25% 0.25% 0.22% 0.11% 0.07% 98.87% 013 0.02% 0.13% 0.27% 0.20% 0.18% 0.44% 0.40% 98.36% 014 0.16% 0.15% 0.15% 0.00% 0.25% 0.40% 0.25% 98.64% 015 0.09% 0.09% 0.11% 0.13% 0.27% 0.51% 0.11% 98.69% 016 0.27% 0.24% 0.24% 0.18% 0.05% 0.07% 0.29% 98.65% 0

[0072] Cheese analogue compositions comprising k-casein, zein, and faba bean hybrid micelle were prepared according to Table 3 using conventional micelle induction and their ability to stretch was determined as set out in Table 14.Table 11 composition CaC12 MgC12 KJCitrate K2HPO4 kcasein zein faba water stretch length (cm)1 0.28% 0.04% 0.16% 0.41% 0.36% 0.24% 0.40% 98.12% 02 0.37% 0.27% 0.14% 0.31% 0.28% 0.40% 0.56% 97.67% 03 0.31% 0.33% 0.33% 0.50% 0.52% 0.20% 0.20% 97.60% 04 0.21% 0.24% 0.29% 0.18% 0.16% 0.60% 0.52% 97.81% 05 0.24% 0.06% 0.06% 0.06% 0.04% 0.32% 0.24% 98.98% 0.6197186 0.36% 0.23% 0.33% 0.23% 0.44% 0.00% 0.36% 98.06% 0.845077 0.20% 0.05% 0.34% 0.21% 0.32% 0.04% 0.60% 98.23% 08 0.00% 0.20% 0.06% 0.16% 0.00% 0.16% 0.48% 98.94% 09 0.39% 0.33% 0.18% 0.23% 0.24% 0.36% 0.00% 98.27% 010 0.08% 0.13% 0.28% 0.12% 0.08% 0.28% 0.04% 99.00% 1.59154911 0.35% 0.10% 0.27% 0.39% 0.20% 0.52% 0.16% 98.01% 012 0.41% 0.09% 0.49% 0.51% 0.48% 0.12% 0.08% 97.82% 013 0.20% 0.18% 0.47% 0.41% 0.40% 0.48% 0.44% 97.42% 014 0.43% 0.22% 0.40% 0.26% 0.56% 0.44% 0.28% 97.41% 015 0.37% 0.16% 0.38% 0.42% 0.60% 0.56% 0.12% 97.39% 016 0.35% 0.27% 0.31% 0.26% 0.12% 0.08% 0.32% 98.29% 0.422535

Claims

CLAIMS1. A hybrid micelle comprising k-casein or a functionally equivalent variant thereof and a plant protein, wherein the plant protein is soluble under basic conditions and insoluble under neutral conditions, and forms a cross-linked micellar network with kappa-casein2. The hybrid micelle of claim 1, wherein the plant protein comprises a proline content of at least about 10%.

3. The hybrid micelle of claim 1, wherein the plant protein is a structural protein from cereal grains.

4. The hybrid micelle of claim 1, wherein the plant protein is selected from the group of gluten, glutenins, gliadins, zein, hordein, secalin, kafirin, avenin, rice protein, legume protein, pea protein and combinations thereof.

5. The hybrid micelle of any one of claims 1-4, wherein the k-casein is bovine k-casein.

6. The hybrid micelle of any one of claims 1-5, wherein the amount of plant protein to k- casein is at a ratio in the range of 1 :20 to 1 :2.

7. The hybrid micelle of any one of claims 1-6, wherein the amount of plant protein ranges from 0.1 to 1% w / v of the micelle solution, and the amount of k-casein ranges from 1 to 2% w / v of the micelle solution.

8. The hybrid micelle of any one of claims 1-7, wherein the cross-linked micellar network forms in the presence of divalent cations.

9. The hybrid micelle of claim 8, wherein the divalent cation is a calcium cation, a magnesium cation or a mixture thereof.

10. The hybrid micelle of claim 8 or 9, wherein the plant protein comprises gluten and the divalent cation is calcium.

11. The hybrid micelle of any one of claims 1-10, wherein the cross-linked micellar network is formed at a pH at or below the isoelectric points of kappa casein and the plant protein.

12. Curd prepared from hybrid micelles as defined in any one of claims 1-11.

13. A food product comprising curd as defined in claim 12 in combination with one or more of a fat, a structuring agent, and a plasticising agent, and optionally flavorants, colorants, preservatives, anti-oxidants, nutrients and / or fillers.

14. The food product of claim 13, which is a beverage, yogurt, cheese, sour cream, or cottage cheese.

15. The food product of claim 13, which is a cheese product.

16. The food product of claim 15, comprising gluten.

17. The food product of claim 15 or 16, wherein the cheese product exhibits stretch.

18. The food product of claim 17, which exhibits stretch of at least 3 cm.

19. A method of preparing a hybrid micelle comprising: i) combining a plant protein and k- casein at a pH that results in solubilization of both the plant protein and k-casein to form a protein solution; and ii) adjusting the pH of the protein solution to result in dispersion of the proteins into kappa casein-plant protein hybrid micelles.

20. The method of claim 19, additionally comprising the step of stabilizing the kappa caseinplant protein hybrid micelle by addition of a cation solution.

21. The method of claim 19 or 20, wherein the plant protein and k-casein are solubilized at a pH of at least about 11.

22. The method of any one of claims 19 to 21, wherein in step ii) the pH is adjusted to a pH at or below the isoelectric pH of the protein and k-casein to form the hybrid micelles.

23. The method of any one of claims 19-22, wherein the amount of plant protein to k-casein is at a ratio in the range of 1 :20 to 1 :2.

24. The method of any one of claims 19-23, wherein the plant protein comprises a proline content of at least about 10%.

25. The method of any one of claims 19-24, wherein the plant protein is gluten.

26. The method of claim 25, wherein in step ii) the pH is adjusted to a pH of about 5.8.

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