Methods and compositions for producing plant-based dairy product substitutes

Abstract

The invention provides method for producing a composition containing oil body and dairy proteins by making an extract from a plant cell or plant expressing the dairy proteins, in close proximity to, or physical connection with, an oil body in the plant cell, plant part or 5 plant. In some embodiments the dairy proteins are expressed as fusions with oil-body associated proteins, or with components with affinity for oil body associated proteins. The invention provides methods for producing oil bodies functionalized with dairy proteins. The invention further provides constructs for expressing the fusion proteins, and cells and plants comprising the constructs, fusion proteins and functionalized oil bodies. The 10 invention provides the isolated functionalized oil bodies and fusion proteins and methods for their use in the production of various products including plant based dairy substitutes. The invention also provides various products comprising the functionalized oil bodies and fusion proteins.

Description

[0001] METHODS AND COMPOSITIONS FOR PRODUCING PLANT-BASED DAIRY

[0002] PRODUCT SUBSTITUTES

[0003] FIELD OF INVENTION

[0004] The invention is in the field plant-based dairy product substitutes, their production, and use in the food and beverage industry.

[0005] BACKGROUND TO THE INVENTION

[0006] Dairy farming and related industries are major contributors to anthropogenic climate change. Dairy represents around 10% of all global human Greenhouse Gas emissions. Moreover, nitrous oxide emissions from fertilizers used to grow feed for livestock are significant sources of greenhouse gas emissions, higher than those caused by fertilizer used on food crops. Manure and effluent waste produced by dairy farming leads to significant eutrophication. Nutrient runoff from livestock operations can lead to pollution of potable water resources in nearby and distant communities. Consequently, a more sustainable and equivalent non-animal substitute resource for dairy is required.

[0007] There is an increased demand for plant-based alternatives to traditional dairy products that can have similar taste, smell, feel, digestibility, functionality, and nutritional value as traditional dairy products.

[0008] After protein, lipid is the second most essential component of milk. In raw milk, lipid is present as fat globules with a variable size (0.1 to 15 pm, average 4 pm). During the homogenization of milk, the size of fat globules is reduced to less than 1pm.

[0009] Plant-based dairy substitutes contain fat or oils to mimic the mouthfeel of bovine milk. This lipid can come from the same source as the protein, as in soy-based "milks", or can be added from another source, as in the case of oat and other grain-based "milks". Fats or oils can also be combined with recombinantly expressed dairy proteins to produce dairy alternatives. Typical sources of oil for addition to plant-based dairy substitutes include coconut, com, soybean, canola, palm, rapeseed, safflower and sunflower. Homogenization is usually applied to ensure reduction of oil droplet size and thus enhancement of creaming stability.

[0010] The first step in the production of plant-based milk substitutes typically involves grinding the raw material, such as cereals, oilseeds, etc., which may be either dry or wet, followed by dispersion in water. During processing, various salts, emulsifiers and stabilizers, and oil as described above are added in an attempt to mimic the creamy mouthfeel and physical stability of dairy milk. Similarly, such components may be added to recombinantly expressed dairy proteins to produce dairy product substitutes.

[0011] Drawbacks to current processes and products include:

[0012] 1. inefficiencies resulting from the need to produce and combine protein and oil, and other additives, from separate sources,

[0013] 2. that the oils used do not truly match animal dairy fats for flavour and texture,

[0014] 3. that the oils used need to be stabilized using emulsifiers (e.g. exogenously added emulsifiers).

[0015] 4. that relative to bovine dairy products, existing plant-based dairy alternatives have sub-optimum functionality with respect to: a) whipping and foaming properties of cream substitutes, b) stability of cream or milk substitutes in beverages, such as coffee c) emulsification when cream substitutes are incorporated into other products such as ice cream and beverages, d) rheological and gelation properties in products such as cream filling, cheese or yoghurt gels, and baked goods, e) most plant proteins have lower protein quality and hence lower nutritional value compared with dairy proteins f) alternative dairy products also don’t have the correct short chain fatty acids in the lipid fractions

[0016] It is therefore an object of the invention to provide improved methods and compositions for producing plant-based dairy substitutes 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

[0017] The invention provides compositions and methods for producing plant-based dairy substitutes. The invention involves the expression of dairy proteins and analogues thereof, in plants, wherein the expression is spatially and / or developmentally coordinated or overlapping with the biosynthesis of oil bodies in the plants. The dairy proteins, and analogues, are produced in close proximity, or in connection with the oil bodies, such that an extract containing both the oil bodies and dairy proteins / analogues can be conveniently and efficiently produced and economically processed for convenient storage, transport and formulation. The invention further facilitates production and use of oil bodies functionalized with dairy proteins in accordance with the invention, and their isolation from the plant cells or plants. The invention further provides recombinantly expressed fusion proteins. The fusion proteins may conveniently be expressed in plant cells or plants, or may be expressed in other systems for purification and addition to plant extracts during processing. The extracts, compositions, functionalized oil bodies, fusion proteins and emulsions produced also have beneficial properties for use in production of plant-based dairy substitutes, as discussed further herein.

[0018] The invention thus enables the safe, economic, ecologically sustainable, and humane production of plant-derived dairy substitutes, such as cream substitutes, coupled with the high nutritional values and functionalities of traditional milk proteins for commercial use, such as used in food compositions.

[0019] The invention also facilitates a “new plant-based dairy making system” comprised of interacting and interrelated elements that enable proprietarily designed dairy-protein- oilseed (or other plant part) crushing and mixing in ratios enabling the creation of a myriad of 'dairy milk / cream compositions', each with differing dairy product forming properties ready to be formulated into diverse dairy structures and derivative products including cheeses, ice creams, milks etc. The protein-oil design uniquely enables this process, and the process is similarly designed to facilitate this. The elements of this 'Dairy Seed System’ include and comprise: a) 'single dairy proteins’, of different types (e.g. alpha / beta / kappa / lactoferrin, etc.) required for dairy product making, attached uniquely, to oil bodies expressed in uniquely designed and cultivated plants / seeds. This is important to maintain plants’ energy balance and protein production capacity while allowing selection of seeds with the optimized concentration per dairy protein; b) harvestable and easily transportable fresh and easily extractable (without expensive and energy consumptive dehydration or fractionation as is the state of the art in animal dairy) as described herein; c) and flexibly co-processed in the crushing and extraction stage as well as post extraction, specifically because, different individual dairy proteins are expressed in single oilseeds / plants enabling the flexible mixing possibilities to create a wide possibility of blends to obtain dairy milk / creams with differing properties such as those required especially for making a wide array of dairy products requiring differing fats and protein profiles. d) and because the protein-oleo nexus and their specific localisations in plants cells are designed to specific create dairy compositions and emulsions.

[0020] This disclosure describes plants engineered to express unique protein-oil bonds, in different seeds / plant organs, and their 'conception as a system', enabling the following unique benefits which are new to the dairy making industry and introduce a new state of the art: a) a unique processing and extraction efficiency b) which has an energy usage saving, c) a time saving, d) a process complexity reduction lowering chances for error and wastage, e) a resources (water and additives) saving and f) thus an economic and financial improvement g) and an environmental and sustainability footprint improvement.

[0021] The ‘system1would not work, but for, the design of a 'new type of dairy protein-oil plant / seed' which links the protein and oil body for the purposes of extracting 'a dairy milk / cream composition’ which enables unique formulation preparedness and flexibility as a "formulation ready dairy ingredient”. The system also requires that only one type of dairy protein be expressed associated with, or connected to, the oil body at a known concentration so that it can be multifariously mixed with other similarly designed single protein-oil body types in other oil seed / plant genus and species to be able to 'control the mixture' and effect the creation of a myriad of milk / cream composition mixes.

[0022] In animal dairy systems milk is collected, then water is dried off at a high energy cost. Large amounts of water are wasted and large amounts of protein run off are wasted and this causes pollution. Then the remaining powder needs to be separated into its component fractions before it can be reformulated, and rehydrated into formulations. The present “dairy system” component of this disclosure circumvents these steps of wasted time, energy, resources and costs. It is designed to reduce these inefficiencies in animal dairy processing. The elements of the design in parts, and as a composed systemic whole, are conceived and ordered for the purpose of obtaining a designed simplicity in the matter of human endeavour and in the application of resources, energy and time.

[0023] / . METHODS

[0024] 1.1 Method for producing a composition comprising an oil body (OB) and a dairy protein (DP)

[0025] In one aspect, the invention provides a method for producing a composition containing at least one oil body and at least one dairy protein, analogue, or part thereof, the method comprising the step of making an extract from a plant cell, plant tissue, plant organ, plant part or plant expressing the dairy protein, analogue, or part thereof, in close proximity to, or physical connection with, an oil body in the plant cell, plant tissue, plant organ, plant part or plant, to produce the composition.

[0026] In one embodiment the plant tissue is an oleaginous tissue. In a further embodiment the plant organ is an oleaginous organ. In a further embodiment the plant organ is a reproductive oleaginous organ.

[0027] In a preferred embodiment the plant part is a seed. In one embodiment the dairy protein, analogue, or part thereof, is expressed in a cell containing an oil body.

[0028] 1.1.1 Expression / production of DP in the cytosol

[0029] In one embodiment the dairy protein, analogue, or part thereof, is expressed in the cytosol of a cell containing an oil body.

[0030] In one embodiment the dairy protein or analogue or part thereof is co-extracted with the oil body.

[0031] 1.1.2 Expression / production of DP in subcellular location

[0032] In a further embodiment the dairy protein, analogue, or part thereof, is localized in a subcellular location in the cell containing an oil body.

[0033] In a further embodiment the subcellular location is selected from: the cell wall or apoplast, a vacuole, a chloroplast, a mitochondrion, a peroxisome and endoplasmic reticulum.

[0034] In one embodiment, directing the dairy protein, analogue, or part thereof to a subcellular location reduces the potential for interference with other cellular processes. In a further embodiment subcellular location also reduces the potential for deleterious effects on plant / plant cell metabolism and growth.

[0035] In one embodiment the dairy protein or analogue or part thereof is co-extracted with the oil body.

[0036] 1.1.3 Expression of a fusion of DP and oil body associated protein (OBAP)

[0037] In a further embodiment the dairy protein, analogue, or part thereof, is expressed as a fusion comprising the dairy protein, analogue, or part thereof and an oil body-associated protein. In one embodiment the fusion, is localized in a subcellular location in the cell as described above.

[0038] 1.1.4 OBAP embedded in OB membrane

[0039] In a further embodiment the oil body-associated protein of the fusion is embedded in the phospholipid membrane of an oil body thereby physically connecting the dairy protein, analogue, or part thereof, to the oil body.

[0040] In a further embodiment the dairy protein or analogue, or part thereof, is physically connected to the oil body because it is fused to the oil body-associated protein, and the dairy protein or analogue, or part thereof, is co-extracted with the oil body.

[0041] In these embodiments the dairy protein, analogue or part thereof are physically connected to the oil body and can be co-extracted with the oil body.

[0042] 1.1.5 Expression of a fusion of DP and component with affinity for OBAP

[0043] In a further embodiment the dairy protein, analogue, or part thereof, is expressed as a fusion comprising the dairy protein, analogue, or part thereof and a component that has affinity for an oil body-associated protein.

[0044] In one embodiment the fusion, is localized in a subcellular location in the cell as described above.

[0045] In one embodiment the fusion of the dairy protein, analogue, or part thereof and a component that has affinity for an oil body-associated protein can be more effectively targeted to the subcellular location in the cell than can a fusion of dairy protein, analogue, or part thereof and an oil body-associated protein (as for example, in 1.1.3 above).

[0046] 1.1.6 DP affinity linked to an OBAP in the OB membrane

[0047] In this embodiment the dairy protein, analogue, or part thereof becomes affinity linked to the oil body via the affinity of the component to an oil body associated protein in the phospholipid membrane of the oil body. 1.1.7 Component with affinity for OBAP

[0048] In one embodiment the component that has affinity for the oil body-associated protein is a hydrophilic portion of an oil body-associated protein. In a further embodiment the hydrophilic portion is the C-terminal hydrophilic portion of an oil body-associated protein.

[0049] In a further embodiment the component that has affinity for the oil body-associated protein is a single-chain Fv antibody (scFv) with specific affinity for the oil-body associated protein.

[0050] 1.1.8 Co-expressing component with affinity for both DP and OBAP

[0051] In a further embodiment the dairy protein, analogue, or part thereof, is co-expressed with a component that has affinity for both the dairy protein, analogue, or part thereof and the an oil body-associated protein.

[0052] 1.1.9 DP affinity linked to an OBAP in the phospholipid membrane via component

[0053] In this embodiment the dairy protein, analogue, or part thereof becomes affinity linked to the oil body via the affinity of the component to both the dairy protein, analogue, or part thereof, and an oil body associated protein in the oil body. The component therefore acts as an intermediate affinity linking the dairy protein, analogue, or part thereof can become to the oil body.

[0054] In these embodiments the dairy protein, analogue or part thereof are physically connected to the oil body and can be co-extracted with the oil body.

[0055] In certain embodiments the part of the dairy protein or analogue, is the hydrophilic domain of a beta-casein protein or analogue thereof. In further embodiments the hydrophilic domain of a beta-casein protein is present as a tandem repeat / s. 1.2 Production of functionalized oil bodies

[0056] In preferred embodiments of the method for producing the composition, the dairy protein, analogue, or part thereof becomes physically linked, or affinity linked, to the oil body to produce an oil body that is functionalized with the dairy protein, analogue, or part thereof. These functionalized oil bodies have beneficial properties for the production of dairy alternatives, as discussed further herein.

[0057] The functionalizing of the oil bodies may occur in vivo.

[0058] Alternatively, the functionalizing may occur during the method for producing the composition. For example, this may occur when a dairy protein, analogue, or part thereof, or fusion, comes into contact with an oil body following disruption of the cell. This disruption may for example release the dairy protein, analogue, or part thereof, or fusion thereof, from a subcellular location, and then come into contact with an oil body.

[0059] In a preferred embodiment the composition produced by the method comprises at least one functionalized oil body as described above. Such functionalized oil bodies produced by the method, can be described as isolated functionalized oil bodies, with isolated meaning separated from the plant cell, plant tissue, plant organ, plant part or plant from which it is extracted.

[0060] In a preferred embodiment the composition produced by the method therefore comprises at least one isolated functionalized oil body as described above.

[0061] Expression and subcellular localization of the dairy proteins, analogues and parts thereof, and the functionalizing of oil bodies, in accordance with the invention, is illustrated in Figures 2, 3 and 4.

[0062] 1.3 Method including a step to enrich for oil bodies and dairy proteins / functionalized oil bodies

[0063] In a further embodiment the method includes a step to enrich for oil bodies, or functionalized oil bodies, in producing the composition. In one embodiment the enrichment step is based on separation of liquids by density.

[0064] In certain embodiments, method to enrich for oil bodies or functionalized oil bodies includes: vortexing to separate oil-water fractions, and centrifugation.

[0065] In one embodiment the extract is subjected to centrifugation, and a fraction containing the oil body and dairy protein, analogue or part thereof, or functionalized oil bodies is collected to produce the composition.

[0066] In further embodiment the composition comprises oil, or oil bodies, or functionalsied oil bodies, at a concentration of at least 1% w / w, preferably at least 2% w / w, preferably at least 5% w / w, preferably at least 10% w / w, preferably at least 20% w / w, preferably at least 30% w / w, preferably at least 40% w / w, preferably at least 50% w / w, preferably at least 60% w / w, preferably at least 70% w / w, preferably at least 80% w / w.

[0067] In further embodiment the composition comprises the dairy protein, analogue or part thereof at a concentration of at least 0.5 mg / 100 mg, preferably at least 0.2 mg / 100 mg, preferably at least 0.3 mg / 100 mg, preferably at least 0.4 mg / 100 mg, preferably at least 0.5 mg / 100 mg, preferably at least 0.6 mg / 100 mg, preferably at least 0.7 mg / 100 mg, preferably at least 0.8 mg / 100 mg, preferably at least 0.9 mg / 100 mg, preferably at least 1.0 mg / 100 mg, preferably at least 1.1 mg / 100 mg, preferably at least 1.2 mg / 100 mg, preferably at least 1.3 mg / 100 mg, preferably at least 1.4 mg / 100 mg, preferably at least

[0068] 1.5 mg / 100 mg, preferably at least 1.6 mg / 100 mg, preferably at least 1.7 mg / 100 mg, preferably at least 1.8 mg / 100 mg, preferably at least 1.9 mg / 100 mg, 2.0 mg / 100 mg, preferably at least 2.1 mg / 100 mg, preferably at least 2.2 mg / 100 mg, preferably at least 2.3 mg / 100 mg, preferably at least 2.4 mg / 100 mg, preferably at least 2.5 mg / 100 mg, preferably at least 2.6 mg / 100 mg, preferably at least 2.7 mg / 100 mg, preferably at least 2.8 mg / 100 mg, preferably at least 2.9 mg / 100 mg, 3.0 mg / 100 mg, preferably at least 3.1 mg / 100 mg, preferably at least 3.2 mg / 100 mg, preferably at least 3.3 mg / 100 mg, preferably at least 3.4 mg / 100 mg, preferably at least 3.5 mg / 100 mg, preferably at least

[0069] 3.6 mg / 100 mg, preferably at least 3.7 mg / 100 mg, preferably at least 3.8 mg / 100 mg, preferably at least 3.9 mg / 100 mg, preferably at least 4.0 mg / 100 mg. 1.4 Mixed starting material

[0070] Those skilled in the art will understand that different starting plant material (plant cells, plant tissues, plant organ, plant part, seeds) expressing different dairy proteins in accordance with the invention can be used as mixed starting material, thus producing a composition comprising an oil body and one or more different dairy proteins.

[0071] In addition, a combination of plant material (plant cells, plant tissues, plant organ, plant part, seeds) expressing dairy proteins in accordance with the invention, and plant material from other sources that don't express dairy proteins in accordance with the invention, can be used as starting material. In such embodiments the composition produced comprises oil bodies and dairy proteins, or functionalized oil bodies, in accordance with the invention, and further includes oil or oil bodies from different sources. Additional starting material could for example include safflower, canola or soybean material that does not express dairy proteins, parts or analogues thereof.

[0072] The invention thus facilitates a system combining a ‘dairy cropping system’ and a ‘dairy processing and formulation system‘ for efficient and economic product making through mixed ratio processing of differing plant materials containing oil bodies and dairy proteins. Plant parts or seeds, can be mixed at a desired ratio, to produce a desired ratio of dairy proteins and oil bodies depending on the requirements of the ultimate product. Such a system is illustrated in Figure 7.

[0073] 1.5 Physical method steps

[0074] In a preferred embodiments, the starting material from which the composition containing at least one oil body and at least one dairy protein, analogue, or part thereof is extracted is seeds.

[0075] In one embodiment the method includes the step of de-hulling the seeds.

[0076] In a further embodiment the method includes the step of physically disrupting the seed, de-hulled seed, or other starting material. Methods for disrupting seeds, and other plant material, are known in the art and include for example, grinding, bead beating, blending, milling, crushing using a colloid mill, processing with single or twin-screw press.

[0077] In a further embodiment the method includes the step of soaking the seeds, de-hulled seeds prior to disruption, and the disruption of the soaked or soaking seed produces a slurry.

[0078] In one embodiment the soaking is in water, an aqueous buffer or an aqueous extraction buffer.

[0079] In one embodiment the seeds or de-hulled seeds are soaked in the water, aqueous buffer or aqueous extraction buffer for at least 30 minutes, preferably at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 4 hours, preferably at least 5 hours, preferably at least 6 hours, preferably at least 7 hours, preferably at least 8 hours, preferably at least 9 hours, preferably at least 10 hours, preferably at least 11 hours, preferably at least 12 hours, preferably at least 13 hours, preferably at least 14 hours, preferably at least 15 hours, preferably at least 16 hours, preferably at least 17 hours, preferably at least 18 hours, preferably at least 19 hours, preferably at least 20 hours, preferably at least 21 hours, preferably at least 22 hours, preferably at least 23 hours, preferably at least 24 hours.

[0080] In a further embodiment the seeds or de-hulled seeds, or other plant material is / are disrupted to produce a powder that is subsequently suspended in water, an aqueous buffer or an aqueous extraction buffer, to produce a slurry.

[0081] In one embodiment the powder is soaked in the water, aqueous buffer or aqueous extraction buffer for at least 30 minutes, preferably at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 4 hours, preferably at least 5 hours, preferably at least 6 hours, preferably at least 7 hours, preferably at least 8 hours, preferably at least 9 hours, preferably at least 10 hours, preferably at least 11 hours, preferably at least 12 hours, preferably at least 13 hours, preferably at least 14 hours, preferably at least 15 hours, preferably at least 16 hours, preferably at least 17 hours, preferably at least 18 hours, preferably at least 19 hours, preferably at least 20 hours, preferably at least 21 hours, preferably at least 22 hours, preferably at least 23 hours, preferably at least 24 hours.

[0082] In a further embodiment the method includes a step to remove particulate matter from the slurry.

[0083] In a further embodiment the method includes the step of filtering the slurry to remove particulate matter and produce a filtered extract.

[0084] In a further embodiment, the slurry or filtered extract is subjected a step to enrich for oil bodies, or functionalized oil bodies.

[0085] In one embodiment, the slurry or filtered extract is subjected to centrifugation.

[0086] In one embodiment the centrifugation is at at least 5000 g, preferably at least 6000 g, preferably at least 7000 g, preferably at least 8000 g, preferably at least 9000 g, preferably at least 10000 g.

[0087] In one embodiment the centrifugation is for at least 10 minutes, preferably at least 11 minutes, preferably at least 12 minutes, preferably at least 13 minutes, preferably at least 14 minutes, preferably at least 15 minutes, preferably at least 16 minutes, preferably at least 17 minutes, preferably at least 18 minutes, preferably at least 19 minutes, preferably at least 20 minutes, preferably at least 30 minutes.

[0088] In another embodiment at larger scale, these separations are performed using continuous- flow decantation centrifugation to separate solids from liquids. The liquid phase is then concentrated for oil bodies, or functionalized oil bodies using continuous flow centrifuges employed routinely in the dairy industry such as a Sharpies tubular bowl model AS- 16 or a disk-stack centrifuge such as Westfalia model SA7 capable of 17, 000-20, 000g for rapid separations with short dwell -times.

[0089] In a further embodiment after centrifugation, a fraction containing the oil bodies and dairy proteins, analogues or parts thereof is collected. In a further embodiment the oil bodies and dairy proteins or analogues, or parts thereof, are subjected to one or more washing steps by mixing the fraction containing the oil body and dairy protein, analogue or part thereof with water or an aqueous solution, and repeating the centrifugation and collection steps, one or multiple times.

[0090] A fraction, preferably the final fraction, containing the oil bodies and dairy proteins or analogues, or functionalized oil bodies, is the "composition comprising an oil body and a dairy protein" in accordance with the invention. The composition can also be described as a "plant-derived dairy milk" when relatively dilute for oil / oil bodies / functionalized oil bodies, or a "plant-derived dairy cream" when relatively concentrated for oil / oil bodies / functionalized oil bodies.

[0091] Certain preferred embodiments of the method are described in the Examples.

[0092] An art-skilled worker will understand that the order in which the steps are performed can be varied, and that considerations of solubility, pH, water binding capacity, temperature and structural integrity should be taken into account, together with the final desired use of the extracts / compositions / creams when deciding on the purification technique to be used.

[0093] 1.6 Plants used in the method

[0094] 1.6.1 Plant comprises construct

[0095] In one embodiment the plant used in the method comprises a genetic construct for expression of at least one dairy protein or analogue thereof, or part thereof, in close proximity to, or physical connection with, an oil body in a plant.

[0096] 1.6.2 Promoter in construct

[0097] In one embodiment the construct comprises a promoter operably linked to a polynucleotide encoding the dairy protein, analogue or part thereof. In one embodiment the promoter drives expression that is spatially and / or developmentally coordinated with, or at least overlapping with, biosynthesis of an oil body.

[0098] In one embodiment the promoter is from a gene encoding a protein that is strongly expressed in seeds or other tissues or organs capable of oil body biogenesis.

[0099] In one embodiment the promoter is a seed-specific promoter. Preferred seed-specific promoters include but are not limited to seed-storage protein promoters. Preferred seedstorage protein promoters include those from phaseolin, conglycinin, glycinin or cruciferin genes.

[0100] Alternatively, in certain embodiments, if the oil bodies are produced in somatic tissues, such as leaves, a promoter from a photosynthetic gene such as RuBP carboxylase or chlorophyll a / b binding protein can be employed.

[0101] In one embodiment the promoter is from a gene encoding a protein that is expressed in a plant cell that produces oil bodies.

[0102] In one embodiment the promoter is from a gene encoding a protein that is expressed in close proximity to, or physical association with, an oil body in a plant.

[0103] In one embodiment the promoter is from a gene encoding an oil body-associated protein.

[0104] In one embodiment the promoter is from a gene encoding a protein that is integral to an oil body.

[0105] In one embodiment the promoter is from a gene encoding a protein selected from an oleosin, a caleosin, and a steroleosin.

[0106] In one embodiment the promoter is from a gene encoding an oleosin.

[0107] In one embodiment the construct drives expression / production of the dairy protein, analogue or part thereof in the cytosol of a cell containing an oil body. 1.6.3 Subcellular targeting signal peptide

[0108] In a further embodiment the construct includes a nucleic acid encoding a signal peptide for directing the dairy protein, analogue or part thereof or fusion to a subcellular location in the cell containing an oil body.

[0109] In this embodiment the construct encodes a signal peptide to direct localization of the dairy protein, analogue or part thereof, or fusion, to the subcellular location.

[0110] In a further embodiment the subcellular location is selected from: the cell wall or apoplast, a vacuole, a chloroplast, a mitochondrion, a peroxisome and endoplasmic reticulum.

[0111] In certain embodiments the signal peptide is one that directs localization to such a subcellular location.

[0112] 1.6.4 Construct encoding a translational fusion of a dairy protein and an oil body- associated protein

[0113] In one embodiment the construct includes a nucleic acid encoding an oil body-associated protein operably linked to the polynucleotide encoding the dairy protein, analogue or part thereof.

[0114] In this embodiment the construct expresses a fusion comprising: a) the oil body-associated protein, and b) the dairy protein or analogue or part thereof.

[0115] In one embodiment in the fusion, the oil body-associated protein makes up the N-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the C- terminal portion of the fusion.

[0116] In a further embodiment the construct includes a nucleic acid encoding a protease cleavage site. In one embodiment the nucleic acid encoding the cleavage site is positioned between the nucleic acid encoding the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0117] The construct may also optionally include a nucleic acid encoding a flexible linker between the nucleic acid encoding the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0118] In one embodiment the construct above is used to express the dairy protein, analogue or part thereof fused to an oil body-associated protein embedded in the phospholipid membrane of an oil body.

[0119] In a further embodiment the construct includes a nucleic acid encoding a signal peptide for targeting the fusion to a subcellular location, as discussed above.

[0120] 1.6.5 Construct encoding translational fusion of a dairy protein and component with affinity for an oil body-associated protein

[0121] In one embodiment polynucleotide encoding the dairy protein, analogue or part thereof is operably linked to at least one nucleic acid encoding a component that has affinity for an oil body-associated protein.

[0122] In one embodiment in the fusion, the component that has affinity for an oil body- associated protein makes up the N-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the C-terminal portion of the fusion.

[0123] In a further embodiment in the fusion, the component that has affinity for an oil body- associated protein makes up the C-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the N-terminal portion of the fusion.

[0124] In one embodiment the component that has affinity for the oil body-associated protein is a hydrophilic portion of an oil body-associated protein. In one embodiment the hydrophilic portion is the C-terminal hydrophilic portion of an oil body-associated protein. In a further embodiment the hydrophilic portion is the N-terminal hydrophilic portion of an oil body-associated protein.

[0125] In a further embodiment the component that has affinity for the oil body-associated protein is a single-chain Fv antibody (scFv) with specific affinity for the oil-body associated protein.

[0126] In a further embodiment the construct includes two nucleic acids encoding components with affinity for an oil body-associated protein.

[0127] In one embodiment the construct expresses a fusion of: a) a first component with affinity for an oil body-associated protein b) a dairy protein or analogue or part thereof c) a second component with affinity for an oil body-associated protein

[0128] In one embodiment the first and / or second component that has affinity for the oil body- associated protein is a hydrophilic portion of an oil body-associated protein. In one embodiment the hydrophilic portion is the C-terminal hydrophilic portion of an oil body- associated protein. In a further embodiment the hydrophilic portion is the N-terminal hydrophilic portion of an oil body-associated protein.

[0129] In a further embodiment the construct includes at least one nucleic acid encoding a protease cleavage site.

[0130] In one embodiment the nucleic acid encoding the cleavage site is positioned between the nucleic acid encoding the component that has affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0131] In a further embodiment the construct includes at least two nucleic acids encoding a protease cleavage site.

[0132] In one embodiment the nucleic acids encoding the cleavage sites are positioned between the nucleic acids encoding the first and second components that have affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof. The construct may also optionally include a nucleic acid encoding a flexible linker between at least one of the nucleic acids encoding the component that has affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0133] In one embodiment the construct above is used to express the dairy protein, analogue or part thereof fused to the at least one component that has affinity for the oil body- associated protein. At least one of the components that has affinity for the oil body- associated protein in the fusion protein, has affinity for the hydrophilic region of a native oil body-associated protein embedded in the phospholipid membrane of an oil body. The dairy protein, analogue or part thereof (in the fusion protein) thereby becomes associated or affinity -linked the oil body.

[0134] In one embodiment polynucleotide encoding the dairy protein, analogue or part thereof is operably linked to a nucleic acid encoding a component that has affinity for an oil body- associated protein.

[0135] In one embodiment in the fusion, the component that has affinity for an oil body- associated protein makes up the N-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the C-terminal portion of the fusion.

[0136] In a further embodiment in the fusion, the component that has affinity for an oil body- associated protein makes up the C-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the N-terminal portion of the fusion.

[0137] In one embodiment the component that has affinity for the oil body-associated protein is a hydrophilic portion of an oil body-associated protein. In a further embodiment the hydrophilic portion is the C-terminal hydrophilic portion of an oil body-associated protein.

[0138] In a further embodiment the construct includes a nucleic acid encoding a protease cleavage site. In one embodiment the nucleic acid encoding the cleavage site is positioned between the nucleic acid encoding the component that has affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0139] The construct may also optionally include a nucleic acid encoding a flexible linker between the nucleic acid encoding the component that has affinity for the oil body- associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0140] In one embodiment the construct above is used to express the dairy protein, analogue or part thereof fused to the component that has affinity for the oil body-associated protein. The component that has affinity for the oil body-associated protein in the fusion protein, has affinity for the hydrophilic region of a native oil body-associated protein embedded in the phospholipid membrane of an oil body. The dairy protein, analogue or part thereof (in the fusion protein) thereby becomes associated affinity linked the oil body.

[0141] In a further embodiment the construct includes a nucleic acid encoding a signal peptide for targeting the fusion to a subcellular location, as discussed above.

[0142] In certain embodiments the part of the dairy protein or analogue, is the hydrophilic domain of a beta-casein protein or analogue thereof. In further embodiments the hydrophilic domain of a beta-casein protein is present as a tandem repeat.

[0143] In one embodiment, the method includes the step of transforming the plant with the construct.

[0144] 2. COMPOSITION COMPRISING AN OIL BODY AND A DAIRY PROTEIN,

[0145] OR FUNCTIONALIZED OIL BODIES

[0146] In a further aspect the invention provides a composition, or extract, comprising at least one of: a) at least one oil body and at least one dairy protein, analogue, or part thereof, in accordance with the invention, and b) at least one a functionalized oil body in accordance with the invention This composition can also be described as a "plant-derived dairy milk" or "plant-derived dairy cream" . When relatively more dilute for oil / oil bodies / functionalized oil bodies, the composition can be described as a "plant-derived dairy milk" . When relatively more concentrated for oil / oil bodies / functionalized oil bodies, the composition can be described as a "plant-derived dairy cream"

[0147] In a further embodiment the composition, extract, "plant-derived dairy milk" or "plant- derived dairy cream" is produced by a method of the invention.

[0148] In further embodiment the composition, extract, plant-derived dairy milk", or "plant- derived dairy cream" comprises oil or oil bodies or functionalized oil bodies, at a concentration of at least 1% w / w, preferably at least 2% w / w, preferably at least 5% w / w, preferably at least 10% w / w, preferably at least 20% w / w, preferably at least 30% w / w, preferably at least 40% w / w, preferably at least 50% w / w, preferably at least 60% w / w, preferably at least 70% w / w, preferably at least 80% w / w.

[0149] In further embodiment the composition , extract, plant-derived dairy milk" or "plant- derived dairy cream" comprises the dairy protein, analogue or part thereof at a concentration of at least 0. 1 mg / 100 mg, preferably at least 0.2 mg / 100 mg, preferably at least 0.3 mg / 100 mg, preferably at least 0.4 mg / 100 mg, preferably at least 0.5 mg / 100 mg, preferably at least 0.6 mg / 100 mg, preferably at least 0.7 mg / 100 mg, preferably at least 0.8 mg / 100 mg, preferably at least 0.9 mg / 100 mg, preferably at least 1.0 mg / 100 mg, preferably at least 1.1 mg / 100 mg, preferably at least 1.2 mg / 100 mg, preferably at least 1.3 mg / 100 mg, preferably at least 1.4 mg / 100 mg, preferably at least 1.5 mg / 100 mg, preferably at least 1.6 mg / 100 mg, preferably at least 1.7 mg / 100 mg, preferably at least 1.8 mg / 100 mg, preferably at least 1.9 mg / 100 mg, 2.0 mg / 100 mg, preferably at least 2.1 mg / 100 mg, preferably at least 2.2 mg / 100 mg, preferably at least 2.3 mg / 100 mg, preferably at least 2.4 mg / 100 mg, preferably at least 2.5 mg / 100 mg, preferably at least 2.6 mg / 100 mg, preferably at least 2.7 mg / 100 mg, preferably at least 2.8 mg / 100 mg, preferably at least 2.9 mg / 100 mg, 3.0 mg / 100 mg, preferably at least 3.1 mg / 100 mg, preferably at least 3.2 mg / 100 mg, preferably at least 3.3 mg / 100 mg, preferably at least 3.4 mg / 100 mg, preferably at least 3.5 mg / 100 mg, preferably at least 3.6 mg / 100 mg, preferably at least 3.7 mg / 100 mg, preferably at least 3.8 mg / 100 mg, preferably at least 3.9 mg / 100 mg, preferably at least 4.0 mg / 100 mg

[0150] 3. ISOLA TED FUNCTIONALIZED OIL BODIES

[0151] In a further aspect the invention provides an isolated functionalized oil body. The oil body is functionalized with the dairy protein, analogue or part thereof, in accordance with the invention.

[0152] 3.1 Composition comprising isolated functionalised oil bodies

[0153] In a further aspect the invention provides a composition comprising an isolated functionalized oil body, in accordance with the invention.

[0154] In further embodiment the composition comprises functionalized oil bodies at a concentration of at least 1% w / w, preferably at least 2% w / w, preferably at least 5% w / w, preferably at least 10% w / w, preferably at least 20% w / w, preferably at least 30% w / w, preferably at least 40% w / w, preferably at least 50% w / w, preferably at least 60% w / w, preferably at least 70% w / w, preferably at least 80% w / w.

[0155] In further embodiment the composition comprises the dairy protein, analogue or part thereof at a concentration of at least 0.1 mg / 100 mg, preferably at least 0.2 mg / 100 mg, preferably at least 0.3 mg / 100 mg, preferably at least 0.4 mg / 100 mg, preferably at least 0.5 mg / 100 mg, preferably at least 0.6 mg / 100 mg, preferably at least 0.7 mg / 100 mg, preferably at least 0.8 mg / 100 mg, preferably at least 0.9 mg / 100 mg, preferably at least 1.0 mg / 100 mg, preferably at least 1.1 mg / 100 mg, preferably at least 1.2 mg / 100 mg, preferably at least 1.3 mg / 100 mg, preferably at least 1.4 mg / 100 mg, preferably at least 1.5 mg / 100 mg, preferably at least 1.6 mg / 100 mg, preferably at least 1.7 mg / 100 mg, preferably at least 1.8 mg / 100 mg, preferably at least 1.9 mg / 100 mg, 2.0 mg / mL, preferably at least 2.1 mg / mL, preferably at least 2.2 mg / mL, preferably at least 2.3 mg / mL, preferably at least 2.4 mg / 100 mg, preferably at least 2.5 mg / 100 mg, preferably at least 2.6 mg / 100 mg, preferably at least 2.7 mg / 100 mg, preferably at least 2.8 mg / 100 mg, preferably at least 2.9 mg / 100 mg, 3.0 mg / 100 mg, preferably at least 3.1 mg / 100 mg, preferably at least 3.2 mg / 100 mg, preferably at least 3.3 mg / 100 mg, preferably at least 3.4 mg / 100 mg, preferably at least 3.5 mg / 100 mg, preferably at least 3.6 mg / 100 mg, preferably at least 3.7 mg / 100 mg, preferably at least 3.8 mg / 100 mg, preferably at least 3.9 mg / 100 mg, preferably at least 4.0 mg / 100 mg.

[0156] 3.2 Emulsion

[0157] In a further aspect the invention provides an emulsion derived from a composition or isolated functionalized oil body, of the invention.

[0158] In a further embodiment the emulsion comprised at least one dairy protein physically linked to an oil body associated protein.

[0159] 4. CONSTRUCTS

[0160] In a further aspect the invention provides a genetic construct for expressing at least one dairy protein or analogue thereof, or part thereof, or fusion.

[0161] In one embodiment the genetic construct is for expressing at least one dairy protein or analogue thereof, or part thereof, or fusion in close proximity to, or physical connection with, an oil body in a plant.

[0162] 4.1 Promoter

[0163] In one embodiment the construct comprises a promoter operably linked to a polynucleotide encoding the dairy protein, analogue or part thereof.

[0164] In one embodiment the promoter drives expression that is spatially and / or developmentally coordinated with, or at least overlapping with, biosynthesis of an oil body.

[0165] In one embodiment the promoter is from a gene encoding a protein that is strongly expressed in seeds or other tissues or organs capable of oil body biogenesis. In one embodiment the promoter is a seed-preferred promoter. In a further embodiment the promoter is a seed-specific promoter.

[0166] Preferred seed specific promoters include but are not limited to seed-storage protein promoters. Preferred seed-storage protein promoters include those from phaseolin, conglycinin, glyclinin or cruciferin genes.

[0167] Alternatively, in certain embodiments, if the oil bodies are to be produced in somatic tissues such as leaves, a promoter from a photosynthetic gene such as RuBP carboxylase or chlorophyll a / b binding protein can be employed.

[0168] In one embodiment the promoter is from a gene encoding a protein that is expressed in a plant cell that produces oil bodies.

[0169] In one embodiment the promoter is from a gene encoding a protein that is expressed in close proximity to, or physical association with, an oil body in a plant.

[0170] In one embodiment the promoter is from a gene encoding an oil body-associated protein.

[0171] In one embodiment the promoter is from a gene encoding a protein that is integral to an oil body.

[0172] In one embodiment the promoter is from a gene encoding a protein selected from an oleosin, a caleosin, and a steroleosin.

[0173] In one embodiment the promoter is from a gene encoding an oleosin.

[0174] In one embodiment the construct drives expression / production of the dairy protein, analogue or part thereof in the cytosol of a cell containing an oil body. 4.2 Subcellular targeting signal peptide

[0175] In a further embodiment the construct includes a nucleic acid encoding a signal peptide for directing the dairy protein, analogue or part thereof or fusion to a subcellular location in the cell containing an oil body.

[0176] In this embodiment the construct encodes a signal peptide to direct localization of the the dairy protein, analogue or part thereof, or fusion, to the subcellular location.

[0177] In a further embodiment the subcellular location is selected from: the wall or apoplast, a vacuole, a chloroplast, a mitochondrion, a peroxisome, and endoplasmic reticulum.

[0178] In certain embodiments the signal peptide is one that directs localization to such a subcellular location.

[0179] 4.3 Construct encoding a translational fusion of a dairy protein and an oil body- associated protein

[0180] In one embodiment the construct includes a nucleic acid encoding an oil body-associated protein operably linked to the polynucleotide encoding the dairy protein, analogue or part thereof.

[0181] In this embodiment the construct expresses a fusion comprising: a) the oil body-associated protein, and b) the dairy protein or analogue or part thereof.

[0182] In one embodiment in the fusion, the oil body-associated protein makes up the N-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the C- terminal portion of the fusion.

[0183] In a further embodiment the construct includes a nucleic acid encoding a protease cleavage site. In one embodiment the nucleic acid encoding the cleavage site is positioned between the nucleic acid encoding the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0184] The construct may also optionally include a nucleic acid encoding a flexible linker between the nucleic acid encoding the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0185] In one embodiment the construct above is used to express the dairy protein, analogue or part thereof fused to an oil body-associated protein embedded in the phospholipid membrane of an oil body.

[0186] In a further embodiment the construct includes a nucleic acid encoding a signal peptide for targeting the fusion to a subcellular location, as discussed above.

[0187] 4.4 Construct encoding translational fusion of a dairy protein and at least one component with affinity for an oil body-associated protein

[0188] In one embodiment polynucleotide encoding the dairy protein, analogue or part thereof is operably linked to at least one nucleic acid encoding a component that has affinity for an oil body-associated protein.

[0189] In one embodiment in the fusion, the component that has affinity for an oil body- associated protein makes up the N-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the C-terminal portion of the fusion.

[0190] In a further embodiment in the fusion, the component that has affinity for an oil body- associated protein makes up the C-terminal portion of the fusion, and the dairy protein or analogue or part thereof makes up the N-terminal portion of the fusion.

[0191] In one embodiment the component that has affinity for the oil body-associated protein is a hydrophilic portion of an oil body-associated protein. In one embodiment the hydrophilic portion is the C-terminal hydrophilic portion of an oil body-associated 1 protein. In a further embodiment the hydrophilic portion is the N-terminal hydrophilic portion of an oil body-associated protein.

[0192] In a further embodiment the construct includes two nucleic acids encoding components with affinity for an oil body-associated protein.

[0193] In one embodiment the construct expresses a fusion of: a) a first component with affinity for an oil body-associated protein b) a dairy protein or analogue or part thereof c) a second component with affinity for an oil body-associated protein

[0194] In one embodiment the first and / or second component that has affinity for the oil body- associated protein is a hydrophilic portion of an oil body-associated protein. In one embodiment the hydrophilic portion is the C-terminal hydrophilic portion of an oil body- associated protein. In a further embodiment the hydrophilic portion is the N-terminal hydrophilic portion of an oil body-associated protein.

[0195] In a further embodiment the first and / or second component that has affinity for the oil body-associated protein is combined epitope, which contains sequences from both the N- and C-terminal hydrophilic portion of an oil body associated protein.

[0196] In a further embodiment the first and / or second component that has affinity for the oil body-associated protein is a single-chain Fv antibody (scFv) with specific affinity for the an oil-body associated protein.

[0197] In a further embodiment the construct includes at least one nucleic acid encoding a protease cleavage site.

[0198] In one embodiment the nucleic acid encoding the cleavage site is positioned between the nucleic acid encoding the component that has affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0199] In a further embodiment the construct includes at least two nucleic acids encoding a protease cleavage site. In one embodiment the nucleic acids encoding the cleavage sites are positioned between the nucleic acids encoding the first and second components that have affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0200] The construct may also optionally include a nucleic acid encoding a flexible linker between at least one of the nucleic acids encoding the component that has affinity for the oil body-associated protein and the polynucleotide encoding the dairy protein or analogue or part thereof.

[0201] In one embodiment the construct above is used to express the dairy protein, analogue or part thereof fused to the at least one component that has affinity for the oil body- associated protein. At least one of the components that has affinity for the oil body- associated protein in the fusion protein, has affinity for the hydrophilic region of a native oil body-associated protein embedded in the phospholipid membrane of an oil body. The dairy protein, analogue or part thereof (in the fusion protein) thereby becomes associated affinity linked the oil body.

[0202] In a further embodiment the construct includes a nucleic acid encoding a signal peptide for targeting the fusion to a subcellular location, as discussed above.

[0203] In certain embodiments the part of the dairy protein or analogue, is the hydrophilic domain of a beta-casein protein or analogue thereof. In further embodiments the hydrophilic domain of a beta-casein protein is present as a tandem repeat.

[0204] In one embodiment the construct is for use in a method of the invention.

[0205] In a further embodiment the construct is when used in a method of the invention.

[0206] In a further embodiment the invention provides a method of producing a construct of the invention, for use in a method of the invention. 4.5 Fusion protein

[0207] In a further aspect the invention provides a protein or fusion protein expressed by a construct of the invention.

[0208] 5. CELL COMPRISING CONSTRUCT

[0209] In a further aspect the invention provides a cell comprising a construct of the invention.

[0210] In a further embodiment the invention provides a cell comprising an expression product of a construct of the invention.

[0211] 6. PLANT CELL COMPRTSING CONSTRUCT

[0212] In a further aspect the invention provides a plant cell comprising a construct of the invention.

[0213] In a further embodiment the invention provides a plant cell comprising an expression product of a construct of the invention.

[0214] 7. PLANT OR PLANT PART COMPRISING CONSTRUCT

[0215] In a further embodiment the invention provides a plant, or plant part, comprising a construct of the invention, or a cell of the invention.

[0216] In a further embodiment the invention provides a plant, or plant part, comprising an expression product of a construct of the invention or cell of the invention.

[0217] In a preferred embodiment the plant part is a seed. 8. METHODS USING THE CONSTRUCTS, CELLS AND PLANTS

[0218] 8.1 Method for expressing a dairy protein in close proximity to, or physical association with, an oil body in a plant, using a construct of the invention

[0219] In a further aspect the invention provides a method for expressing at least one dairy protein or analogue thereof or part thereof, in close proximity to, or physical connection with, an oil body in a plant, the method including the step of expressing the dairy protein from a construct of the invention.

[0220] In a further embodiment the method includes the step of transforming the plant with a construct of the invention.

[0221] 8.2 Method for producing a plant expressing at least one dairy protein in close proximity to, or physical association with, an oil body in a plant

[0222] In a further aspect the invention provides a method for producing a plant expressing at least one dairy protein or analogue thereof, or part thereof, in close proximity to, or physical association with, an oil body in a plant.

[0223] In one embodiment the method includes the step of expressing the dairy protein, analogue thereof, or part thereof from a construct of the invention.

[0224] In a further embodiment the method includes the step of transforming the plant with a construct of the invention.

[0225] 8.3 Method for producing composition with oil body (OB) and dairy protein (DP)

[0226] In a further aspect the invention provides a method for producing a composition containing at least one oil body and at least one dairy protein or analogue, or part thereof, the method comprising the step of making an extract from a cell, plant or plant part of the invention or a plant produced by a method of the invention, to produce the composition.

[0227] Embodiments of the invention are as described in Section 1 above. 8.4 Production of functionalized oil bodies

[0228] In preferred embodiments of the method for producing the composition, the dairy protein, analogue, or part thereof becomes physically linked, or affinity linked, to the oil body to produce an oil body that is functionalized with the dairy protein, analogue, or part thereof. These functionalized oil bodies have beneficial properties for the production of dairy alternatives, as discussed further herein.

[0229] The functionalizing of the oil bodies may occur in vivo.

[0230] Alternatively the functionalizing may occur during the method for producing the composition. For example this may occur when a dairy protein, analogue, or part thereof, or fusion , comes into contact with an oil body following disruption of the cell. This disruption may for example release the dairy protein, analogue, or part thereof, or fusion thereof, from a subcellular location, and then come into contact with an oil body.

[0231] In a preferred embodiment the composition produced by the method comprises at least one functionalized oil body as described above. Such functionalized oil bodies produced by the method, can be described as isolated functionalized oil bodies, with isolated meaning separated from the plant cell, plant tissue, plant organ, plant part or plant from which it is extracted.

[0232] In a preferred embodiment the composition produced by the method therefore comprises at least one isolated functionalized oil body as described above.

[0233] 8.5 Composition comprising an oil body and a dairy protein

[0234] In a further aspect the invention provides a composition or extract comprising at least one of: a) at least one oil body and at least one dairy protein or analogue, or part thereof, in accordance with the invention, and b) at least one a functionalized oil body in accordance with the invention This composition can also be described as a "plant-derived dairy milk" or "plant-derived dairy cream" . When relatively more dilute for oil / oil bodies / functionalized oil bodies, the composition can be described as a "plant-derived dairy milk" . When relatively more concentrated for oil / oil bodies / functionalized oil bodies, the composition can be described as a "plant-derived dairy cream"

[0235] In a further embodiment the composition, extract, "plant-derived dairy milk" or "plant- derived dairy cream" is produced by a method of the invention.

[0236] In further embodiment the composition, extract, plant-derived dairy milk", or "plant- derived dairy cream" comprises oil, or oil bodies, or functionalized oil bodies, at a concentration of at least 1% w / w, preferably at least 2% w / w, preferably at least 5% w / w, preferably at least 10% w / w, preferably at least 20% w / w, preferably at least 30% w / w, preferably at least 40% w / w, preferably at least 50% w / w, preferably at least 60% w / w, preferably at least 70% w / w, preferably at least 80% w / w.

[0237] In further embodiment the composition the composition, extract, plant-derived dairy milk" or "plant-derived dairy cream" comprises the dairy protein, analogue or part thereof at a concentration of at least 0.1 mg / 100 mg, preferably at least 0.2 mg / 100 mg, preferably at least 0.3 mg / 100 mg, preferably at least 0.4 mg / 100 mg, preferably at least 0.5 mg / 100 mg, preferably at least 0.6 mg / 100 mg, preferably at least 0.7 mg / 100 mg, preferably at least 0.8 mg / 100 mg, preferably at least 0.9 mg / 100 mg, preferably at least 1.0 mg / 100 mg, preferably at least 1.1 mg / 100 mg, preferably at least 1.2 mg / 100 mg, preferably at least 1.3 mg / 100 mg, preferably at least 1.4 mg / 100 mg, preferably at least

[0238] 1.5 mg / 100 mg, preferably at least 1.6 mg / 100 mg, preferably at least 1.7 mg / 100 mg, preferably at least 1.8 mg / 100 mg, preferably at least 1.9 mg / 100 mg, 2.0 mg / 100 mg, preferably at least 2.1 mg / 100 mg, preferably at least 2.2 mg / 100 mg, preferably at least 2.3 mg / 100 mg, preferably at least 2.4 mg / 100 mg, preferably at least 2.5 mg / 100 mg, preferably at least 2.6 mg / 100 mg, preferably at least 2.7 mg / 100 mg, preferably at least 2.8 mg / 100 mg, preferably at least 2.9 mg / 100 mg, 3.0 mg / 100 mg, preferably at least 3.1 mg / 100 mg, preferably at least 3.2 mg / 100 mg, preferably at least 3.3 mg / 100 mg, preferably at least 3.4 mg / 100 mg, preferably at least 3.5 mg / 100 mg, preferably at least

[0239] 3.6 mg / 100 mg, preferably at least 3.7 mg / 100 mg, preferably at least 3.8 mg / 100 mg, preferably at least 3.9 mg / 100 mg, preferably at least 4.0 mg / 100 mg. 8.6 Isolated functionalized oil bodies

[0240] In a further aspect the invention provides an isolated functionalized oil body. The oil body is functionalized with the dairy protein, analogue or part thereof, in accordance with the invention.

[0241] In a further aspect the invention provides a composition comprising an isolated functionalized oil body, in accordance with the invention.

[0242] In further embodiment the composition, comprises functionalized oil bodies at a concentration of at least 1% w / w, preferably at least 2% w / w, preferably at least 5% w / w, preferably at least 10% w / w, preferably at least 20% w / w, preferably at least 30% w / w, preferably at least 40% w / w, preferably at least 50% w / w, preferably at least 60% w / w, preferably at least 70% w / w, preferably at least 80% w / w.

[0243] In further embodiment the composition the composition comprises the dairy protein, analogue or part thereof at a concentration of at least 0. 1 mg / 100 mg, preferably at least 0.2 mg / 100 mg, preferably at least 0.3 mg / 100 mg, preferably at least 0.4 mg / 100 mg, preferably at least 0.5 mg / 100 mg, preferably at least 0.6 mg / 100 mg, preferably at least 0.7 mg / 100 mg, preferably at least 0.8 mg / 100 mg, preferably at least 0.9 mg / 100 mg, preferably at least 1.0 mg / 100 mg, preferably at least 1.1 mg / 100 mg, preferably at least

[0244] 1.2 mg / 100 mg, preferably at least 1.3 mg / 100 mg, preferably at least 1.4 mg / 100 mg, preferably at least 1.5 mg / 100 mg, preferably at least 1.6 mg / 100 mg, preferably at least

[0245] 1.7 mg / 100 mg, preferably at least 1.8 mg / 100 mg, preferably at least 1.9 mg / 100 mg, 2.0 mg / 100 mg, preferably at least 2.1 mg / 100 mg, preferably at least 2.2 mg / 100 mg, preferably at least 2.3 mg / 100 mg, preferably at least 2.4 mg / 100 mg, preferably at least 2.5 mg / 100 mg, preferably at least 2.6 mg / 100 mg, preferably at least 2.7 mg / 100 mg, preferably at least 2.8 mg / 100 mg, preferably at least 2.9 mg / 100 mg, 3.0 mg / 100 mg, preferably at least 3.1 mg / 100 mg, preferably at least 3.2 mg / 100 mg, preferably at least

[0246] 3.3 mg / 100 mg, preferably at least 3.4 mg / 100 mg, preferably at least 3.5 mg / 100 mg, preferably at least 3.6 mg / 100 mg, preferably at least 3.7 mg / 100 mg, preferably at least

[0247] 3.8 mg / 100 mg, preferably at least 3.9 mg / 100 mg, preferably at least 4.0 mg / 100 mg. 8.7 Isolated fusion protein

[0248] In a further embodiment the invention provides an isolated fusion protein of the invention, or produced in a method of the invention.

[0249] 8.8 Methods and use of fusion proteins

[0250] In a further embodiment the invention provides a method of use of an isolated fusion protein of the invention in the production of a food, beverage, ingredient, nutraceutical or supplement.

[0251] 8.9 Emulsion

[0252] In a further aspect the invention provides an emulsion derived from a composition or isolated functionalized oil body, of the invention.

[0253] In a further embodiment the emulsion comprised at least one dairy protein physically linked to an oil body associated protein.

[0254] 9. METHOD FOR PRODUCING A FOOD ORBEVERAGE

[0255] In a further embodiment the invention provides a method for producing a food or beverage product, the method comprising processing at least one functionalized oil body, composition, extract, plant-derived dairy milk or plant-derived dairy cream, or emulsion of the invention to produce the dairy product substitute.

[0256] In a preferred embodiment the food or beverage product is a dairy product substitute.

[0257] 9.1 Method for producing dairy product substitute

[0258] In a further embodiment the invention provides a method for producing a dairy product substitute, the method comprising processing at least one functionalized oil body, composition, extract, plant-derived dairy milk or plant-derived dairy cream, or emulsion of the invention to produce the dairy product substitute. In a further embodiment the invention provides a method for producing a dairy product substitute, the method comprising adding at least one functionalized oil body, composition, extract, plant-derived dairy milk or plant-derived dairy cream, or emulsion of the invention to another composition to produce the dairy product substitute.

[0259] Although not preferred, in one embodiment the method comprises the step of treating the at least one functionalized oil body, composition, extract, plant-derived dairy milk or plant-derived dairy cream, of the invention with a protease to release the dairy protein or analogue, or part thereof from the oil body-associated protein.

[0260] When this step is applied, the dairy protein or analogue, or part thereof is separated from the oil body.

[0261] Preferably the method does not comprise protease treatment to release the dairy protein or analogue, or part thereof from the oil body-associated protein.

[0262] Numerous different types of dairy product substitute can be produced using the composition, or extract, or plant-derived dairy cream of the invention as starting material, or as an ingredient.

[0263] In one embodiment the dairy product substitute is selected from a milk substitute, a cream substitute, a whipping cream substitute, a mayonnaise substitute, an ice cream substitute, a cheese substitute, and a yoghurt substitute.

[0264] In one embodiment the method involves adjusting the lipid content. Preferably, the lipid is oil.

[0265] In one embodiment the method involves adding other ingredients to the composition, or extract, or plant-derived dairy cream of the invention. In one embodiment the method may include adding at least one emulsifier. In a further embodiment the method may include adding at least one stabiliser. In a further embodiment the method may include adding at least one flavor compound. In a further embodiment the method may include adding at least one colour compound. In one embodiment the method includes an ultra-high temperature (UHT) processing step.

[0266] In a further embodiment the method includes a pasteurization step.

[0267] 10. FOOD OR BEVERAGE

[0268] In a further embodiment the invention provides a food or beverage product comprising at least one functionalized oil body, composition, extract, plant-derived dairy milk or plant- derived dairy cream, emulsion, or fusion protein of the invention.

[0269] 10.1 Dairy product substitute

[0270] In a preferred embodiment the food or beverage product is a dairy product substitute.

[0271] In one embodiment the dairy product substitute is selected from a milk substitute, a cream substitute, a whipping cream substitute, a mayonnaise substitute, an ice cream substitute, a cheese substitute, and a yoghurt substitute.

[0272] In one embodiment the dairy product substitute comprises at least one additional component selected from at least one emulsifier, at least one stabilizer, at least one flavor compound, at least one colour compound, at least one preservative, at least one sweetening compound, at least one nutritional compound (i.e. vitamin, mineral, antioxidant, bioactive or other source of additional nutritional benefit).

[0273] In a further embodiment the invention the dairy product substitute is produced by a method of the invention.

[0274] 11. NUTRACEUTICAL OR SUPPLEMENT

[0275] In a further embodiment the invention provides a nutraceutical or supplement product comprising at least one functionalized oil body, composition, extract, plant-derived dairy milk or plant-derived dairy cream, emulsion or fusion protein of the invention. In one embodiment the nutraceutical or supplement product is in the form of at least one of: a powder, a capsule, a tablet, a gel and a liquid.

[0276] In one embodiment the nutraceutical or supplement product comprises at least one additional components selected from at least one emulsifier, at least one stabilizer, at least one flavor compound, at least one colour compound, at least one preservative, at least one sweetening compound, at least one nutritional compound (i.e. vitamin, mineral, antioxidant, bioactive or other source of additional nutritional benefit).

[0277] DETAILED DESCRIPTION OF THE INVENTION

[0278] The invention provides compositions and methods for producing plant-based dairy substitutes. The invention involves the expression of dairy proteins and analogues thereof, and parts thereof in plants, wherein the expression is spatially and / or developmentally coordinated with the biosynthesis of oil bodies in the plants. The dairy proteins, and analogues, are produced in close proximity, or physical association with the oil bodies, such that an extract containing both the oil bodies and dairy proteins / analogues, or parts thereof can be conveniently and efficiently produced. The extract also has beneficial properties for use in production of plant-based dairy substitutes.

[0279] Oil bodies are naturally occurring lipid particles, and consist mainly of triacylglycerols surrounded by a phospholipid monolayer incorporating proteins such as oleosins, caleosins and steroleosins. The proteins embedded in the phospholipid monolayer provide stability to the oil bodies when they are dispersed in water and subjected to various treatments. Oil bodies occur largely in seeds but also occur in other plant parts, including leaves.

[0280] In accordance with the invention dairy proteins or analogues or parts thereof can be expressed as fusion proteins with oil body-associated proteins such as oleosins or with components with affinity for oil body-associated proteins, such as oleosins, so that the dairy proteins become physically anchored, or affinity linked, to the oil body through their connection, or affinity linking, to the oleosins. This facilitates convenient co- purification of oil bodies and milk proteins to provide compositions that can be used in the production of dairy product substitutes. Alternatively, if the dairy proteins are expressed in close proximity to the oil bodies, such as in the cytoplasm or cell walls of oil body containing cells, the dairy proteins can similarly be co-purified with the oil bodies. In such dairy product substitutes, the dairy protein component is provided by recombinant expression of dairy proteins and analogues, or parts thereof, while the oil component is provided by the oil bodies.

[0281] In further accordance with the invention the oil bodies also provide enhanced dairy texture and mouthfeel to plant derived dairy product substitutes, relative to that provided by traditionally used plant oils.

[0282] In further accordance with the invention, because of their high-surface activity, the presence of dairy proteins, such as caseins or analogues, or parts thereof, in close proximity to oil bodies in the dairy product substitutes, enhances functionalities such as the aeration of cream substitutes which facilitates whipping and foaming properties. This aids emulsification when such cream substitutes are incorporated into other products, such as ice cream and beverages.

[0283] In further accordance with the invention, the inclusion of whey protein (such as - lactoglobulins) in the oil body / dairy protein preparations improves rheological and gelation properties in products such as cream fillings, cheeses, yoghurt gels, and baked goods. This is due to the ability of whey protein to undergo heat-induced aggregation and gelation.

[0284] The invention thus enables the safe, economic, ecologically sustainable, and humane production of plant-derived dairy substitutes, such as cream substitutes, coupled with the high nutritional values and functionalities of traditional milk proteins for commercial use, such as used in the food and beverage industry.

[0285] Definitions

[0286] The term "close proximity" and grammatical equivalents thereof, means close enough within the plant tissue that the dairy protein, analogue or part thereof, can be co-purified with the oil body, following disruption of the plant material. Preferably the "close proximity" means within the same cell. The "close proximity" may allow in vivo association of the dairy protein, analogue or part thereof, with the oil body. Alternatively, this may occur during the process for producing the composition in accordance with the invention.

[0287] The term "physically connected" and grammatical equivalents thereof, means that the dairy protein, analogue or part thereof, is connected to the oil body via connection to an oil body associated protein. In this embodiment the dairy protein, analogue or part thereof, may be part of a fusion protein with at least part of an oil body associated protein that is embedded in the phospholipid membrane of the oil body.

[0288] The term "affinity linked" and grammatical equivalents thereof, means that the dairy protein, analogue or part thereof, is associated the oil body via affinity to an oil body associated protein. In this embodiment the dairy protein, analogue or part thereof, may be part of a fusion comprising a component that has affinity to the oil body associated protein that is embedded in the phospholipid membrane of the oil body.

[0289] The term "oleaginous tissue" means and oil producing tissue.

[0290] The term "oleaginous organ" means and oil producing organ. Example of oleaginous organ include fruits (olive, avocado, almonds, etc.), seeds (canola, safflower, soybean, etc.), vegetative organs over-expressing DGAT.

[0291] The term "emuslifer" as used herein means a substance or compound that plays a crucial role in stabilizing emulsions. Emulsions are mixtures of two or more unmixable substances, such as oil and water, that are combined into a stable, uniform solution with the help of an emulsifier. Emulsifiers have both hydrophilic and hydrophobic properties. This dual nature allows them to bridge the gap between substances that would otherwise separate, creating a stable and homogenous mixture. Emulsifiers are commonly used in various industries, including food, cosmetics, and pharmaceuticals, to ensure consistent product formulations and desired properties. Dairy proteins and analogues for use in the invention

[0292] The terms “dairy protein / s” and “milk protein / s” can be used interchangeably.

[0293] 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. A 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.

[0294] 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.

[0295] 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. 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.

[0296] 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.

[0297] As ingredients in food products, milk proteins introduce a range of sensory characteristics to the food products and the person ingesting them, 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.

[0298] 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.

[0299] 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.

[0300] In one embodiment the milk fat globule membrane protein is selected from a mucin, butyrophilin, a fatty-acid binding protein and xanthine dehydrogenase.

[0301] In one embodiment the whey protein is selected from, beta-lactoglobulin, alphalactalbumin, lactoferrin and albumin.

[0302] Examples of dairy proteins for use in the invention are shown in Table 1 below and their sequences are provided in the Sequence Listing. Table 1. Examples of milk or dairy proteins known in the art Source of dairy proteins

[0303] The dairy or milk proteins for use in the invention may be from any suitable source.

[0304] 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.

[0305] In a preferred embodiment the dairy or milk proteins for use in the invention are from bovine organisms.

[0306] Dairy protein analogues

[0307] 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.

[0308] In one embodiment the dairy protein or analogues 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 dairy protein of the type described above, or a sequence described in Table 1.

[0309] Parts of dairy proteins or analogues

[0310] The parts of the dairy proteins or analogues are polypeptide fragments of the dairy proteins or analogues.

[0311] 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. In some embodiments, the part may be a functional domain of a dairy protein. For example the functional domain may be: a hydrophobic domain of a beta casein protein, or a hydrophilic domain of a beta-casein.

[0312] In a preferred embodiment, the part is a hydrophilic domain of a beta-casein.

[0313] In one embodiment the hydrophilic domain of a beta-casein has at least 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.

[0314] Oil bodies

[0315] As discussed above, 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).

[0316] Oil-body associated proteins

[0317] 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.

[0318] 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), caoleosins (Hsieh and Huan, 2004, Endoplasmic reticulum, oleosins, and oil seeds in tapetum cells. Plant Physiology, 136:3427-3434).

[0319] Oleosins and their incorporation into oil bodies

[0320] As mentioned earlier, oleosins are integrally associated with oil bodies. Oleosins are specifically expressed during seed maturation and are incorporated into the membrane of the ER by a signal recognition particle. During oleosin and oil bodies biogenesis, nascent oleosin polypeptides will remain on the ER, N- and C-terminal peptides are exposed on the cytoplasmic side, and hairpins are hidden between the ER phospholipid bilayers. This ER-associated semi-stable oleosin should be temporary and the oleosin later diffuses to the budding oil body surface (Huang et al., 2017).

[0321] In accordance with the invention dairy proteins and analogues can be targeted to oil bodies by fusing them directly to the N or C terminal of oleosins.

[0322] Table 2. Examples of oleosins and caleosins known in the art

[0323] Modified oleosins

[0324] In some embodiments the invention involves the use of oleosins such as those described that are modified to alter or improve their functional properties.

[0325] 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).

[0326] 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.

[0327] In some embodiments the invention therefore involves the use of oleosins including at least one artificially introduced cysteine (WO2011 / 053169).

[0328] In one embodiment the oleosin protein, or 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 a sequence described in Table 2.

[0329] Coordinated expression of dairy proteins or dairy protein / oil body associated protein fusions with oil body biosynthesis

[0330] In some embodiments expression of the dairy protein / oil body associated protein fusions is developmentally coordinated with biosynthesis of the oil body.

[0331] In some embodiments expression is driven by a promoter controlling such expression.

[0332] In one embodiment the promoter is from a gene encoding a protein that is strongly expressed in seeds or other tissues or organs capable of oil body biogenesis.

[0333] In one embodiment the promoter is a seed-specific promoter. Preferred seed specific promoters include but are not limited to seed-storage protein promoters. Preferred seed seed-storage protein promoters include those from phaseolin, conglycinin, glycinin or cruciferin genes. Some non-limiting examples are provided in Table 3 below.

[0334] Table 3. Seed preferred promoters In one embodiment the seed preferred promoter 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 seed preferred promoter of the type described above, or a sequence described in Table 3.

[0335] Alternatively, in certain embodiments, if the oil bodies are produced in somatic tissues such as leaves, a promoter from a photosynthetic gene such as RuBP carboxylase or chlorophyll a / b binding protein can be employed.

[0336] In some embodiment the promoter is from a gene encoding an oil-body associated protein.

[0337] In some embodiment the oil-body associated protein is selected from an oleosin, caleosin or steroleosin as herein described.

[0338] In a preferred embodiment the promoter is from a gene encoding an oleosin.

[0339] Table 4. Oleosin promoters

[0340] In one embodiment the oleosin promoter 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 promoter of the type described above, or a sequence described in Table 4.

[0341] Protein targeting with signal peptides

[0342] In some embodiments expression of the dairy protein or dairy protein / oil body associated protein fusions is targeted to the cell wall.

[0343] In some embodiments, targeting dairy protein reduces the chances of pleiotropic effects and interfering with oil body formation.

[0344] In some embodiments expression of a signal peptide is used to facilitate targeting.

[0345] Table 5. Cell wall signal peptides

[0346] In one embodiment the signal peptide 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 signal peptide in Table 5.

[0347] Hydrophilic domains of oil body-associated proteins

[0348] Some embodiments of the invention involve use of hydrophilic domains of oil body- associated proteins. In some embodiments the dairy protein, analogue, or part thereof, is fused to a hydrophilic region of an oil body-associated protein, which is associated with a corresponding hydrophilic region of an oil body-associated protein embedded in the phospholipid membrane of an oil body, and the dairy protein or analogue, or part thereof, is co-extracted with the oil body.

[0349] This approach reduces the likelihood of interference with normal oil body formation, stability and composition.

[0350] Oil body-associated proteins include oleosins, encoding oleosins, caleosins and steroleosins as described herein.

[0351] Oleosin has short amphipathic N- and C-terminal peptides flanking a conserved hydrophobic hairpin domain, which penetrates and stabilizes the oil body. A combination of the hydrophobic hairpin and part of the N-terminal region of oleosins are necessary for oleosin targeting to the ER and moving onto budding oil bodies and extracting them to cytosol (Huang and Huang, Plant Physiol. 2017; 174(4): 2248-2260). On the other hand, the C-terminal region of oleosins is not essential for the oleosin targeting to the oil bodies. Additionally, the C-terminal region has a unique affinity towards other oleosin proteins thus may act as a binding tag. To alleviate the interference, the dairy proteins are fused to the C-terminal domain of oleosins. Consequently, the dairy proteins fused to C- terminals part of oleosin will bind to the oil body and will be presented to the cytoplasm.

[0352] Table 6. C-terminal hydrophilic domains of oleosins and caleosins

[0353] In one embodiment the hydrophilic domain 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 6.

[0354] In some embodiments the components with affinity to oil body associated proteins are fusions of the N- and C- terminal hydrophilic domains of oleosins. Examples are shown in Table 7 below.

[0355] Table 7. Fusions N- and C- terminal domains of oleosins 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 7.

[0356] Single-chain Fv antibody (scFv) with specific affinity against an oil-body associated protein

[0357] Single chain antibody with specificity for an oil body. Nucleic acid sequences encoding single chain antibodies with specificity for an oil body may be prepared from hybridoma cell lines expressing monoclonal antibodies raised against an oil body protein. In one embodiment, the single chain antibody specifically binds an oleosin, as described by Alting-Mees et al. (2000) IBC's International Conference on Antibody Engineering, Poster #1 (US07547821B2).

[0358] Polypeptide sequence identity

[0359] 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.

[0360] 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. Sci. 23, 403-5.)

[0361] Methods for producing polynucleotides of the invention 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 encoding a dairy protein. 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).

[0362] The polynucleotides of the invention, encoding the modified dairy proteins of the invention, may be codon optimised to resemble the codon usage of the cell or organism in which the modified dairy 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.

[0363] Methods for producing constructs and vectors

[0364] 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.

[0365] 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).

[0366] Methods for producing host cells comprising polynucleotides, constructs or vectors

[0367] The invention provides a host cell which comprises a genetic construct or vector of the invention.

[0368] Host cells may be selected from but not limited to bacterial, fungal, insect, mammalian or plant cells.

[0369] 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).

[0370] Methods for producing plant cells and plants comprising constructs and vectors

[0371] 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.

[0372] 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. Imperial College Press, London.

[0373] Methods for genetic manipulation of plants

[0374] 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). For 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.

[0375] 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. 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

[0376] 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.

[0377] 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.

[0378] 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 etal., 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 etal., 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 l l(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.

[0379] The polynucleotides of the invention, and their encoded plant-derived dairy 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 Rycke, R. , Van Montagu, M. and Angenon, G. (1997) An Agrobacterium- mediated transient gene expression system for intact leaves. Plant Sci. 122, 101-108).

[0380] Plant cells and plants

[0381] The plant cells and plants in which the modified dairy proteins are expressed, and from which various sequences for use in the invention are sourced, may be from any plants species.

[0382] In one embodiment the plant cell or plant, is from a gymnosperm plant species.

[0383] In a further embodiment the plant cell, or plant, is from an angiosperm plant species.

[0384] In a further embodiment the plant cell, or plant, is from a from dicotyledonous plant species.

[0385] In a further embodiment the plant cell, or plant, is from a monocotyledonous plant species.

[0386] In a further embodiment the plant cell, or plant, is from an oil seed species.

[0387] In one embodiment the plant or plant cell is selected from but not limited to: soybean, tobacco, pea, chickpea, bean, lupin, fava bean, lentils or any other legume, tapioca, rice, barley, wheat, oats, maize, tomato, potato, canola, oil seed rape, sunflower, safflower, coconut, almond and / or hemp.

[0388] Use of the plant-derived compositions / plant-derived dairy milks / creams of the invention in food and beverages

[0389] The plant-derived compositions / plant-derived dairy milks / creams of the invention may be processed into, used as ingredients in, food and beverage products, incorporating one or many of the functional characteristics found in native milk and dairy proteins. Such products may include but are not limited to milk, butter, buttermilk, cheese, cream, icecream, whey, casein, yoghurt, milk powders, infant formulas, sports drinks or pet and animal dairy products or baked goods and confectionaries.

[0390] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[0391] 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.

[0392] 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.

[0393] 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").

[0394] BRIEF DESCRIPTION OF THE DRAWINGS

[0395] Various embodiments of the invention are now illustrated with reference to the accompanying figures, in which:

[0396] Figure 1 shows a schematic representation of constructs according to the invention.

[0397] Figure 2 shows a schematic representation of various fusion proteins and their impact on oleosin / oil body structure according to the invention.

[0398] Figure 3 shows a further schematic representation of various dairy proteins and fusion of oil body associated proteins in different cellular location in accordance with the invention, and production of isolated functionalized oil bodies in accordance with the invention.

[0399] Figure 4 shows a further schematic representation of various dairy proteins and fusions in different cellular location in accordance with the invention, and production of isolated functionalized oil bodies in accordance with the invention.

[0400] Figure 5 shows the process of different GM seed sources that contain different dairy proteins and their consequent mixture and process to provide desired starting material (milk, cream and protein streams) for downstream formulations. Three potential basic streams can be produced: A cream - oil fraction enriched with oil-bodies associated with dairy proteins; milk stream - a diluted version of the cream stream; soluble fraction - a plant protein meal. Figure 6 shows a flow chart illustrating embodiments of methods and processing of basic ingredients of the invention to produce various product compositions in accordance with the invention.

[0401] Figure 7 shows a traditional dairy production system (top) compared to a Dairy Crop System in accordance with the invention (bottom).

[0402] Figure 8 shows a schematic representation of pET28a(+) vectors.

[0403] Figure 9 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

[0404] - 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

[0405] - recombinant in inclusion bodies; Sup - refolded protein fractions; pel - refolded proteins in the pellet.

[0406] Figure 10 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 Ipg 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.

[0407] Figure 11 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). I g of positive control (Sigma Aldrich beta-lactoglobulin and aCSN).

[0408] Figure 12 shows SDS-PAGE analysis of beta-casein binding to oil bodies. Beta-casein was spiked into oil bodies at increasing concentrations (0, 10, 50, 100, 250, and 500 pg) and the supernatant and oil fractions were analyzed by SDS-PAGE. The gel was stained with a Coomassie stain. Positive control: a pure beta-casein (Ipg). Sup - supernatant fraction; oil

[0409] - oil bodies fraction. Figure 13 shows SDS-PAGE analysis of beta-casein binding to oil bodies. Beta-casein was spiked into oil bodies at different concentrations (50, 100, and 250 pg) and the supernatant and oil fractions were analyzed by SDS-PAGE. The gel was stained with a Coomassie stain. Black arrows - expected size of Beta-casein (27kDa), Oleosin:BC2 (50kDa), C- Ole:BC2 (35kDa), NC-01e:BC2 (41kDa) and C-Ole:BC2:C-Ole (41kDa). Sup - supernatant fraction; oil - oil bodies fraction.

[0410] Figure 14 shows SDS-PAGE analysis of beta-casein binding to oil bodies. 250 pg Betacasein (BC2 and BC3) was spiked into oil bodies, and the supernatant and oil fractions were analyzed by SDS-PAGE. Left - Coomassie stain, right - Western Blot with betacasein antibodies. Expected size of Beta-casein, BC2 and BC3 (25kDa), Sup - supernatant fraction; OB - oil bodies fraction.

[0411] Figure 15 shows (A) SDS-PAGE (Coomassie) and (B) western blot analysis of betalactoglobulin (native and 5C-A mutant) binding to oil bodies. 250 pg Beta-lactoglobulin (native and mutant) with and w / o C-terminal oleosin was spiked into oil bodies, and the supernatant and oil fractions were analyzed by SDS-PAGE. Expected size of WT, native and mutant Beta-lactoglobulin (20kDa), and C-terminal Oleosin: lactoglobulin (native and mutant) (27kDa). Sup - supernatant fraction; OB - oil bodies fraction.

[0412] Figure 16 shows a schematic representation of the binary vectors.

[0413] Figure 17 shows Subcellular Localization and Expression Patterns of Oleosin-Fused mCherry Proteins in Transgenic Tobacco Leaves. Each Image (1A to 1H) consists of four subimages - Top left: Bright field image of transiently expressed leaf; Top right: Chloroplast band; Bottom left: BODIPY staining; Bottom right: mCherry band.

[0414] (A) Construct 1: Cytoplasmic Expression. mCherry is expressed in the cytoplasm; (B) Construct 2: Cell Wall Expression. mCherry is expressed in the cell wall.; (C) Construct 3 : Cytoplasmic and Oil Body Binding mCherry is expressed in the cytoplasm and binds to oil bodies (indicated by arrows); (D) Construct 4: Cytoplasmic and Oil Body Binding (N-terminal fusion). mCherry is expressed in the cytoplasm and binds to oil bodies (indicated by arrows); (E) Construct 5: Limited Expression (Not in the Cell Wall). Minimal or no mCherry expression, not localized in the cell wall; (F) Construct 6: Limited Expression, absent in the Cell Wall; Minimal or no mCherry expression, absent in the cell wall; (G) Construct 7: Limited Expression. Minimal or no mCherry expression, not present in the cell wall; (H) Construct 8: Limited Expression, oil body localization, absent from the cell wall; (I) Construct 9: Limited Expression, localization in the cytoplasm and co-localize with oil-bodies; (J) Construct 10: High expression, cell wall localization; (K) Construct 11 : Limited Expression. Minimal or no mCherry expression; (L) Construct 12: High Expression, cell wall localization; (M) Construct 13: Limited Expression. Minimal or no mCherry expression; (N) Construct 14: Limited Expression, cell wall localization.

[0415] Figure 18 shows Detection of the C-terminal-oleosin:beta-casein (BC3) fusion protein in transgenic Arabidopsis T1 seeds. Proteins extracted from wild-type (WT) and T1 seeds (lines 4, 6, 7, 8, 9, 11, 12 &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).

[0416] Figure 19 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.

[0417] Figure 20 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. Figure 21 shows extracted cream from wild-type Arabidopsis seeds (left) and transgenic seeds (right).

[0418] Figure 22 shows the coffee whitener preparation - flow diagram.

[0419] EXAMPLES

[0420] Various embodiments of the invention are now illustrated with the following non-limiting examples.

[0421] Example 1: Expression of beta casein fused to oleosin and oleosin affinity peptides in E. coli.

[0422] Vector preparation and transformation into E. coli

[0423] 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: 98) and BC3 (SEQ ID NO: 87), 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 enhance the stability.

[0424] For the present experiments, the applicant generated twenty five distinct constructs, as illustrated in Figure 8 and Table 8. The backbone of the constructs are as follows: Backbone 1: Asses non-fused dairy proteins (either native beta-casein, BC2, BC3, alphacasein S 1 and beta-lactoglobulin) 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.

[0425] Backbone 3: In this experiment, the applicants assessed the fusion of a dairy protein with the hydrophilic C-terminal segment of oleosin.

[0426] 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).

[0427] 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.

[0428] 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: 95).

[0429] 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.

[0430] A total of 25 vectors were produced and transformed into E. Colt strain BL21 (DE3). All the vectors are presented in Table 8.

[0431] Table 8: List of pET28a(+) vectors transformed into E. Coli

[0432] 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.

[0433] 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.

[0434] 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 9). 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 10 shows that beta-lactoglobulin proteins (fused and nonfused) were successfully refolded and obtained at the expected size.

[0435] 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 11. Example 2: Oil Bodies Binding Analysis of recombinant fused and non-fused dairy proteins

[0436] Oil Body Purification:

[0437] Oil bodies were purified from safflower seeds (10g) using an adapted flotation centrifugation method inspired by Tzen et al. (1990). Initially, safflower seeds were ground to a fine consistency in a mortar and pestle, using 50mL of deionized water (DDW). Subsequently, the safflower milk was filtered through a strainer into a Falcon tube and then centrifuged at 10,000 x g for 20 minutes at 4°C. The resulting oil pad (white oil bodies phase at the top) was carefully collected into a new Eppendorf tube. The oil pad was resuspended in a grinding buffer comprising 0.6M sucrose and lOmM Na2POi (pH 7.5) at a ratio of 1ml per 300mg of oil bodies. The homogenate was subjected to centrifugation at 10,000 x g for 20 minutes at 4°C. Oil bodies, located at the top of the homogenate, were collected and subsequently washed with ImL of Ionic buffer containing 0.6M sucrose, 2M sodium chloride, and lOmM Na2?O4 (pH 7.5). Following another round of centrifugation at 10,000 x g for 20 minutes at 4°C, the oil bodies at the top were collected. To facilitate further purification, the collected oil bodies were resuspended in 0.5mL of 9M urea and agitated on a shaker for 10 minutes at 80 RPM, at room temperature. This step was followed by the addition of 0.5mL of lOmM Na2POi (pH 7.5), after which the homogenate underwent centrifugation under the same conditions. The oil bodies were again collected from the top and subjected to a final resuspension in 0.5mL of DDW. Their total weight was determined, resulting in a final weight of 1308mg.

[0438] Analysis of Beta-Casein Binding to Oil Bodies:

[0439] To investigate the binding capacity of beta-casein to oil bodies, purified oil bodies resuspended in lOmM Na2PC>4 (pH 7.5), were subjected to spiking with varying concentrations of bovine beta-casein (Sigma), including 10, 50, 100, 250, and 500 qg, in a total volume of 1 mL. Following spiking, the oil bodies were centrifuged at 10,000 x g for 10 minutes, aiming to separate the oil and aqueous phases. The oil bodies were resuspended in 1 mL phosphate buffer. The resulting oil phase and supernatant were then subjected to analysis using SDS-PAGE gels. The obtained results indicated that with increasing amounts of added beta-casein, a higher quantity of protein was detected in the oil fraction. However, even at the highest spike concentration of 500 pg, only a small proportion of the total beta-casein was recovered in the oil phase (Figure 12). This observation suggests a relatively low binding affinity of native beta-casein for the oil bodies under these conditions.

[0440] To understand if beta casein fused to oleosin or to peptides with oleosin affinity have improved binding capacity to oil bodies, the applicants repeated the same experiments and spiked increased concertation for oleosin :beta-casein (BC2) and the different oleosin- aflfinity peptide fused to beta-casein (BC2) into safflower oil-bodies. Purified safflower oil bodies were used to analyze the oil body binding capacity of refolded oleosin-casein fusion proteins. 100 mg of oil bodies in 1 mb of 10 mM sodium phosphate buffer, pH 7.5, were mixed with refolded proteins obtained from the four constructs described in Figure 8 at concentrations of 50-250 pg. As a control, bovine beta-casein (Sigma) was added at 100 and 250 pg. Another control was the addition of 250 pg of BC2 and BC3 (expressed and purified as described WO2023119200. Figure 14). The mixtures were centrifuged at 10,000 x g for 20 min at 4°C to separate the oil body and aqueous phases. The oil bodies were resuspended in 1 mb phosphate buffer. Westen blot analysis performed using anti-Bovine beta casein from Rabbit (1: 10,000 dilution and incubation ON at 4°C), then anti-rabbit- HRP conjugate antibody from Goat (1: 10,000 dilution, incubated 1 hour at RT). Detection performed using chemiluminescence and ECL solution.

[0441] The relative band intensities in the oil body and supernatant fractions were used to compare the oil body binding capacity of the oleosimcasein and oleosin affinity peptide: casein fusions to that of beta-casein.

[0442] Native beta-casein (from Sigma) displayed only weak association with the oil bodies. In contrast, the oleosin-casein fusions showed significantly increased binding, where most protein found in the oil-bodies fraction (Figure 13).

[0443] A similar analysis was performed for Beta-lactoglobulin and C terminal oleosin: Betalactoglobulin and its corresponding mutants with five cysteine to alanine substitutions. Beta-lactoglobulin from sigma was used as a control. Native Beta-lactoglobulin and modified beta-lactoglobulins did not display any association with the oil bodies. In contrast, the oleosin and c-terminal oleosin fusion proteins of the different iterations of beta-lactoglobulins showed significantly increased binding, where most protein found in the oil-bodies fraction (Figure 15). These results demonstrate that oleosin fusion and oleosin-affinity peptide fusions allows targeting of recombinant dairy proteins to plant oil bodies. The data supports oleosin fusion as a strategy to modulate stability and nutritional properties of engineered dairy proteins in plant oils.

[0444] A similar analysis was performed for Alpha-casein, Oleosimalpha casein and C terminal oleosin: Alpha-casein. Alpha-casein from sigma was used as a control. Alpha casein showed a binding affinity to oil bodies and the fusion proteins did not result in enhanced binding affinity. These findings underscore the varying interactions between different dairy proteins and oil bodies. The limited binding of native beta-casein highlights the necessity of fusion strategies for its efficient incorporation into oil bodies. In contrast, the inherent affinity of alpha-casein for oil bodies suggests potential flexibility in protein selection, potentially bypassing the need for fusion in certain applications. The successful enhancement of beta-lactoglobulin binding through fusion further validates the versatility of this approach, enabling the targeted association of a wider range of dairy proteins with oil bodies. These observations collectively emphasize the adaptability of the invention, allowing for tailored strategies based on the specific properties of the chosen dairy protein, ultimately contributing to the efficient and versatile production of plant-based dairy substitutes.

[0445] Example 3: Methodology for Investigating the Expression, Subcellular Localization, and Oil Body Binding of Oleosin-Fused Visual Marker Proteins in Transgenic Tobacco Leaves

[0446] The applicants sought to understand whether the fusion of oleosin sequences to dairy proteins could facilitate their binding to oil bodies, as well as enable them to accumulate in different organelles and pass through the secretory system. To address this, the applicants developed a comprehensive methodology utilizing transgenic Nicotiana benthamiana plants engineered to accumulate oil bodies in their leaves. The applicant’s objective was to elucidate the expression, subcellular localization, and oil body interactions of visual marker proteins fused to oleosin, with and without signal peptides.

[0447] Signal Peptide Utilization: To direct the accumulation of proteins into distinct cellular compartments, such as the cell wall or apoplast, the applicants harnessed signal peptides derived from diverse plant sources. The applicant’s focus was on the utilization of two signal peptides: the Arabidopsis endoglucanase CEL1 signal peptide (accession number Atlg70710) and the tobacco pathogenesis-related protein R signal peptide (accession number P07052). Additionally, the applicants explore the potential for fusion between these milk proteins or analogues and oil body-associated proteins, thus facilitating their binding to oil bodies during plant material processing.

[0448] Plant Material and Transformation:

[0449] As an experimental foundation, the applicants employed transgenic tobacco (Nicotiana benthamii) plants renowned fortheir remarkable capacity to accumulate oil and oil bodies within their leaves. These plants were genetically engineered to express essential lipid biosynthesis genes, namely WRINKLED 1 (WRIT), LEAFY COTYLEDON 3 (LEC2), and acyl-CoA: diacylglycerol acyltransferase (DGAT1). The transformation process adhered to a well-established protocol as delineated in Vanhercke et al., 2014 (Plant Biotechnology Journal (2014) 12, pp. 231-239).

[0450] Transient Expression and Construct Details:

[0451] To evaluate the behavior of visual marker proteins fused to oleosin, the applicants transiently introduced constructs into tobacco leaves. These constructs included variations with and without signal peptides to discern their impact on expression, localization, and oil body interactions. The complete expression cassettes (promoter, coding region, and terminator) were cloned in the multiple cloning site (Hindlll) of the pCAMBIA0390 plant transformation vectors (Hajdukiewicz et al., 1994). The constructs were accurately infiltrated into the three uppermost infiltratable leaves of the transgenic tobacco plants, ensuring uniform distribution along one side of the leaf midrib. A total of eight binary vectors were synthesized which differed in the fusion protein design and signal peptide (see Figure 16 for the backbone structure and Table 9 for the construct list). Table 9: Constructs for transient expression and description of different fusion protein Sample Collection:

[0452] The applicant’s methodology dictated the collection of leaf samples at a precise time point: four days post-infdtration. From each infiltrated leaf, three 3.5mm leaf discs were excised, ensuring comprehensive coverage across diverse regions within the infiltrated area.

[0453] BODIPY Staining:

[0454] For the purpose of subcellular localization and the visualization of oil bodies, the excised leaf discs were immediately immersed in a solution containing 0.4 pg / ml BODIPY 515. Vacuum infiltration techniques were employed to ensure uniform staining, followed by a 15-minute incubation at room temperature with periodic agitation. Subsequent to staining, the leaf discs underwent two rinses with deionized water before being mounted in fresh deionized water.

[0455] Microscopy Imaging:

[0456] To capture high-resolution images of the stained leaf discs, the applicantsemployed a Leica SP8 microscope. Anon-sequential scan setting was implemented, with specific laser and emission settings detailed in the previous section. Imaging was conducted using a 40x / l . 10 objective lens, enabling the acquisition of six images / stacks per construct. These meticulously executed procedures lay the foundation for a robust evaluation of the expression, subcellular localization, and interactions with oil bodies of visual marker proteins fused to oleosin, both with and without signal peptides.

[0457] Results:

[0458] A series of oleosin-fluorescent protein fusions were transiently expressed in tobacco leaves to evaluate subcellular localization and oil body targeting. Leaves were co-infiltrated with constructs inducing oil body formation through WRI1, LEC2, and DGAT1 overexpression. Localization was imaged by confocal microscopy 4 days after infiltration (Figure 17):

[0459] • Cytoplasmic mCherry Expression: Construct 1 with mCherry alone showed consistent cytoplasmic localization. This served as a control for cytosolic protein distribution.

[0460] • Apoplast Targeting: Construct 2 test the addition of the Arabidopsis cell signal peptide to mCherry restricted fluorescence to the apoplast surrounding mesophyll cells. This control confirmed secretion of proteins into the extracellular space. • Oil Body Targeting by Oleosin C-terminal fusion: Joining mCherry to oleosin sequence led to strong colocalization of fluorescence with BODIPY-stained oil bodies. Pixel intensity correlation analysis indicated a high degree of overlap between the two channels. This demonstrates effective oil body targeting can be achieved by C-terminal oleosin fusion.

[0461] • Oil Body Targeting by Oleosin N-terminal fusion: Fusion of mCherry to oleosin by N-terminal fusion showed weaker oil body association based on overlay and pixel intensity correlation. Some fluorescence remained cytosolic. This suggests C- terminal placement is preferred for robust oil body anchoring.

[0462] • Weaker or no expression of cell wall targeted full oleosin fused to mCherry: The applicant’s experiments utilizing signal peptides demonstrated that oleosin- fused proteins, whether C- or N-terminal fusion, showed little to no expression and no secretion into the apoplast. It is suggested that the oleosin hydrophobic nature may hinder their passage through the secretory system. This observation provides valuable insights into the behavior of oleosin fusion proteins within plant cells.

[0463] • Oil Body Targeting by Oleosin Affinity Peptide Fusions: The fusion of mCherry to oleosin affinity peptide sequences resulted in distinct localization patterns. The C-terminal hydrophilic part of oleosin fused to mCherry exhibited partial colocalization of mCherry fluorescence with BODIPY-stained oil bodies. In contrast, constructs involving the N+C hydrophilic parts of oleosin or small chain oleosin antibody fused to mCherry displayed low or no expression.

[0464] • Cell Wall Targeting Oleosin Affinity Peptides Fused to mCherry: Unlike full- length oleosin fusion to mCherry, constructs designed for cell wall taigeting exhibited high expression levels and clear localization in the cell wall. This observation underscores the potential of utilizing affinity peptides for achieving high accumulation in organelles without potentially interfering with oil body formation, as may occur during cytoplasmic expression. This design allows for straightforward processing post-harvest, facilitating the binding of these affinity peptides with oil bodies after processing.

[0465] Advantages of Using Oleosin Affinity Peptide Fusions Compared to Full Oleosin Fusion:

[0466] • Enhanced Subcellular Targeting Precision: Oleosin affinity peptide fusions offer a higher degree of precision in subcellular targeting compared to full oleosin fusion. Full oleosin fusion cannot go through the secretory system, limiting its potential to target different organelles. In contrast, affinity peptides allow for specific targeting to oil bodies or other organelles of interest.

[0467] • Versatility: Oleosin affinity peptides can be tailored to taiget different organelles or cellular compartments, providing versatility for a wide range of biotechnological applications. Full oleosin fusion cannot achieve this level of specificity and adaptability for targeted subcellular localization.

[0468] • Minimized Interference with Cellular Processes: Full oleosin fusion can potentially interfere with normal cellular processes due to the large size and hydrophobic nature of oleosin. This interference restricts its ability to be targeted to different organelles within the cell. In contrast, affinity peptide fusions are typically smaller and have a reduced impact on cellular functions, making them more suitable for applications where interference should be minimized.

[0469] • Facilitated Downstream Processing: Affinity peptide fusions simplify downstream processing steps. After harvest, the fusion proteins can be efficiently extracted from the plant material using the affinity peptides, streamlining purification and extraction processes.

[0470] Example 4: Seed specific expression of beta casein fused to oleosin

[0471] DNA constructs arre synthesized to test both the incorporation of dairy proteins into oil bodies and the localization in plant cells (Table 10). The elements presented in the Table are stacked from 5 ’ to 3 ’ of the DNA insert. The coding region is inserted into pHGHPB binary plasmid which contain the basta resistance gene (bar gene). The coding region is flanked either by the phaseolin promoter and 3’UTR (SEQ ID NO: 1\, Phaseolus vulgaris acc. J01263. 1) or soybean glycinin G1 promoter and 3'UTR (SEQ ID NO: 72; Glycine max acc. P04776). All the binary vectors were transferred into A. tumefaciens LB 4404 Transformation vectors were constructed using different combinations of promoters, signal proteins and protein fusions. The promoter and gene fragments are inserted into a binary vector. The construct compositions are described in Table 10 below.

[0472] Table 10: Transformation vectors prepared for oilseed transformation

[0473] Transformation into Arabidopsis, Camelina and Canola can be performed as described previously [Clough and Benet, 1998, Plant J. 16:735-743; Liu et al., 2012, In Vitro Cell.Dev.Biol. — Plant. 48:462-468; De Block et al., 1989, Plant Physiol. 91:694-701],

[0474] More than 15 independent transformants are generated for each binary vector, are propagated in vitro and transferred to the greenhouse. The presence of the transgene can be confirmed by PCR on genomic DNA using specific primers for genes encoding the dairy protein. The binary vectors are used as a template for positive control. Example 5: Seed specific expression of beta casein fused to oleosin affinity peptides DNA constructs are synthesized to test both the incorporation of dairy proteins into oil bodies and the localization in plant cells (Table 11). The elements presented in the Table are stacked from 5 ’ to 3 ’ of the DNA insert. The coding regions are inserted into pHGHPB binary plasmid which contain the basta resistance gene (bar gene). Coding region is flanked either by the phaseolin promoter and 3’UTR (SEQ ID NO: 7 \ , Phaseolus vulgaris acc. J01263. 1) or soybean glycinin G1 promoter and 3'UTR (SEQ ID NO: 72; Glycine max acc. P04776). All the binary vectors were transferred into A. tumefaciens LB 4404. Table 11: Transformation vectors prepared for oilseed transformation

[0475] Transformation into Arabidopsis, Camelina and Canola can be performed as described previously [Clough and Benet, 1998, Plant J. 16:735-743; Liu et al., 2012, In Vitro Cell.Dev.Biol. — Plant. 48:462-468; De Block et al., 1989, Plant Physiol. 91:694-701],

[0476] More than 15 independent transformants are generated for each binary vector, are propagated in vitro and transferred to the greenhouse. The presence of the transgene can be confirmed by PCR on genomic DNA using specific primers for genes encoding the dairy protein. The binary vectors are used as a template for positive control.

[0477] Example 6: Arabidopsis T1 Seeds Selection, Protein Extraction, and Detection

[0478] 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.

[0479] As shown in Figure 18, 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.

[0480] Example 7: Producing the composition comprising an oil body and a dairy protein, or a "plant-derived dairy milk" or a "plant-derived dairy cream". extraction by mechanical di (bench-top scale):

[0481] • The dehulled seeds from the plants produced by the methods of the invention can be soaked overnight in Milli-Q water at a seed-to-water ratio of 1:4 (w / w).

[0482] • The soaked dehulled seeds can then be crushed for 5 min using a hand blender to produce a slurry.

[0483] • The shiny can then be filtered using a 200 pm nylon mesh fabric to obtain a seed extract.

[0484] • Oil bodies and milk proteins / analogues can then be obtained by centrifugation, 10,000 g for 20 min at 20°C.

[0485] • The fraction containing the milk proteins / analogues and oil bodies can then be separated.

[0486] • Washing steps may be added to remove the exogenous proteins. For this purpose, repeated centrifugal washings can be performed by mixing the fraction containing the milk proteins / analogues with milli-Q water at a fraction-to-water ratio of 1:4, followed by centrifugation and filtration as described above. The resulting fraction can be described according to the invention as the "composition comprising an oil body and a dairy protein", and can also be described as a "plant-derived dairy milk" or "plant-derived dairy cream".

[0487] Aqueous extraction by mechanical disruption (pilot plant scale):

[0488] • The dehulled seeds from the plants produced by the methods of the invention can be soaked overnight in Milli-Q water at a seed-to-water ratio of 1:4 (w / w).

[0489] • Then, the dehulled, soaked seeds can be crushed using a colloid mill at a frequency of 30 Hz to produce a sluny.

[0490] • To remove the solids, the resulting sluny can be passed through a clarifier to obtain a seed extract.

[0491] • Oil bodies and milk proteins / analogues can then be obtained by centrifugation of the extract at 10,000 g for 20 min at 20°C.

[0492] • The fraction containing the milk proteins / analogues and oil bodies can then be separated.

[0493] • Washing steps may be added to remove the exogenous proteins. For this purpose, repeated centrifugal washings can be performed by mixing the fraction containing the milk proteins / analogues with milli-Q water at a fraction-to-water ratio of 1:4, followed by centrifugation and filtration as described above.

[0494] The resulting fraction can be described according to the invention as the "composition comprising an oil body and a dairy protein", and can also be described as a "plant-derived dairy milk" or "plant-derived dairy cream".

[0495] A flow chart summarizing these processes is shown in Figure 5.

[0496] Example 8: Oil Body Extraction from Plant Seeds

[0497] High-speed oil body extraction

[0498] Approximately 50 mg of both transgenic and wild-type (WT) Arabidopsis seeds were utilized in accordance with the precise amounts listed in Table 12. 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.

[0499] Table 12: Sample Description

[0500] The results of the oil body extraction procedure revealed the presence of four distinct layers, as depicted in Figure 19A. 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 19C). 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 (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 20). 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 20). 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.

[0501] Low -speed oil body extraction

[0502] 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 21).

[0503] Example 9: Production of dairy product substitutes

[0504] A. Production of a whipping cream substitute:

[0505] A whipping cream formulation using the present invention (functionalized cream preparation) will be prepared as follows.

[0506] 1. The base formulations will be prepared by mixing appropriate proportions of functionalised "plant-derived dairy milk / cream" (with up to 40% lipid), sugar, stabilizers, and emulsifiers.

[0507] 2. The preparation will be homogenized at 200 bar to produce a stable emulsion.

[0508] 3. The mixture is heat treated by UHT at 135-150°C for 2-4 s, or by pasteurization at 75- 85 °C for 1-3 min to achieve the desired safety and shelf-life.

[0509] 4. The mixture is cooled down to 15°C and packed in aseptic bottles or UHT containers.

[0510] 5. The formulation will be assessed by measuring viscosity, whipping ability, foam stability, texture, and liquid separation. These will be determined using standard methodologies commonly applied by those knowledgeable in the preparation of whipping cream products.

[0511] 6. The levels of ingredients and processing conditions will be optimised to achieve the desired product characteristics, similar to that of whipping dairy cream and an improvement upon plant-based whipping creams.

[0512] B. Production of a yoghurt substitute:

[0513] A stirred yoghurt formulation using the present invention (functionalised cream preparation) will be prepared as follows. a) The base formulations will be prepared by mixing appropriate proportions of functionalised "plant derived dairy milk / cream" (with up to 20% lipid), plant proteins, sugar, and water. b) This will be heat treated (i.e. UHT at 135-150°C for 2-4 s, or by pasteurization at 75- 85°C for 1-3 min) and homogenized (i.e. 200 bar). c) The mixture will then be fermented with plant-based yoghurt cultures to reduce the pH to below 5.0 to form a gel. d) Pectin, modified starch or gellan gum or other polysaccharides will then be mixed with sheared gel to achieve desired consistency, viscosity and texture. e) Flavours andfruit preparations may also be added at this stage. f) The formulation will be assessed by measuring viscosity, texture, water separation and particle size. These will be determined using standard methodologies commonly applied by those knowledgeable in the preparation of yoghurt products. g) The composition of ingredients and processing conditions will be optimised to achieve the desired product characteristics, similar to that of dairy-based yoghurt and an improvement upon standard plant-based yoghurt formulations.

[0514] C. Production of a cheese substitute:

[0515] A cheese formulation using the present invention (functionalised cream preparation) will be prepared as follows.

[0516] 1. Appropriate proportions of functionalised "plant-derived dairy milk / cream" (with up to 30% lipid), plant proteins, salt, calcium, phosphate, and water will be mixed to prepare a “cheese milk”. 2. Cheese milk will be coagulated by to form a curd mass, using a combination of acidification (direct or by adding plant-based cheese cultures) and enzymatic hydrolysis.

[0517] 3. Heat treatment and calcium addition could also be applied to achieve the desired curd structure.

[0518] 4. The curd will then be separated into a semisolid mass.

[0519] 5. Hydrocolloid gums, starches, emulsifying salts and flavouring agents could be added to the curd, under shear conditions to achieve the desired texture, meltability and flavour profile.

[0520] 6. The cheese formulation will be assessed by measuring rheological properties, texture, and flavour profiles. These will be determined using standard methodologies commonly applied by those knowledgeable in the preparation of cheese products.

[0521] 7. The formulations and processing methods will be optimised to achieve the desired product characteristics, similar to that of dairy cheese and improved upon standard plant-based cheese formulations.

[0522] D. Production of an oat milk substitute:

[0523] An oat milk formulation using the present invention (functionalised cream preparation) will be prepared as follows.

[0524] • Rolled oats will be mixed with water at a ratio of 1 :4 or 1 : 6 oats to water (w / w).

[0525] • The mixture will be transferred to a tank at 80-95°C.

[0526] • Amylases will be added to the mixture following the instructions of the enzyme manufacturer to induce liquification. The reaction can take 1-3 h, depending on the enzyme.

[0527] • The mixture will be cooled to 60-65°C and transferred to another tank at 50-60°C for 15-60 min to induce saccharification. The mixture will be heated to 80°C for 30 min to inactivate the enzymes.

[0528] • The fibre will be separated from the liquid fraction by centrifugation or using a decanter.

[0529] • The liquid fraction will be transferred to a blending tank and mixed with 3-5% functionalised "plant-derived dairy milk / cream", 0.1% salt, and 0.1% flavorings.

[0530] • This formulation will be homogenized at 200 bar followed by UHT treatment (135- 150°C for 2-4 s). • The beverage will be cool down to 10°C and packed into UHT containers.

[0531] • The oat milk formulation will be assessed by measuring rheological properties, texture, and flavour profiles. These will be determined using standard methodologies commonly applied by those knowledgeable in the preparation of oat milk products.

[0532] • The formulations and processing methods will be optimised to achieve the desired product characteristics, more similar to that of dairy milk than standard oat milk formulations.

[0533] The schematic representation of these processes (with a plant-based milk and whipping cream for example) is shown in Figure 5.

[0534] Functionalised "plant-derived dairy milk / cream" can also be used as an ingredient in ice cream, frozen desserts, cakes, toppings, cream fillings, beverages, and sauces, as a replacement for dairy cream.

[0535] E. Production of a coffee whitener:

[0536] The coffee whitener formulation consisted of vegetable oil (10%), emulsifier (lecithin, 1%), sugar (Maltodextrin, 5%), protein (Sodium Caseinate, oil-bodies functionalized with equivalent to 1.8% protein, or recombinant oleosimbeta-casein proteins, 1.8%), Dipotassium phosphate (0.2%), Xanthan gum (0.15%), and water to make up the volume (500ml). Initially, a 0.15% Xanthan gum prepared in 100 milliliters of water. Sugar and Dipotassium phosphate were then added to this mixture. Subsequently, the chosen protein source (Sodium Caseinate, oil-bodies functionalized with equivalent to 1.8% protein, or recombinant oleosimbeta-casein proteins) was hydrated in 50 milliliters of water and added to the mixture. Liquified vegetable oil with lechitin preheated to 60°C and added to the mixture. The mixture was then heated to 60°C and blended using a hand blender (ultratorrex) at high speed for 2 minutes. To further homogenize the formulation, a two-stage homogenizer was employed, subjecting the mixture to pressures of 200 and 50 bar.

Claims

1. CLAIMS:

1. A method for producing a composition containing at least one oil body and at least one dairy protein, analogue, or part thereof, the method comprising the step of making an extract from a plant cell, plant part or plant expressing the dairy protein, analogue, or part thereof, in close proximity to, or physical connection with, an oil body in the plant cell, plant part or plant, to produce the composition.

2. The method of claim 1 wherein dairy protein, analogue, or part thereof, is expressed in a plant cell containing an oil body.

3. The method of claim 1 or 2 wherein dairy protein, analogue, or part thereof, is targeted to a subcellular location of a plant cell containing an oil body.

4. The method of any preceding claim wherein the oil body and at least one dairy protein, analogue, or part thereof are co-purified.

5. The method of any preceding claim wherein the dairy protein, analogue, or part thereof is physically connected to the oil body in the composition.

6. The method of any preceding claim wherein the oil body is functionalized with the dairy protein, analogue, or part thereof in the composition.

7. A method of any preceding claim wherein the composition comprises oil, oil bodies, or functionalized oil bodies, at a concentration of at least 1% w / w.

8. A method of any preceding claim wherein the composition comprises the dairy protein, analogue or part thereof at a concentration of at least 0.1 mg / 100 mg.

9. A composition containing at least one of: a) at least one oil body and at least one dairy protein, analogue, or part thereof, and b) at least one functionalized oil body, produced by the method of any preceding claim.

10. An isolated functionalised oil body that is functionalised with at least one dairy protein, analogue, or part thereof.

11. A composition comprising the isolated functionalised oil body of claim 10.

12. The composition of any preceding claim comprising oil bodies, or functionalized oil bodies, at a concentration of at least 1% w / w.

13. The composition of any preceding claim comprising the dairy protein, analogue or part thereof at a concentration of at least 0. 1 mg / 100 mg.

14. An emulsion derived from a composition, or isolated functionalised oil body, of any preceding claim.

15. A construct for expression of at least one dairy protein or analogue thereof, or part thereof, in close proximity to, or physical connection with, an oil body in a plant, the genetic construct comprising a promoter operably linked to a nucleic acid encoding: a) a fusion of an oil body-associated protein and the dairy protein, analogue or part thereof, or b) a fusion between a component that has affinity for an oil body-associated protein and the dairy protein, analogue or part thereof.

16. The construct of claim 15 further comprising a nucleic acid encoding a signal peptide for directing the dairy protein, analogue or part thereof or fusion to a subcellular location in the cell containing an oil body.

17. A construct comprising a nucleic acid encoding: a) a fusion of an oil body-associated protein and a dairy protein, analogue or part thereof, or b) a fusion between a component that has affinity for an oil body-associated protein and a dairy protein, analogue or part thereof.

18. A fusion protein comprising: a) an oil body-associated protein and the dairy protein, analogue or part thereof, or b) a component that has affinity for an oil body-associated protein and a dairy protein, analogue or part thereof.

19. A cell, plant cell, plant, plant part, seed containing the genetic construct of claim any preceding claim.

20. An extract from the cell, plant cell, plant, plant part, seed of claim 19 comprising at least one oil body and at least one dairy protein, analogue, or part thereof, or at least one functionalized oil body.

21. A composition comprising at least one oil body and at least one dairy protein, analogue, or part thereof, or at least one functionalized oil body derived from the extract of claim 20.

22. The composition of claims 21 comprising oil, oil bodies, or functionalized oil bodies, at a concentration of at least 1% w / w.

23. The composition of claims 21 or 22 comprising the dairy protein, analogue or part thereof at a concentration of at least 0.1 mg / 100 mg.

24. An isolated functionalised oil body that is functionalised with at least one dairy protein, analogue, or part thereof, that is purified from a cell, plant cell, plant, plant part, seed of claim 19.

25. A method for expressing at least one dairy protein or analogue thereof or part thereof, in close proximity to, or physical connection with, an oil body in a plant, the method including the step of expressing the dairy protein from a construct of claim 15 in a plant.

26. A method for producing a plant expressing at least one dairy protein or analogue thereof, or part thereof, in close proximity to, or physical association with, an oil body in the plant, wherein the method includes the step of expressing the dairy protein, analogue thereof, or part thereof from a construct of claim 15 in a plant.

27. A method for producing a composition containing at least one oil body and at least one dairy protein or analogue, or part thereof, the method comprising the step of making an extract from a cell, plant or plant part claim 19 to produce the composition.

28. The method of any one of claims 25 to 27 wherein at least one oil body functionalized with the at least one dairy protein or analogue, or part thereof, is produced.

29. A composition produced by the method of claim 27 or 28 comprising at least one of: a) at least oil body and at least one dairy protein or analogue, or part thereof, and b) at least one functionalized oil body.

30. An isolated functionalized oil body produced by the method of any preceding claim.

31. An emulsion derived from a composition or functionalized oil body of, or produced by the method of, any preceding claim.

32. A food or beverage ingredient comprising the functionalised oil body, composition, or emulsion of any preceding claim.

33. A food or beverage comprising the functionalised oil body, composition, emulsion, fusion protein, or ingredient of any preceding claim.

34. A dairy product alternative comprising the functionalised oil body, composition, emulsion, fusion protein, or ingredient of any preceding claim.

35. The dairy product alternative of claim 34 that is selected from a milk substitute, a cream substitute, a whipping cream substitute, a mayonnaise substitute, an ice cream substitute, a cheese substitute, and a yoghurt substitute.

36. A nutraceutical or supplement comprising the functionalised oil body, composition, emulsion, fusion protein, or ingredient of any preceding claim.

37. A nutraceutical or supplement comprising the functionalised oil body, composition, emulsion, fusion protein, or ingredient of any preceding claim.

38. A production process comprising: a) Producing, by the method of any preceding claim, a composition containing at least one of: i. at least one oil body and at least one dairy protein, analogue, or part thereof, and ii. an isolated functionalized oil body, and b) Processing the composition of a) into a food or beverage product, or a nutraceutical or supplement39. The production process of claim 38 where the food or beverage is a dairy product alternative.

40. The production process of claim 39 wherein the dairy product alternative is selected from a milk substitute, a cream substitute, a whipping cream substitute, a mayonnaise substitute, an ice cream substitute, a cheese substitute, and a yoghurt substitute.

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