Methods and compositions for producing emulsions

Abstract

The invention provides methods for enhancing emulsification properties of a first protein, by fusing the first protein (preferably a dairy protein) with an oil body-associated protein, or a protein with affinity for an oil body-associated protein, to produce a fusion protein with at least one enhanced emulsification property relative to the first protein. The invention also provides fusion proteins produced by the methods, polynucleotides encoding and cells and plants expressing, the fusion proteins, and extracts from the cells and plants comprising the fusion proteins. The invention also methods for using the fusion proteins to produce emulsions, and sequestering proteins into such emulsions, and the emulsions and protein-enriched emulsions produced. The invention also provides for the use of the fusion proteins and emulsions in compositions, and for producing compositions, including food and beverage products, particularly in dairy, and dairy product substitutes, particularly in plant-based dairy product substitutes.

Description

[0001] METHODS AND COMPOSITIONS FOR PRODUCING EMULSIONS

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] The contents of Australian provisional patent application number 2024904196 filed 18 December 2024, are incorporated herein by reference in their entirety.

[0004] FIELD OF INVENTION

[0005] The invention is in the field of emulsifiers, their production, and use, particularly in the food and beverage industry, particularly in dairy products, and particularly in plant-based dairy product substitutes.

[0006] BACKGROUND TO THE INVENTION

[0007] Emulsifiers

[0008] Emulsifiers are surface-active (or amphiphilic) substances often used to stabilise emulsions, colloids and foams. They are capable of adsorbing at the interface of mutually insoluble phases (e.g. oil and water, air and water) because of their amphiphilic nature. The hydrophilic region is oriented to the aqueous phase, whereas the lipophilic region of the emulsifier seeks the lipid or air phase (Hartel & Hasenhuettl, 2019; McClements, 2016).

[0009] Common emulsifiers in the food industry

[0010] In the food industry, the primary emulsifiers include small-molecule surfactants, phospholipids, and amphiphilic biopolymers. The effectiveness of these emulsifiers in forming and stabilising emulsions varies significantly based on their molecular and physicochemical properties. Examples in each category:

[0011] Small-molecule surfactants (Norn, 2014): mono- and diglycerides, derivatives of mono / diacylglycerides (e.g. acetylated, lactylated, succinylated), salts of fatty acids, polyglycerol esters of fatty acids, Sorbitan esters and polysorbates, sucrose esters, Sodium and Calcium Stearoyl Lactylate, and diacetyltartaric esters of monoacylglycerols (DATEM).

[0012] Phospholipids (McClements, 2016): lecithins (e.g. soy lecithin, sunflower lecithin, egg lecithin). Amphiphilic biopolymers (McClements, 2016):

[0013] • Proteins: milk proteins, meat and fish proteins, egg proteins, plant proteins.

[0014] • Polysaccharides: gum Arabic, modified starches, modified celluloses, etc.

[0015] Table 1. Small-molecule surfactants listed as direct food additives.

[0016] Reproduced from Hartel & Hasenhuettl, 2019.

[0017] Milk proteins as emulsifiers

[0018] Milk proteins function as surface active substances in emulsions because of their amphiphilic structure and they contribute to the stability of the oil droplets by a combined effect of electrostatic and steric stabilisation mechanisms (Dalgleish, 1997). The behaviour of the oil droplets will depend on the type of milk protein adsorbed at the interface and the surface load.

[0019] Milk protein as emulsifiers

[0020] Caseins have high surface activity, i.e. they adsorb rapidly at the interface of liquids at low concentrations and reduce surface tension. This means caseins create small -droplet size emulsion that is stable to gravitational separation. Caseins are heat stable. Therefore, casein-stabilised emulsions are known to have high stability against heat treatment, mostly at neutral pH.

[0021] Whey proteins also create small droplet size emulsions because of its high surface activity at low protein levels. These emulsions are therefore quite stable to gravitational separation (creaming). Drawbacks / disadvantages of current emulsifiers

[0022] Ideally, an emulsifier should (1) be capable of rapidly adsorbing to the surface of freshly formed droplets during homogenization; (2) be capable of reducing the interfacial tension by a significant amount; and (3) be capable of forming an interfacial coating that is either resistant to rupture and / or provides a sufficiently stability during food processing and storage (McClements, 2016).

[0023] However, current options in the food industry have several drawbacks including significant variability in emulsification performance, which makes it difficult to select which emulsifiers are more efficient / suitable for different food applications. Current emulsifiers also have different sensitivity to pH, ionic strength, temperature (heating), solvent composition, mechanical shearing and dehydration. These factors are critical during food production, transportation and storage and can compromise the quality of the product.

[0024] Although current emulsifiers meet certain product applications requirements, they can be costly. The food industry is always looking for cheaper alternatives. Either emulsifiers produced by inexpensive processes, or new emulsifiers that provide improved functional properties using less material will be preferred.

[0025] Most of the existing emulsifiers that are commonly used in food manufacturing are not “clean label”, which leads to rejection by consumers. The need for new emulsifiers that meet clean label demands and perform well in various applications have ongoing challenges for research and development in the food industry.

[0026] Some emulsifiers can also alter the sensory properties of food products, potentially leading to undesirable flavours or textures if not used correctly.

[0027] It is therefore an object of the invention to provide improved emulsifiers, and methods for their production and use that overcome one or more of the deficiencies of the prior art and / or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION

[0028] The applicants have surprisingly found that the emulsification properties of proteins can be enhanced by fusing them to an oil body-associated protein (OBP) or a protein with affinity for an oil body-associated protein (AOBP). The applicants have shown that such fusion proteins have enhanced emulsification properties relative to the proteins alone. The applicants have also shown that the fusion proteins can be used to produce emulsions that are protein -enriched relative to emulsions produced using protein alone.

[0029] For example, the applicants have shown that that dairy proteins fused OBPs or AOBPs have enhanced emulsification properties relative to the dairy protein alone, and that the fusion proteins can be used to produce emulsions that are protein-enriched relative to emulsions produced using dairy protein alone.

[0030] The invention provides methods for producing and using such fusion proteins, methods for using such fusion proteins to form emulsions and protein-enriched emulsions, emulsions and protein-enriched emulsions, comprising such fusion proteins, and compositions comprising such fusion proteins and emulsions. The invention also provides such fusion proteins, emulsions and compositions. These are particularly useful for the food and beverage industry and particularly useful in plant-based dairy product substitutes.

[0031] The invention also provides foods, beverages, dairy products, dairy product substitutes and plant-based dairy product substitutes, and ingredients for the above, comprising such fusion proteins and emulsions. Beneficial properties of the foods, beverages, dairy products, dairy product substitutes and plant-based dairy product substitutes, and ingedients, are described further herein. Methods for enhancing emulsification properties and producing fusion proteins

[0032] In the first aspect the invention provides a method for enhancing at least one emulsification property of a first protein, the method comprising fusing the first protein to second protein selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP), to produce a fusion protein with at least one enhanced emulsification property relative to the first protein.

[0033] In a further aspect the invention provides a method for producing a fusion protein with at least one enhanced emulsification property by fusing a first protein to second selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP) wherein the fusion protein has at least one enhanced emulsification property relative to the first protein.

[0034] In a further embodiment the method includes a step of measuring at least one emulsification property.

[0035] In a further embodiment the method includes a step of selecting the fusion protein based on measuring at least one enhanced emulsification property of the fusion protein relative to the first protein.

[0036] The enhanced emulsification properties are described herein

[0037] In a further aspect the invention provides a fusion protein produced by a method of the invention. In one embodiment the fusion protein comprises a first protein fused to a second protein selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP).

[0038] In a further embodiment, the fusion protein has at least one enhanced emulsification property relative to the first protein.

[0039] In a further aspect the invention provides a fusion protein comprising a first protein fused to a second protein selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP).

[0040] In a further embodiment, the fusion protein has at least one enhanced emulsification property relative to the first protein.

[0041] Polynucleotide encoding fusion protein

[0042] In a further aspect the invention provides a polynucleotide encoding a fusion protein of any preceding claim.

[0043] In one embodiment the polynucleotide forms part of a construct.

[0044] Cell, plant cell, plant or plant part comprising fusion protein or polynucleotide encoding a fusion protein

[0045] In a further aspect the invention provides a cell, plant cell, plant or plant part comprising at least one of: a) a fusion protein of any preceding claim, and b) a polynucleotide encoding a fusion protein of any preceding claim. Extract from cell, plant cell, plant or plant part

[0046] In a further aspect the invention provides an extract of the cell, plant cell, plant or plant part, comprising the fusion protein.

[0047] Use of fusion proteins

[0048] In a further aspect the invention provides the use of a fusion protein of the invention as an emulsifier.

[0049] In a further aspect the invention provides a method of using a fusion protein of the invention as an emulsifier.

[0050] Methods for producing emulsions.

[0051] In a further aspect the invention provides a method for producing an emulsion the method comprising combining: a) a first component, b) a second component, and c) a fusion protein of the invention.

[0052] In one embodiment the first component and the second component are substantially immiscible. In a further embodiment the first component and the second component are immiscible.

[0053] In one embodiment the first component is a hydrophilic component.

[0054] In a further embodiment the second component is a hydrophobic component.

[0055] In a further embodiment the hydrophilic component is an aqueous phase. In a further embodiment the hydrophobic component is a lipid phase.

[0056] Emulsion

[0057] In a further aspect the invention provides an emulsion produced by a method of the invention.

[0058] In a further aspect the invention provides an emulsion comprising a fusion protein of the invention.

[0059] In a further aspect the invention provides an emulsion comprising: a) a first component, b) a second component, and c) a fusion protein of the invention.

[0060] In one embodiment the emulsion is stabilized by the fusion protein of the invention.

[0061] In a further embodiment the emulsion has at least one enhanced property relative to an emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0062] Methods for sequestering protein from a protein source and producing a protein-enriched emulsion

[0063] In a further aspect the invention provides a method for sequestering protein from a protein source into an emulsion, the method comprising performing the method for producing an emulsion in the presence of the protein source, the method resulting in a protein-enriched emulsion.

[0064] In a further aspect the invention provides a method for producing a protein-enriched emulsion, the method comprising performing the method for producing an emulsion in the presence of a protein source. Protein enriched emulsion

[0065] In a further aspect the invention provides a produced by a method of the invention.

[0066] Method of producing a composition

[0067] In a further aspect the invention provides a method for producing a composition, the method providing the step of using a fusion protein of the invention as an emulsifier.

[0068] In one embodiment the fusion protein is in, or derived from, the extract of the invention.

[0069] In a further aspect the invention provides a method for producing a composition, the method comprising the step of incorporating an emulsion, or protein-enriched emulsion of the invention into the composition.

[0070] In one embodiment the composition is selected from a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed.

[0071] In a preferred embodiment the composition is a plant-based dairy product substitute.

[0072] In a further embodiment the composition is an ingredient for use in a food product selected from a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed.

[0073] In one embodiment, the composition is an ingredient for use in formulations in which milk proteins are used for other functional properties. In one embodiment the functional property is gelation.

[0074] In one embodiment, the composition is an ingredient for use in a soft drink. In a preferred embodiment, the composition is an ingredient for use in a plant-based dairy product substitute.

[0075] In one embodiment the composition, or an emulsion in the composition, is stabilized by the fusion protein.

[0076] In a further embodiment the composition, or the emulsion in the composition, has enhanced stability relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0077] Compositions

[0078] In a further aspect the invention provides a composition produced by a method of the invention.

[0079] In a further aspect the invention provides a composition comprising at least one of: a) a fusion protein of the invention, b) an emulsion of the invention, c) a protein-enriched emulsion of the invention, and d) an extract of the invention.

[0080] In one embodiment the composition is selected from a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed.

[0081] In a preferred embodiment the composition is a plant-based dairy product substitute.

[0082] In a further embodiment the composition is an ingredient for use in at least one of a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed. In a further preferred embodiment the composition is an ingredient for use in a plant-based dairy product substitute.

[0083] In a further embodiment the composition, or an emulsion in the composition, is stabilized by the fusion protein.

[0084] In a further embodiment the composition, or the emulsion in the composition, has enhanced stability relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0085] In a further embodiment the composition, or the emulsion in the composition, is protein enriched relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0086] DETAILED DESCRIPTION

[0087] The fusion proteins of the invention, produced by a method of the invention, used in a method the invention, or found in emulsions, extracts and compositions of the invention, comprise first protein fused to a second protein selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP).

[0088] The first protein, used to produce the fusion protein in accordance with the invention, may be any protein.

[0089] The term “protein” as used herein is intended to cover chains of amino acids of any length and can be used interchangeably with “peptides” and “polypeptides”.

[0090] Preferably the first protein is a protein that is useful as a food or beverage ingredient. In one embodiment the first protein is selected from a meat protein, a fish protein, an egg protein, a plant protein and a dairy protein.

[0091] In a preferred embodiment the first protein is a dairy protein or an analogue thereof.

[0092] Dairy proteins and analogues for use in the invention

[0093] The terms “dairy protein / s” and “milk protein / s” can be used interchangeably.

[0094] Dairy contains two main groups of proteins, namely caseins and whey proteins. There are four types of caseins, denoted as as 1- casein, as2- casein, p-casein and K-caseins which represent approximately 37, 10, 35 and 12% of the whole casein respectively. Each of the four caseins exhibits variability in the degree of phosphorylation and glycosylation. All caseins are phosphorylated: most of the asl- casein molecules contain 8 PO4 residues but some contain 9; as2- casein contains 10, 11, 12 or 13 mol PO4 / mol; P-casein usually contains 5 mol PO4 / mol but occasionally 4 mol PO4 / mol; K-casein contains 1 mol PO4 / mol. K-casein is the only casein which is normally glycosylated and contains galactose, galactosamine and N-acetyl neuraminic acid. Further heterogeneity in caseins arises from the occurrence of genetic polymorphism, which is due to either substitutions or, rarely, deletions of amino acids in the caseins because of mutations causing changes in base sequences in the genes.

[0095] In comparison to typical globular proteins, the structures of caseins are quite unique. An interesting feature of all caseins is the amphiphilicity of the primary structures. All caseins have regions that are acidic, basic or neutral and hydrophilic or hydrophobic. The caseins, compared to typical globular proteins which have mainly a-helical and P-sheet structures, contain less secondary structures. All major caseins also interact with each other to form various complexes of different sizes and shapes. Caseins bind calcium, and the extent of binding is directly related to the number of phospho-serine residues in the molecule. Whey proteins are even more heterogeneous group of proteins than the caseins. The principal fractions of whey proteins are P-lactoglobulin, bovine serum albumin, a- lactalbumin and immunoglobulins which account for more than 95% of the proteins in the whey fraction. Unlike the caseins, the whey proteins possess high level of secondary, tertiary and in most cases, quaternary structures. Several other proteins are found in small quantities in whey and these include P-microglobulin, lactoferrin and transferrin, which are both iron binding proteins, protease peptones and a group of acyl glycoproteins.

[0096] An additional dairy protein of interest is lactoferrin, particularly bovine lactoferrin (bLF). Bovine lactoferrin isolated from bovine colostrum and milk is a potential antioxidant with benefits including anti -microbial and anti-inflammatory properties.

[0097] Milk proteins, especially whey and casein proteins, are used in a wide variety of functional and nutritional applications and have a range of properties that make them particularly suitable in the formation of and incorporation within food-grade materials. They are effective encapsulating materials, possess film-forming and emulsifying properties that allow them to act as stabilisers in emulsion-based systems, act as carriers of other materials and may be easily formed into a dried state.

[0098] As ingredients in food products, milk proteins introduce a range of sensory characteristics to the food products, including inducing satiety, improved mouth- feel, viscosity and structure, flavour and they act as substrates for other flavours and aromatic compounds in multi-ingredient compositions.

[0099] In one embodiment, the dairy protein for use in the invention is selected from a casein protein, lactoferrin, and a milk fat globule membrane protein, and a whey protein.

[0100] In one embodiment the casein protein is selected from asl- casein, as2- casein, p-casein, and K-casein. In one embodiment the casein protein is bovine A2 p-casein. In one embodiment the bovine A2 P-casein has proline (P) at position 67 in its amino- acid sequence. In one embodiment the milk fat globule membrane protein is selected from a mucin, butyrophilin, a fatty-acid binding protein and xanthine dehydrogenase.

[0101] In one embodiment the whey protein is selected from, beta-lactoglobulin, alpha- lactalbumin, lactoferrin and albumin.

[0102] Examples of dairy proteins for use in the invention are shown in Table 2 below and their sequences are provided in the Sequence Listing. Table 2. Examples of milk or dairy proteins known in the art

[0103] Source of dairy proteins

[0104] The dairy or milk proteins for use in the invention may be from any suitable source.

[0105] In one embodiment the dairy or milk proteins for use in the invention may be from a source selected from sheep, deer, camel, goat, human and bovine organisms.

[0106] In a preferred embodiment the dairy or milk proteins for use in the invention are from bovine organisms.

[0107] Dairy protein analogues

[0108] Dairy proteins such as those described above can also be modified to altered to improve their functional properties. Such modified dairy proteins can be referred to as dairy protein analogues.

[0109] In one embodiment the dairy protein or analogues for use in the invention has at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78% , preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88% , preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% , preferably at least 99% amino acid sequence identity to a known dairy protein of the type described above, or a sequence described in Table 2.

[0110] Parts of dairy proteins or analogues

[0111] The parts of the dairy proteins or analogues are polypeptide fragments of the dairy proteins or analogues.

[0112] In a preferred embodiment the part comprises at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% of full-length sequence of the dairy protein or analogue.

[0113] In some embodiments, the part may be a functional domain of a dairy protein. For example, the functional domain may be selected from: a hydrophobic domain of a beta casein protein, or a hydrophilic domain of a beta-casein.

[0114] In a preferred embodiment, the part is a hydrophilic domain of a beta-casein.

[0115] In one embodiment the hydrophilic domain of a beta-casein has at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to the hydrophilic domain of bovine beta-casein as set forth in SEQ ID NO:59. Second protein

[0116] The second protein in the fusion proteins of the invention, produced by a method of the invention, used in a method the invention, or found in emulsions and compositions of the invention, is selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP).

[0117] In one embodiment, the second protein in the fusion protein is an oil body-associated protein (OBP).

[0118] Oil bodies

[0119] Oil bodies are naturally occurring lipid particles, found predominantly in the seeds of certain plants. Oil bodies biosynthesis is initiated at the ER membrane in a process that transforms fatty acids and sterols into triglycerides and sterol esters respectively. Since these molecules are hydrophobic, they are captured within the interface of the two phospholipid monolayers of the ER membrane. At a certain lipid concentration, the mass of triglycerides and sterol esters are then separated from the bilayer ER surrounded by a phospholipid monolayer (Dhiman et al., 2020).

[0120] Oil body-associated proteins (OBPs)

[0121] As discussed above, oil bodies are surrounded by a phospholipid monolayer incorporating proteins such as oleosin, caleosins and steroleosins that provide stability to the oil bodies when they are dispersed in water and subjected to various treatments.

[0122] Oil body-associated proteins for use in the invention are well-known to those skilled in the art and include for example oleosins (Shao et al 2019, New insights into the role of seed oil body proteins in metabolism and plant development, Front. Plant Sci., https: / / doi.org / 10.3389 / fpls.2019.01568), steroleosins (Lin et al 2002, Steroleosin, a sterol-binding dehydrogenase in seed oil bodies. Plant Physiol. 128: 1200-1211), and caleosins (Hsieh and Huan, 2004, Endoplasmic reticulum, oleosins, and oil seeds in tapetum cells. Plant Physiology, 136:3427-3434). Preferably the OBP is selected from an oleosin and a caleosin.

[0123] Table 3. Examples of oleosins and caleosins known in the art

[0124] In one embodiment the OBP, for use in the invention has at least 90%, preferably at least

[0125] 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity a protein sequence of Table 3.

[0126] In a further embodiment the OBP is an oleosin.

[0127] Oleosins have three functional domains consisting of an amphipathic N-terminal arm, a highly conserved central hydrophobic core (~72 residues) and a C-terminal amphipathic arm. The accepted topological model is one in which the N- and C-terminal amphipathic arms are located on the outside of the OBs and the central hydrophobic core is located inside the OB (Huang, 1992; Loer and Herman, 1993; Murphy 1993). The negatively charged residues of the N- and C-terminal amphipathic arms are exposed to the aqueous exterior whereas the positively charged residues are exposed to the OB interior and face the negatively charged lipids. Thus, the amphipathic arms with their outward facing negative charge are responsible for maintaining the OBs as individual entities via steric hinderance and electrostatic repulsion both in vivo and in isolated preparation (Tzen et al., 1992). The N-terminal amphipathic arm is highly variable and as such no specific secondary structure can describe all examples. In comparison the C-terminal arm contains a a-helical domain of 30-40 residues (Tzen et al., 2003). The central core is highly conserved and thought to be the longest hydrophobic region known to occur in nature; at the centre is a conserved 12 residue proline knot motif which includes three spaced proline residues (for reviews see Frandsen et al., 2001; Tzen et al., 2003). The secondary, tertiary and quaternary structure of the central domain is still unclear. Modelling, Fourier Transformation-Infra Red (FT-IR) and Circular Dichromism (CD) evidence exists for a number of different arrangements (for review see Roberts et al., 2008).

[0128] The properties of the major oleosins is relatively conserved between plants and is characterised by the following:

[0129] • 15-25kDa protein corresponding to approximately 140-230 amino acid residues.

[0130] • The protein sequence can be divided almost equally along its length into 4 parts which correspond to a N-terminal hydrophilic region, two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region.

[0131] • The topology of oleosin is attributed to its physical properties which includes a folded hydrophobic core flanked by hydrophilic domains. This arrangement confers an amphipathic nature to oleosin resulting in the hydrophobic domain being embedded in the phospholipid monolayer (Tzen et al., 1992) while the flanking hydrophilic domains are exposed to the aqueous environment of the cytoplasm.

[0132] In one embodiment the oleosin for use in the invention has at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to a known oleosin of the type described above, or to an oleosin protein sequence of Table 3.

[0133] Modified oleosins

[0134] In some embodiments the invention involves the use of oleosins such as those described that are modified to alter or improve their functional properties.

[0135] For example, oleosins can be engineered to contain up to 13 Cys residues in each amphipathic arm. This enhances stability of the oil bodies. Heterologous overexpression of cysteine-oleosin resulted in reduced triacylglycerol turnover and enhanced oil content in oilseeds (Winichayakul et al., 2013).

[0136] In accordance with the invention, use of such modified oleosins is beneficial to the process of producing dairy product substitutes such as but not limited to, cream substitutes.

[0137] In some embodiments the invention therefore involves the use of oleosins including at least one artificially introduced cysteine (WO2011 / 053169).

[0138] In one embodiment the modified oleosin protein, for use in the invention has at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to a known oleosin of the type described above, or to an oleosin sequence described in Table 3.

[0139] In further embodiment, the second protein in the fusion protein is a protein with affinity for an oil body-associated protein (AOBP).

[0140] Protein with affinity for an oil body-associated protein (AOBP)

[0141] In one embodiment the AOBP is the C-terminal hydrophilic portion of an oil body- associated protein as described herein.

[0142] The hydrophilic, and hydrophobic regions / arms of oleosins can be easily identified by those skilled in the art using standard methodology (for example: Kyte and Doolitle (1982).

[0143] Furthermore, while the hydrophilic domains of oleosins are relatively variable, the central hydrophobic core is well conserved. Thus, those skilled in the art can also readily identify the hydrophilic domains, as flanking the conserved central hydrophobic core. Table 4. Examples of C-terminal hydrophilic domains of oleosins and caleosins

[0144] In one embodiment the C-terminal hydrophilic portion of an oil body-associated protein for use in the invention has at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to a hydrophilic domain in Table 4. In some embodiments the AOBP is a fusion of the N- and C- terminal hydrophilic domains of oleosins. Examples are shown in Table 5 below. Table 5. Examples of fusions ofN- and C- terminal domains of oleosins

[0145] In one embodiment the fusions of the N- and C- terminal hydrophilic domains of oleosins for use in the invention has at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% , preferably at least 99% amino acid sequence identity to a fusions of the N- and C- terminal hydrophilic domains of oleosins in Table 5.

[0146] Arrangement of fusion protein

[0147] In one embodiment in the fusion, the first protein makes up the N-terminal portion of the fusion, and the second protein makes up the C-terminal portion of the fusion.

[0148] In a further embodiment in the fusion, the first protein makes up the C-terminal portion of the fusion, and the second protein makes up the N-terminal portion of the fusion. Examples of such arrangements are as illustrated as follows: first protein-second protein second protein-first protein

[0149] The fusion may also include a third protein fused to the other terminus of the first protein.

[0150] Examples of such arrangements are as illustrated as follows: third protein-first protein-second protein second protein-first protein-third protein.

[0151] Preferably the third protein (like the second protein) is also either: a) an oil body-associated protein (OBP), or b) a protein with affinity for an oil body-associated protein (AOBP) as herein described.

[0152] The emulsions of the invention may also have different structures. For example, oil-in- water (o / w), water-in-oil (w / o), water-in-oil-in-water (w / o / w).

[0153] The continuous phase (also known as the external phase or dispersion medium) may be the hydrophobic component or the hydrophilic component.

[0154] The dispersed phase (also known as the internal phase) which is made of small droplets dispersed in the continuous phase, may be the hydrophobic component or the hydrophilic component.

[0155] Enhanced emulsification properties of the fusion proteins

[0156] In various embodiments, the enhanced emulsification properties of the fusion proteins are selected from but not limited to: • being a stronger emulsion than the first protein,

[0157] • the capacity to produce a more stable emulsion than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0158] • the capacity to produce an emulsion with smaller oil droplet size than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0159] • the capacity to produce an emulsion with improved interfacial stability relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0160] • the capacity to produce an emulsion with increased resistance to creaming relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0161] • the capacity to produce an emulsion with increased resistance to phase separation relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0162] • an altered isoelectric point relative to that of the first protein

[0163] • the capacity to produce a more stable emulsion under acidic conditions (pH below 5) than an emulsion produced using the first protein as an emulsifier in place of the fusion protein, and

[0164] • the capacity to produce a more stable emulsion under basic conditions (pH above 5) than an emulsion produced using the first protein as an emulsifier in place of the fusion protein.

[0165] In various embodiments, the enhanced emulsification property of the fusion protein as described herein is enhanced by at least 1%, preferably at least 2%, more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, relative to the first protein..

[0166] In a preferred embodiment the fusion protein is a stronger emulsifier than the first protein. Preferably the fusion protein has at least 1%, more preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more preferably at least 15%, more preferably at least 16%, more preferably at least 17%, more preferably at least 18%, more preferably at least 19%, more preferably at least 20%, more preferably at least 21%, more preferably at least 22%, more preferably at least 23%, more preferably at least 24%, more preferably at least 25%, more preferably at least 26%, more preferably at least 27%, more preferably at least 28%, more preferably at least 29%, more preferably at least 30%, more preferably at least 31%, more preferably at least 32%, more preferably at least 33%, more preferably at least 34%, more preferably at least 35%, more preferably at least 36%, more preferably at least 37%, more preferably at least 38%, more preferably at least 39%, more preferably at least 30%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70% stronger emulsifier activity than the first protein alone.

[0167] Strength of emulsifier

[0168] The term "strong emulsifiers" as used herein, and grammatical equivalents thereof, refers to emulsifiers that excel in forming and maintaining highly stable emulsions when compared to weaker emulsifiers. Emulsifiers can vary in their emulsifying capabilities, and strong emulsifiers are particularly effective at preventing the separation of unmixable components over extended periods of time. These emulsifiers often possess a strong affinity for both the hydrophilic and hydrophobic phases in an emulsion, leading to robust interfacial interactions. Strong emulsifiers are especially valuable in applications where emulsion stability is critical, such as in the production of long -shelf-life food products, pharmaceutical formulations, or high-performance cosmetics.

[0169] Emulsifier strength is determined by one (or more) of the following: Emulsion stability: The primary measure of an emulsifier's strength is its ability to create and maintain a stable emulsion overtime. This is often evaluated through visual inspection of the emulsion's appearance, such as creaminess, phase separation, or the presence of droplets. The longer an emulsion remains stable, the stronger the emulsifier is considered to be.

[0170] In various embodiments, emulsification, and the strength of an emulsifier, can be determined by one or more of: a) Emulsion droplet size: The size of the oil droplets dispersed in the water phase (or vice versa) is a critical parameter. Smaller droplets generally indicate a more effective emulsifier, as they provide better stability and a smoother texture. This can be measured using techniques like dynamic light scattering (DLS) or laser diffraction. b) Turbidity or optical density: Measuring the turbidity or optical density of an emulsion can provide an indirect assessment of its stability. A more stable emulsion will have lower turbidity or optical density, indicating fewer large droplets or phase separation. c) Creaming or sedimentation rate: Emulsion strength can also be evaluated by monitoring the rate at which creaming (rise of oil phase) or sedimentation (settling of solid particles) occurs over time. A strong emulsifier will slow down these processes. d) Zeta potential: Zeta potential is a measure of the electrostatic repulsion between particles in an emulsion. A higher absolute zeta potential indicates greater repulsion, which can contribute to emulsion stability. Techniques like electrophoretic mobility or electrophoretic light scattering are used to measure zeta potential. d) Interfacial tension: Emulsifier strength can be assessed by measuring the interfacial tension between the oil and water phases in the presence of the emulsifier. A lower interfacial tension indicates a better ability to reduce the forces that drive phase separation.

[0171] In a preferred embodiment, emulsification, and the relative strength of an emulsifier is determined by measuring oil droplet size in the emulsion, as in a) above.

[0172] In a further embodiment the fusion protein is capable of forming a protein-enriched emulsion as herein described.

[0173] Production of the fusion protein

[0174] The fusion protein may be conveniently produced by recombinant expression via in vitro or in vivo systems.

[0175] Suitable in vivo systems include but are not limited to bacterial, yeast, fungal, plant cellular, animal cellular, whole plant and whole animal systems.

[0176] Preferably the expression is in plant cells or whole plants.

[0177] Such recombinant expression requires suitable constructs with polynucleotides encoding the fusion proteins, and genetic elements (such as promoters and terminators) for regulating expression, known to those skilled in the art.

[0178] A promoter may be used to drive expression of the fusion protein in a particular cell or tissue within an organism, such as a plant. Production of the fusion proteins may also be directed to desired subcellular location via use of suitable signal peptides, known to those skilled in the art. Suitable elements encoding signal peptides, can therefore be incorporated into the constructs for expressing the fusion proteins.

[0179] Polynucleotides and constructs

[0180] In a further aspect the invention provides a polynucleotide encoding the fusion proteins.

[0181] In a further aspect the invention provides a construct for expressing the fusion proteins.

[0182] Preferably the construct comprises a promoter operably linked to the polynucleotide encoding the fusion protein.

[0183] Preferably the construct comprises a terminator operably linked to the polynucleotide encoding the fusion protein.

[0184] Cells and plants comprising polynucleotides and constructs

[0185] In a further aspect the invention provides a cell comprising the polynucleotide and / or construct.

[0186] In one embodiment the cell expresses the fusion protein.

[0187] Plants comprising polynucleotides and constructs

[0188] In a further aspect the invention provides a plant comprising polynucleotides and / or constructs.

[0189] In one embodiment the plant expresses the fusion protein. Isolated fusion proteins

[0190] In a further aspect the invention provides the fusion proteins that are isolated or purified from such expression systems.

[0191] In some embodiments the fusion proteins of the invention, produced by a method of the invention, used in a method the invention, or found in emulsions and compositions of the invention are isolated or purified from such expression systems.

[0192] Production of emulsion

[0193] In a further aspect the invention provides a method for producing an emulsion the method comprising combining: a) a first component, b) a second component, and c) a fusion protein of the invention.

[0194] In one embodiment the first component and the second component are substantially immiscible. In a further embodiment the first component and the second component are immiscible.

[0195] In one embodiment the first component is a hydrophilic component.

[0196] In a further embodiment the second component is a hydrophobic component.

[0197] Hydrophilic component

[0198] In some embodiments the hydrophilic component is selected from but not limited to a waterbased solution, a plant-based aqueous solution or extract, an aqueous carbohydrate solution, and an aqueous protein solution. Water based solutions include but are not limited to purified water, distilled water, or buffered saline solutions.

[0199] The plant-based aqueous solutions or extracts may be selected from but not limited to plant milks. The plant milk can be selected from but not limited to almond milk, oat milk, soy milk, rice milk, cashew milk, hemp milk, or pea protein milk.

[0200] The carbohydrate in the aqueous carbohydrate solutions may be selected from but not limited to sucrose, glucose, fructose, maltodextrin, or trehalose.

[0201] The aqueous protein solution includes but is not limited to pea protein hydrolysate, soy protein isolate, and rice protein concentrate.

[0202] Hydrophobic component

[0203] In some embodiments the hydrophobic component is selected from but not limited to a plant oil, a medium-chain triglyceride (MCT), an essential oil or flavor oil, and a lipid-based bioactive.

[0204] In some embodiments the plant oil is selected from but not limited to avocado oil, canola oil, coconut oil, flaxseed oil, olive oil, palm oil, rice bran oil, safflower oil, soybean oil, sunflower oil and vegetable oil.

[0205] In some embodiments the medium -chain triglyceride (MCT) is selected from but not limited to fractionated cocoa butter, coconut oil or palm kernel oil.

[0206] In some embodiments the essential oils or flavor oil is selected from but not limited to eucalyptus oil, orange oil, peppermint oil, and vanilla bean oil.

[0207] In some embodiments the lipid-based bioactive is selected from but not limited to fish oil, algal oil, and omega-3 fatty acid concentrates. The emulsion may contain additives, selected from but not limited to: o Nutritional enhancers selected from but not limited to: vitamins (A, D, E, K, B- complex), minerals (calcium, magnesium, potassium, zinc), or amino acids (lysine, methionine). o Stabilizers selected from but not limited to: xanthan gum, guar gum, carrageenan, or pectin for improved viscosity and texture. o Further emulsifiers selected from but not limited to: lecithin, mono- and diglycerides, or polysorbates (if needed for additional stabilization). o Flavorings selected from but not limited to: natural or artificial flavors such as vanilla, chocolate, strawberry, or coffee. o Sweeteners selected from but not limited to: stevia, aspartame, monk fruit extract, agave syrup, or honey. o Colorants selected from but not limited to: natural pigments like beta-carotene, chlorophyll, or beet extract. o Preservatives selected from but not limited to: ascorbic acid, citric acid, or tocopherols for shelf-life extension.

[0208] In some embodiments the emulsions are produced using isolated or purified recombinantly expressed fusion proteins.

[0209] In other embodiments the fusion proteins are recombinantly expressed in an organism, and an extract is made from the organism or part thereof that contains the fusion protein. In some embodiments the emulsion is produced within such an extract, or within such an extract when combined with other components such as those described herein.

[0210] Suitable organisms include plants. Suitable plant parts include seeds. Properties of the emulsion

[0211] In various embodiments the emulsion has enhanced properties relative to an emulsion produced using the first protein in place of the fusion protein.

[0212] In various embodiments, the enhanced properties are selected from but not limited to:

[0213] • a stronger emulsion than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0214] • a more stable emulsion than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0215] • smaller oil droplet size than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0216] • improved interfacial stability relative to that of an emulsion produced using the fist protein as an emulsifier in place of the fusion protein,

[0217] • increased resistance to creaming relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0218] • increased resistance to gravitational separation relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0219] • increased resistance to phase separation relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0220] • more stable emulsion under acidic conditions (pH below 5) than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0221] • increased resistance to Ostwald ripening than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0222] • increased resistance to ethanol-induced flocculation than an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0223] • increased surface shear viscosity relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0224] • increased heat stability relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein, • increased mechanical shearing resistance relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein,

[0225] • presence of a thicker interfacial layer covering the oil droplets relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein.

[0226] In various embodiments, the enhanced property of the emulsion as described herein is enhanced by at least 1%, preferably at least 2%, more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, relative to that of an emulsion produced using the first protein as an emulsifier in place of the fusion protein.

[0227] In a preferred embodiment the emulsion is a stronger emulsion than an emulsion produced using the first protein as an emulsifier in place of the fusion protein.

[0228] Emulsion strength can be assessed as described above.

[0229] Amount of fusion protein required to produce an emulsion

[0230] In some embodiments, as a consequence of the increased emulsification strength of the fusion protein of the invention relative that of the first protein, less of the fusion protein is required to produce an emulsion, than the amount of the first protein that would be required to produce an equivalent emulsion.

[0231] In one embodiment at 1% less, more preferably at least 2% less, more preferably at least 3% less, more preferably at least 4% less, more preferably at least 5% less, more preferably at least 6% less, more preferably at least 7% less, more preferably at least 8% less, more preferably at least 9% less, more preferably at least 10% less, more preferably at least 11% less, more preferably at least 12% less, more preferably at least 13% less, more preferably at least 12% less, more preferably at least 13% less, more preferably at least 14% less, more preferably at least 15% less, more preferably at least 16% less, more preferably at least 17% less, more preferably at least 18% less, more preferably at least 19% less, more preferably at least 20% less, more preferably at least 21% less, more preferably at least 22% less, more preferably at least 23% less, more preferably at least 24% less, more preferably at least 25% less, more preferably at least 26% less, more preferably at least 27% less, more preferably at least 28% less, more preferably at least 29% less, more preferably at least 30% less, more preferably at least 31% less, more preferably at least 32% less, more preferably at least 33% less, more preferably at least 34% less, more preferably at least 35% less, more preferably at least 36% less, more preferably at least 37% less, more preferably at least 38% less, more preferably at least 39% less, more preferably at least 30% less, more preferably at least 41% less, more preferably at least 42% less, more preferably at least 43% less, more preferably at least 44% less, more preferably at least 45% less, more preferably at least 46% less, more preferably at least 47% less, more preferably at least 48% less, more preferably at least 49% less, more preferably at least 50% less, more preferably at least 51% less, more preferably at least 52% less, more preferably at least 53% less, more preferably at least 54% less, more preferably at least 55% less, more preferably at least 56% less, more preferably at least 57% less, more preferably at least 58% less, more preferably at least 59% less, more preferably at least 60% less, more preferably at least 61% less, more preferably at least 62% less, more preferably at least 63% less, more preferably at least 64% less, more preferably at least 65% less, more preferably at least 66% less, more preferably at least 67% less, more preferably at least 68% less, more preferably at least 69% less, more preferably at least 70% less of the fusion protein is required to produce an emulsion, than the amount of the first protein that would be required to produce an equivalent emulsion.

[0232] In one embodiment an equivalent emulsion has equivalent stability.

[0233] In a further embodiment the emulsion has an oil concentration of at least 1%, preferably at 2%, more preferably at 5%, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 80% by weight.

[0234] In a further embodiment the emulsion is protein enriched as herein described.

[0235] Method for sequestering protein from a protein source and producing a protein-enriched emulsion

[0236] In a further aspect the invention provides a method for sequestering protein from a protein source into an emulsion, the method comprising performing a method for producing an emulsion in accordance with the invention, in the presence of the protein source, the method resulting in a protein-enriched emulsion.

[0237] In a further aspect the invention provides a method for producing a protein-enriched emulsion, the method comprising performing the method for producing an emulsion in accordance with the invention, in the presence of a protein source.

[0238] This in one embodiment the method comprising combining: a) a first component, b) a second component, and c) a fusion protein of the invention in the presence of a protein source.

[0239] In one embodiment the first component and the second component are substantially immiscible. In a further embodiment the first component and the second component are immiscible. Protein source

[0240] In one embodiment the protein source is present as part of the first component.

[0241] In one embodiment the first component is a protein-containing hydrophilic component.

[0242] In some embodiments the protein-containing hydrophilic component is selected from but not limited to a water-based solution, a plant-based aqueous solution or extract, an aqueous carbohydrate solution, and an aqueous protein solution.

[0243] The plant-based aqueous solutions or extracts may be selected from but not limited to plant milks. The plant milk can be selected from but not limited to almond milk, oat milk, soy milk, rice milk, cashew milk, hemp milk, or pea protein milk.

[0244] The aqueous protein solution includes but is not limited to pea protein hydrolysate, soy protein isolate, and rice protein concentrate.

[0245] The method therefore provides for capture of protein present in the first component into the emulsion produced.

[0246] In some embodiments the protein is a water-soluble protein.

[0247] In some embodiments the protein is extracted into, or added to, the hydrophilic component from another source, prior to performance of the method to produce the protein-enriched emulsion.

[0248] In a further embodiment the protein source is present as part of the second component.

[0249] In a further embodiment the second component is a hydrophobic component. In some embodiments the hydrophobic component is selected from but not limited to a protein-containing plant oil, a medium-chain triglyceride (MCT), an essential oil or flavor oil, and a lipid-based bioactive.

[0250] In some embodiments the plant oil is selected from but not limited to avocado oil, canola oil, coconut oil, flaxseed oil, olive oil, palm oil, rice bran oil, safflower oil, soybean oil, sunflower oil and vegetable oil.

[0251] In some embodiments the medium -chain triglyceride (MCT) is selected from but not limited to fractionated cocoa butter, coconut oil or palm kernel oil.

[0252] In some embodiments the essential oils or flavor oil is selected from but not limited to eucalyptus oil, orange oil, peppermint oil, and vanilla bean oil.

[0253] In some embodiments the lipid-based bioactive is selected from but not limited to fish oil, algal oil, and omega-3 fatty acid concentrates.

[0254] In some embodiments the protein is a lipid soluble protein.

[0255] In some embodiments the protein is extracted into, or added to the hydrophobic component from another source, prior to performance of the method to produce the protein enriched emulsion.

[0256] In some embodiments the protein that is incorporated into the protein-enriched emulsion may be added to one or both of the first and second components, or to a combination of the first and second components, prior to formation of the protein-enriched emulsion. In one embodiment the added protein in another extract from a plant or plant part. In some embodiments the plant part is a seed. In a further embodiment the added protein is in plant material from which other components have been extracted. In one embodiment the material is a protein cake or protein meal.

[0257] In one embodiment the protein cake or protein meal is obtained after the extraction of oil from plant material. Such cakes and meals are rich in protein. In one embodiment the plant material is a seed or seeds. Preferred seed are oil seeds. In a further embodiment the plant material is a nut or nuts.

[0258] The method therefore provides the opportunity to capture, or “up-cycle”, the protein present in such protein cake, protein meal, or other plant material, to create valuable protein-rich emulsions and compositions as herein described.

[0259] In one embodiment the emulsion in the composition, is protein-enriched relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0260] In a further embodiment the emulsion is protein enriched by at least 1%, preferably at least 2%, more preferably at least 5%, more preferably at least 10, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, more preferably at least 150%, more preferably at least 200%, more preferably at least 300%, more preferably at least 400%, more preferably at least 500%, more preferably at least 600%, more preferably at least 700%, more preferably at least 900%, more preferably at least 1000% relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein. Protein enriched emulsion

[0261] In a further aspect the invention provides a protein-enriched emulsion produced by a method of the invention.

[0262] In one embodiment the emulsion is protein -enriched relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0263] In a further embodiment the emulsion is protein enriched by at least 1%, preferably at least 2%, more preferably at least 5%, more preferably at least 10, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, more preferably at least 150%, more preferably at least 200%, more preferably at least 300%, more preferably at least 400%, more preferably at least 500%, more preferably at least 600%, more preferably at least 700%, more preferably at least 900%, more preferably at least 1000% relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0264] Method for producing compositions

[0265] In a further aspect the invention provides a method for producing a composition, the method providing the step of using a fusion protein of the invention as an emulsifier.

[0266] In various embodiments, the compositions comprise the components of the emulsions of the invention as herein described.

[0267] In further embodiments the compositions possess the enhanced emulsification properties of the fusion proteins and emulsions of the invention as herein described. In one embodiment the composition is selected from but not limited to a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional composition, a pharmaceutical composition, an animal feed, or an ingredient for use in such compositions.

[0268] In one embodiment the food product is selected from but not limited to a baked good, confectionary, pet food, dairy product, milk, cream, creamer, margarine, ice cream, milk powder, infant formula, salad dressing, mayonnaise, cheese, yoghurt, whipping cream, and whipped toppings.

[0269] In one embodiment the dairy product substitutes is selected from but not limited to a milk substitute, butter milk substitute, a cream substitute, a creamer substitute, a margarine, a butter substitute, a whipping cream substitute, a mayonnaise substitute, an ice cream substitute, a cheese substitute, and a yoghurt substitute.

[0270] Preferred dairy product substitutes are plant-based dairy product substitutes.

[0271] In one embodiment the plant-based dairy product substitutes is selected from but not limited to a plant-based milk substitute, a plant-based cream substitute, a plant-based creamer substitute, a plant-based whipping cream substitute, a plant-based mayonnaise substitute, a plant-based ice cream substitute, a plant-based cheese substitute, and a plantbased yoghurt substitute.

[0272] Composition comprising fusion protein and emulsions of the invention

[0273] The invention provides composition produced by methods of the invention and compositions comprising fusion proteins, emulsions, protein-enriched emulsions, and extracts of the invention.

[0274] In various embodiments, the compositions comprise the components of the emulsions of the invention as herein described. In one embodiment the composition is selected from but not limited to a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional composition, a pharmaceutical composition, an animal feed, or an ingredient for use in such compositions.

[0275] In one embodiment the food product is selected from but not limited to a baked good, confectionary, pet food, dairy product, milk, cream, creamer, margarine, ice cream, milk powder, infant formula, salad dressing, mayonnaise, cheese, yoghurt, whipping cream, and whipped toppings.

[0276] In one embodiment the dairy product substitutes is selected from but not limited to a milk substitute, butter milk substitute, a cream substitute, a creamer substitute, a margarine, a butter substitute, a whipping cream substitute, a mayonnaise substitute, an ice cream substitute, a cheese substitute, and a yoghurt substitute.

[0277] Preferred dairy product substitutes are plant-based dairy product substitutes.

[0278] In one embodiment the plant-based dairy product substitutes is selected from but not limited to a plant-based milk substitute, a plant-based cream substitute, a plant-based creamer substitute, a plant-based margarine, a plant-based butter substitute, a plant-based whipping cream substitute, a plant-based mayonnaise substitute, a plant-based ice cream substitute, a plant-based cheese substitute, and a plant-based yoghurt substitute.

[0279] The nutritional or pharmaceutical composition may further include:

[0280] • Nutritional additives selected from but not limited to: probiotics (e.g., Lactobacillus rhamnosus, Bifidobacterium longum), prebiotics (inulin, galactooligosaccharides), or dietary fibers (resistant starch, cellulose).

[0281] • Therapeutic agents selected from but not limited to: anti-inflammatory compounds (e.g., curcumin, ibuprofen), antioxidants (e.g., resveratrol, ascorbic acid), or drug carriers (lipophilic or hydrophilic drugs). • Bioactives selected from but not limited to: omega-3 fatty acids (e.g., DHA, EP A), plant sterols, or herbal extracts (e.g., ginseng, green tea polyphenols).

[0282] • Excipients selected from but not limited to solubility enhancers.

[0283] • Delivery system modifiers selected from but not limited to: cyclodextrins or liposomes for drug encapsulation, nanoparticles or microemulsions for sustained release.

[0284] • Sensory enhancers selected from but not limited to: effervescence agents (e.g., sodium bicarbonate, citric acid) for pharmaceutical powders.

[0285] Properties of the compositions

[0286] In various embodiments the compositions possess the enhanced emulsification properties of the fusion proteins and emulsions of the invention as herein described.

[0287] In one embodiment the composition has increased stability against coalescence during temperature cycling than does composition produced using the first protein as an emulsifier in place of the fusion protein.

[0288] In one embodiment the increased stability reduces rebodying or plasticization.

[0289] Plasticization or rebodifying of dairy cream refers to the process of altering the texture and consistency of cream to enhance its stability and usability in various applications.

[0290] These processes are crucial in producing whipped creams, spreads, and other dairy products where a stable texture is desired. Plasticization can extend the shelflife of dairy products.

[0291] In a further embodiment use of fusion proteins as emulsifiers leads to the formation of oil droplet clusters (e.g. at high emulsification pressure conditions).

[0292] In certain embodiments these oil droplets clusters can be used to develop new food textures that would normally be achieved by combining several ingredients in a food formulation.

[0293] In one embodiment the fusion proteins improve the stability of products. Such products include but are not limited to UHT / pasteurised plant-based beverages, coffee whiteners, and cream liqueurs.

[0294] In a further embodiment the fusion proteins improve foaming ability and stability.

[0295] These properties in key in applications such as dairy desserts (e.g. mousses), whipping cream and ice cream.

[0296] In a further embodiment, fusion proteins, in which the first protein is a casein, improve heat stability of emulsion-based beverages such as plant-based protein drinks that often undergo heat treatments such as pasteurisation and UHT.

[0297] In plant-based milks (e.g. almond, soy, oat) the fusion proteins preferably establish and maintain stable emulsions with reduced phase separation during storage.

[0298] In vegan creamers the fusion proteins preferably establish and maintain enhanced shelf-life and improved texture under varying temperature conditions (e.g., hot and cold beverages).

[0299] In salad dressings the fusion proteins preferably establish and maintain increased viscosity and uniform consistency without separation over extended periods.

[0300] In ice cream alternatives the fusion proteins preferably establish and maintain superior air incorporation and freeze-thaw stability, for example in plant-based formulations.

[0301] In one embodiment the composition, or the emulsion in the composition, is protein enriched relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0302] In a further embodiment the composition, or the emulsion in the composition, is protein enriched by at least 1%, preferably at least 2%, more preferably at least 5%, more preferably at least 10, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, more preferably at least 150%, more preferably at least 200%, more preferably at least 300%, more preferably at least 400%, more preferably at least 500%, more preferably at least 600%, more preferably at least 700%, more preferably at least 900%, more preferably at least 1000% relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

[0303] Polypeptide sequence identity

[0304] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www.ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0305] A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem.

[0306] Sci. 23, 403-5.)

[0307] Methods for producing polynucleotides for use in the invention

[0308] Methods for producing polynucleotides (that can be used to express the proteins and analogues of the invention) are well known to those skilled in the art, and include use of cloning and recombinant DNA technologies. These technologies may involve modification of an existing polynucleotide. Alternatively, the polynucleotide can be synthesised in its entirety by methods commonly used by those skilled in the art, and available commercially as a service from numerous well-known providers (e.g. GeneArt, Thermo Fisher Scientific).

[0309] The polynucleotides of the invention, encoding the proteins of the invention, may be codon optimised to resemble the codon usage of the cell or organism in which the proteins are expressed. Codons may be optimised using known tools (such as www.genewiz.com). This may result in improved gene expression and increased the translational efficiency, by accommodating the codon bias of the host.

[0310] Methods for producing constructs and vectors

[0311] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and / or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.

[0312] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).

[0313] Methods for producing host cells comprising polynucleotides, constructs or vectors

[0314] The invention provides a host cell which comprises a genetic construct or vector of the invention.

[0315] Host cells may be selected from but not limited to bacterial, fungal, insect, mammalian or plant cells.

[0316] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al. , Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification). Methods for producing plant cells and plants comprising constructs and vectors

[0317] The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention, or used in the methods of the invention. Plants comprising such cells also form an aspect of the invention.

[0318] Methods for transforming plant cells, plants and portions thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants.

[0319] Imperial College Press, London.

[0320] Methods for genetic manipulation of plants

[0321] A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297; Hellens et al., 2000, Plant Mol Biol 42: 819-32; Hellens et al., Plant Meth 1: 13). Lor example, strategies may be designed to increase expression of a polynucleotide / polypeptide in a plant cell, organ and / or at a particular developmental stage where / when it is normally expressed or to ectopically express a polynucleotide / polypeptide in a cell, tissue, organ and / or at a particular developmental stage which / when it is not normally expressed. The expressed polynucleotide / polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

[0322] Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.

[0323] The promoters suitable for use in the constructs of this invention are functional in plant cells that contain oil bodies. Preferably the promoters drive expression that is spatially and / or developmentally coordinated with the biosynthesis of oil bodies. In one embedment the promoters are from genes encoding oil body associated proteins. Suitable oil body- associated genes include those encoding oleosins, caleosins and steroleosins as described herein

[0324] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.

[0325] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance.

[0326] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial No. 5, 159, 135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent Nos. 5, 187, 073 and 6. 020, 539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci.104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, Oi l ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant Cell Rep. 24( l):45-51); Primus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink 2005 Plant Cell Rep. 2006 ;25(2): 117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep.22(l):38-45); strawberry (Oosumi et al., 2006 Planta. 223(6): 1219-30; Folta et al., 2006 Planta Apr 14; PMID: 16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995;44: 129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407-412), Canola (Brassica napus L.). (Cardoza and Stewart, 2006 Methods Mol Biol. 343:257-66), safflower (Orlikowska et al, 1995, Plant Cell Tissue and Organ Culture 40:85-91), ryegrass (Altpeter et al., 2004 Developments in Plant Breeding 11 (7):255-250), rice (Christou et al., 1991 Nature Biotech. 9:957-962), maize (Wang et al., 2009 In: Handbook of Maize pp. 609- 639) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25,5: 425-31). Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.

[0327] The polynucleotides of the invention, and their encoded proteins and analogues, can also be conveniently expressed and test via transient expression in leaves of transgenic plants, such as Nicotian benthamiana, using Agrobacterium infiltration (Kapila, J. , De Ry eke, R. , Van Montagu, M. and Angenon, G. (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. 122, 101-108).

[0328] Plant cells and plants

[0329] The plant cells and plants in which the fusion proteins are expressed, and from which various sequences, extracts, oils, proteins, protein cakes, and protein meals, for use in the invention are sourced, may be from any plant species.

[0330] In one embodiment the plant cell or plant, is from a gymnosperm plant species.

[0331] In a further embodiment the plant cell, or plant, is from an angiosperm plant species.

[0332] In a further embodiment the plant cell, or plant, is from a from dicotyledonous plant species.

[0333] In a further embodiment the plant cell, or plant, is from a monocotyledonous plant species.

[0334] In a further embodiment the plant cell, or plant, is from an oil seed species. In one embodiment the plant or plant cell is selected from but not limited to: almond, avocado, barley, bean, camelina, canola, chickpea, coconut, com, cowpea, fava bean, grapeseed, hemp, lentils or any other legume, lupin, maize, mung bean, oats, oil seed rape, pea, pomegranate, potato, quinoa, rice, sesame, safflower, soybean, sunflower, tapioca, tobacco, tomato, wheat.

[0335] In a further embodiment the plant is a nut. In one embodiment the nut is selected from a almonds, pecans, walnuts, cashews, pistachios, peanuts, kola nuts, palm nuts, hazelnuts, filberts, Brazil nuts, macadamia nuts, chestnuts, groundnuts and shea nuts.

[0336] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[0337] Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[0338] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

[0339] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. The term “comprising” as used in this specification means “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that orthose prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. In some embodiments, the term "comprising" (and related terms such as "comprise and "comprises") can be replaced by "consisting of' (and related terms "consist" and "consists").

[0340] In the description in this specification, reference may be made to subject matter which is not within the scope of the appended claims. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the presently appended claims

[0341] BRIEF DESCRIPTION OF THE DRAWINGS

[0342] Various embodiments of the invention are now illustrated with reference to the accompanying Figures, in which:

[0343] Figure 1 shows a schematic representation of pET28a(+) vectors.

[0344] Figure 2 shows SDS-PAGE (Coomassie stain) analysis of beta-casein protein fused to oleosin (oleosin:BC2; C-Ole:BC2 - BC2 fused to C-terminal part of oleosin; NC-Ole:BC2

[0345] - N and C terminals of oleosin fused to BC2; and C-Ole:BC2:C-Ole - BC2 flanked from both ends with C-terminal part of oleosin) and 0.5 and Ipg of positive control (Sigma Aldrich beta-casein). Black arrows - expected size of Beta-casein (27kDa), Oleosin:BC2 (50kDa), C-Ole:BC2 (35kDa), NC-Ole:BC2 (41kDa) and C-Ole:BC2:C-Ole (41kDa). Pre

[0346] - recombinant in inclusion bodies; Sup - refolded protein fractions; pel - refolded proteins in the pellet.

[0347] Figure 3 shows SDS-PAGE (Coomassie stain) analysis of beta-lactoglobulin proteins non fused (BLG) fused to C-terminal part of oleosin (C-term oleosin: BLG) and 1 pg of positive control (Sigma Aldrich beta-lactoglobulin). Black arrows - expected size of Beta- lactoglobulin (18.5kDa), C-Ole: BLG (27kDa). Black squares represent refolded recombinant proteins.

[0348] Figure 4 shows SDS-PAGE (Coomassie stain) analysis of the purified proteins including beta-lactoglobulin and alpha-casein proteins. Non-fused (BLG and aCSN) and fused to C- terminal part of oleosin (C-term oleosimBLG, C term ole:aCSN) and the full-length oleosin (Ole:aCSN). Ipg of positive control (Sigma Aldrich beta-lactoglobulin and aCSN).

[0349] Figure 5 shows (A) SDS-PAGE analysis of beta-casein binding to oil bodies. 100 p g Betacasein was emulsified with 20% of oil, and the supernatant and emulsion fractions were analyzed by SDS-PAGE. The gel was stained with a Coomassie stain. Expected size of Beta-casein, BC2 and BC3 (25kDa), Oleosin:BC2 and Oleosin:BC3 (50kDa). Sup - supernatant fraction; Emu - emulsion fraction.

[0350] Figure 6 shows (A) SDS-PAGE analysis of beta-lactoglobulin (native and 5C-A mutant) binding to oil bodies. 100 pg of Beta-lactoglobulin (native and mutant) with and w / o C- terminal oleosin was emulsified with 20% of oil, and the supernatant and emulsion fractions were analyzed by SDS-PAGE. The gel was stained with a Coomassie stain. Expected size of WT, native and mutant Beta-lactoglobulin (20kDa), and C-terminal OleosimP lactoglobulin (native and mutant) (27kDa). Sup - supernatant fraction; Emu - emulsion fraction.

[0351] Figure 7 shows (A) SDS-PAGE analysis of alpha-casein binding to oil. 100 pg Alphacasein was emulsified with 20% of oil, and the supernatant and emulsion fractions were analyzed by SDS-PAGE. The gel was stained with a Coomassie stain. Expected size of Alpha-casein (25kDa), OleosimaCSN (47kDa) and C-terminal Oleosin: aCSN (32kDa). Sup - supernatant fraction; Emu - emulsion fraction.

[0352] Figure 8 shows in A. Oil droplet size distribution for emulsions prepared from beta-casein obtained from Sigma and oleosimbeta-casein BC2 (El 1) in different concentration of safflower oil; B. Oil droplet size distribution for emulsions prepared from beta-casein obtained from Sigma and C-terminal oleosimbeta-casein BC2 (E12) in different concentration of safflower oil. For both Figures - Beta-casein:black line - 10% safflower oil; black dash line - 20% safflower; Black dotted line - 30% safflower oil. Recombinant oleosimbeta-casein BC2 / C -terminal oleosin: beta-casein BC2 (E11 / E12) (gray lines) at different oil concentrations (10%, 20%, and 30%). The y-axis shows the volume percentage of oil droplets, and the x-axis shows the oil droplet size in micrometers (pm).

[0353] Figure 9 shows Volume-weighted mean droplet diameters (D4,3, pm) of 10%, 20%, 30% safflower oil concentration emulsions formed with A. beta-casein (BC-Sigma) and oleosimbeta-casein (El l); B. C-terminal oleosimbeta-casein (E12). For both images: Black line - beta-casein (Sigma); dashed line - recombinant oleosimbeta-casein BC2 or recombinant C-terminal oleosimbeta-casein BC2.

[0354] Figure 10 shows Preferential Adsorption of Recombinant OleosimBeta-Casein BC2 and C-terminal OleosimBeta-Casein BC2 Proteins at the Oil / Water Interface of Emulsions. A) Illustrates the separation of the emulsion into two phases, cream (oil layer) and aqueous layer, achieved by centrifugation. B) Presents an SDS-PAGE gel stained with Coomassie blue, revealing the presence of beta-casein and oleosimbeta-casein (oleosin:BC2) proteins originating from their respective sources, including inclusion bodies for the recombinant protein (S), soluble protein in buffer for Sigma beta-casein, the emulsion (E), and the aqueous phase (A) after cream separation. C) Presents an SDS- PAGE gel stained with Coomassie blue, revealing the presence of beta-casein and C- terminal oleosimbeta-casein (C-Ole:BC2) proteins originating from their respective sources, including inclusion bodies for the recombinant protein (S), soluble protein in buffer for Sigma beta-casein, the emulsion (E), and the aqueous phase (A) after cream separation.

[0355] Figure 11 shows detection of the C-terminal-oleosimbeta-casein (BC2) fusion protein in transgenic Arabidopsis T1 seeds. Proteins extracted from wild-type (WT) and T1 seeds (lines 4, 6, 7, 8, 9, 11, 12 and 13) expressing construct #3 were analyzed by SDS-PAGE- stain-free detection (A) and western blotting (B) with anti-beta casein antibodies. A distinct band at the expected molecular weight of is observed in the transgenic lines but not WT (top arrow), indicating accumulation of the fusion protein. The band intensity is higher than the positive control (0.1 and 0.5 pg purified beta-casein standard from Sigma - bottom arrow).

[0356] Figure 12 shows in A illustration depicting the four distinct layers formed after oil body extraction and centrifugation of Arabidopsis seeds. These include the upper oil body emulsion (OB), supernatant (Sup), middle oil body emulsion (OB-m), and pellet. B) Photographs of the OB, OB-m, Sup, and pellet fractions obtained from wild-type (WT), wild-type spiked with beta-casein (WT + P-CSN), and transgenic lines 6 and 9 expressing the oleosimbeta-casein fusion. C) Nile red fluorescence staining verifying oil body content in the upper OB layers.

[0357] Figure 13 shows western blotting analysis for Beta casein of Arabidopsis seed extraction. The samples tested are wild type (WT), WT+ beta-casein (WT+ P-CSN), Line 9 and Line 6. Black arrows - expected size of Beta-casein (27kDa), C-terminal Oleosin:BC3 (37kDa). Sup - supernatant fraction; OB- oil bodies fraction, OB-m - middle layer emulsion oil bodies and Pel - pellet.

[0358] Figure 14 shows A) Extracted cream from wild-type Arabidopsis seeds (left) and transgenic seeds (right). B) Confocal micrograph of respective layers obtained from transgenic seed slurry after centrifugation. The proteins and fats are stained using Fast Green FCF and Nile Red, respectively. Arrows are differentiating between protein and oil aggregates C) A confocal micrograph for the cream layer obtained from transgenic and wild-type Arabidopsis seed. For each sample, the fats staining (Nile Red) is shown on the left (I) and both fats and proteins staining (Nile Red + Fast Green) is shown on the right (II). EXAMPLES

[0359] Various embodiments of the invention are now illustrated with the following non-limiting examples.

[0360] Example 1: Expression of beta casein fused to oleosin and oleosin affinity peptides in E. coli.

[0361] Vector preparation and transformation into E. coli

[0362] To evaluate the functionality of dairy proteins fused with either oleosin or an oleosin affinity peptide, the applicant employed bovine beta-casein or a beta-casein mutants, referred to as BC2 (SEQ ID NO: 87) and BC3 (SEQ ID NO: 78), Alpha Casein SI (SEQ ID NO: 1) and Beta-lactoglobulin (SEQ ID NO: 39) as representatives of dairy proteins. The beta-casein mutants, BC2 and BC3, have been previously characterized (see W02023119200). In the BC3 variant, the five phospho-serine residues have been substituted with the negatively charged amino acids aspartic acid (D) or glutamic acid (E) residues. This substitution of phospho-serine residues with aspartic acid (D) or glutamic acid (E) was designed to mimic the negative charge of the phosphate group, thereby facilitating calcium binding and lowering the isoelectric point of the protein. The specific mutations introduced were S15D, S17E, S18D, S19E, and S35D. In the BC2 variant, S17, S19 and S35 residues have been substituted with aspartic acid (D). This substitution also facilitates calcium binding and enhances stability.

[0363] For the present experiments, the applicant generated twenty five distinct constructs, as illustrated in Figure 1 and Table 6. The backbone of the constructs are as follows:

[0364] Backbone 1: Asses non-fiised dairy proteins (either native beta-casein, BC2, BC3, alphacasein S 1 and beta-lactoglobulin)

[0365] Backbone 2: These constructs involve testing the fusion of a dairy protein (either native beta-casein, BC2, BC3, alpha-casein SI and beta-lactoglobulin) protein with oleosin at the C-terminal end. Backbone 3: In this experiment, the applicants assessed the fusion of a dairy protein with the hydrophilic C-terminal segment of oleosin.

[0366] Backbone 4: To further explore the fusion possibilities, the applicants fused a dairy protein with a peptide that encompasses both hydrophilic segments of oleosin (N-terminal and C- terminal).

[0367] Backbone 5: This experiment involved fusing a dairy protein with the C-terminal portion of oleosin, utilizing segments from both the N-terminal and C-terminal ends of the betacasein protein.

[0368] All of these fragments were cloned into the pET28a (+) vector. The oleosin sequence utilized as the basis for these constructs is derived from Brassica napus oleosin (BnOLE - SEQ ID NO: 49; Acc. #P29110). Specifically, the sequence for the C-terminal oleosin is based on the last 56 amino acids of AtOLE (At4g25140 SEQ ID NO: 51), while the sequence for the N+C terminals of oleosin is a fusion of the first 45 amino acids of AtOLE with the last 56 amino acids of AtOLE (to form SEQ ID NO: 84).

[0369] These meticulously designed constructs serve as the foundation for the present experimental investigations into the fusion of dairy proteins with oleosin, allowing the applicant to examine various fusion strategies and their potential applications.

[0370] A total of 25 vectors were produced and transformed into E. Colt strain BL21 (DE3). All the vectors are presented in Table 6.

[0371] Table 6: List of pET28a(+) vectors transformed into E. Coli

[0372] The transformed BL21 (DE3) were incubated at 37 °C to form single colonies on an LB agar plate with 50 pg / mL kanamycin. A selected colony was used to grow overnight in 10 mL LB medium containing 50 pg / mL kanamycin at 37 °C to provide the starter. The starter was then diluted 100 times in the growing medium (0.5L LB medium in a 2L container) and grown at 37°C until the OD600 reached 0.5-0.6. Protein synthesis was induced with 0.1 and 0.4 mM IPTG at 18°C and 24°C overnight (Beta CSN proteins induced with 0.4mM IPTG at 18°C O.N; constructs contained alpha-casein SI proteins and Beta- lactoglobulin induced with 0.4mM IPTG 24°C O.N. ; C-terminal oleosin-Beta lactoglobulin induced with O.lmM IPTG 24°C O.N.). The samples were then centrifuged at 4°C and 8,000 rpm for 10 min, with the pellets separated and kept at -80°C.

[0373] Bacteria pellets were lysed using a 1: 10 ratio of cooled lysis buffer (25mM Tris buffer pH=8-9, 50 mM NaCl, DNase, Lysozyme, and protease inhibitor) - e.g. pellet from 50mL resuspend with 5mL lysis buffer. Cell disruption performed using a sonicator (Sonics vibra cell, Labotal) - Program: Time - 30 sec, Pulse - 5 sec On, 5 sec Off, Amplitude - 30%. Twice for each sample. The lysate centrifuge for 20min at 4°C, 12,000 rpm for separation of the soluble and insoluble fractions.

[0374] All beta-casein, the oleosin alpha-casein and C-terminal oleosin-Beta lactoglobulin recombinant proteins accumulated in inclusion bodies and required dissolving and refolding steps as described: The lysate pellets were resuspended in 5mL of 25mM Tris buffer pH=9 and centrifuged for 20 minutes at 4°C and 12,000 rpm. The supernatant was discarded and the pellet was resuspended again in 5mL of 20mM NaOH. After centrifugation, the supernatant was slowly titrated back to a pH of ~8 using 0. IM HC1, with continuous stirring. The applicants observed the expected recombinant proteins in the right sizes (Figure 2). The lysate pellets of Beta-lactoglobulin protein were resuspended in 5mL of 25mM Tris buffer pH=9 with 8M Urea and incubated O.N. at room temperature. The samples were refolded by removal of the urea using gradient dialysis at room temperature every 1 / 2 hour from 6M to 0 urea in 25mM Tris pH=9. The samples were centrifuged for 20 minutes at 4°C and 12,000 rpm. The supernatant was then loaded on His-Trap HP ImL column (Cytiva). The protein eluted from the column by an increasing gradient of imidazole (up to 250mM). The purity of the protein was identified by SDS- PAGE and all purified fractions were pooled and dialyzed into the storage buffer (DDW or 25mM TrisHCl pH=9). Figure 3 shows that beta-lactoglobulin proteins (fused and nonfused) were successfully refolded and obtained at the expected size. The lysate supernatants of alpha-casein were incubated at 80 °C for 30 min. Then, centrifuged for 20 minutes at 25°C and 12,000 rpm. The supernatant was then loaded on His-Trap HP ImL column (Cytiva). The protein eluted from the column by an increasing gradient of imidazole (up to 250mM). The purity of the protein was identified by SDS-PAGE and all purified fractions were pooled and dialyzed into the storage buffer (25mM Tris pH=9, 50mM NaCl). The final purified products of native alpha casein, c-terminal, and full oleosimalpha casein are presented in Figure 4.

[0375] Example 2: Binding Analysis of Safflower oil and recombinant Alpha-casein, BetaCasein and Beta-lactoglobulin, Oleosin:beta-casein\alpha-casein and oleosin affinity peptides fused to beta-casein, alpha-casein and beta-lactoglobulin

[0376] To understand if the recombinant proteins fused to oleosin or to oleosin peptides have improved affinity to pure safflower oil, the applicants created an emulsion with 20% Safflower oil and 100 pg of the recombinant proteins. As a control, bovine beta-casein, alpha-casein, and Beta-lactoglobulin (Sigma) were used.

[0377] Sample preparation of lOOug of recombinant proteins in 25mM Tris buffer pH=9. 200pL of pure safflower oil was added the volume was adjusted to a final of ImL with 25 mM Tris buffer pH=9. For emulsification, the solution was sonicated (Sonics vibra cell, Labotal) - Program: Time - 30 sec, Pulse - 1 sec On, 1 sec Off, Amp - 30%. The samples were centrifuged at 12,000 x rpm, 20 min at 4C. Separation of the aqueous and emulsion phases by syringe and needle (pulling lOOuL of supernatant, taking the needle out, and letting the remaining supernatant pour out). Resuspend that oil\emulsion with ImL buffer.

[0378] The relative band intensities in the emulsion versus supernatant fractions provided an estimate of the affinity of the recombinant proteins for the oleosimprotein / oleosin affinity peptide: protein fusions compared to the native sequence and the Bovine milk proteins purchased from Sigma.

[0379] All proteins displayed some association with the oil. The majority of the beta-casein remains in the aqueous phase and part of it is adsorbed in the emulsion (Figure 5). Alpha casein showed higher affinity to the oil with a greater fraction of the native protein absorbed into the emulsion. On the other hand, the oleosin-casein fusions (of both beta and alpha) showed significantly increased affinity, where all the proteins (OleosimAlpha casein and Oleosimbeta casein) were found in the emulsion fraction (Figures 5, 7). The oleosin affinity peptide showed an improved affinity of beta-lactoglobulin to safflower oil with a higher fraction of the protein in the emulsion fraction compared to the aqueous phase (Figure 6). The oleosin C-terminal peptide fused to alpha casein did not show a significant improvement in the oil binding affinity (Figure 7).

[0380] These results demonstrate that oleosin fusion and oleosin-affinity peptide fusions allow the improved extraction of recombinant dairy proteins to the plant oil phase, enriching the plant cream with dairy proteins. This data supports oleosin fusion as a strategy to modulate the stability and nutritional properties of engineered dairy proteins in plant oils.

[0381] Example 3: Emulsion stability of recombinant beta-casein fused to oleosin or to oleosin affinity peptides

[0382] Materials

[0383] The applicants compared Oleosimbeta-casein, C-terminal Oleosimbeta casein fusion proteins from E. colt in inclusion bodies and bovine beta-casein was obtained commercially (Sigma). Solutions of 20 mM and 0.5 mM NaOH were prepared in distilled water.

[0384] Emulsion Preparation

[0385] Aqueous dispersions of the fusion proteins (oleosin:BC2, C-terminal oleosin:BC2) and bovine beta-casein (2% w / v) were made by resuspending the proteins in 0.2 mM NaOH solution. For each protein, emulsions containing 10%, 20% and 30% (v / v) safflower oil were prepared. The oil and aqueous phases were pre-mixed using an Ultra-Turrax homogenizer for 30 seconds. Emulsions were then homogenized by sonication using a microtip probe sonicator (Sonics Vibra Cell, Labotal). Sonication was done at 30% amplitude with 5 sec on / off pulses for a total duration of 30 sec. The sonication was repeated twice for each emulsion. Particle Size Analysis

[0386] The size distribution of oil droplets in the emulsions was measured by static light scattering using a Mastersizer 2000 instrument (Malvern Instruments). Prior to analysis, emulsions were diluted 1 : 1 with SDS solution. Approximately 250 pL of diluted emulsion was loaded into the sample unit for each measurement. Measurements were taken in triplicate for each emulsion. Particle size distribution data was exported and analyzed using Excel and GraphPad Prism software. Statistical comparisons between emulsions were done by t-tests and AN OVA in Prism.

[0387] Particle Size Distribution of Emulsions

[0388] The particle size distribution of emulsions prepared with oleosin-beta casein fusion protein (Ell) or beta casein protein at 10%, 20%, and 30% (v / v) oil concentrations is shown in Figure 8A. All emulsions contained 0.6% (v / v) protein in the aqueous phase as estimated by the Kjeldahl method. For emulsions with 10% and 20% oil, the size distribution of oil droplets stabilized by the oleosin-beta casein fusion protein was similar to that of droplets stabilized by beta casein (Figure 8). The majority of oil droplets ranged from 0.5 to 10 pm in diameter, with a small population of droplets less than 0.5 pm and greater than 10 pm (see dashed line in Figure 8A). This suggests the emulsion properties of the oleosin-beta casein fusion protein are comparable to those of bovine beta casein. Increasing the oil concentration resulted in larger oil droplet sizes for both beta casein and oleosin-beta casein emulsions. At fixed protein concentration, higher oil content increases the total surface area of the oil droplets. With limited emulsifier present, the average droplet size increases. This expected correlation between oil percentage and droplet size was observed for both proteins.

[0389] The particle size distribution of emulsions prepared with C-Ole:BC2 fusion proteins (E 12) or beta casein protein at 10%, 20%, and 30% (w / w) oil concentrations is shown in Figure 8B. All emulsions contained 0.6% (w / w) protein in the aqueous phase as estimated by the Kjeldahl method. The 10% emulsions stabilized with E12 inclusion bodies and beta casein show very similar patterns, with most oil droplets ranging between 0.5 and 10 pm (see dashed square in Figure 8B). This result also indicates that E12 and beta casein have similar emulsifying ability at this oil concentration. However, when the oil concentration increases to 20 and 30%, the particle size distributions of E12 show the presence of larger oil droplets (mostly between 10 to 100 pm), suggesting less emulsifying ability. This suggests the emulsion properties of the C-terminal oleosin-beta casein fusion protein are comparable to those of bovine beta casein at 10% oil. Increasing the oil concentration resulted in larger oil droplet sizes for both beta casein and oleosin-beta casein emulsions. At fixed protein concentration, higher oil content increases the total surface area of the oil droplets. With limited emulsifier present, the average droplet size increases. This expected correlation between oil percentage and droplet size was observed for both proteins. In summary, recombinant oleosimbeta casein and oleosin affinity peptides fused to beta casein can effectively stabilize oil-in-water emulsions similar to beta casein protein alone.

[0390] Effect of Protein Type on Mean Oil Droplet Size

[0391] The mean volume-weighted oil droplet diameter (D4,3 pm) for emulsions stabilized by oleosimbeta casein (BC2) and C-terminal oleosimbeta casein (BC2) fusion protein or beta casein at 10%, 20%, and 30% oil concentrations is shown in Figure 9. The oleosin-beta casein emulsions exhibited smaller mean oil droplet sizes compared to the beta casein emulsions at 10% and 20% oil concentrations. At 10% oil, the oleosin-beta casein emulsion had a D4,3 of 2. 1 pm while the beta casein emulsion had a D4,3 of 2.5 pm. Similar differences were seen at 20% and 30% oil concentrations (Figure 9A). The smaller droplet sizes produced by oleosin-beta casein fusion protein suggest it has equivalent or improved emulsification properties compared to standard beta casein. This enhanced functionality could be due to the emulsion stabilizing effects of the oleosin domain in the fusion protein. In conclusion, recombinant oleosin-beta casein created smaller emulsion oil droplets and therefore may have advantages as an emulsion stabilizer relative to beta casein alone.

[0392] The C-terminal oleosin-beta casein emulsions (E12) similar smaller mean oil droplet sizes compared to the beta casein emulsions at 10% and 20% oil concentration. The droplet sizes produced by C-terminal oleosin-beta casein fusion protein suggest it has equivalent emulsification properties compared to standard beta casein (Figure 9B). Adsorption of Oleosin-Beta Casein and oleosin affinity peptides fused to beta casein to Oil Droplet Interface

[0393] To confirm the oleosin-beta casein and fusion protein acts as an emulsifier by adsorbing to the oil-water interface, emulsions were centrifuged to separate the oil droplet-containing cream phase from the aqueous phase. The cream and aqueous phases were analyzed by SDS-PAGE with Coomassie staining (Figure 10). A prominent protein band was observed in the solution and emulsion samples for the both oleosimbeta casein and c-terminal oleosimbeta-casein inclusion bodies. As expected, this protein band was absent in the aqueous phases after centrifugation but present in the cream phases. This demonstrates the oleosin-beta casein fusion protein is associating with the oil droplets during emulsification. The lack of protein in the aqueous phase indicates the oleosin-beta casein and c-terminal oleosimbeta-casein is surface active and absorbs to the oil-water interface to stabilize the oil droplets. The SDS-PAGE results support the role of the recombinant protein as an emulsifier in the prepared emulsions.

[0394] Example 4: Arabidopsis T1 Seeds Selection, Protein Extraction, and Detection

[0395] T1 Arabidopsis seeds were obtained after transformation with construct #3 and following basta selection. The transgenic seeds were analyzed for accumulation of C-terminal- oleosimbeta-casein fusion proteins. Proteins were extracted from ground seeds in a carbonate buffer with p-mercaptoethanol and protease inhibitors. Samples were centrifuged and the supernatant was analyzed by SDS-PAGE and western blotting. After gel electrophoresis, proteins were transferred to a nitrocellulose membrane. The membrane was blocked then incubated with anti -beta casein primary antibody (1: 10,000 dilution) overnight at 4°C, followed by washing. HRP-conjugated secondary antibody (1 : 10,000) was applied for 1 hour at room temperature. Signals were detected using ECL substrate and imaging on a ChemiDoc system.

[0396] As shown in Figure 11 , a distinct band of the expected molecular weight for the C-terminal- oleosimbeta-casein fusion was successfully detected in T1 seeds from transgenic lines, but not in wild-type controls. The band signal was at the expected size and higher compared to the positive-control beta-casein obtained from Sigma. This demonstrates accumulation of the engineered protein taigeted to seed oil bodies, confirming stable transgene expression and inheritance in the Arabidopsis lines generated.

[0397] Example 5: Oil Body Extraction from Plant Seeds

[0398] High-speed oil body extraction Approximately 50 mg of both transgenic (T1 seeds of lines 6 and 9, expressing C-terminal oleosin:BC2 fusion protein) and wild-type (WT) Arabidopsis seeds were utilized in accordance with the precise amounts listed in Table 7. These seeds were soaked in deionized distilled water (DDW) for one hour. Subsequently, the water was carefully removed, and 1 mb of grinding buffer (comprising 10 mM sodium phosphate, pH=7.5, and 0.6 M sucrose) was added. The seeds were then subjected to grinding using the Tissuelyser with four rounds of 30 seconds each at a speed of 300 RPM. Following grinding, the samples underwent centrifugation at 4°C, at a force of 10,000 RCF for a duration of 20 minutes. From this centrifugation, samples were collected from four distinct phases, namely the upper emulsion (OB), supernatant, middle emulsion (OB-m), and pellets. In order to serve as a control, 150 pg of Beta casein was spiked into the WT seeds prior to the grinding process. The OB and OB-m layers were stained with Nile-Red (Img / mL in methanol, diluted 1:9 dye to sample ratio) to verify fat content. Each layer was sampled and loaded on SDS-PAGE and Western Blot performed using Beta-Casein antibodies.

[0399] Table 7: Sample Description

[0400] The results of the oil body extraction procedure revealed the presence of four distinct layers, as depicted in Figure 12A. These layers included atop layer rich in oil bodies, a middle layer of supernatant (Sup) primarily containing soluble proteins, a middle emulsion housing oil bodies (OB-m), and a pellet consisting of insoluble proteins, debris, and cell wall material. For the WT, an especially prominent layer of oil bodies (OB) or fat was observed at the top, a phenomenon corroborated by Nile-Red staining (Figure 12C). In contrast, the spiked betacasein exhibited a thinner layer of oil bodies, with most of the oil bodies sedimenting to form the OB-m layer. Interestingly, the transgenic plants expressing the recombinant C-terminal oleo sin: Beta-casein (BC2) (Lines 6 and 9) displayed minimal or very thin top layers of oil bodies. As confirmed by Nile Red staining, most of the oil bodies had sedimented to the bottom. This observation suggests that the C-terminal oleosimbeta-casein fusion protein stabilizes the oil bodies, promoting the formation of a single unified layer of oil bodies. The samples loaded on SDS-PAGE gel showed that beta-casein and the transgenic fused proteins were mostly found in the oil body fractions (OB and OB-m - Figure 13).

[0401] Western blot analysis detected a distinct band at ~35 kDa, corresponding to the expected size of the C-terminal oleosin: beta-casein fusion protein, in both the OB and OB-m fractions from transgenic lines (line 9) or only at OB-m and pellet (line 6). This band was absent in WT (Figure 13). Some fainter bands were observed in the supernatant likely due to partial solubility. Beta-casein was detected in both OB-m and OB fraction of spiked WT.

[0402] Low -speed oil body extraction

[0403] Approximately 2-3 grams of both transgenic (T3 seeds of line 12) and wild-type (WT) Arabidopsis seeds were soaked in a 1:5 ratio (w / w) with buffer (comprising 50 mM Sodium bicarbonate) overnight. The soaked seeds were ground using a Tissuelyser at 30 Hz frequency for 2 minutes, repeating for 3 rounds. Following grinding, the samples underwent centrifugation at a force of 3,000 RCF for 10 minutes. The transgenic seed cream forms in the middle layer, while the Arabidopsis wild-type seed cream forms in the top layer with an aqueous layer on the top and a broken seeds layer at the bottom (Figure 14A). Confocal microscopy analysis and protein content estimation by micro-BCA Confocal laser scanning microscopy measurements were performed for all the different layers (figure 14B). The cream was diluted to 1:4 (and undiluted sample also) were stained using Nile Red (1 mg / mL in acetone) for lipids and Fast Green FCF (1 mg / mL in Milli-Q water) for proteins. 200 pL of the cream was mixed with 12 pL of Nile Red and 6 pL of Fast Green. The stained sample was placed on a concave microscope slide, covered with a 0.17 mm thick cover slip, and immediately examined. Imaging was performed using a 63 mm oil immersion objective lens. For lipids, Nile Red was excited at 488 nm (Argon laser), and the emission was measured in the 500-580 nm range. For proteins, Fast Green was excited at 633 nm (He-Ne laser), and the emission was measured in the 650-700 nm range. The images were processed using ImageJ software.

[0404] The cream layer from centrifuged transgenic Arabidopsis seed slurry shows fat in the protein matrix in the middle layer. In contrast, the cream obtained from wild-type seeds shows no protein (Figure 14C). The interaction of protein and fat in the cream from transgenic seeds increases the cream's density, causing the cream layer to settle down. This demonstrates the interaction of protein and fat in transgenic seeds due to genetic modification, unlike the wild-type cream where such interaction is absent.

[0405] Protein estimation using BCA assay was conducted. The BCA analysis showcases that the aqueous phase contains around 0.024 % protein while the cream phase contains 10 % protein, signifying that most protein went to the cream.

[0406] The altered density and sedimentation pattern of oil bodies in transgenic Arabidopsis seeds indicates that the C-terminal oleosin: beta-casein fusion protein stably associates with oil bodies, increasing their density. The oil body layer thickness measurements, confocal microscopy and western blotting confirms preferential partitioning of the C-terminal oleosimbeta-casein protein into the oil body fraction compared to the spiked native betacasein and to the WT Arabidopsis oil bodies fraction. Overall, these results validate the ability of oleosin affinity peptides fused to dairy proteins to serve as a tool for modification and sequestration of proteins into plant oil bodies for food and industrial uses. REFERENCES

[0407] Dalgleish, D. G. (1997). Adsorption of protein and the stability of emulsions. Trends in Food Science & Technology, 8 (1), 1-6.

[0408] Frandsen GI, Mundy J, Tzen JT (2001) Oil bodies and their associated proteins, oleosin and caleosin. Physiol Plant 112: 301-307.

[0409] Hartel, R. W., & Hasenhuettl, G. L. (2019). Food emulsifiers and their applications (Third edition ed.). Springer.

[0410] Huang AHC (1992) Oil bodies and oleosins in seeds. Annu Rev Plant Physiol Plant Mol Biol 43: 177-200.

[0411] J Kyte, R F Doolittle (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol; 157(1): 105-32.

[0412] Loer DS, Herman EM (1993) Cotranslational integration of soybean (Glycine max) oil body membrane protein oleosin into microsomal membranes. Plant Physiol 101: 993-998.

[0413] McClements, D. J. (2016). Food emulsions: principles, practices, and techniques (Third edition ed.). CRC Press LLC.

[0414] Murphy DJ (1993). Structure, function and biogenesis of storage lipid bodies and oleosins in plants. Progress in Lipid Research, 32(3): 247-280.

[0415] Norn, V. (2014). Emulsifiers in food technology (Second edition ed.). Wiley Blackwell.

[0416] Tzen JTC, Lie GC, Huang AHC. (1992) Characterization of the charged components and their topology on the surface of plant seed oil bodies. J Biol Chem 267: 15626-15634.

[0417] Winichayakul et al., (2013). In Vivo Packaging of Triacylglycerols Enhances Arabidopsis Leaf Biomass and Energy Density. Plant Physiol.; 162(2):626-639.

Claims

CLAIMS1. A method for enhancing at least one emulsification property of a first protein, the method comprising fusing the first protein to second protein selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP), to produce a fusion protein with at least one enhanced emulsification property relative to the first protein.

2. A method for producing a fusion protein with at least one enhanced emulsification property, the method comprising fusing a first protein to second selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP) wherein the fusion protein has at least one enhanced emulsification property relative to the first protein.

3. The method of claim 1 or 2 that includes a step of selecting the fusion protein based on measuring at least one enhanced emulsification property of the fusion protein relative to the first protein.

4. A fusion protein produced by a method of any preceding claim.

5. A fusion protein comprising a first protein fused to a second protein selected from: a) an oil body-associated protein (OBP), and b) a protein with affinity for an oil body-associated protein (AOBP). wherein the fusion protein has at least one enhanced emulsification property relative to the first protein.

6. A polynucleotide encoding a fusion protein of any preceding claim.

7. A cell, plant cell, plant or plant part comprising at least one of:a) a fusion protein of any preceding claim, and b) a polynucleotide encoding a fusion protein of any preceding claim.

8. An extract of the cell, plant cell, plant or plant part, comprising the fusion protein.

9. Use of the fusion protein of any preceding claim as an emulsifier.

10. A method of using a fusion protein of any preceding claim as an emulsifier.

11. A method for producing an emulsion the method comprising combining: a) a first component, b) a second component, and c) a fusion protein of any preceding claim.

12. The method of claim 11 in which the first component and the second component are substantially immiscible.

13. The method of claim 11 or 12 in which the first component is a hydrophilic component.

14. The method of any one of claims 11 to 12 in which the second component is a hydrophobic component.

15. An emulsion produced by a method of any one of claims 9 to 14.

16. An emulsion comprising a fusion protein of any preceding claim.

17. A emulsion comprising: a) a first component, b) a second component, and c) a fusion protein of any preceding claim.

18. The emulsion of claim 17 in which the first component is a hydrophilic component.

19. The emulsion of claim 17 or 18 in which the second component is a hydrophobic component.

20. The emulsion of any one of claims 15 to 19 that is stabilized by the fusion protein.

21. The emulsion of any one of claims 15 to 20 that has at least one enhanced property relative to an emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

22. A method for sequestering protein from a protein source into an emulsion, the method comprising performing the method of claim 11 in the presence of the protein source, the method resulting in a protein-enriched emulsion.

23. A method for producing a protein-enriched emulsion, the method comprising performing the method of claim 11 in the presence of a protein source.

24. A protein-enriched emulsion produced by the method of claim 22 or 23.

25. A method for producing a composition, the method providing the step of using a fusion protein of any preceding claim as an emulsifier.

26. The method of claim 25 in which the fusion protein is in, or derived from, the extract of claim 8.

27. A method for producing a composition, the method comprising the step of incorporating the emulsion, or protein-enriched emulsion of any preceding claim into the composition.

28. The method of claim 25 or 27 in which the composition is selected from a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed.

29. The method of claim 25 or 27 in which the composition is an ingredient for use in at least one of a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed.

30. The method of claim 28 or 29 in which the composition is a plant-based dairy product substitute or an ingredient for use in a plant-based dairy product substitute.

31. The method of any one of claims 25 to 30 in which the composition, or an emulsion in the composition, is stabilized by the fusion protein.

32. The method of any one of claims 25 to 31 in which the composition, or the emulsion in the composition, has enhanced stability relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

33. A composition produced by a method of any one of claims 25 to 32.

34. A composition comprising at least one of: a) a fusion protein of any preceding claim, or produced by the method of any preceding claim, b) an emulsion of any preceding claim, or produced by the method of any preceding claim, c) a protein-enriched emulsion of any preceding claim, and d) an extract of any preceding claim.

35. The composition of claim 33 or 34 that is selected from: a) a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation, and an animal feed, and b) an ingredient for use in at least one of a food product, a beverage product, a dairy product, a dairy product substitute, a plant-based dairy product substitute, a nutritional supplement, a pharmaceutical formulation and an animal feed.

36. The composition of claim 35 that is a plant-based dairy product substitute, or an ingredient for use in a plant-based dairy product substitute.

37. The composition of any preceding claim in which the composition, or an emulsion in the composition, is stabilized by the fusion protein.

38. The composition of any preceding claim in which the composition, or the emulsion in the composition, has enhanced stability relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

39. The composition of any preceding claim in which the composition, or the emulsion in the composition, is protein enriched relative to a composition or emulsion in which the first protein is used as an emulsifier in place of the fusion protein.

Images