Composition of recombinant casein with plant material

Abstract

This invention relates to compositions comprising: (a) micelles dispersed in an aqueous solution, wherein the micelles comprise recombinant proteins of αS1 casein, αS2 casein, β casein, and κ casein; and (b) plant-derived material. A method for preparing the compositions is further provided.

Description

[0001] Cross-references to related applications This application claims priority to Israeli Patent Application No. 308452, filed November 9, 2023, entitled “Composition of Recombinant Caseins and Plant Material,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of recombinant proteins and their use in the production of milk-like micelles in the presence of plant materials. Background Technology

[0003] Cow's milk accounts for the vast majority of the global dairy market, while in the United States, plant-based alternatives account for $1 billion and lactose-free milk is estimated to be worth $700 million. Four specific caseins are known in cow's milk: αS1 casein, αS2 casein, β-casein, and κ-casein. Milk produced by mammals is a highly complex fluid comprising thousands of components (e.g., if all triglycerides were identified). While many consider mammal-produced milk (such as cow's milk) to be an ideal source of nutrition, various milk alternatives to mammal-produced milk (e.g., cow's milk), such as plant-based or nut-based milks (e.g., soy milk, almond milk, or coconut milk), are sought after due to reasons related to the allergenicity of mammal-produced milk, lactose intolerance of certain components, personal preferences, and perceived environmental benefits of reducing the dairy industry.

[0004] Existing dairy milk alternatives (such as soy milk, almond milk, or coconut milk) fall short in both flavor and functionality; moreover, the industrial and cultural importance of dairy milk stems in large part from its practicality in derivative products (such as cheese, yogurt, cream, or butter). While non-dairy plant-based milks address environmental and health concerns (and simultaneously provide a desired flavor for a small segment of the population), almost none of them can be used to create such derivative products.

[0005] There remains a great need for dairy substitutes or compositions that have the desired flavor and performance characteristics, such as micelle-like particles made from recombinant casein and plant materials. Summary of the Invention

[0006] According to a first aspect, a composition is provided comprising: (a) micelles dispersed in an aqueous solution, wherein the micelles comprise recombinant proteins of αS casein, β casein and κ casein; and (b) plant-derived material.

[0007] According to another aspect, a method for preparing the composition of the present invention is provided, the method comprising mixing recombinant proteins of αS casein, β casein and κ casein with plant-derived materials to prepare the composition.

[0008] In some embodiments, the recombinant proteins of αS casein, β casein and κ casein are present in the composition at a weight / weight ratio (w / w) of 2:2:1 to 6:6:1.

[0009] In some embodiments, the αS casein includes αS1 casein, αS2 casein, or both.

[0010] In some embodiments, the recombinant protein is a plant-based recombinant protein.

[0011] In some embodiments, the plant-derived material includes extracts, homogenates, any fractions thereof, or any combination thereof derived from plants.

[0012] In some embodiments, the composition further comprises calcium ions.

[0013] In some embodiments, the calcium ions are present in the composition at a concentration ranging from 2 mM to 100 mM.

[0014] In some embodiments, the calcium ions are present in the composition in the form of CaCl2.

[0015] In some embodiments, the composition further comprises phosphate ions, polyphosphate ions, or combinations thereof.

[0016] In some embodiments, the composition further comprises at least one ion selected from the following: Zn 2+ Mg 2+ Cu 2+ Na + K + Fe 2+ Fe 3+ and any combination thereof.

[0017] In some embodiments, the composition comprises a plurality of micelles of the micelles, and is characterized in that the average zeta potential ranges between -17 mV and 6 mV.

[0018] In some embodiments, the composition is characterized by an average zeta potential range between -14 mV and -8 mV.

[0019] In some embodiments, the plurality of micelles are characterized by an average particle size range between 100 nm and 250 nm, between 300 nm and 500 nm, or both.

[0020] In some embodiments, the micelles are substantially similar to micelles in milk, optionally wherein the milk is cow's milk.

[0021] In some embodiments, the plurality of micelles are characterized in that any one of the following is at least 90% identical to the latex micelles: size, average size or maximum size, diameter, average diameter or maximum diameter, taste, flavor, odor, sensory properties or any combination thereof.

[0022] In some embodiments, the method includes obtaining a plurality of micelles dispersed in an aqueous solution, wherein each of the plurality of micelles comprises a recombinant protein of the αS casein, β casein, and κ casein.

[0023] In some embodiments, the recombinant proteins of αS casein, β casein and κ casein are mixed in the plurality of micelles of the composition at a final weight / weight ratio (w / w) of 2:2:1 to 6:6:1.

[0024] In some embodiments, the mixing is carried out in the presence of at least one ion selected from the group consisting of: Ca 2+ Phosphate, polyphosphate, Zn 2+ Mg 2+ Cu 2+ Na + K + Fe 2+ Fe 3+ and any combination thereof.

[0025] In some embodiments, the mixing includes mixing under at least one of the following conditions: heating, cooling, ultrasonic treatment, electrolysis, and any combination thereof.

[0026] In some embodiments, the recombinant protein of the plurality of micelles of the composition is a plant-based recombinant protein.

[0027] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of any conflict, the patent specification (including definitions) shall prevail. Furthermore, these materials, methods, and examples are exemplary only and are not intended to be limiting.

[0028] Other embodiments of the invention and its full scope will become apparent from the detailed description given below. However, it should be understood that while preferred embodiments of the invention have been indicated, the detailed description and specific examples are given by way of example only, and various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description. Attached Figure Description

[0029] Figures 1A-1C Charts showing DLS studies of individual α-casein (1A), β-casein (1B), and κ-casein (1C).

[0030] Figures 2A-2C Includes charts showing isothermal titration calorimetry (ITC) studies, which show the critical micelle concentrations of individual α-casein (2A), β-casein (2B), and κ-casein (2C).

[0031] Figure 3 Includes charts showing ITC studies involving the titration of α-casein into β-casein.

[0032] Figure 4 Includes charts from an ITC study showing micellar assembly obtained by adding κ-casein to a mixture of α-casein and β-casein.

[0033] Figure 5 Includes tables and charts from the DLS study, which showed that all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) assembled into micelles (without any other additives).

[0034] Figures 6A-6B Charts and tables, including those from the ITC (6A) and DLS (6B) studies, show that all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) assemble into micelles. A saturated calcium phosphate solution was titrated into the casein mixture.

[0035] Figures 7A-7B Charts and tables, including those from the ITC (7A) and DLS (7B) studies, show that all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) assemble into micelles. The casein mixture was titrated into a saturated calcium phosphate solution.

[0036] Figures 8A-8DThe chart includes a diagram from the DLS study, which tested the effects of different calcium ion sources on the assembly of all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) into micelles. (8A) Casein only; (8B) Casein with calcium phosphate; (8C) Casein with calcium chloride; and (8D) Casein with both calcium chloride and calcium phosphate.

[0037] Figure 9 Includes photographs and images of protein blot analysis showing the effects of heat treatment and the addition of food-grade salt on the assembly of all four casein species (αS1 casein, αS2 casein, β casein, and κ casein) into micelles. A - Casein solution only; B - Casein solution with added salt; M - Molecular size marker.

[0038] Figure 10 Includes charts and tables from the DLS study, which shows the effects of heat treatment and the addition of food-grade salt on the assembly of all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) into micelles.

[0039] Figure 11 Includes tables, charts, and photographs of a Nile Red staining assay that shows the effect of added salt on the assembly of all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) into micelles.

[0040] Figure 12 This includes a table summarizing the zeta potential measurements of individual casein micelles and micelles formed in the presence of additives, including all casein (αS1 casein, αS2 casein, β casein, and κ casein).

[0041] Figure 13 Includes micrographs obtained by cryogenic transmission electron microscopy (TEM), showing micelles containing all caseins (αS1 casein, αS2 casein, β casein, and κ casein) formed in the presence of saturated calcium phosphate and 0.2% calcium chloride. Scale bars = 0.5 µm (top left), 200 nm (top right), or 100 nm (bottom).

[0042] Figure 14 Includes charts showing the interaction between lettuce protein and casein and their potential impact on micellization.

[0043] Figures 15A-15CThe chart includes a diagram from an ITC study showing the interaction of recombinant casein with plant material in the absence of additives (15A) or in the presence of additives (15B-15C). (15A) All caseins (αS1 casein, αS2 casein, β casein, and κ casein) and lettuce extract; (15B) All caseins, lettuce extract, calcium phosphate, and calcium chloride; and (15C) All caseins, lettuce extract, and calcium chloride.

[0044] Figures 16A-16C The charts include those from the DLS study, which showed micellization of recombinant casein in the presence of plant material and additives. (16A) Lettuce extract and calcium chloride; (16B) Micelleization of all casein in lettuce extract; and (16C) Micelleization of all casein in lettuce extract and calcium chloride.

[0045] Figures 17A-17C Figures include those from the DLS study, which showed micellization of recombinant casein in the presence of plant material and regulated salt concentrations. (17A) Micelleization of all casein in lettuce extract; and (17B) Micelleization of all casein in calcium phosphate and calcium chloride; and (17C) Micelleization of all casein in lettuce extract and calcium chloride.

[0046] Figures 18A-18B Includes cryo-TEM micrographs showing micellar mimicry of natural latex in plant material from recombinant casein. (18A) Natural latex micelles (control); and (18B) Micelle formation of all casein in lettuce extract and calcium chloride. Scale bar = 100 nm.

[0047] Figure 19 Includes a table summarizing the average size (nm) and zeta potential (mV) of “milk-like” micelles or particles obtained with the following samples: (1) (α+β+κ) + lettuce extract + 0.1% CaCl2 in phosphate buffer; (2) (α+β+κ) + lettuce extract + 0.2% CaCl2 in phosphate buffer; (3) (α+β+κ) + lettuce extract + 0.25% CaCl2 in phosphate buffer; and (4) lettuce extract in phosphate buffer, serving as a control.

[0048] Figure 20 Includes a cryo-TEM micrograph showing micelles obtained from Noga lettuce expressing four casein proteins. Scale bar = 200 nm.

[0049] Figure 21Includes tables and photographs from a DLS study showing the effect of CaCl2 concentration on micellization. CaCl2 was applied at concentrations of 0 mM (negative control), 18 mM, 45 mM, 72 mM, 90 mM, 108 mM, and 180 mM.

[0050] Figure 22 Includes tables and photographs from a DLS study showing the effect of lettuce material concentration on micellization. Noga lettuce material was applied at concentrations of 0.742%, 1.484%, 2.968%, 4.452%, and 5.935%. 0% served as a negative control.

[0051] Figure 23 Includes tables and photographs from a DLS study showing the effect of oat material concentration on micellization. Oat material was applied at concentrations of 0.742%, 1.484%, 2.226%, 2.968%, 4.452%, and 5.935%. 0% served as a negative control. Detailed Implementation

[0052] Composition According to a first aspect, a composition is provided comprising: (a) micelles dispersed in an aqueous solution, wherein the micelles comprise at least one recombinant casein; and (b) a plant-derived material.

[0053] In some embodiments, the aqueous solution is or includes a medium. As used herein, the terms "solution" and "medium" are used interchangeably.

[0054] In some embodiments, the at least one recombinant casein is selected from αS casein, β casein, κ casein, or any combination thereof. In some embodiments, the at least one recombinant casein includes αS casein and β casein. In some embodiments, the at least one recombinant casein includes αS casein and κ casein. In some embodiments, the at least one recombinant casein includes αS casein, β casein, and κ casein.

[0055] In some embodiments, αS casein includes αS1 casein, αS2 casein, or both.

[0056] In some embodiments, the at least one recombinant casein includes: αS1 casein, αS2 casein, β casein, and κ casein.

[0057] In some embodiments, the composition comprises (a) micelles dispersed in an aqueous solution, wherein the micelles comprise recombinant αS1 casein, αS2 casein, β casein and κ casein; and (b) plant-derived material.

[0058] In some embodiments, the composition includes recombinant αS1 casein, αS2 casein, β casein and κ casein; and (b) plant-derived material.

[0059] In some embodiments, the recombinant protein comprises the amino acid sequence of mammalian casein. In some embodiments, the mammal is a domesticated mammal. In some embodiments, the mammal is a domesticated mammal. In some embodiments, the domesticated mammal is or includes bovine animals (common cattle). Bos taurus In some embodiments, mammals include or are human subjects. In some embodiments, the recombinant protein includes the amino acid sequence of bovine casein, or its analogues having at least 70%, 80%, 90%, 95%, or 99% sequence homology or identity (or any value and range thereof). Each possibility represents a separate embodiment of the invention.

[0060] The amino acid sequence of mammalian casein will be obvious to those skilled in the art, as is provided in Uniprot.

[0061] In some embodiments, bovine αS1 casein comprises an amino acid sequence as disclosed in Protein Master Registry Number P02662, or its analogues having at least 70%, 80%, 90%, 95%, or 99% sequence homology or identity (or any value and range thereof). Each possibility represents a separate embodiment of the invention.

[0062] In some embodiments, bovine αS2 casein comprises an amino acid sequence as disclosed in Protein Master Registry Number P02663, or its analogues having at least 70%, 80%, 90%, 95%, or 99% sequence homology or identity (or any value and range thereof). Each possibility represents a separate embodiment of the invention.

[0063] In some embodiments, bovine β-casein comprises an amino acid sequence as disclosed in Protein Master Registry Number P02666, or its analogues having at least 70%, 80%, 90%, 95%, or 99% sequence homology or identity (or any value and range thereof). Each possibility represents a separate embodiment of the invention.

[0064] In some embodiments, bovine κ casein comprises an amino acid sequence as disclosed in Protein Master Registry Number P02668, or its analogues having at least 70%, 80%, 90%, 95%, or 99% sequence homology or identity (or any value and range thereof). Each possibility represents a separate embodiment of the invention.

[0065] In some embodiments, the micelles comprise recombinant proteins of αS-casein, β-casein, and κ-casein in a weight / weight ratio (w / w) of 1:1:1 to 10:10:1, 2:2:1 to 10:10:1, 3:3:1 to 10:10:1, 4:4:1 to 10:10:1, 5:5:1 to 10:10:1, 6:6:1 to 10:10:1, 7:7:1 to 10:10:1, 2:2:1 to 8:8:1, 3:3:1 to 5:5:1, 2:2:1 to 6:6:1, or 2:2:1 to 9:9:1. Each possibility represents a separate embodiment of the invention.

[0066] In some embodiments, the composition comprises recombinant proteins of αS-casein, β-casein, and κ-casein in a weight / weight ratio (w / w) of 1:1:1 to 10:10:1, 2:2:1 to 10:10:1, 3:3:1 to 10:10:1, 4:4:1 to 10:10:1, 5:5:1 to 10:10:1, 6:6:1 to 10:10:1, 7:7:1 to 10:10:1, 2:2:1 to 8:8:1, 3:3:1 to 5:5:1, 2:2:1 to 6:6:1, or 2:2:1 to 9:9:1. Each possibility represents a separate embodiment of the invention.

[0067] In some embodiments, αS casein comprises αS1 casein and αS2 casein in a weight ratio of 3:1 to 1:3, 2:1 to 1:2, or 1:1. Each possibility represents a separate embodiment of the invention.

[0068] As used herein, the term "recombinant protein" refers to a protein encoded by recombinant DNA and therefore not naturally occurring. The term "recombinant DNA" refers to a DNA molecule formed through laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of vectors, plasmids, or viruses used to express recombinant proteins in cells.

[0069] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to polymers containing amino acid residues. In another embodiment, the terms “peptide,” “polypeptide,” and “protein” as used herein encompass natural peptides, peptide mimics (generally including non-peptide bond or other synthetic modifications), and peptide analogs such as peptide-mimics and semi-peptides, or any combination thereof. In another embodiment, the described peptides, polypeptides, and proteins are modified to make them more stable or more capable of penetrating cells in vivo. In one embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to polymers containing naturally occurring amino acids. In another embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to polymers containing amino acids for which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0070] As used herein, the term "analyte" refers to a polypeptide that is similar to, but not identical to, the polypeptide of the present invention, and that is still capable of binding succinic acid or still includes a succinic acid-binding pocket. Analytes may have deletions or mutations in the amino acid sequence that result in an amino acid sequence different from that of the polypeptide of the present invention. It should be understood that all analogs of the polypeptide of the present invention will still be capable of binding succinic acid or still include a succinic acid-binding pocket. Furthermore, analogs may be fragments of the polypeptide of the present invention; however, in this case, the fragment must include at least 50 consecutive amino acids of the polypeptide of the present invention.

[0071] As used herein, the term "analyte" includes any peptide having a substantially identical amino acid sequence to one of the sequences specifically shown herein, but in which one or more residues have been conservedly substituted with functionally similar residues and exhibiting the capabilities disclosed herein. Examples of conserved substitutions include: a nonpolar (hydrophobic) residue such as isoleucine, valine, leucine, or methionine replacing another residue; a polar (hydrophilic) residue replacing another residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine; a basic residue such as lysine, arginine, or histidine replacing another residue; or an acidic residue such as aspartic acid or glutamic acid replacing another residue. Each possibility represents a separate embodiment of the invention.

[0072] In some embodiments, the recombinant protein is a plant-based recombinant protein. In some embodiments, the recombinant protein is expressed in plant cells, plant cell lines, plant tissues, or any combination thereof. In some embodiments, the recombinant protein is extracted or isolated from plant cells, plant cell lines, plant tissues, or any combination thereof.

[0073] In some embodiments, the plant is a leafy green vegetable. In some embodiments, the plant is lettuce. In some embodiments, the leafy green vegetable is or includes lettuce. In some embodiments, the plant is a grain. In some embodiments, the plant is cereal grain. In some embodiments, the plant is oats (…). Avena sativa In some implementations, the plant is lettuce ( ). Lactuca sativa In some implementations, the plant is a leafy green vegetable, lettuce, or both.

[0074] In some embodiments, plant-derived materials include any material produced, secreted, or both from a plant or its tissues or cells. In some embodiments, plant-derived materials include fractions, portions, or both of plant material. In some embodiments, plant-derived materials include extracts, homogenates, exudates, isolates, any fractions thereof, or any combination thereof derived from plants.

[0075] In some embodiments, the composition further includes calcium ions.

[0076] In some embodiments, calcium ions are in the form of calcium chloride (CaCl2) or comprise calcium chloride (CaCl2). In some embodiments, calcium ions are present in the compositions of the present invention in the form of CaCl2.

[0077] In some embodiments, the composition includes calcium ions in concentrations ranging from 0 mM to 1000 mM, 10 mM to 100 mM, 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 60 mM to 100 mM, 5 mM to 100 mM, 10 mM to 90 mM, 15 mM to 90 mM, 20 mM to 90 mM, 25 mM to 90 mM, 30 mM to 90 mM, 10 mM to 90 mM, or 40 mM to 90 mM.

[0078] In some embodiments, the composition further comprises phosphate ions, polyphosphate ions, or combinations thereof.

[0079] In some embodiments, the composition further comprises at least one ion selected from the following: Zn 2+ Mg 2+ Cu 2+ Na + K + Fe 2+ Fe 3+ Or any combination thereof.

[0080] In some embodiments, the composition comprises a plurality of micelles.

[0081] As used in this article, the term "multiple" covers any integer equal to or greater than 2.

[0082] In some embodiments, compositions comprising a plurality of micelles, as disclosed herein, are characterized by an average zeta potential range of -30 to 30 mV, -25 to 30 mV, -20 to 20 mV, -17 to 10 mV, -10 to 10 mV, -15 to 10 mV, -14 to 5 mV, -13 to 1 mV, -20 to 0 mV, -11 to 20 mV, -20 to -12 mV, -20 to -13 mV, -20 to -15 mV, -18 to -12 mV, -17 to -13 mV, -16 to -11 mV, -17 to -6 mV, -17 to -10 mV, -15 to -10 mV, -14 to -8 mV, -15 to -12 mV, or -14 to -12 mV. Each possibility represents a separate embodiment of the invention.

[0083] In some embodiments, the plurality of micelles are characterized by an average particle size range of about 100 nm to about 500 nm, 150 nm to about 500 nm, 200 nm to about 500 nm, 250 nm to about 500 nm, 300 nm to about 500 nm, 350 nm to about 500 nm, 400 nm to about 500 nm, 150 nm to about 400 nm, 200 nm to about 400 nm, 150 nm to about 350 nm, or 100 nm to about 300 nm. Each possibility represents a separate embodiment of the invention.

[0084] In some embodiments, the plurality of micelles are characterized by an average particle size range of about 100 nm to about 300 nm, and 300 nm to about 500 nm. In some embodiments, the plurality of micelles are characterized by an average particle size range of about 120 nm to about 290 nm, and 310 nm to about 500 nm. In some embodiments, the plurality of micelles are characterized by an average particle size range of about 100 nm to about 250 nm, and 300 nm to about 500 nm.

[0085] In some embodiments, the plurality of micelles is characterized by at least two micelle subgroups. In some embodiments, the at least two subgroups of the plurality of micelles differ from each other based on average particle size or differ according to average particle size.

[0086] In some embodiments, the first subgroup of the at least two subgroups of the plurality of micelles is characterized by an average particle size range of 100 nm to 300 nm, 110 nm to 290 nm, 120 nm to 280 nm, 100 nm to 200 nm, 100 nm to 270 nm, or 100 nm to 250 nm. Each possibility represents a separate embodiment of the invention.

[0087] In some embodiments, the second subgroup of the at least two subgroups of the plurality of micelles is characterized by an average particle size range of 300 nm to 500 nm, 310 nm to 490 nm, 320 nm to 500 nm, 330 nm to 500 nm, 350 nm to 500 nm, or 400 nm to 500 nm. Each possibility represents a separate embodiment of the invention.

[0088] In some embodiments, the plurality of micelles, as disclosed herein, comprising at least two subgroups, are characterized by sterility, a reduced rate of spoilage, or a tendency to spoilage (e.g., due to microbial infection, contamination, proliferation, or similar processes) compared to control micelles. In some embodiments, the control micelles do not contain at least two subgroups of the plurality of micelles.

[0089] In some implementations, the average particle size is determined by dynamic light scattering (DLS).

[0090] As used in this article, the terms “micelles” and “particles” are used interchangeably.

[0091] In some embodiments, the micelles of the compositions of the present invention are substantially similar to those in milk. In some embodiments, the milk includes or consists of cow's milk. In some embodiments, the micelles of the compositions of the present invention are substantially similar to those in milk, optionally wherein the milk is or includes cow's milk.

[0092] As used herein, the term "substantially similar" means at least 70%, 80%, 90%, 95%, or 99% identical, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0093] In some embodiments, micelles substantially similar to those representing the compositions of the present invention have sizes, average or maximum sizes, diameters, average or maximum diameters, tastes, flavors, odors, sensory properties, or any combination thereof that are substantially similar to micelles in a milk.

[0094] In some embodiments, the composition further comprises an organic acid. The types of organic acids will be apparent to those skilled in the art. Non-limiting examples of organic acids include, but are not limited to, acetic acid, lactic acid, citric acid, maleic acid, or any combination thereof.

[0095] In some embodiments, the composition is an edible composition. In some embodiments, the composition is a dairy substitute. In some embodiments, the composition is a pre-dairy substitute composition / product / ingredient, for example, used as an ingredient and / or starting material in the preparation of a dairy substitute.

[0096] In some embodiments, the composition is a dry composition. In some embodiments, the composition is dried. In some embodiments, the composition is lyophilized or freeze-dried. In some embodiments, the invention considers any drying means and methods known to those skilled in the art.

[0097] Preparation method According to another aspect, a method for preparing the composition of the present invention is provided.

[0098] According to another aspect, a method for preparing a composition is provided, the composition comprising: (a) micelles dispersed in an aqueous solution, wherein the micelles comprise at least one recombinant casein; and (b) plant-derived material.

[0099] In some embodiments, the micelles comprise recombinant proteins of αS casein, β casein, and κ casein.

[0100] In some embodiments, the recombinant proteins of αS casein, β casein, and κ casein are mixed in a final weight / weight ratio (w / w) of 1:1:1 to 10:10:1, 2:2:1 to 10:10:1, 3:3:1 to 10:10:1, 4:4:1 to 10:10:1, 5:5:1 to 10:10:1, 6:6:1 to 10:10:1, 7:7:1 to 10:10:1, 2:2:1 to 8:8:1, 3:3:1 to 5:5:1, 2:2:1 to 6:6:1, or 2:2:1 to 9:9:1. Each possibility represents a separate embodiment of the invention.

[0101] In some embodiments, mixing is performed to obtain a plurality of micelles dispersed in an aqueous solution. In some embodiments, each of the plurality of micelles comprises a recombinant protein of αS casein, β casein, and κ casein.

[0102] In some embodiments, the recombinant protein of the plurality of micelles (such as the compositions of the present invention) is a plant-based recombinant protein.

[0103] In some embodiments, mixing is carried out in the presence of at least one ion selected from the following: Ca 2+ Phosphate, polyphosphate, Zn 2+ Mg 2+ Cu 2+Na + K + Fe 2+ Fe 3+ and any combination thereof.

[0104] In some embodiments, mixing includes mixing the recombinant protein with calcium chloride, calcium phosphate, or both.

[0105] In some embodiments, mixing includes mixing under at least one of the following conditions: heating, cooling, ultrasonic treatment, electrolysis, or any combination thereof.

[0106] In some embodiments, mixing includes sequential mixing in the following order: (1) casein recombinant protein; (2) calcium ions or salts thereof (e.g., CaCl2, calcium phosphate or both); and (3) plant material.

[0107] In some embodiments, mixing includes sequential mixing in the following order: (1) casein recombinant protein; (2) plant material; and (3) calcium ions or salts thereof (e.g., CaCl2, calcium phosphate, or both).

[0108] In some embodiments, mixing includes sequential mixing in the following order: (1) calcium ions or their salts (e.g., CaCl2, calcium phosphate, or both); (2) casein recombinant protein; and (3) plant material.

[0109] In some embodiments, mixing includes sequential mixing in the following order: (1) calcium ions or their salts (e.g., CaCl2, calcium phosphate, or both); (2) plant material; and (3) casein recombinant protein.

[0110] In some embodiments, mixing includes sequential mixing of: (1) plant material; (2) calcium ions or salts thereof (e.g., CaCl2, calcium phosphate or both); and (3) casein recombinant protein.

[0111] In some embodiments, mixing includes sequential mixing in the following order: (1) plant material; (2) casein recombinant protein; and (3) calcium ions or salts thereof (e.g., CaCl2, calcium phosphate, or both).

[0112] In some embodiments, the method includes a step prior to mixing, comprising mixing recombinant proteins of αS casein, β casein and κ casein to obtain a recombinant casein mixture.

[0113] In some embodiments, the mixing includes simultaneously mixing casein recombinant protein; calcium ions or their salts (e.g., CaCl2, calcium phosphate, or both); and plant material.

[0114] In some implementations, mixing is performed under heating conditions.

[0115] In some embodiments, the method further includes the step of determining the size, shape, diameter, zeta potential, or any combination thereof of the prepared composition or the micelles included therein.

[0116] In some embodiments, the determination is made based on any analytical method known to those skilled in the art. Non-limiting examples of applicable analytical methods include, but are not limited to, dynamic light scattering (DLS), electron microscopy, laser diffraction, size exclusion chromatography, gel electrophoresis, field flow fractionation, or any combination thereof, some of which are illustrated below.

[0117] In some embodiments, the method further includes the step of drying the composition.

[0118] The means and methods used for drying are common and will be obvious to those skilled in the art. Non-limiting examples of such drying methods include, but are not limited to, hot air drying, contact drying, infrared drying, freeze drying, fluidized bed drying, or high-frequency drying, to name just a few.

[0119] In some embodiments, a method is provided for preparing an edible composition, food composition, food product, or any combination thereof from the compositions of the present invention. In some embodiments, the method includes providing the compositions of the present invention. In some embodiments, the edible composition, food composition, food product, or any combination thereof is a dairy product substitute. In some embodiments, the edible composition, food composition, food product, or any combination thereof is a pre-dairy product substitute composition / product / ingredient. In some embodiments, the method includes mixing the compositions of the present invention with at least one additional ingredient to prepare the edible composition, food composition, or food product.

[0120] According to another aspect, a method for preparing a food product requiring a certain amount of latex micelles is provided, comprising replacing the amount of latex micelles required for preparing the food product with an equal amount of micelles of the composition of the present invention.

[0121] As used herein, the term "dairy substitute" refers to a composition that effectively replaces milk in a food composition and provides milk-like functionality. In some embodiments, among objects of edible dairy substitutes, for example, compared to a dairy product control, providing milk-like functionality results in reduced or significantly reduced allergenicity, sensitivity (or oral sensitivity), intolerance, or any combination thereof.

[0122] General considerations As used in this article, the term “about” refers to ±10%.

[0123] The terms “comprises,” “comprising,” “includes,” “including,” “having,” and their conjugates mean “including but not limited to.”

[0124] The term "consisting of" means "including and limited to".

[0125] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or portions, but only if such additional ingredients, steps, and / or portions do not substantially alter the essential and novel features of the claimed composition, method, or structure.

[0126] The term “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described as “exemplary” is not necessarily to be construed as preferred or superior to other implementations and / or as excluding the inclusion of features of other implementations.

[0127] The term “optionally” is used herein to mean “provided in some embodiments but not in others”. Any specific embodiment of the invention may include a number of “optional” features unless these features conflict with each other.

[0128] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. For example, the terms “a compound” or “at least one compound” may include multiple (kinds) of compounds, including mixtures thereof.

[0129] Throughout this application, various embodiments of the invention are presented in a range format. It should be understood that the range format is used merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, a description of a range should be considered as specifically disclosing all possible subranges and individual numerical values ​​within those ranges. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0130] Whenever a range of values ​​is indicated herein, it is intended to include any referenced numbers (fractions or integers) within the indicated range. The phrases “range / amplitude” between the first indicated number and the second indicated number and “range / amplitude” from the first indicated number to the second indicated number are used interchangeably herein and are intended to include the first and second indicated numbers and all fractions and integers in between.

[0131] As used herein, the term “method” refers to the manner, means, techniques and procedures used to accomplish a given task, including but not limited to those known or readily developed by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine from known manner, means, techniques and procedures.

[0132] It should be understood that, for clarity, certain features of the invention described in a single embodiment may also be provided in combination in individual embodiments. Conversely, for brevity, various features of the invention described in a single embodiment may also be provided individually or in any suitable sub-combination or suitably provided in any other described embodiment of the invention. Certain features described in various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0133] Various embodiments of the invention have been described for illustrative purposes, but these descriptions are not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein has been chosen to best illustrate the principles of these embodiments, their practical application or improvement relative to commercially available technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0134] The various embodiments and aspects of the invention described above and claimed in the claims section below are experimentally supported in the following examples.

[0135] Other objects, advantages, and novel features of the invention will become apparent to those skilled in the art upon examination of the following embodiments, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the invention described above and claimed in the claims section below is experimentally supported in the following embodiments.

[0136] Example In general, the nomenclature used in this paper and the laboratory procedures used in this invention encompass molecular, biochemical, microbiological, and recombinant DNA technologies. Such technologies are described in detail in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Vol. I-III Ausubel, RM, ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vol. I-4, Cold Spring Harbor Laboratory Press, New York (1998); The methodologies described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III, Cellis, JE, ed. (1994); “Culture of Animal Cells - A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition; “Current Protocols in Immunology”, Volumes I-III, Coligan, JE, ed. (1994); Stites et al.(eds.), “Basic and Clinical Immunology” (8th eds.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), “Strategies for Protein Purification and Characterization - A Laboratory Course Manual” CSHL Press (1996); all of these are incorporated by reference. Other general references are provided throughout this document.

[0137] Material Twenty (20) mg / ml of PBS solution (Sigma) for each casein.

[0138] Romaine (longleaf lettuce) Lactuca sativa var. longifolia) in PBS or citrate-phosphate buffer.

[0139] Oatmeal Avena sativa The solution in PBS or citrate-phosphate buffer.

[0140] Genetically modified romaine lettuce powder extract, in which the lettuce expresses recombinant bovine casein.

[0141] Saturated calcium phosphate solution.

[0142] Calcium chloride.

[0143] Magnesium chloride.

[0144] 0.1 M citric acid solution.

[0145] 0.2 M disodium hydrogen phosphate.

[0146] Nile red (1 mg / ml).

[0147] method Isothermal titration calorimetry (ITC) ITC is used to directly measure the heat released or consumed during a bimolecular reaction. Using ITC measurements, the inventors obtain data on (a) heat flux and (b) enthalpy of reaction (ΔH).

[0148] Dynamic light scattering (DLS) Using DLS, the inventors analyzed aggregates in macromolecules to determine protein size. Scattered light was used to determine the diffusion coefficient and particle size using the Stokes-Einstein equation. Information about (a) zeta potential; intensity; (c) volume; and (d) particle count was obtained using DLS.

[0149] Micellar construction The stepwise construction of casein micelles was accomplished by combining α-casein and β-casein in solution, followed by the addition of κ-casein.

[0150] All four casein proteins were assembled into micelles by heat treatment with the addition of food-grade salt. Half (0.5) g of the standard casein mixture (Sigma) was added to 10 ml of double-distilled water (DDW) or 10 ml of plant extract, or 10 ml of transgenic plant extract heated to 50°C in a water bath. The casein mixture was allowed to heat and swell with constant stirring. The pH was adjusted to 6.5–7.1 and the solution was further heated to 70°C. When the casein was completely dissolved, the solution was cooled and food-grade salt and CaCl2 were added. The pH was adjusted again and the resulting “milk” was pasteurized in an autoclave.

[0151] Nile Red (NR) Staining In short, 25 μl of NR (1 mg / ml) was added to 1 ml of each test sample. The samples were incubated for 15 minutes, centrifuged at 2,000 rpm for 2 minutes on a benchtop centrifuge, and measured in 96-well plates at an excitation wavelength of 559 nm. Furthermore, after 24 hours of incubation, the samples were examined and imaged using a fluorescence binocular microscope.

[0152] Western blot (WB) The “milk” sample before and after micellization was separated by 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Invitrogen), and then transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked to prevent nonspecific antibody binding and incubated with an anti-casein antibody (abcam) and a second horseradish peroxidase (HRP) conjugated antibody.

[0153] Direct imaging cryogenic transmission electron microscopy (cryo-TEM) Technion in Israel uses in-house standard procedures for cryogenic TEM analysis.

[0154] Example As a preliminary analysis, the inventors demonstrated that individual casein proteins can form micelles (Figures 1-2), but not in an efficient manner, as shown by the low to no peaks of higher molecular weight protein aggregates in the figures below.

[0155] For "emulsion-like" micelles to form, appropriate interactions between various casein proteins are required. Therefore, the inventors titrated α-casein into a solution containing β-casein. According to ITC research, the inventors demonstrated that protein-protein interactions occur between α-casein and β-casein, such as an S-shaped increase in the enthalpy of the reaction. Figure 3 Furthermore, the inventors investigated the ability of α, β, and κ casein to assemble micelles. The results showed that the enthalpy of the reaction decreased in an S-shape over time, indicating that κ casein interacts with α and β casein. Figure 4 ).

[0156] In their DLS study, the inventors further investigated the assembly of all four casein proteins (αS1 casein, αS2 casein, β casein, and κ casein) into micelles in the absence of any additives (e.g., salts or ions). The results showed that particles of the desired micelle size were indeed obtained. Figure 5 (The left peak in the upper and lower figures). In this respect, the volume of these particles is quite low compared to the volume of free proteins (one or more). Figure 5 (See the right box in the image below).

[0157] The inventors then investigated the effect of salt addition on the assembly of all four casein types (i.e., αS1 casein, αS2 casein, β casein, and κ casein) into micelles. Furthermore, the order of addition was tested, e.g., titrating salt into the casein mixture and vice versa. The results showed that when a saturated solution of calcium phosphate was titrated into the casein mixture, particles of the desired micelle size were observed, as before (but with a lower volume compared to the free protein (one or more); Figure 6). Furthermore, the results also indicated that when calcium and phosphate were unrestricted, the equilibrium between individual casein and micellar casein was shifted towards micellar organization when the casein mixture was titrated into a saturated solution of calcium phosphate, as observed in both strength and volume (Figure 7).

[0158] The inventors further investigated different methods of adding calcium ions to casein mixtures. They tested the addition of calcium phosphate, calcium chloride, and combinations thereof. The results showed that obtaining protein aggregates with sizes comparable to latex micelles (e.g., average 200-250 nm) required the addition of specific proportions of different salts (Figure 8). Under heating conditions, the inventors further demonstrated visible changes in the casein mixtures after salt titration. Figure 9 This color change is accompanied by an increase in molecular weight, as demonstrated by Western blot analysis. Figure 9 According to DLS analysis, titrating food-grade salt into a mixture containing all four caseins described above under heating conditions provided particles of the desired micelle size; however, contrary to previous attempts described above, these were the dominant particle groups in the sample. Figure 10 To further characterize these micelles, the inventors further stained them with Nile Red. The results showed that the micelles disclosed herein exhibit hydrophobic properties similar to those of commercially available cow's milk. Figure 11 ).

[0159] Using zeta potential analysis, the inventors demonstrated that the observed zeta potential of the casein mixture was higher than that of either α-casein or β-casein alone. This result may indicate that κ-casein, which has the highest zeta potential among the three caseins, is located on the surface of the particles. Figure 12 Using cryogenic TEM, the inventors demonstrated, based on the average size (200-250 nm) of α, β, and κ caseins, that they form protein aggregates equivalent to latex micelles when combined with saturated calcium phosphate and 0.2% CaCl2. Figure 13 ).

[0160] Furthermore, the inventors sought to investigate whether plant material interacts with casein, thereby influencing micellization. To this end, the inventors conducted an ITC study on the interaction between a mixture of individual casein (4:4:1) and any of the following: (a) buffer solution only; (b) a saturated solution of calcium phosphate; (c) 1% lettuce extract; and (d) 2% Noga lettuce extract. Significant interactions were observed in the presence of saturated calcium phosphate solution and 2% lettuce extract. Figure 14 Furthermore, the inventors have demonstrated that the highest percentage of lettuce extract that can be added to solution without interfering with the micellization reaction is 3%. Figure 22 Furthermore, the inventors' DLS studies also showed that the highest percentage of oat extract that can exist in solution without interference is the same as that of lettuce extract (3%). Figure 23 ).

[0161] Based on ITC and DLS analyses (Figures 15–17), micelles of all four casein species disclosed herein, with appropriate particle size and volume, were demonstrated to be produced in the presence of plant materials (e.g., lettuce extract and oat extract) and calcium salts (one or more). Furthermore, using cryo-TEM, the inventors also demonstrated that casein micellization occurred in lettuce extract with the addition of calcium chloride, producing micelles equivalent to those in natural cow's milk with an average size of 200–250 nm (Figure 18). To the inventors' knowledge, this is the first evidence of casein micelle formation from individual casein proteins in vitro in the presence of plant extracts.

[0162] in conclusion Therefore, any amino acid chain of any sequence or length will inherently undergo aggregation at certain points and under certain conditions, depending on concentration, pH, etc., thus producing aggregate-like structures. Therefore, co-expression of multiple proteins (such as casein) in a cell may provide aggregate structures that include these multiple casein proteins. Nevertheless, it will be apparent to a skilled technician that the spontaneously formed aggregate structures in plant cells are not complex and delicate “milk-like” micelles.

[0163] Therefore, in order to form latex-like micelles that further incorporate plant material, the inventors designed to express each individual casein in a separate plant, then isolate and mix them in vitro or in vitro in the presence of plant material to achieve controlled and appropriate interactions. Consequently, the inventors titrated individually expressed α-casein (S1 and S2 combinations), β-casein, and κ-casein in the presence of plant material and found that specific weight / weight ratios of casein (ranging from 2:2:1 to 6:6:1) were advantageous in obtaining micelles of appropriate size that further incorporated plant material.

[0164] Furthermore, to emphasize the importance of independently expressed casein in vitro or in vitro mixed with plant material, the inventors further demonstrate that exogenous supplementation of calcium ions, specifically calcium chloride at concentrations of 2 mM to 100 mM, is advantageous in obtaining “emulsion-like” micelles (e.g., average 100–250 nm, characterized by a zeta potential of -20 mV to -5 mV). Figure 19 The inventors discovered that at least 2 mM of CaCl2 effectively initiates the reaction, while concentrations above 100 mM result in very noticeable calcium precipitation, and therefore are not recommended, as demonstrated by dynamic light scattering (DLS) analysis. In DLS analysis, primarily large insoluble particles were observed, possibly including protein aggregates, but not micelles. Figure 21 ).

[0165] Although the invention has been specifically described, those skilled in the art will understand that various modifications and variations can be made. Therefore, the invention should not be construed as limited to the specifically described embodiments, and the scope and concept of the invention will be more readily understood by referring to the appended claims.

Claims

1. A composition comprising: a. Micelles dispersed in an aqueous solution, wherein the micelles comprise recombinant proteins of αS casein, β casein and κ casein; and b. Plant-derived materials.

2. The composition according to claim 1, wherein the recombinant proteins of αS casein, β casein and κ casein are present in the composition in a weight / weight ratio (w / w) of 2:2:1 to 6:6:

1.

3. The composition according to claim 1 or 2, wherein the αS casein comprises: αS1 casein, αS2 casein, or both.

4. The composition according to any one of claims 1 to 3, wherein the recombinant protein is a plant-based recombinant protein.

5. The composition according to any one of claims 1 to 4, wherein the plant-derived material comprises plant extracts, homogenates, any fractions thereof, or any combination thereof.

6. The composition according to any one of claims 1 to 5, further comprising calcium ions.

7. The composition of claim 6, wherein the calcium ions are present in the composition at a concentration ranging from 2 mM to 100 mM.

8. The composition according to claim 6 or 7, wherein the calcium ions are present in the composition in the form of CaCl2.

9. The composition according to any one of claims 1 to 8, further comprising phosphate ions, polyphosphate ions, or combinations thereof.

10. The composition according to any one of claims 1 to 9, further comprising at least one ion selected from the group consisting of Zn 2+ Mg 2+ Cu 2+ Na + K + Fe 2+ Fe 3+ and any combination thereof.

11. The composition according to any one of claims 1 to 10, comprising a plurality of micelles of the micelles, and characterized in that the average zeta potential ranges between -17 mV and 6 mV.

12. The composition according to claim 11, characterized in that... The average zeta potential ranges from -14 mV to -8 mV.

13. The composition according to claim 11 or 12, wherein the plurality of micelles are characterized in that the average particle size ranges between 100 nm and 250 nm, between 300 nm and 500 nm, or both.

14. The composition according to any one of claims 1 to 13, wherein the micelles are substantially similar to micelles in milk, optionally wherein the milk is cow's milk.

15. The composition according to any one of claims 11 to 14, wherein the plurality of micelles are characterized in that any one of the following is at least 90% identical to the latex micelles in size, average size or maximum size, diameter, average diameter or maximum diameter, taste, flavor, odor, sensory properties or any combination thereof:

16. A method for preparing the composition according to any one of claims 1 to 15, the method comprising mixing recombinant proteins of αS casein, β casein and κ casein with plant-derived material to prepare the composition.

17. The method of claim 16, further comprising obtaining a plurality of micelles dispersed in an aqueous solution, wherein each of the plurality of micelles comprises a recombinant protein of the αS casein, β casein and κ casein.

18. The method of claim 17, wherein the recombinant proteins of αS casein, β casein and κ casein are mixed in the plurality of micelles of the composition at a final weight / weight ratio (w / w) of 2:2:1 to 6:6:

1.

19. The method according to any one of claims 16 to 18, wherein the mixing is carried out in the presence of at least one ion selected from the group consisting of: Ca 2+ Phosphate, polyphosphate, Zn 2+ Mg 2+ Cu 2+ Na + K + Fe 2+ Fe 3+ and any combination thereof.

20. The method according to any one of claims 16 to 19, wherein the mixing comprises mixing under at least one condition selected from: heating, cooling, ultrasonic treatment, electrolysis, and any combination thereof.

21. The method according to any one of claims 17 to 20, wherein the recombinant protein of the plurality of micelles of the composition is a plant-based recombinant protein.

Images