Process for producing a protein product, and a protein product

Abstract

Disclosed are processes for producing protein products, including protein concentrates from biomass material comprising Rubisco protein. Also disclosed are protein concentrates with advantageous functional properties, related protein products, and food products comprising protein concentrates.

Description

PROCESS FOR PRODUCING A PROTEIN PRODUCT, AND A PROTEIN PRODUCTFIELD OF THE INVENTION

[0001] The present invention broadly relates to processes for producing protein concentrates from biomass material comprising Rubisco protein. The invention also relates to protein concentrates, related protein products, and food products comprising protein concentrates described herein.BACKGROUND TO THE INVENTION

[0002] Ribulose-l,5-bisphosphate carboxylase / oxygenase, known as RuBisCo or Rubisco, is the most abundant protein in the world, being present in all green leaves, as well as algae and cyanobacteria. Rubisco plays a critical role in photosynthesis as the first enzyme involved in fixation of atmospheric carbon dioxide into energy rich molecules.

[0003] Leaf protein was identified as a potential food source in the 1940s by British biochemist and virologist Norman Pirie during the Second World War. In the 1950s this protein fraction was characterized as being principally comprised of Rubisco, a white, cytoplasmic protein with a large molecular weight. In the 1970s researchers at the USDA Western Regional Research Centre undertook studies to prepare Rubisco as a leaf protein concentrate from alfalfa in a process they called "Pro-Xan".

[0004] Thus, the study of Rubisco protein, and of processes for preparing Rubisco protein from green leaves, has continued for more than 80 years. Today, there is an increasing need to provide food proteins to the world's growing population at a low carbon cost. It is clear that Rubisco protein is a promising prospect for meeting this need. Despite this, advances from the laboratory to the factory have been slow, and most research is still investigative rather than directed to solving processing problems which occur when attempting to produce Rubisco compositions at commercial scale, or to create new Rubisco compositions with properties which are functionally useful in the food industry.

[0005] Production of Rubisco protein and Rubisco containing protein products from biomass material comprising Rubisco protein is difficult as the protein subunits are prone to disassociation, denaturation, and precipitation. Denaturation of the Rubisco protein is undesirable as this can have an adverse effect on its functionalproperties, both physical and organoleptic. Providing a product comprising Rubisco protein for human consumption also provides challenges in terms of scale-up for industrial methods, complicated processing steps for removal of impurities, food safety, yield, and product purity.

[0006] Other methods for extracting protein from leaf material involve complicated pH changes throughout the process (pH 11 to pH 4.5), complicated protein extraction techniques, multiple solvent washes, and / or processes that would provide residual solvent in the food product, which are clearly not suitable for food applications and also not practical on a commercial scale (WO 2011 / 075671, Yang et al. J. Agric. Food Chem. (52) 2223-2225, (2004)).

[0007] One of the main processing concerns in relation to the production of Rubisco protein products is the removal of undesirable colours and flavours to provide a "white" protein product while retaining functional properties, both physical and organoleptic. For example, the use of acid precipitation has been found to have an adverse effect on emulsion properties (B.P. Lamsal et al., LWT 40 (2007) 1520-1526). Retention of emulsion properties is desirable for use in production of food products using the protein product. Use of organic solvents, such as alcohols, during processing of the protein material can have an adverse effect on the structure and functional properties of the protein, as discussed in Yoshikawa et al., Int J Biol Macromol. 2012 Apr l;50(3):865-71. WO 2011 / 075671, for example, does not employ acid precipitation of RuBisCO due to expected and demonstrated (Comparative Example 1) reduced protein content following its use. The process of WO 2011 / 075671 instead utilises activated carbon to remove undesirable compounds such as chlorophyll and carotenes.

[0008] These undesirable colours and flavours are either inherent to the biomass source or are generated during the harvesting stage through disruption of cellular structure, lysis of organelles and chloroplasts, or through the action of released enzymes or oxidation upon cellular compounds. Undesirable colours and pigments include chlorophyll responsible for at dark green colour of leaves, carotenoids, and polyphenols and flavones which may oxidise during processing and interact with protein to impart a brown discolouration.

[0009] Volatile compounds with high odour or taste such as the aldehyde trans- 2-hexenal, hexanal, and 2,6-nonadienal are formed in in response to plant wounding by hydroperoxide lyase. Benzaldehyde, furfural, and 2-methylbutanal are formed via amino acid catabolism via protease, peptidases, transaminases, keto-acid decarboxylases, keto-acid dehydrogenases and alcohol dehydrogenases.

[0010] Astringent-tasting non-volatile compounds and coloured compounds can be generated from phenolic secondary metabolites following cell wall rupture due to the action of the enzymes identified previously and due to polyphenol oxidase.

[0011] Current commercial methods for removing impurities when preparing a Rubisco protein product at industrial scale include crystallisation, filtration and chromatographic techniques which may use organic solvents and processing aids including resins, adsorbents, polyvinylpyrrolidone (PVPP), polyethylene glycol (PEG), and activated carbon, for example. These methods are used as they are less likely to denature or aggregate the Rubisco protein, but these methods can be complicated in terms of process steps and equipment used, resulting in high production costs and low efficacy. Separation of volatile and astringent organic molecules from the proteins can be challenging as the molecules may associate via hydrophobic, covalent, or non- covalent interactions which have higher affinity which cannot be disrupted via filtration processes.

[0012] The ability for a protein product used in the production of food products to create stable emulsions is particularly desirable. Emulsification is the most important process in the manufacturing of many formulated foods. Protein emulsifying activity is the ability of the protein to participate in emulsion formation and to stabilize the newly created emulsion. The emulsifying capacity is the ability of the protein solution or suspension to emulsify oil.

[0013] The food industry is increasingly looking to develop foods intended to reduce negative impacts on the environment, human health, and animal welfare. One focus is the replacement of animal-sourced ingredients (e.g. from meat, fish, eggs, or milk), with ingredients from alternative sources (e.g. plants, algae, and microbes). One of the issues with replacement of animal-sourced protein ingredients is the ability of alternative sources, such as biomass material comprising Rubisco protein, to provide a protein product that can at least replicate the emulsifying capacity of animal-sourced proteins in food production.

[0014] It is an object of the present invention to provide a Rubisco protein concentrate suitable for human consumption; and / or a Rubisco protein concentrate with functional properties suitable for further commercial processing; and / or a Rubisco protein concentrate and / or related protein products substantially free of undesirable colours and / or flavours; and / or a Rubisco protein concentrate with favourable emulsion properties; and / or a method for preparing a Rubisco protein concentrate; and / or to at least provide the public with a useful choice.

[0015] Other objects of the invention may become apparent from the following description which is given by way of example only.

[0016] Any discussion of documents, acts, materials, devices, articles, or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.SUMMARY OF THE INVENTION

[0017] In a first aspect, the invention provides a Rubisco protein concentrate having a water content of at least about 50% w / w.

[0018] In a further aspect, the invention provides a Rubisco protein concentrate, comprising: a water content between 50% - 95%; a dry matter content between 5% - 50% w / w, wherein the dry matter comprises Rubisco protein in an amount between 70% - 90% w / w; and ethanol, wherein the ethanol is present at a concentration of less than about 0.5%.

[0019] Preferably, the Rubisco protein concentrate comprises: less than about 0.5% total saponins; and / or less than about 0.5% flavones and polyphenols compounds; and / orCIE colour values of a* > 0; b* > 10, and L* > 50.

[0020] Preferably, the Rubisco protein concentrate comprises: less than about 0.5% total saponins; and / or less than about 0.5% flavones and polyphenols compounds; and / orCIE colour values of a* > -1; b* > 10, and L* > 50.

[0021] In a further aspect, the invention provides a protein powder comprising Rubisco protein in an amount between 70% - 90%, having one or more of the following features: less than about 0.5% total saponins; and / or less than about 0.5% flavones and polyphenols compounds; and / orCIE colour values of a* > 0; b* > 10, and L* > 50.

[0022] In a further aspect, the invention provides a protein powder comprising Rubisco protein in an amount between 70% - 90%, having one or more of the following features: less than about 0.5% total saponins; and / or less than about 0.5% flavones and polyphenols compounds; and / orCIE colour values of a* > -1; b* > 10, and L* > 50.

[0023] In a further aspect, the invention provides a process for producing a protein concentrate from a biomass material comprising Rubisco protein, the process comprising steps of: i. providing a solid protein fraction prepared from biomass material comprising Rubisco, preferably wherein the solid protein fraction comprises at least 50% w / w Rubisco protein, ii. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol,Hi. separating the solid protein fraction from the first solvent or solvent mixture; iv. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol, v. separating the solid protein fraction from the second solvent or solvent mixture, and vi. washing the solid protein fraction with water to produce a protein concentrate.

[0024] In a further aspect, the invention provides a process for producing a protein concentrate from a biomass material comprising Rubisco protein, the process comprising steps of:1. providing a clarified juice from a biomass material comprising Rubisco protein;2. precipitating protein from the clarified juice to produce a liquid and a solid protein fraction,3. separating the liquid from the solid protein fraction;4. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol,5. separating the solid protein fraction from the first solvent or solvent mixture;6. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol, and7. separating the solid protein fraction from the second solvent or solvent mixture, and8. washing the solid protein fraction with water to produce a protein concentrate to produce a protein concentrate.

[0025] In a further aspect, the invention provides a process for producing a protein product from a biomass material comprising Rubisco protein, the process comprising steps of: a. providing a clarified juice from a biomass material comprising Rubisco protein; b. precipitating protein from the clarified juice to produce a liquid and a solid protein fraction, c. separating the liquid from the solid protein fraction; d. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol,e. separating the solid protein fraction from the first solvent or solvent mixture; f. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol, and g. separating the solid protein fraction from the second solvent or solvent mixture, to produce a protein product.

[0026] In a further aspect, the invention provides a process for producing a protein concentrate from a juice of a biomass material comprising Rubisco protein, the protein concentrate having a water content of at least about 50% (w / w), the process comprising steps ofA. Providing a juice from a biomass material comprising Rubisco protein;B. Preparing a solid protein fraction from the juice; andC. Reducing the water content of the solid protein fraction to produce the protein concentrate.

[0027] In a further aspect, the invention provides a process for producing a protein concentrate from a juice of a biomass material comprising Rubisco protein, the protein concentrate having an emulsion activity of at least about 70%, the process comprising steps of1. Providing a juice from a biomass material comprising Rubisco protein;2. Preparing a solid protein fraction from the juice; and3. Reducing the water content of the solid protein fraction to produce the protein concentrate, wherein, throughout the process, the water content of the solid protein fraction is maintained at at least about 50% (w / w).

[0028] In a further aspect, the invention provides a process for preparing an emulsion, the process comprising homogenizing a Rubisco protein concentrate of the invention, an aqueous phase, and an oil phase.

[0029] In a further aspect, the invention provides a process for preparing an emulsion, the process comprising homogenizing a Rubisco protein concentrate prepared by the process of the invention, an aqueous phase, and an oil phase.

[0030] In a further aspect, the invention provides a food product comprising a Rubisco protein concentrate prepared by the process of the invention, or a Rubisco protein concentrate of the invention.

[0031] In a further aspect, the invention provides use of a Rubisco protein concentrate prepared by the process of the invention, or a Rubisco protein concentrate of the invention in the preparation of a food product,

[0032] In a further aspect, the invention provides a method of preparing a food product comprising combining a Rubisco protein concentrate prepared by the process of the invention, or a Rubisco protein concentrate of the invention with additional ingredients.

[0033] The following embodiments and preferences may relate alone or in any combination of any two or more to any of the above aspects.

[0034] In various embodiments, the solid protein fraction is prepared by: a. providing a clarified juice from a biomass material comprising Rubisco protein; and b. precipitating protein from the clarified juice to produce a liquid and a solid protein fraction.

[0035] In various embodiments, providing a clarified juice comprises steps of i. subjecting juice from a biomass material comprising Rubisco protein to a mild heat treatment to coagulate a green protein fraction; and ii. separating the juice from the coagulated green protein fraction to provide a clarified juice.

[0036] In various embodiments, the clarified juice is prepared by subjecting juice from a biomass material comprising Rubisco protein to a mild heat treatment to coagulate a green protein fraction; and separating the juice from the coagulated green protein fraction to provide a clarified juice.

[0037] In various embodiments, the process further comprises a step of washing the solid protein fraction with water to produce a protein concentrate.

[0038] In various embodiments, step (b) is an acid precipitation step.

[0039] In various embodiments, the biomass material comprising Rubisco protein is a plant material, a plant or part of a plant. In various embodiments, the biomass material comprising Rubisco protein is alfalfa.

[0040] In various embodiments, the first solvent or solvent mixture is the same as the second solvent or solvent mixture.

[0041] In various embodiments, the first solvent or solvent mixture is different to the second solvent or solvent mixture.

[0042] In various embodiments, the first solvent or solvent mixture has a higher water content than the second solvent or solvent mixture.

[0043] In various embodiments, the process comprises further lower alcohol extraction steps.

[0044] In various embodiments, the process comprises further water wash steps.

[0045] In various embodiments, wherein the water wash step(s) occur(s) after either or both separation step(s).

[0046] In various embodiments, the process further comprises the step of removing a portion of the water from the protein concentrate.

[0047] In various embodiments, removing a portion of the water from the protein concentrate comprises subjecting the protein concentrate to a vacuum.

[0048] In various embodiments, removing a portion of the water from the protein concentrate comprises subjecting the protein concentrate to a vacuum during mixing.

[0049] In various embodiments, the protein concentrate is not subjected to a drying step.

[0050] In various embodiments, the protein concentrate has a water content of at least about 50% w / w.

[0051] In various embodiments, the dry matter content of the protein concentrate is at least about 80% protein (w / w).

[0052] In various embodiments, the protein concentrate has a lower alcohol content of about 2% to about 0.1% (w / w). In various embodiments, the protein concentrate has a lower alcohol content of about 2% to about 0.01% (w / w).

[0053] In various embodiments, the lower alcohol is ethanol.

[0054] In various embodiments, one or more of the lower alcohol solvent extraction steps is a continuous countercurrent extraction step.

[0055] In various embodiments, the lower alcohol extraction steps are repeated until the alcohol extraction liquid fraction has an absorbance at 664m of less than 0.05 Absorbance units in a 1 cm pathlength.

[0056] In various embodiments, the lower alcohol extraction steps are repeated until the until the protein concentrate has CIE colour values of a* > 0; b* > 10, and L* > 50.

[0057] In various embodiments, the lower alcohol extraction steps are repeated until the until the protein concentrate has CIE colour values of a* > -1; b* > 10, and L* > 50.

[0058] In various embodiments, step A comprises steps of:I. subjecting juice from a biomass material comprising Rubisco protein to a mild heat treatment to coagulate a green protein fraction; andII. separating the juice from the coagulated green protein fraction to provide a clarified juice.

[0059] In various embodiments, step B comprises steps of:I. subjecting the clarified juice to an acid precipitation step to produce a liquid and a solid protein material;II. separating the liquid from the solid protein material.

[0060] In various embodiments, step B further comprises steps of:I. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol; andII. separating the solid protein fraction from the first solvent or solvent mixture.

[0061] In various embodiments, step B further comprises a step of:I. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol; andII. separating the solid protein fraction from the second solvent or solvent mixture.

[0062] In various embodiments, the lower alcohol is ethanol.

[0063] In various embodiments, the protein concentrate comprises a lower alcohol content of about 1.5% to about 0.1% (w / w).

[0064] In various embodiments, the protein concentrate is a stable suspension.

[0065] In various embodiments, the protein concentrate is frozen.

[0066] In various embodiments, the protein concentrate has not been dried and reconstituted.

[0067] In various embodiments, the protein concentrate has an emulsion activity of at least about 70%.

[0068] In various embodiments, the protein concentrate is able to emulsify25.2 g oil per gram of protein. In various embodiments, the protein concentrate is able to emulsify 40 g oil per gram of protein, such as 50 g, or 55 g oil per gram of protein.

[0069] In various embodiments, the protein concentrate comprises less than about 0.5 % total saponins.

[0070] In various embodiments, the protein concentrate comprises less than about 0.5% flavones and polyphenols compounds.

[0071] In various embodiments, the protein concentrate has CIE colour values of a* > 0; b* > 10; and L* > 50.

[0072] In various embodiments, the protein concentrate has CIE colour values of a* > -1; b* > 10; and L* > 50.

[0073] In various embodiments, the process further comprises a step of homogenizing the Rubisco protein concentrate, an aqueous phase, and an oil phase, to prepare an emulsion.

[0074] In various embodiments, the amount of Rubisco protein concentrate used to prepare the emulsion is 0.05 - 5% (w / w).

[0075] In various embodiments, the amount of oil used to prepare the emulsion is 50 - 90% (w / w).

[0076] In various embodiments, the emulsion is prepared with a ratio of about 1 :2.6:6.3 to about 1 :54.5: 108 (w / w) protein:oil:aqueous phase.

[0077] In various embodiments, the amount of oil used to prepare the emulsion is about 26-70% (w / w) with respect to the Rubisco protein concentrate:aqueous phase components. In various embodiments, the amount of Rubisco protein concentrate used to prepare the emulsion between 0.5-10% with respect to the oil and aqueous phase components. In various embodiments, the amount of oil used to prepare the emulsion is about 26-70% (w / w) with respect to the Rubisco protein concentrate:aqueous phase components, and the amount of Rubisco protein concentrate used to prepare the emulsion between 0.5-10% with respect to the oil and aqueous phase components.

[0078] In a further aspect, the invention provides a Rubisco protein concentrate having a water content of at least about 50% (w / w), CIE colour values of a* > -1; b*> 10; and L* > 50, an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate), an oil holding capacity of between about 2.5 and about 6.9g / g, and a water holding capacity of between about 2.5 and about 3.5 g / g.

[0079] Preferably, the Rubisco protein concentrate has a water content between about 50% and about 99.5% w / w, more preferably between about 65% and about 85% w / w, between about 50% and 75%, or between 80% and 99.5% w / w.

[0080] Preferably, the Rubisco protein concentrate has CIE colour values of a*> 0; b* > 10; and L* > 50.

[0081] Preferably, the Rubisco protein concentrate has an oil holding capacity of between about 3.0 and about 6.0 g / g.

[0082] Preferably, the Rubisco protein concentrate has an oil holding capacity of between about 3.0 and about 4.0 g / g.

[0083] Preferably, the Rubisco protein concentrate has a water holding capacity of between about 2.7 and about 3.4g / g.

[0084] Preferably, the Rubisco protein concentrate has a lower alcohol content, preferably ethanol, of less than about 0.5%, more preferably between about 0.5% and about 0.1%.

[0085] Preferably, the Rubisco protein concentrate comprises less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds.

[0086] Preferably, the Rubisco protein concentrate has a water content between about 50% and about 95% w / w and a dry matter content between 5% - 50% w / w, wherein the dry matter comprises Rubisco protein in an amount between 70% - 90% w / w.

[0087] Preferably, the Rubisco protein concentrate has an emulsion activity of between about 70% and about 90% (when measured at 5% protein inclusion rate).

[0088] Preferably, the Rubisco protein concentrate has an emulsion activity of between about 52% and about 60%, preferably between about 53% and 56% (when measured at 1% protein inclusion rate).

[0089] Preferably, the Rubisco protein concentrate has a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably between about 90% and 99.2% w / w and is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein.

[0090] Preferably, the Rubisco protein concentrate has a foaming stability after 30 minutes of between about 55% and about 90%, more preferably between about 58% and 86%.

[0091] In a further aspect, the invention provides a food product comprising an emulsion of Rubisco protein:oil:aqueous phase in a ratio of about 1:2.5:6.3 to about 1 :54.5: 108 (w / w), wherein the emulsion is formed by combining:(i) a Rubisco protein concentrate having a water content of at least about 70% (w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, an emulsion activity of greater than about 70% (when measured at 5% protein inclusionrate), an oil holding capacity of between about 2.5 and about 6.9g / g, and a water holding capacity of between about 2.5 and about 3.5 g / g;(ii) an edible oil; and(iii) water.

[0092] Preferably, the oil holding capacity of the Rubisco protein concentrate is between about 3.1 and about 3.6 g / g

[0093] Preferably, the water holding capacity of the Rubisco protein concentrate is between about 2.7 and about 3.4 g / g.

[0094] Preferably, the edible oil is an oil or fat selected from one or more of canola, coconut, rice, sunflower, olive, rice bran, soybean, rape, vegetable, palm, and flax oil, and animal fats.

[0095] Preferably, the food product has a lower alcohol content, preferably ethanol, of less than about 0.5%.

[0096] Preferably, the food product comprises less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds.

[0097] Preferably, the Rubisco protein concentrate has a water content between about 70% and about 99.5% w / w, more preferably between about 70% and about 95% or between about 70% and about 85% w / w or between 80% and about 99.2%w / w, or between about 90% and 99.2% w / w.

[0098] Preferably, the Rubisco protein concentrate has a water content between about 70% and about 95% w / w and a dry matter content between 5% - 50% w / w, wherein the dry matter comprises protein in an amount between 70% - 90% w / w.

[0099] Preferably, the Rubisco protein concentrate has a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably between about 90% and 99.2% w / w and is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein.

[0180] Preferably, the Rubisco protein concentrate has an emulsion activity of between about 70% and about 90% (when measured at 5% protein inclusion rate).

[0101] Preferably, the Rubisco protein concentrate has an emulsion activity of between about 53% and 56% (when measured at 1% protein inclusion rate).

[0102] Preferably, the food product includes one or more additional ingredients selected from a lipid, a carbohydrate, a protein, a flavour, a vitamin, a mineral, a milk product, water, a food additive, a colour, a fruit preparation, or any combination of any two or more of these ingredients.

[0103] Preferably, the emulsion is formed and added to the other food product ingredients.

[0104] Preferably, the emulsion is formed with one or more of the other food product ingredients.

[0105] Preferably, the food product is selected from emulsified foods such as mayonnaises, dressings, sauces, condiments, and high-fat fillings; dairy alternatives such as plant-based milks, yoghurts, cheeses, spreads, creams, and creamers; frozen and / or chilled whipped items such as fruit ices, ice creams, mousses, custards and desserts; confectionery items such as meringues, candies and prepared and / or instantised beverages such as sports drinks, shakes, juices; plant-based egg alternatives such as egg analogues, replacers, and substitutes; formed, emulsified and / or extruded plant-based meat and fish analogues such as burgers, sausages, hot dogs, salami, cold cuts, cuts, strips and pieces, and frozen portions and meat snacks; bakery items such as breads, cakes, wraps, biscuits, crackers, bars, and pastries; extruded products such as snack foods, pasta, and breakfast cereals.

[0106] In a further aspect, the invention provides an emulsifying extender composition comprising a Rubisco protein concentrate having a water content between 50% - 99.5%; CIE colour values of a* > -1; b* > 10, and L* > 50; an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate); an oil holding capacity of between about 2.5 and about 6.9g / g; a water holding capacity of between about 2.5 and about 3.5 g / g; a dry matter content between 5% - 50% w / w, and ethanol; wherein the dry matter comprises protein in an amount between 70% - 90% w / w, and wherein the ethanol is present at a concentration of less than about 0.5%.

[0107] Preferably, the emulsifying extender composition comprises less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds

[0108] Preferably, the Rubisco protein concentrate has CIE colour values of a* > 0; b* > 10, and L* > 50.

[0109] Preferably, the Rubisco protein concentrate has an oil holding capacity of between about 3.1 and about 3.6 g / g.

[0110] Preferably, the Rubisco protein concentrate has a water holding capacity of between about 2.7 and about 3.4g / g.

[0111] Preferably, the Rubisco protein concentrate has a water content between about 50% and about 95%, more preferably between about 65% and about 85% w / w, or between 80% and about 99.2%w / w, or between about 90% and 99.2% w / w.

[0112] Preferably, the Rubisco protein concentrate has a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably has a water content of between about 90% and 99.2% w / w and is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein.

[0113] Preferably, the Rubisco protein concentrate has an emulsion activity of between about 70% and about 90% (when measured at 5% protein inclusion rate).

[0114] Preferably, the Rubisco protein concentrate has an emulsion activity of between about 53% and 56% (when measured at 1% protein inclusion rate).

[0115] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0116] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highestvalue enumerated are to be considered to be expressly stated in this application in a similar manner.

[0117] 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.

[0118] To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.

[0119] Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples.BRIEF DESCRIPTION OF THE FIGURES

[0120] The present invention will be described by way of example only and with reference to the accompanying Figures.

[0121] Figure 1 is a schematic of a process according to the invention.

[0122] Figure 2 is a schematic of a batch-mode countercurrent extraction process according to the invention.

[0123] Figure 3 is a schematic of a process design for countercurrent washing.

[0124] Figure 4 shows an image of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel with Coomassie blue staining showing the purification and retention of protein content achieved using Example 1 for purification of the Rubisco proteins. Rubisco large subunit, ~ 55kDa, Rubisco small subunit, ~15 kDa). Lane 1 = Standard, Ladder (PagerRuler Prestained adder 10-250kDa); Lane 2 = Juice from biomass material comprising Rubisco; Lane 3 = Clarified juice; Lane 4 = Clarified juice after acid precipitation; Lane 5 = Acid precipitation pellet; Lane 6 = Ethanolextraction 1 pellet; Lane 7 = Ethanol extraction 2 pellet: Lane 8 = water wash 1 supernatant; Lane 9 = water wash 1 pellet; Lane 10 = water wash 2 supernatant; Lane 11 = water wash 2 pellet; Lane 12 = blank.

[0125] Figure 5 shows an image of a native non-denaturing polyacrylamide gel electrophoresis (Native PAGE) plate with Coomassie blue staining shows the purification protein content achieved using Example 1 for purification of the Rubisco proteins. Rubisco native oligomeric molecule weight ~ 550kDa. Lane 1 = Ladder NativeMark Protein standard; Lane 2 = Juice from biomass material comprising Rubisco; Lane 3 = Clarified juice; Lane 4 = Clarified juice after acid precipitation; Lane 5 = Acid precipitation pellet; Lane 6 = Ethanol extraction 1 pellet; Lane 7 = Ethanol extraction 2 pellet: Lane 8 = water wash 1 pellet; Lane 9 = water wash 2 pellet; Lane 10 = blank.

[0126] Figure 6 shows the hardness of patties prepared using methylcellulose (MC) or Rubisco protein concentrate, as evidenced by compressive force.

[0127] Figure 7 shows the chewiness of patties prepared using methylcellulose (MC) or Rubisco protein concentrate, as evidenced by force measurements.DEFINITIONS

[0128] The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains.

[0129] The general chemical and biological terms used herein have their usual meanings.

[0130] Examples of definitions of common terms in microbiology, molecular biology and biochemistry can be found in Methods for General and Molecular Microbiology, 3rdEdition, C. A. Reddy, et al. (eds.), ASM Press, (2008); Encyclopedia of Microbiology, 2nded., Joshua Lederburg, (ed.). Academic Press, (2000); Microbiology By Cliffs Notes, I. Edward Alcamo, Wiley, (1996); Dictionary of Microbiology and Molecular Biology, Singleton et al. (2d ed.) (1994); Biology of Microorganisms lit h ed. Brock et al., Pearson Prentice Hall, (2006); Biodiversity of Fungi: Inventory and Monitoring Methods, Mueller et al. Academic Press, (2004); Genes IX, Benjamin Lewin, Jones & Bartlett Publishing, (2007); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994); andMolecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995).

[0131] The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting each statement in this specification and claims that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise", "comprised" and "comprises" are to be interpreted in the same manner.

[0132] As used herein the term "and / or" means "and" or "or", or both.

[0133] As used herein "(s)" following a noun means the plural and / or singular forms of the noun.

[0134] The term "juice" as used herein refers to the juice obtained by pressing, crushing, macerating, or otherwise removing liquid from a biomass material comprising Rubisco, such as a plant, or algae.

[0135] Functional properties have been described as relating to how ingredients behave during preparation and cooking of foods (physical properties), and how they affect the look, texture, odour, colour, and taste of the finished food product (organoleptic properties). The term "functional properties" as used herein in relation to a protein composition or a protein concentrate refers to both physical properties such as solubility, emulsion activity, emulsion stability, foam stability, and gel strength, and organoleptic properties such as flavour, texture, colour, odour, palpability, and sensory perception.

[0136] As used herein, the term "protein fraction" is used to refer to protein solids suspended or distributed in a liquid phase, and the term "protein material" is used to refer to protein solids from which a liquid solvent or supernatant has been removed.

[0137] As used herein the term "protein concentrate" or "Rubisco protein concentrate" and the like, is used to refer to the protein product (solid protein fraction) that has been concentrated from a Rubisco-containing biomass after it has undergone a water washing step (as discussed further below) and optionally a further step to reduce alcohol and water content (as discussed further below). Related protein products will include powder protein products produced by removal of water from the protein concentrate using evaporation techniques as would be known to skilled worker.[0013S] As used herein, the term "clarified juice" refers to a juice derived from biomass material comprising Rubisco, and from which the green chlorophyll-associated proteins have been removed, for example by steps of coagulation (for example, using mild heat treatment or flocculation) and then separation. Any clarified juice can be used in the invention. Clarified juice is also know in the art as "brown juice".

[0139] As used herein, the term "lower alcohol" refers to a saturated or unsaturated compound having 1-4 carbon atoms, and one or more hydroxyl groups. A preferred lower alcohol is ethanol. Other lower alcohols suitable for use in the invention include, without limitation, methanol, isopropanol (2-propanol), and glycerol. A preferred solvent comprises 80 - 100% lower alcohol, for example 85, 90 or 95%, with the remainder being water. Another preferred solvent comprises 40 - 100% lower alcohol, for example from 40 to 100%, 50 to 100%, 60 to 100% lower alcohol content, with the remainder being water.

[0140] As used herein the term "water content" means the quantity of the total water contained in the product, expressed as a percentage of the total weight. Water content is measured as the mass lost when a weighted sample is heated at 100 °C for 2 hours or more, until a constant weight is achieved.

[0141] As used herein the term "dry matter" means residual total non-volatiles solids of a preparation when water has been evaporated. Dry matter is measured as the residual mass when a weighted sample is heated at 100 °C for 2 hours or more, until a constant weight is achieved.

[0142] As used herein the term "stable suspension" means a dispersion of solute particles which do not dissolve but are suspended throughout the bulk of a medium, and is stable with respect to for example, enzyme activity and microbial growth.

[0143] The term "GRAS" refers to a food or food ingredient Generally Recognised As Safe for humans, and is a United States Food and Drug Administration (FDA) designation under sections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act.

[0144] As used herein, the term "cytosolic proteins" means the soluble liquid phase proteins from the cytoplasm, produced by juicing the plant and algal cells, for example, glutamate synthase, transketolase, glycine dehydrogenase, cobalamin- independent methionine synthase, serine hydroxymethyltransferase, and ferredoxin- NADP reductase.

[0145] As used herein, the term "Rubisco" refers to the protein ribulose-1,5- bisphosphate carboxylase / oxygenase as extracted from photosynthetic organelles of plant, algae, and microorganisms, using commercial feasible methods known to those in the art. As will be appreciated by someone skilled in the art, an extract from a plant will contain a range of proteins and the use of the word Rubisco does not imply the protein is solely Rubisco protein. The protein extract from plant leaves may also be variously referred to as leaf protein concentrate (LPC) and leaf protein isolate (LPI). In the present invention, the Rubisco protein has a purity of least about 80% to about 90% of the total protein, for example from about 84 to about 86%.

[0146] As used herein, the term "extraction step" refers to the steps of contacting the solid protein fraction with a solvent or solvent mixture comprising a lower alcohol and separating the solid protein fraction from the solvent or solvent mixture.DETAILED DESCRIPTION

[0147] As discussed in more detail herein, the invention broadly relates to a process for producing a protein concentrate from a biomass material comprising Rubisco protein. The invention also broadly relates to protein concentrates comprising Rubisco protein and related protein products comprising Rubisco protein.

[0148] The biomass material is preferably freshly harvested and not prepared from dried and reconstituted material.

[0149] The biomass material is preferably harvested leaf material that is then pressed, ground, or the like, to produce a juice containing the Rubisco protein. The harvested leaf material is preferably freshly harvested. The juice is preferably cooled prior to further processing as is discussed further herein. The invention also broadly relates to a protein concentrate produced by the process. The invention also broadly relates to a Rubisco containing protein concentrate that has desirable physical and organoleptic properties. The invention also particularly relates to a Rubisco containing protein concentrate having the emulsifying capacity desirable for use in food production. The invention also relates to Rubisco containing protein powders having desirable properties that may be produced from the protein concentrate.Biomass material

[0150] The invention can be used to produce a Rubisco protein concentrate from biomass material comprising Rubisco protein, including a plant material, a plantor part of a plant, or from algae. In various embodiments, the biomass meets the FDA standard for GRAS (Generally Recognised As Safe for humans).

[0151] The biomass material comprising Rubisco protein is preferably a plant material, a plant(s), or part of a plant(s), and / or an algae, that contains Rubisco protein. Preferably the plant material will be the leaves of a plant or plants. For example, a food crop plant, a fodder crop plant such as alfalfa, a plant part which is a part of a food crop plant, including a plant waste material such as the tops of tubers, or any non-food crop plant or a part thereof. Preferably, the plant material comprises green leaves.

[0152] In various embodiments, the plant material, the plant, or the plant part can be from the family Fabaceae, such as alfalfa, kudzu or forage peas; from the family Gramineae or Poaceae, such as oats; from the family Lemnoideae, such as duckweed; or spinach; from the family Brassicaceae, such as kale; or roadside crops, cocksfoot, Italian ryegrass, Raphno, clover, Persian clover, rapeseed leaves, carrots, radishes, Jerusalem artichoke; from the family Amaranthaceae, such as beets, spinach, fodder beet; triticale, white clover, barley, tobacco, chicory, sugar cane, or fava bean leaves.

[0153] In a preferred embodiment, the plant is alfalfa (Medicago sativa, also called lucerne). As a legume, alfalfa does not require high levels of synthetic nitrogen input, reducing costs and negative environmental consequences of nitrogen application. It has year-round growth and fields do not need to be tilled and replanted every year. In another embodiment, the plant is oats (Avena sativa), which are a good catch-crop for managing excess nitrogen and soaking up excess nutrients.

[0154] In various embodiments, the biomass material comprising Rubisco protein is algae. In various embodiments, the algae is selected from the classes Bacillariophyceae, Chloropyceae, Cyptophyceae, Dinophyceae, Euglenophyceae and Rhodophyceae.Harvesting and juicing

[0155] Biomass comprising Rubisco protein may be harvested according to known methods.

[0156] The harvested biomass is then subjected to a juicing step which can use any suitable form of maceration device, such as a screw press, macerator, cutting mill, hammer mill, microfluidizer, blender, sonicator or similar device, to physically disruptthe biomass to release the liquid component (juice) of the feed material from the dry matter component (pulp).

[0157] The pulp waste can be used for animal feed or silage.

[0158] The juice produced is then preferably cooled using a suitable refrigeration system, such as a heat exchanger. The juice is preferably cooled to a temperature of less than about 12 °C but more preferably to less than about 7 °C, or more preferably to less than about 4 °C, but above the freezing point of the liquid.

[0159] A dosing composition may also be added to the juice. The dosing composition can be added before, during or after the juicing and / or cooling step. The dosing composition preferably comprises a reducing agent, such as sodium metabisulfite, Vitamin C, sodium sulfite, alkali metal sulfites, known reducing agents such as dithiothreitol, glutathione, chelating agents such as EDTA, EGTA, enzyme inhibitors for inhibition of proteases and lyases.

[0160] The harvesting and juicing occurs at neutral pH, for example from pH 6- 8, preferably pH 6-7.5. This pH range includes the natural pH range of the extracts. An increase at pH during harvesting and juicing is associated with undesirable yellow colour in the final product.Coagulation of green protein fraction

[0161] In various embodiments, the juice is subjected to a mild heat treatment, or heating and chil li ng / cooli ng step, to coagulate a first green protein fraction containing chlorophyll and chlorophyll-containing proteins; and then separating the juice (supernatant) from the coagulated first green protein fraction to provide a clarified juice with other plant proteins in solution. The proteins remaining in the clarified juice comprise predominantly Rubisco protein (usually about 50-95% w / w, for example about 60-95%, about 70-95%, or about 75-95%), and also comprise other cytosolic proteins, such as, by way of non-limiting examples, glutamate synthase, transketolase, glycine dehydrogenase, cobalamin-independent methionine synthase, serine hydroxymethyltransferase, and ferredoxin-NADP reductase.

[0162] In a preferred embodiment, the juice is held at elevated temperature, for example in a range of between about 40 °C to about 75 °C, between about 40 °C to about 70 °C, between about 40 °C to about 65 °C, between about 40 °C to about 60 °C, between about 45 °C to about 75 °C, about 45 °C to about 70 °C, between about 45 °C to about 65 °C, or between about 45 °C to about 60 °C, for a period oftime between 15 seconds and 2 hours, in order to coagulate the green protein fraction from the juice. Temperature can be monitored during this step, for example using an in-line temperature sensor, and maintained within a desired temperature range for example using a heat exchanger such as a shell in tube, tube-in-tube, immersed coil, spiral, or plate heat exchanger; direct steam injection; or other known heating methods. After the heating step the juice is cooled to less than about 12°C, for example less than about 11 °C, 10 °C, 9 °C, 8°C, 7°C, 6°C, 5°C, more preferably to less than about 4°C, in order to control microbial growth and enzymatic activity. Cooling temperature can be achieved using a heat exchanger or method such as mentioned above for the heating step. The temperature increase and decrease can be performed over a period of time between 15 seconds and 30 minutes, but is preferably achieved over a time period of about 20 seconds.

[0163] Alternatively, the coagulation can be carried out by flocculation. For example, a chemical or salt coagulant to modify the solution ionic strength such as ammonium sulfate can be added. Alternatively, a chemical or salt coagulant to destabilise the protein ionisation such as calcium chloride can be added at an example concentration of 20 mM which forms flocs in solution that then coagulate together. A pH-mediated flocculation can be used, such as by acid addition to below pH 6.0, or by base addition to above pH 8.0. Alternatively, a chemical flocculating agent such as polylysine, chitosan, polyacrylamides, iron sulfates, or other cationic or anionic polymers may be added.

[0164] The coagulation step occurs at neutral pH, for example from pH 6-8 or pH 6-7.5.Separation of green protein fraction (coagulate) from liquid to produce clarified juice (supernatant)

[0165] Once coagulation has occurred, the green protein fraction (coagulate) can be separated from the liquid supernatant using known methods. The separated green protein material is generally in the form of a green sludge, pellet, or similar (depending on the method used). The liquid is a clarified juice comprising Rubisco proteins and other cytosolic proteins which have not coagulated and remain in solution. The separation can be achieved using a separation apparatus such as a laboratory centrifuge, disc stack centrifuge (i.e. industrial separator), clarifier, decanter, sedicanter, filter press / coarse filter, settling tank, or hydrocyclone. Typically, juice containing the coagulated green protein fraction can be fed to the separation apparatus via a pump as would be known in the art. For example, an industrialseparator (when used) spins coagulated material out into the discharge to form a green protein material as a "pellet" leaving a clarified juice which comprises the Rubisco protein. The clarified juice will be at a neutral pH (about pH6 - about pH 7.5 to 8) that encompasses the natural pH of the Rubisco protein. This provides advantages in processing and simplifies the Rubisco protein extraction process.

[0166] The heating and cooling steps (or flocculation step), followed by the separation step described above, provide a clarified juice from which the green protein fraction has been removed. These methods of providing a clarified juice are described by way of example only and, while preferred, the invention is not limited to methods which use a clarified juice which is provided by carrying out these steps.

[0167] Optionally, membrane filtration or separation may be performed to concentrate the clarified juice. Filtration may be performed by microfiltration, ultrafiltration, diafiltration and / or other filtration methods known in the art, whereby removing water, small molecules, and ash from clarified juice and increasing the concentration of protein. Advantageously, the concentration of the clarified juice will also remove small molecules which may provide buffering capacity thus reducing the amount of acid required in the subsequent precipitation steps, and reducing the volume of the clarified juice, which increases processing efficiency and through-put.Precipitation

[0168] Following separation of the green protein fraction from the juice to produce the clarified juice, the process comprises the step of precipitating the proteins that remain in solution in the clarified juice. This step is preferably an acid precipitation step. It has surprisingly been found that despite the expected adverse effect of acid precipitation on the functional properties of the protein, desirable properties are retained, meaning this simple and effective way of purifying the proteins from the clarified juice as described further herein is a viable and preferred option. In other embodiments, the process for precipitating the protein from the clarified juice can be via further coagulation and flocculation steps using methods, such chemical or salt coagulants, pH mediated flocculation, higher temperatures (> 60°C), or flocculating agents as previously discussed.

[0169] Acid may be added to the clarified juice to precipitate the dissolved protein, including Rubisco protein, from the clarified juice. This produces a solid protein fraction comprising Rubisco protein, which is suspended in a liquid (supernatant). Acid addition can be carried out by any appropriate method known to a skilled worker, such as drop-wise in a small scale or in-line addition.

[0170] In various embodiments, acid is added to bring the pH of the clarified juice to within a range of from about 3 to about 5.5, or about 3 to about 5, or about 3 about 4.5, or about 3.5 to about 5.5, or about 3.5 to about 5, or about 3.5 to about 4.5, or about 4 to about 5.5, or about 4 to about 5, or about 4 to about 4.5, for example a pH of 4.5.

[0171] Any suitable acid can be used for this step. Since the end product is intended for human or animal consumption the acid is preferably food safe. Examples of suitable acids include, without limitation, acetic acid, ascorbic acid, citric acid, lactic acid, propionic acid, succinic acid, sorbic acid, tartaric acid, phosphoric acid, sulfuric acid, or hydrochloric acid, all of which are available as food safe acids. Preferably, the acid is phosphoric acid.Separation of precipitated solid protein material from liquid (supernatant)

[0172] The process then comprises the step of separating the solid protein fraction precipitated in the acid precipitation step from the liquid supernatant.

[0173] Once precipitated, the solid protein fraction can be separated from its supernatant using standard separation techniques which use apparatus such as a centrifuge, disc stack centrifuge (i.e. industrial separator), clarifier, decanter, sedicanter, filter press / coarse filter, settling tank, or hydrocyclone. While it would be possible to use other separation methods such as microfiltration (see Example 15), ultrafiltration, diafiltration, plate and frame membrane separation, dead-end filtration, activated carbon absorption, or column chromatography, these methods are less preferred, since they are more complex or require specialized equipment or reagents, and / or present additional challenges in terms of process rate or scale.

[0174] The separation step produces a solid protein material which may be light green in colour, and a supernatant comprising soluble compounds including sugars, carbohydrates, ash, and some fats. If the separation method is a centrifuge, the solid protein material can for example be described as a pellet. The solid protein material comprises Rubisco protein and cytosolic proteins, but also comprises components such as flavonoids and colour compounds which are undesirable in a consumer product.Lower alcohol extraction steps

[0175] In various embodiments, the process then comprises the following steps to remove the flavonoids and colour compounds from the solid protein material:• contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol,• separating the solid protein fraction from the first solvent or solvent mixture;• contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol, and• separating the solid protein fraction from the second solvent or solvent mixture.

[0176] Once precipitated in the acid precipitation step and separated out of solution, the protein material is typically not white in appearance, and may be pale green or creamy in appearance, due to a residual presence of colour compounds such as chlorophyll, carotenoids, or polyphenol compounds that were not previously removed. Such colour in the protein material is undesirable from the point of view of a consumer food product. Food manufacturers wish to control colour in their products and in some cases colour can be unattractive to consumers. Thus, there is a distinct advantage in providing a protein material which is white in colour, or neutral in colour.

[0177] The protein material can also have an "off" flavour or additional flavour, such as a grassy or "plant" flavour, or a sour, bitter, or astringent flavour. This is due to the retention of small molecules and flavour components within the protein matrix, including volatile compounds (small organic molecules, acids, fatty acids, alcohol, esters, aldehydes, etc.), flavonoids, and glycosylated molecules. Such flavours are undesirable from the point of view of a consumer food product, and thus there is a distinct advantage in providing a protein material which is flavourless.

[0178] The protein material can also have a grassy, sour, bitter, or astringent odour. Compounds contributing to odour are predominantly volatile compounds (small organic molecules, acids, fatty acids, alcohol, esters, aldehydes, etc). This is undesirable from the point of view of a consumer food product, and thus there is a distinct advantage in providing a protein material which is odour free.

[0179] As discussed above, current Rubisco preparation methods are chosen as they are less likely to denature or aggregate the Rubisco protein, but these methods can be complicated in terms of process steps and equipment used, resulting in high production costs and low efficacy.

[0180] The use of acid precipitation is effective in providing leaf protein concentrates but has been found to have an adverse effect on food functionality such emulsion properties (B.P. Lamsal et al., LWT 40 (2007) 1520-1526).

[0181] Use of organic solvents, such as alcohols, ketones, ester, or petroleum ethers during processing of the protein materials has been reported to be effective in decolorizing leaf protein concentrates (Wang, et al. J. Food Sci. 1976, 41 (2), 286; Bray, et al. J. Sci. Food Agric. 1978, 29 (10), 839-846) and removing or modifying the flavour and aroma profiles of plant proteins, but has been reported to denature the proteins and to have an adverse effect on the structure and functional properties of the protein, such that it is generally considered that "extraction with solvents denatures the proteins and further destroys their functionality" (Fiorentini, R.; et al. Qual. Plant. Plant Foods Hum. Nutr. 1983, 32 (3-4), 335-350). In addition, use of organic solvents has also been reported to lead to a reduction in protein solubility by 30-40%, reduced oil holding capacity by 15-20%, and reduced emulsion stability by ~28% in extracted pea proteins (Wang, et al., Food Res. Int. 2020, 132, 109085); or a weakened the gel network and reduced gel strength which influence product development performance (Tan, et al., Food Bioprod. Process. 2023, 141, 73-80).

[0182] Many times, processes and patents directed towards acid precipitation or solvent based washing of leaf protein concentrates are focused on the protein recovery yield for the former, or the removal of volatiles and astringents for the latter, but do not report the resultant functional properties of the prepared protein concentrates, despite the importance of such functionality attributes to favour human consumption.

[0183] The inventors have surprisingly found that the process of the present invention which includes a sequence of extraction steps, each using a lower alcohol solvent, and each followed by a protein separation step (as discussed below), provides a simplified, economically viable, process for producing a protein concentrate from a protein material comprising Rubisco which surprisingly has desirable functional properties (physical and organoleptic) and in particular which has desirable emulsifying capacity.

[0184] Even more surprisingly, despite being subjected to an acid precipitation step, which is preferably used, and multiple lower alcohol extraction steps, both of which would be expected to detrimentally affect the Rubisco protein concentrate and result in the loss of desired functional properties ((B.P. Lamsal et al., LWT 40 (2007) 1520-1526; Yoshikawa et al., Int J Biol Macromol. 2012 Apr 1 ; 50(3) : 865-71), theresulting protein concentrate surprisingly has desirable functional properties (physical and organoleptic) and in particular has desirable emulsifying capacity.

[0185] The protein product produced following the lower alcohol extraction steps will then be washed with water to remove residual lower alcohol solvent and optionally subjected to evaporation to remove excess water and residual lower alcohol solvent (if any) from the product.

[0186] The inventors have surprisingly found that at least two lower alcohol extraction steps, each extraction step followed by separation of the protein fraction from the lower alcohol solvent, are required to remove the undesired flavour and colour compounds from the solid protein material. This is due to the variety of solubility / hydrophobicity attributes in the solid protein material. The first alcohol extraction acts in part as a dewatering step, removing a significant amount, but not all, of the water entrained in the protein fraction. This results in preferential removal of materials with good solubility in a water / alcohol mixture in this first step, such as flavonoid compounds, and an altered hydrophobicity of the solid protein material. The second alcohol extraction performed on the solid protein material is effective to remove more hydrophobic compounds, including colour compounds, and improve the colour of the protein pellet to close to a white protein material. In some embodiments, the first alcohol extraction produces a brown solvent solution, and a light green solid protein material, and the second alcohol extraction produces a green solvent solution and a white solid protein material.

[0187] The lower alcohol solvent(s) used for each extraction step can be the same or different and can be a mixture. In one embodiment, the first solvent or solvent mixture is the same as the second solvent or solvent mixture. In one embodiment, the first solvent or solvent mixture is different to the second solvent or solvent mixture.

[0188] In various embodiments, the first solvent or solvent mixture has a higher water content than the second solvent or solvent mixture.

[0189] In some embodiments, the solvent is "fresh" for each extraction steps, i.e. solvent in not recycled.

[0190] In some embodiments, one or more of the lower alcohol solvent extraction steps is a supercritical fluid extraction step. Supercritical fluid extraction can be carried out using either a single lower alcohol solvent or a mixture of solvents, with or without the additional use of co-solvents. Examples of supercritical fluids includecarbon dioxide which is a highly selective solvent for extraction of non-polar lipophilic compounds, and supercritical dimethyl ether (DIME) with can extract lipophilic compounds with improved water miscibility under process conditions. Lower alcohols can be used as co-solvents to facilitate extraction.

[0191] The extraction steps can each be carried out by combining the solid protein material and the solvent. For example, the solid protein material and the solvent can be brought together in a vessel and optionally mixed, using any suitable mixing apparatus, for example a high shear mixer or a blender. The ratio for combining the solid protein material and the solvent can suitably be 100: 1 - 1: 10 solvent: protein (wt / wt), for example 10: 1 - 1 : 10 solvent: protein (wt / wt). For example, the ratio can be 3:2 solvent: protein (wt / wt). The solvent solution can then be removed from the solid protein fraction using a suitable separation technique as known to the person skilled in the art, or as described above in relation to the separation of the green protein fraction from liquid.

[0192] Each solvent extraction step can be a batch extraction step or a continuous extraction step, as will be understood by a person skilled in the art. A batch extraction process is a process with a discrete number of extraction steps, each followed by a separation step. In some embodiments, the extraction process can be a batch-mode countercurrent extraction process. A batch-mode countercurrent extraction process is described below and in Figure 2.

[0193] In some embodiments, one or more of the lower alcohol solvent extraction steps is a continuous countercurrent extraction step. A continuous countercurrent extraction step can be carried out using a continuous horizontal helix extractor or a similar apparatus. In some embodiments, two or more of the extraction steps is a continuous countercurrent extraction step, and a separation step is carried out between the first continuous countercurrent extraction step and the second continuous countercurrent extraction step.

[0194] In other embodiments, the solvent solution obtained from the second extraction step is recycled for use in a first extraction step of a subsequent batch of solid protein material, in a manner analogous to a countercurrent extraction. Surprisingly, while the inventors have found that at least two lower alcohol extraction steps are required to remove the flavonoids and other compounds, the re-use of the solvent in this way does not significantly affect the efficiency of the lower alcohol extractions. In a continuous process, the use of multiple steps, phases, effects, or loops, is considered to be two or more extraction steps described above.

[0195] Following the lower alcohol extraction steps, the solid protein fraction is preferably washed with water to remove the alcohol from the protein fraction. This will also remove any residual undesirable components that have been extracted into that alcohol. Water washing can be completed by any method known to a skilled person and should be continued or repeated until the alcohol content of the protein fraction is at acceptable levels. Optionally, the water washed protein fraction can be further concentrated by removing excess water from the fraction. This can be done by any method known to a skilled person such as centrifugation, filtration, partial evaporation, or pressing, or the like.

[0196] In particular, the inventors have surprisingly found that the water content of the solid protein fraction precipitated in the acid precipitation step can be maintained at no lower than about 50% w / w of the separated protein fraction, and that maintaining this level of water content in the protein fraction through-out rest of the process, including the removal of excess water following the water washing step, is important for achieving the desired emulsifying capacity of the protein concentrates. As will be appreciated by a skilled worker, addition of a non-water based solvent to the separated solid protein fraction will cause a decrease in the relative proportion of water content in the solid protein fraction and as such the proportionate water content may be less than 50% during the lower alcohol washing steps (see Example 1). The solid protein fraction is returned to having a water content of at least 50% during the water wash step to remove residual solvent from the solid protein fraction. Therefore, the water content in the protein fraction following the acid precipitation step and the water content in the protein fraction after the water washing step to remove residual alcohol is maintained at no lower than about 50% w / w. The inventors have found that that the water content of the solid protein fraction precipitated in the acid precipitation step and in the solid protein fraction following the water washing step is surprisingly high (as demonstrated in Example 1-5), and typically may be significantly higher than 50%, for example excess of 70%, in excess of 75%, or even in excess of 80% (w / w) water. When it is desired to provide a Rubisco protein concentrate with advantageous functional properties such as a desired emulsion activity, it is important that the water content in the protein fraction is maintained at no lower than 50% (w / w) throughout the process as discussed above, including the step of removal of excess water following the water washing step.

[0197] It is hypothesised that the Rubisco protein structure retains water that is effectively bound in the structure, and it is the retention of this bound water that allows the Rubisco protein concentrates of the invention to achieve the levels of emulsifying capacity shown by concentrates of the invention and that make theproducts desirable for use in food production. Water that is removed from the protein fraction through the process is thought to be free water outside the protein structure. Therefore, when it is desired to obtain favourable emulsion properties in the Rubisco protein concentrate, the protein fraction should not be dried at any stage in the process to a point at which the water content of the protein fraction is lower than about 50% w / w of the protein fraction (except as may occur during the lower alcohol (preferably ethanol) wash step as explained above). In various embodiments, the Rubisco protein concentrate comprises water of which the majority is associated within the protein.[0019S] The water content of the protein fraction can be measured according to known methods and, from a practical perspective, would likely be measured in the final product produced. Measurement could, of course, be done at any or every suitable point in the process where the protein fraction is available for measurement, but this may not be practical in commercial production. Once the process parameters have been set to achieve the desired product, it should not be necessary to measure throughout the process. Should the water content of the protein fraction in the final protein containing product not be adequate, the process would be reviewed to determine where the issue arose. It is also possible that the water content of the protein in the feedstock for the process (the biomass comprising Rubisco protein) may need to be measured as, if this is below required water levels, it may not be suitable for use in the process. Such matters involving water content measurement would be well within the knowledge of a person skilled in this art.

[0199] Known methods for determining the water content of the protein fraction would include: loss on drying (LOD), thermogravimetric analysis (TGA), gas chromatography using a thermal conductivity detector and the Karl Fischer titration, and near-infrared spectroscopy. This list is not intended to be limiting and any suitable test could be used.

[0200] Figure 1 shows an embodiment of the invention, with optional steps in hashed lines. As shown in Figure 1, in one embodiment of the invention, biomass material comprising Rubisco is harvested and juiced and then undergoes a heat and cooling step to separate out a green protein fraction. The remaining solution (clarified juice) undergoes an acid precipitation step and is separated into supernatant and a solid protein fraction. The solid protein fraction is extracted with a lower alcohol twice, followed by an optional water wash. The alcohol supernatant can optionally be reused in the washing steps. The refined solid protein concentrate can then be further processed such as by evaporating to provide a protein concentrate.

[0201] Figure 2 shows a schematic for a batch-mode countercurrent extraction of a protein material (100, 101, 102, 103, 104, 105) using a lower alcohol (200, 201, 202, 203, 204, 205) for five extraction steps (n = 1), as well as a corresponding schematic for a number of extraction steps (n + 3). The batch-mode countercurrent extraction method is advantageous in minimising the required quantities of ethanol to remove colour and flavour components from the initial crude protein precipitated material.

[0202] Referring to the schematic of Figure 2 where n = 1, a higher percentage (at least 60%, preferably at least 95%) lower alcohol (200) is used to perform an extraction on a protein material 105 which has already been subjected to multiple extraction steps using a lower-percentage lower alcohol (201, 202, 203, 204). Thus, the protein material 100 is initially subjected to a first extraction step using the lower- percentage alcohol 204, which as a result of this extraction is decreased in percentage, resulting in lower alcohol 205, and protein material 101. Lower alcohol 205 can optionally be distilled or otherwise purified or recycled to recover a higher- percentage lower alcohol. Protein material 101 is subjected to a second extraction step using the lower-percentage alcohol 203, resulting in lower alcohol 204, and protein material 102. Protein material 102 is subjected to a third extraction step using the lower-percentage alcohol 202, resulting in lower alcohol 203, and protein material 103. Protein material 103 is subjected to a fourth extraction step using the lower- percentage alcohol 201, resulting in lower alcohol 202, and protein material 104. Protein material 103 is subjected to a final extraction step using the highest- percentage alcohol 201 as discussed above, resulting in lower alcohol 201, and protein material 105.

[0203] Referring to the schematic of Figure 2 where n is not specified, the process can be continued iteratively starting with extraction of crude protein material 100 using a final fractional lower alcohol percentage (200 + 3 + n), and ending with extraction of the purified protein material (100 + (3+n) using highest percentage lower alcohol 200.

[0204] The lower alcohol 200 can optionally be 100% purity ethanol, or an otherwise high-percentage ethanol (at least 60% purity). The purified protein material (105) or (100 + (4+n))) is typically a clean white protein material.

[0205] Figure 3 shows a process design that could be used in the process of the invention. Acid is added to clarified juice from biomass material comprising Rubisco in tanks. Once precipitated, the supernatant is separated from the solid protein fraction, which is transferred to another tank for an ethanol extraction (ethanol stripping). Inthe embodiment shown, the protein fraction undergoes three successive ethanol extractions. The used ethanol is sent for recovery via ion exchange and then can be reused. The protein fraction has residual ethanol removed via three successive water washes. The resulting protein concentrate is then sent for further processing. A final dewatering step can be accomplished by for example, filtration, partial evaporation, or pressing.

[0206] In some embodiments the spent lower alcohol can contain polyphenols, flavonoids, secondary metabolites and colour compounds, which may be recovered (e.g. by using a chromatography resin) for secondary purposes such as a dietary supplementation product.

[0207] In general, while the invention uses a plurality of lower alcohol extraction steps, being two or more, for example two, three, four, five, six, seven, or eight lower alcohol extraction steps, the person skilled in the art will appreciate a third or subsequent lower alcohol extraction step is optional and may be carried out in some circumstances, if needed. For example, if earlier processing steps have been completed outside an optimum temperature range or processing time, and the separation step has been less effective at removing colour compounds, third or subsequent lower alcohol extraction steps can be employed to decrease the amount of colour compounds present. In some instances, a third, fourth, fifth, sixth, seventh or eighth extraction step may provide only a marginal benefit over two extraction steps, with the marginal benefit decreasing for each additional extraction step.

[0208] In various embodiments, the extraction steps are repeated until the alcohol extraction liquid fraction has an absorbance at 664nm of less than 0.05 Absorbance units in a 1 cm pathlength. Absorbance is measured in a UV-vis spectrophotometer as known to a skilled worker.

[0209] UV / visible absorption is measured across the range of 250 - 700 nm. A peak at 664nm is characteristic of chlorophyll, chlorophyll by-products and other coloured compounds.

[0210] UV-vis spectrophotometric absorbance was recorded on a SpectraMax iD5 Multi-Mode microplate reader in 96 well black / clear bottom polystyrene plates in absorbance spectrum mode with wavelength scanning from 230 nm to 700 nm. Dilution of samples with water was performed as necessary to bring the maximum absorption to less than 10 AU. Chlorophyll content was quantified at 664 nm. Alternative spectroscopic methods, such as fluorescence excitation / emission can also be used to quantify chlorophyll, flavone, and polyphenol content.

[0211] It was observed that the first ethanol wash step was less effective in extracting coloured compounds than subsequent ethanol wash steps reflecting the need of the first ethanol addition volume to adjust the hydrophobicity of the extraction solvent (by diluting the water entrained within the protein concentrate) to obtain an optimal ethanol:water ratio for extraction of the hydrophobic compounds such as chlorophyll and other coloured compounds Table A.Table A

[0212] The efficiency of colour extraction could be changed through changing the concentration (%) of solvent: water in the extraction solvent, changing the volume of washing solvent composition: protein concentrate used (w / w or v / w), changing the number of solvent washing steps, using counter current extraction or a combination thereof. The efficiency of extraction and decolourisation could be quantified for example by UV-vis spectrophotometric measurement of the extraction solvent or via colour measurement of the protein pellet.

[0213] The colour of the protein pellet can be quantified and defined using the CIELAB colour space values for perceived colour. In various embodiments, the extraction steps are repeated until the protein concentrate has CIE colour values of a* > 0; b* > 10, and L* > 50. In various embodiments, the extraction steps are repeated until the protein concentrate has CIE colour values of a* > -1; b* > 10, and L* > 50.

[0214] Samples can be measured using known apparatus such as a Colorimeter (ColorFlex EZ 45 / 0, Hunter Associates Laboratory, Inc., Reston, VA, USA) or Buchi Proximate NIR (BUCHI Labortechnik AG) and expressed as L* (lightness), a* (red - green), and b* (blue - yellow). As compared with the untreated protein product (which had undergone acid precipitation only, the colour shifts (AE* ) of the treated (protein fraction that has undergone acid precipitation, ethanol extraction and water wash) concentrate resulting from lower alcohol extraction can be calculated as adistance differences among L* , a* , and b* within a colour space, and calculated using coordinate geometry where Lo *, ao *, and bo* is the colour of the protein sample which had undergone acid precipitation only, and L* , a* , and b* is the colour of samples which had undergone acid precipitation, ethanol extraction and water wash steps. A bigger AE* value represents a larger colour difference between the ethanol extraction and water washed protein and the acid precipitated but unwashed protein which had undergone acid precipitation only.

[0215] The protein pellet generated via sequential solvent then water washing was measured for CIELAB values as shown in Table B. Solvent extraction which removes chlorophyll reduces the a* (negative / green) contribution from on average a* = -4.38 in the acid coagulated protein concentrate to a* = -0.34 in the solvent washed protein concentrate.Table B: CIELAB colour measurement of solvent washed protein concentrate resuspended 1:1 in water (L*a*b*)

[0216] For protein pellet the CIELAB a* value, representing the green - magenta spectrum should have a* value > 0; b* representing the blue-yellow spectrum should have a value > 10, and L* representing the lightness value should be > 50.

[0217] The inventors have found that two lower alcohol extraction steps is usually sufficient to produce a Rubisco protein concentrate having the desired purity and functional properties, as shown below in the Examples. Further extraction steps can be run depending on the desired purity.

[0218] In various embodiments, the process comprises the step of at least two extraction cycles wherein the solid protein fraction is extracted with a first and second solvent or solvent mixture comprising a lower alcohol. An extraction cycle comprises contacting the solid protein fraction with a solvent or solvent mixture followed by separating the solid protein fraction from the solvent or solvent mixture. The solvent or solvent mixture comprises a lower alcohol. The solvent or solvent mixture for each extraction step can be the same or different. Optionally, a water solids wash may be performed within the sequence to vary the order of the extraction cycles.

[0219] The invention provides a process for producing a protein concentrate from a biomass material comprising Rubisco protein, the process comprising steps of: a. providing a clarified juice from a biomass material comprising Rubisco protein; b. precipitating protein from the clarified juice to produce a liquid and a solid protein fraction, c. separating the liquid from the solid protein fraction; d. performing at least two extraction cycles wherein the solid protein fraction is subjected to a solvent extraction with a solvent or solvent mixture comprising a lower alcohol until the alcohol extraction solvent has an absorbance at 664nm of less than 0.05 Absorbance units in a 1 cm path length; e. separating the solid protein fraction; and f. washing the solid protein fraction with water to produce a protein concentrate.

[0220] The invention provides a process for producing a protein concentrate from a biomass material comprising Rubisco protein, the process comprising steps of: a. providing a clarified juice from a biomass material comprising Rubisco protein; b. precipitating protein from the clarified juice to produce a liquid and a solid protein fraction, c. separating the liquid from the solid protein fraction;d. performing at least two extraction cycles wherein the solid protein fraction is subjected to a solvent extraction with a solvent or solvent mixture comprising a lower alcohol until the solid protein fraction has CIE colour values of a* > 0; b* > 10, and L* > 50; e. separating the solid protein fraction; and f. washing the solid protein fraction with water to produce a protein concentrate.

[0221] The invention provides a process for producing a protein concentrate from a biomass material comprising Rubisco protein, the process comprising steps of: a. providing a clarified juice from a biomass material comprising Rubisco protein; b. precipitating protein from the clarified juice to produce a liquid and a solid protein fraction, c. separating the liquid from the solid protein fraction; d. performing at least two extraction cycles wherein the solid protein fraction is subjected to a solvent extraction with a solvent or solvent mixture comprising a lower alcohol until the solid protein fraction has CIE colour values of a* > -1; b* > 10, and L* > 50; e. separating the solid protein fraction; and f. washing the solid protein fraction with water to produce a protein concentrate.Water washing step

[0222] In preferred embodiments, the process further comprises a step of washing the solid protein fraction from the lower alcohol extraction steps with water.

[0223] The water washing step is used to remove most of the lower alcohol solvent remaining in the solid protein material. This can help to decrease the alcohol content to a level suitable for human consumption and maintains the water content of the solid protein fraction to no less than 50% w / w as discussed above. A water washing step can be achieved, for example, by bringing the solid protein material andthe water together in a vessel and optionally mixing, using any suitable mixing apparatus, for example a high shear mixer or a blender. The water can be removed from the protein fraction using a suitable separation technique as described herein, or a method as described below.

[0224] It is advantageous to use water at this step rather than other solvents or solutions as water is pH neutral and does not leave residues that are not food safe.

[0225] Preferably, the water washing step is carried out at a temperature of less than about 50 °C, more preferably the water is cooled to between about 8 to about 16 °C. Optionally, the water washing step can be repeated.

[0226] In some embodiments, the process of the invention comprises at least two lower alcohol extraction steps and at least two water washing steps. In some embodiments, the lower alcohol extraction steps and at least two water washing steps can be interspersed or alternated, if desired. For example, where "E" represents an alcohol extraction step and "W" represents a water washing step, in some preferred embodiments the process of the invention comprises steps in sequence of E, E, W, W or W, E, E, W, W. In some other embodiments the process of the invention may comprise steps in sequence of; W, E, E, W, W, W; E, W, E, W; W, W, E, E; or other combinations of these steps.

[0227] In some embodiments, the water washing step can be a continuous countercurrent process.

[0228] A small amount of water and a small amount of the lower alcohol solvent is expected to remain in the washed protein material, which is described below as a protein concentrate. In preferred embodiments, the residual amount of lower alcohol solvent remaining in the washed pellet of protein material is in the range of from about 0.1% to about 1.5%. More preferably, the residual amount of lower alcohol solvent remaining in the washed pellet of protein material is less than about 1%, for example less than about 0.9%, or less than about 0.8%, or less than about 0.7%, or less than about 0.6%, or less than about 0.5%. In various embodiments, the lower alcohol is ethanol. Where it is desired to achieve the preferred emulsion activity for the protein concentrate, the amount of water in the washed protein material should be no lower than 50% w / w as has been discussed previously.

[0229] The washed solid protein product and / or Rubisco protein concentrate can be separated by any means known to a skilled worker, such as standard separation techniques which use apparatus such as a centrifuge, disc stack centrifuge(i.e. industrial separator), clarifier, decanter, sedicanter, filter press / coarse filter, settling tank, hydrocyclone, microfiltration, ultrafiltration, diafiltration, plate and frame membrane separation, dead-end filtration, activated carbon absorption, or column chromatography.Concentration

[0230] In various embodiments, the process further comprises a step of removing a portion of the water from the protein concentrate.

[0231] In various embodiments, removing a portion of the water from the protein concentrate comprises subjecting the protein concentrate to a vacuum.

[0232] In various embodiments, water and residual ethanol is removed using a vacuum scraped-surface evaporator. The evaporator subjects the protein material to a vacuum while mixing. The constant application of shear to the protein material assists with even removal of moisture, to avoid uneven drying and clumping of the protein material. This method is suitable for preparing a protein material with an alcohol content of less than about 0.5%. In other embodiments, the water and residual ethanol is removed using a rotary evaporator, a static simple manual agitation evaporator, a Thermomix evaporator, spinning-cone column, plate evaporator, falling film evaporator, or a Centritherm Evaporator (Flavourtech).

[0233] Other suitable methods for further concentration the protein concentrate will be apparent to a skilled worker, such as partial freeze-drying.

[0234] At any suitable point in the process, the Rubisco protein-containing material, such as the clarified juice, the solid protein fractions, the protein product, or the protein concentrate at any point in the wash cycles, may be stored for a period of time and / or transported to other facilities before continuing the process.Protein concentrate

[0235] Described herein is a process for producing a protein concentrate from a juice of a biomass material comprising Rubisco protein, the protein concentrate having a water content of at least about 50% (w / w), the process comprising steps of a. Providing a juice from a biomass material comprising Rubisco protein; b. Preparing a solid protein fraction from the juice; andc. Reducing the water content of the solid protein fraction to produce a protein concentrate.

[0236] In various embodiments, step (a) comprises steps of: i. subjecting juice from a biomass material comprising Rubisco protein to a mild heat treatment to coagulate a green protein fraction; and ii. separating the juice from the coagulated green protein fraction to provide a clarified juice.

[0237] In various embodiments, step (b), following step (a) ii, comprises steps of: i. subjecting the clarified juice to an acid precipitation step to produce a liquid and a solid protein material; ii. separating the liquid from the solid protein material.

[0238] In various embodiments, step (b) further comprises steps of:Hi. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol; and iv. separating the solid protein fraction from the first solvent or solvent mixture.

[0239] In various embodiments, step (b) further comprises a step of:Hi. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol; and iv. separating the solid protein fraction from the second solvent or solvent mixture.

[0240] In various embodiments, the lower alcohol is ethanol.

[0241] In various embodiments, step (b) further comprises washing the solid protein fraction following contact with the first and / or second solvent or solvent mixture, with water.

[0242] Drying steps, which remove substantially all of the water from the protein, may alter one or more of the secondary, ternary or quaternary proteinstructures. In preferred embodiments of the invention, the protein concentrate has not been subjected to a drying step during its preparation that removes more than 50% of the water in the protein fraction, as has been discussed previously. However, in some embodiments, the protein concentrate that has been prepared using the simplified process described previously can be dried sufficiently to provide a powder with good colour and flavour properties that replicate those achieved in the protein concentrate, but that are otherwise difficult to achieve and which are desirable to consumers. Such a powder also forms an aspect of the present invention. Powder formation can be achieved using any process known in the art such as spray drying, freeze drying, evaporation, or the like.

[0243] Therefore, in an aspect, the invention provides protein powder comprising Rubisco protein in an amount between 70% - 90%, having one or more of the following features:• less than about 0.5% total saponins; and / or• less than about 0.5% flavones and polyphenols compounds; and / or• CIE colour values of a* > 0; b* > 10, and L* > 50.

[0244] In another aspect, the invention provides protein powder comprising Rubisco protein in an amount between 70% - 90%, having one or more of the following features:• less than about 0.5% total saponins; and / or• less than about 0.5% flavones and polyphenols compounds; and / or• CIE colour values of a* > -1; b* > 10, and L* > 50.

[0245] A proximate composition for the Rubisco protein concentrate is provided in Table C, wherein total solids (TS), Crude Protein (CP), Non-protein nitrogen (NPN), Protein (CP minus NPN), Ash, Carbohydrates and Fat are measured using standard analytical methods, as known by those skilled in the art.Table C: Proximate composition for Rubisco protein concentrate

[0246] In various embodiments, the protein concentrate has an ethanol concentration of less than about 2% w / w, for example less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, or less than about 0.8%, or less than about 0.7%, or less than about 0.6%, or less than about 0.5%. Ethanol concentration can be measured by methods known to a skilled worker, such as GC-FID.

[0247] In one such method, quantitation of ethanol in protein concentrates or in supernatant solution samples is performed via gas chromatography with flame ionisation detection (GC-FID). In general, solution phase samples are vigorously agitated before sampling, then solution is sampled via pipette (40 pL) and diluted with internal standard (ISTD) (1000 pL, 6% acetonitrile in water). Cloudy solutions are vigorous agitated before sampling via pipette, weighed and the diluted with internal standard (ISTD) (1000 pL, 6% acetonitrile in water). Solid samples are weighed in triplicated (50-100 mg), diluted into 2 mL of ISTD, and vigorously agitated. Prepared samples are centrifuged (10000 RCF, 5 mins) and the supernatant analysed. GC-FID is performed on an Agilent 8890 system fitted with an Agilent HP5 column (30m x 0.32mm 0.25micron coating). Oven temperature of 35 °C (4 min) ramping to 200 °C at 50 / min, held 1 min then equilibrate for 1 min at 35 °C. Inlet 200 °C, detector 280 °C, air flow 400mL / min, H2 fuel 30mL / min, makeup N2 25 mL / min, column flow (H2 is carrier gas) lml / min (column + makeup = constant). Injection volume 0.1 mL with a split liner installed and a split ratio of 100: 1. Measurements are calibrated (ISTD) against a solution of ethanol (5.00 mL) and acetonitrile (5.00 mL) with 200 mL of water. Analysis is performed in duplicate as independent measurements, with reported standard deviation.

[0248] In various embodiments, the protein concentrate has an ethanol concentration of from about 2.0% to about 0.1% w / w, and any values between these ranges can be selected, for example from about 1.8% to about 0.1% w / w, or about 1.6% to about 0.1%, or about 1.5% to about 0.1%, or about 2.0% to about 0.2% w / w, or about 1.8% to about 0.2% w / w, or about 1.6% to about 0.2%, or about 1.5% to about 0.2%, or about 0.5% to about 0.1% w / w.

[0249] The Rubisco protein concentrate prepared according to the invention has advantageous organoleptic properties, in particular a neutral colour as defined by a pale CIELAB colour space characterisation and a low absorbance spectra of residual extractables colour; a favourable, non-bitter, non-grassy flavour as defined by lowextractable content of polyphenol, glycoside, residual small molecules when measured by LC-MS, and a favourable aroma as defined by low content of extractable volatile compounds as measured by GC-0 or GC-MS.

[0250] The Rubisco protein concentrate described herein has reduced total quantity of flavone, polyphenol, secondary metabolites and lipids as determined by LC-MS methods.

[0251] In one such method, 100 mg of a sample is weighed into a microfuge tube and combined with 800 uL of an extraction solution. The tubes are sonicated for 30 minutes at 40 °C to extract soluble materials, then centrifuged at 10,000 RCF for 5 minutes to sediment the debris. The supernatant is taken and diluted five-fold with water to make the extracts amenable to LCMS, then centrifuged again to sediment any precipitated material. An aliquot of the supernatant is transferred to a glass HPLC vial for analysis. Chromatographic separation is performed with a Thermo Fisher Ultimate 3000RS UPLC system coupled with a diode array detector and an Exploris 480 HRMS. 1-10 uL of extract is injected onto a Waters BEH C18 column (100mm x 2.1 mm, 1.8 um) and separated over a 30 minute gradient with 0.1% formic acid in water and neat acetonitrile mobile phases. The eluent is detected in both positive and negative polarities to detect a wide range of ions, over the m / z range 80-1200. Concentration information on specific analytes is made by reference to a calibration curve of specific representative polyphenolic or saponin compounds in a dilution series from 0.1 - 10 ppm. Representative [M+H] + or [M-H]- ions are used for quantitation, performed using Xcalibur Quan Broswer. To obtain untargeted information and library matches, the same data is processed using Thermo Compound Discoverer software. Extractable compounds include a range of biologically derived alcohols, aldehydes, ketones, furans, alkanes and alkenes acids, hydrocarbons, ketones, sulfur compounds, terpenes, esters, polyunsaturated lipids, amino lipids, sphingolipids, glycosides and amino acids e.g. apigenin, epicatechin, ferulic acid, coumaric acid, catechin, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, quercetin, kaempferol, isoflavones, coumestrol, liquiritigenin, medicagenic acid, bayogenin, saponins, and soyasapogenols.

[0252] In another method, 100 mg of a sample is weighed into a microfuge tube and combined with 900 uL of an extraction solution (90% EtOH + 0.01% HCI). The tubes are sonicated for 60 minutes at 25 °C to extract soluble materials, then centrifuged at 10,000 RCF for 2 minutes to sediment the debris. The supernatant is taken and diluted two-fold with water to make the extracts amenable to LCMS, then centrifuged again to sediment any precipitated material. An aliquot of the supernatantis transferred to a glass HPLC vial for analysis. Chromatographic separation is performed with a Thermo Fisher Ultimate 3000RS UPLC system coupled with a diode array detector and an Exploris 480 HRMS. 1-10 pL of extract is injected onto a Waters BEH C18 column (100mm x 2.1 mm, 1.8 pm) and separated over a 30 minute gradient with using aqueous ammonium formate and methanol mobile phases. The eluent is detected in both positive and negative polarities to detect a wide range of ions, over the m / z range 80-1200 m / z. Concentration information on specific analytes is made by reference to a calibration curve of specific representative polyphenolic or saponin compounds purchased from commercial suppliers in a dilution series from 5 ppb to 1 ppm. Representative [M+H] + or [M-H]- ions are used for quantitation, performed using Xcalibur Quan Browser. To obtain untargeted information and library matches, the same data is processed using Thermo Compound Discoverer software. Extractable compounds include a range of biologically derived alcohols, aldehydes, ketones, furans, alkanes and alkenes acids, hydrocarbons, ketones, sulfur compounds, terpenes, esters, polyunsaturated lipids, amino lipids, sphingolipids, glycosides and amino acids e.g. apigenin, epicatechin, ferulic acid, coumaric acid, catechin, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, quercetin, kaempferol, isoflavones, coumestrol, liquiritigenin, medicagenic acid, bayogenin, saponins, and soyasapogenols.

[0253] Described herein, according to an aspect of the invention, is a Rubisco protein concentrate comprising less than about 0.5 % w / w total saponins, such as less than about 0.4%, 0.3%, 0.2%, 0.1%, or 0.05% total saponins. In various embodiment, the Rubisco protein concentrate from about 0.5% to about 0% total saponins, such as from about 0.4% to about 0%, or about 0.3% to about 0%, or about 0.2% to about 0%, or about 0.1% to about 0%, or about 0.05% to about 0%, or about 0.5% to about 0.01%, or 0.4% to about 0.01%, or about 0.3% to about 0.01%, or about 0.2% to about 0.01%, or about 0.1% to about 0.01% total saponins.

[0254] Using the above LC-HRMS method, saponins were identified and quantified in the juice, clarified juice, precipitated solid protein material and Rubisco protein concentrate following solvent washing. The amounts of saponins quantified in each process step sample was dependent on concentration or dilution steps, such that the apparent increase in saponins for the concentrated clarified juice is due to concentration of volume when compared to the precursor juice. Solvent washing, in preference ethanol washing, was highly effective in removing saponins from the Rubisco protein concentrate to provide a lower saponin content.

[0255] The principal saponins were characterised as Azukisaponin II (diglycoside of soyasapogenol B, >90% saponin content), soyasapogenol B, soyasapogenol E, soyasapogenol C, and Azukisaponin III (collectively 5-8%) which are reported to be non-toxic, non-hemolytic saponin, with minor quantities of medicagenic acid, zanhic acid, and gypsogenic acid characterised at less than 1.0% collective content. The sum of saponins was quantified as about 19.3 mg / kg (see Table D).Table D: Quantitation of saponins during purification

[0256] Described herein, according to an aspect of the invention, is a Rubisco protein concentrate comprising less than about 0.5% w / w flavones and polyphenol compounds such as less than about 0.4%, 0.3%, 0.2%, 0.1%, or 0.05% flavones and polyphenol compounds. In various embodiment, the Rubisco protein concentrate from about 0.5% to about 0% flavones and polyphenol compounds, such as from about 0.4% to about 0%, or about 0.3% to about 0%, or about 0.2% to about 0%, or about 0.1% to about 0%, or about 0.05% to about 0%, or about 0.5% to about 0.01%, or 0.4% to about 0.01%, or about 0.3% to about 0.01%, or about 0.2% to about 0.01%, or about 0.1% to about 0.01% flavones and polyphenol compounds.

[0257] Using the above LC-HRMS method, flavones and polyphenol were identified and quantified in the juice, clarified juice, precipitated solid protein material, combined ethanol wash, combined water washes, and Rubisco protein concentrate. The amounts of flavones and polyphenols quantified in each process step sample was dependent on concentration or dilution steps, such that the apparent increase in flavones and polyphenols for the concentrated clarified juice is due to concentration of volume when compared to the precursor juice. Solvent washing, in preference ethanol washing, was highly effective in removing flavone and polyphenols from the Rubisco protein concentrate to provide a lower total flavone and polyphenol compound content.

[0258] The principal flavone and polyphenols characterised were apigenin, ferulic acid, coumaric acid, and hydroxybenzoic acid, with other individual minor components at less than 1% of the flavone and polyphenol composition. The sum of the flavones and polyphenols in the precipitated solvent washed solid protein material was 548 mg / kg (0.05%), Table E.Table E: Quantitation of flavones and polyphenols during purification

[0259] Saponin and flavones and polyphenols content can be measured by methods known to a skilled worker, such as LC-HRMS.

[0260] The extracted protein pellet has a lower organic odour, the volatile compounds of which can be quantified using headspace mass spectrometry

[0261] Headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) is used to determine differences between volatile compounds in treated (protein fraction that has undergone ethanol extraction and water wash) and untreated (acid precipitation only) samples, i.e. in the juice, clarified juice, precipitated solid protein material and Rubisco protein concentrate following solvent washing. In one such method, samples are placed in 20 mL preconditioned headspace vials. GC / MS analysis is carried out using an Agilent 6890N GC (Shanghai, China) equipped with an Agilent 5975B, VL MS triple axis detector (Palo Alto, CA, USA). Volatile compounds are extracted using a headspace solid phase microextraction (SPME) method. The fibre used is a mixed phase consisting of divinylbenzene / carboxen™ on polydimethylsiloxane (DVB / CAR / PDMS; 1cm, 40pm; Supelco). All samples are subjected to 5 min incubation (equilibration) and 60 min or 30min extraction onto the SPME fibre at 40°C. The fibre is desorbed directly in the injection port of the GC / MS at 270 °C for 5 min; 2 min in splitless mode followed by 3 min with a purge flow of 60mL / min. Helium is the carrier gas at a flow rate of 1.2 mL / min. Methyl isobutyl ketone (1.0 nL / g) is used as an internal standard (IS) insamples. Following desorption, the fibre is conditioned for 2 min at 270°C. Separation is achieved using a ZB-WAX capillary column (Phenomenex, USA) of 60 m x 0.32mm I.D. with a film thickness of 0.5 pm. Helium is the carrier gas at a flow rate of 1.2 mL / min. The oven temperature is initially 40 °C for 4 min, then increased by 5 °C / min to 210 °C, then from 210 to 240°C at 10°C / min and finally held at 240°C for 5 min. The MS parameters are a transfer line temperature of 200 °C; quadrupole temperature at 150 °C with emission current of 35 pA; and an ion source at 230 °C. The run time is recorded in full scan mode (m / z 29-300 mass range). Compounds are identified based on comparison of their mass spectra to the NIST 2014 database supported by comparing retention indices (RI) to reference values, where available, or the Smart Aroma Database (Shimadzu Corporation). Samples are analysed in triplicate with the average relative abundance of each compound reported utilising a deconvoluted peak area (total ion count, TIC) after subtraction of background signals. Relative concentrations of identified compounds are calculated using the peak area of the volatile compound and the peak area of the internal standard (IS), reported as n Mg of sample.

[0262] Volatile compounds that are undesirable in the protein concentrate include, but are not limited to, 2-methyl-butanal, 3-methyl-butanal, 2-methyl-2- pentenal, 2-ethyl-furan, hexanal, D-limonene, (cis)-2-hexenal, (trans)-2-hexenal, 1- octen-3-one, 1-hexanol, (trans)-3-hexen-l-ol, 3-octanol, trans-2-hexen-l-ol, 1-octen- 3-ol, acetic acid, furfural, linalool, benzaldehyde, dimethyl sulfoxide, (trans,trans)-2,6- nonadienal, 4-hydroxy-butanoic acid, 1-nonanol, benzeneacetaldehyde, furanone compounds, 2,5-dimethyl-benzaldehyde, benzyl alcohol, eugenol, 2,4-di-tert- butylphenol, phenylethyl alcohol, 3-octanone, propanal, 3,5,5-trimethyl-2-hexene, 2- ethyl-l-hexanol, methoxy-phenyl-oxime, l-nonen-4-ol, and hexanoic acid.

[0263] Using the HS-SPME-GC-MS method, volatile compounds were quantified during the process (Table F). The process of the invention wherein two ethanol washes of 95-100% ethanol and two subsequent washes of water with final evaporation is used is effective in removing 91.4% of the odour containing volatile molecules to provide a Rubisco protein concentrate with lower organic odour and a low content of volatile compounds.Table F: Quantitation of effectiveness of volatile compound removal during processing as assessed by HS-SPME-GC-MS

[0264] Different concentrations for ethanol (45%, 55%, 65%, 75%, and 95%) were used to extraction volatile compounds from the acid precipitate, wherein two ethanol extractions at the indicated ethanol % concentration were performed, followed by two water washes with quantification of the residual volatile by HS-SPME-GC-MS. Greater than 95% removal of volatile organic compounds was achieved with solvent washing (Table G).Table G: Volatile compound removal via solvent washing as assessed by HS- SPME-GC-MS

[0265] The Rubisco protein concentrate prepared according to the invention has advantageous functional properties and is particularly suitable for preparing a stable emulsion.

[0266] In some embodiments, the protein concentrate is frozen. Optionally, the protein concentrate can be in the form of a frozen block. In other embodiments, the protein concentrate may be stabilized or provided as a suspension which can be stabilized with respect to enzyme activity or microbial growth, for example by providing at reduced temperature, or a pH which discourages enzyme activity or microbial growth. In various embodiments, the Rubisco protein concentrate is a stable suspension.

[0267] Described herein, according to an aspect of the invention, is a Rubisco protein concentrate having a water content of at least about 50% w / w, or at least about 55%, 60%, 65%, 70%, 75%, 80% w / w water. In various embodiments, the protein concentrate has a water content in the range of about 50 to about 95% w / w, for example from about 50 to about 90%, or about 50 to about 85%, or about 50 to about 80%, or about 55 to about 95%, or about 55 to about 90%, or about 55 to about 85%, or about 55 to about 80%, or about 60 to about 95%, or about 60 to about 90%, or about 60 to about 85%, or about 60 to about 80%, or about 65 to about 95%, or about 65 to about 90%, or about 65 to about 85%, or about 65 to about 80%, or about 70 to about 95%, or about 70 to about 90%, or about 70 to about 85%, or about 70 to about 80%, or about 75 to about 95%, or about 75 to about 90%, or about 75 to about 85%, or about 75 to about 80% w / w.

[0268] In various embodiments, the protein concentrate has a total solids content in the range of 15 to 50 %w / w, for example from about 15 to about 45%, from about 15 to about 40%, from about 15 to about 35%, from about 15 to about 30%, from about 15 to about 28%, or about 15 to about 25%, or about 15 to about 23%, or about 18 to about 30%, from about 18 to about 50%, from about 18 to about 45%, from about 18 to about 40%, from about 18 to about 35%, or about 18 to about 28%, or about 18 to about 25%, or about 18 to about 23%, from about 20 to about 50%, from about 20 to about 45%, from about 20 to about 40%, from about 20 to about 35%, or about 20 to about 30%, or about 20 to about 28%, or about 20 to about 25%, or about 20 to about 23% w / w.

[0269] In various embodiments, the protein concentrate comprises at least about 30% w / w protein based on dry matter, or at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% w / w protein based on dry matter. In various embodiments, the protein concentrate comprises from about 30 to about 90% w / w protein based on dry matter, for example from about 35 to about 90%, or about 40 to about 90%, or about 45 to about 90%, or about 50 to about 90%, or about 55 to about 90%, or about 60 to about 90%, or about 30 to about 85%, about 35 to about 85%, or about 40 to about 85%, or about 45 to about 85%, or about 50 to about 85%, or about 55 to about 85%, or about 60 to about 85%, or about 30 to about 80%, about 35 to about 80%, or about 40 to about 80%, or about 45 to about 80%, or about 50 to about 80%, or about 55 to about 80%, or about 60 to about 80% w / w protein based on dry matter.

[0270] In various embodiments, the protein concentrate comprises from 0 to about 30% w / w carbohydrates based on dry matter. In various embodiments, the protein concentrate comprises no sugars.

[0271] In various embodiments, the protein concentrate comprises from about 0 to about 15% w / w total fat based on dry matter. Fat may include monounsaturated fat, mega 3, omega 9, polyunsaturated fat, saturated fat, and unsaturated fat.

[0272] In various embodiments, the protein concentrate comprises from about 0 to about 4% w / w ash based on dry matter.

[0273] Optionally, the protein concentrate may be combined with additives including cryoprotectants, antioxidants, flowing agents, glidants, binding agents, bulking agents, anti-caking agents, lubricants, fillers, sweeteners, excipients including colourants, flavouring agents but not limited thereto.

[0274] In various embodiments, the protein concentrate is not subjected to a drying step.

[0275] In preferred embodiments, the invention produces a protein concentrate with a water content of 70 - 85% (w / w), which has not been subjected to a drying step (for example an evaporation, pressing, spray drying or freeze drying step). The water content of the Rubisco protein concentrate is preferably between 50% - 95% w / w and more preferably between about 65% and about 85%.

[0276] The invention provides a protein concentrate prepared by a process of the invention.Functional Properties

[0277] In various embodiments, the protein concentrate according to the invention comprising Rubisco protein has favourable physical functional properties, such as emulsion activity, acid stability, alkaline stability, gelation characteristics, heat stability, frozen stability, foam ability, and foam stability.

[0278] As discussed above, the presence of these functional properties is surprising as it is generally considered that proteins must be in their undenatured state to impart favourable physical properties, and undenatured proteins are considered critical for success in food replica products as disclosed in W00221007484A1, W02011078671A1 and WO2016054375A1. The industrial manufacture of non-denatured proteins is challenging. Surprisingly, the proteinconcentration comprising Rubisco protein of the invention despite being subject to acid precipitation and solvent washing retains or has superior functionality attributes which are not dependent on preparation of a non-denatured protein product.Emulsion Properties

[0279] In various embodiments, the protein concentrate comprising Rubisco protein has favourable emulsion properties which make it useful in various food applications, such as: as a natural, non-synthetic, sole emulsifying agent in the absence of other emulsifying agents; as an emulsifying extender in the presence of other emulsifying agents, for example, in applications such as an egg yolk or egg white extender; and as a replacement or substitute for synthetic emulsifying agents such as polyoxyethylene derivatives of sorbitan fatty acid esters, mono- and diglycerides derivatives of sugar alcohol fatty acid esters, and modified emulsifying starches. The ability to provide an alternative emulsifying extender option to enhance the emulsification provided by another emulsifier(s) from a natural, non-animal source is a particular advantage of the present invention. The invention therefore also includes an emulsifying extender composition, the composition comprising a Rubisco protein concentrate having a water content of at least about 50% w / w. In particular, the emulsifying extender composition comprises a Rubisco protein concentrate, comprising a water content between 50% - 95%; a dry matter content between 5% - 50% w / w, wherein the dry matter comprises protein in an amount between 70% - 90% w / w; and ethanol, wherein the ethanol is present at a concentration of less than about 0.5%. Preferably, the emulsifying extender composition comprises less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds and / or comprising CIE colour values of a* > 0; b* > 10, and L* > 50. Preferably, the emulsifying extender composition comprises less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds and / or comprising CIE colour values of a* > -1; b* > 10, and L* > 50. The water content of the Rubisco protein concentrate is preferably between about 65% and about 85%. The Rubisco protein concentrate will preferably have a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably between about 90% and 99.2% w / w and is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein.

[0280] In a preferred form the emulsion extender composition comprises a Rubisco protein concentrate having a water content between 50% - 95%; CIE colour values of a* > -1; b* > 10, and L* > 50; an emulsion activity of greater than about70%; an oil holding capacity of between about 2.5 and about 6.9g / g; a water holding capacity of between about 2.5 and about 3.5 g / g; a dry matter content between 5% - 50% w / w, and ethanol; wherein the dry matter comprises protein in an amount between 70% - 90% w / w, and wherein the ethanol is present at a concentration of less than about 0.5%. It is preferred that the Rubisco protein concentrate, and therefore the emulsifying extender composition, has less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds. The Rubisco protein concentrate may have CIE colour values of a* > 0; b* > 10, and L* > 50 and / or an oil holding capacity of between about 3.1 and about 3.6 g / g and / or a water holding capacity of between about 2.7 and about 3.4g / g and / or a water content between about 65% and about 85% w / w (which will also vary the dry matter content accordingly). The Rubisco protein concentrate will preferably have a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably between about 90% and 99.2% w / w and is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein. The Rubisco protein concentrate has also been shown to have an emulsion activity of between about 52% and about 60%, preferably between about 53% and 56%, (when measured at 1% protein inclusion rate).

[0281] Food emulsions are formed by mixing two immiscible liquids to create a uniform suspension in which small droplets of one liquid, called the dispersed phase, are surrounded by droplets of the other liquid phase called the continuous phase. Typically, non-stabilised temporary emulsions are prepared by mixing 3 parts oil to 1 part water by whisking or blending. These temporary emulsions usually separate in less than 1 hour. Emulsions may be favourably stabilized with addition of emulsifying or stabilising agents and salts, which form a film around the dispersed phase or lower the interfacial tension in the emulsion to enable the formation of a semi-permanent or permanent emulsion which may favourably last periods of time from hours to months.

[0282] Emulsion activity is a measure of the weight or volume of oil that can be emulsified by the protein concentrate. Emulsion activity is measured, for example, by making 50 mL of a 5% protein solution in water, and homogenising with 50 mL of vegetable oil. Following homogenisation, the emulsion is centrifuged, and the total volume and emulsion volume measured. The emulsion activity is expressed as the ratio of the emulsion volume to the total volume. In various embodiments, the protein concentrate of the invention has an emulsion activity of greater than about 70%. The protein concentrate of the invention has been found to have an emulsion activity substantially comparable to the emulsion activity of animal protein options.

[0283] Emulsion properties can also be investigated using an emulsion capacity test, which provides information about the extent to which the emulsion can carry oil. For example, an emulsion can be prepared by homogenizing a 1: 10: 10 ratio of protein material:water:oil, and then adding further aliquots of oil to the emulsion and rehomogenizing the emulsion, until the emulsion is observed to split into two distinct phases. In various embodiments, the ratio at which the emulsion splits is in the range 1 : 10:20 - 1: 10:30 protein material:water:oil. In one embodiment, the ratio at which the emulsion splits is 1 : 10:25.2, i.e. the protein concentrate is able to emulsify 25.2 g oil per gram of protein. In various embodiments, the protein concentrate is able to emulsify 40 g oil per gram of protein, such as 50 g, or 55 g oil per gram of protein.

[0284] Acid stability can be measured by putting 20g (or 20 mL) of emulsion in a conical tube (50 mL) and adding 5% phosphoric acid and homogenizing with a glass bar and monitoring with a pH-meter reducing the pH to 0.5 and 1.0 pH units below the starting emulsion pH value. The emulsion is centrifuged at 10,000g for 15 minutes. The syneresis (volume of oil exudated by emulsion) is measured as the mass of oil excluded from the emulsion. The emulsion acid stability is the ratio of the volume of the remaining emulsion phase to the total volume of the solution. The emulsion acid stability has been found to have acid stability substantially comparable to the acid stability of animal protein concentrates.

[0285] Alkaline stability can be measured by putting 20g (or 20 mL) of emulsion in a conical tube (50 mL) and adding 10% sodium hydroxide and homogenizing with a glass bar and monitoring with a pH-meter reducing the pH to 0.5 and 1.0 pH units above the starting emulsion pH value. The emulsion is centrifuged at 10,000g for 15 minutes. The syneresis (volume of oil exudated by emulsion) is measured as the mass of oil excluded from the emulsion. The emulsion alkaline stability is the ratio of the volume of the remaining emulsion phase to the total volume of the solution. The emulsion alkaline stability has been found to have alkaline stability substantially comparable to the alkaline stability of animal protein concentrates.

[0286] The invention provides a process for preparing an emulsion, the process comprising homogenizing a Rubisco protein concentrate of the invention, an aqueous phase, and an oil phase.

[0287] The invention provides a process for preparing an emulsion, the process comprising homogenizing a Rubisco protein concentrate prepared by the process of the invention, an aqueous phase, and an oil phase.

[0288] The invention also provides an emulsion comprising a homogenized combination of a Rubisco protein concentrate of the invention, an aqueous phase, and an oil phase.

[0289] In various embodiments the amount of Rubisco protein concentrate used to prepare the emulsion is in the range 0.05 - 5% (w / w).

[0290] In various embodiments, the amount of oil used to prepare the emulsion is in the range 50 - 90% (w / w).

[0291] In various embodiments, the emulsion is prepared with a ratio of about 1 :2.5:5.3 to about 1 :54.5: 108 (w / w) protein:oil:aqueous phase, for example from about 1 :25: 11.1 to about 1:54.5: 108, or from about 1:30: 14 to about 1:54.5: 108, or from about 1:40:25 to about 1 :54.5: 108, or from about 1:2.5: 5.3 to about 1 :59:33, or from about 1:2.5:5.3 to about 1:40:25.

[0292] In various embodiments, the amount of oil used to prepare the emulsion is about 26-70% (w / w) with respect to the Rubisco protein concentrate:aqueous phase components. In various embodiments, the amount of Rubisco protein concentrate used to prepare the emulsion between 0.5-10% with respect to the oil and aqueous phase components. In various embodiments, the amount of oil used to prepare the emulsion is about 26-70% (w / w) with respect to the Rubisco protein concentrate:aqueous phase components, and the amount of Rubisco protein concentrate used to prepare the emulsion between 0.5-10% with respect to the oil and aqueous phase components.

[0293] In various embodiments, the aqueous phase is principally water.

[0294] In various embodiments, the oil phase is an edible oil or fat including but not limited to canola, coconut, rice, sunflower, olive, rice bran, soybean, rape, vegetable, palm, flax, and animal fats.

[0295] The emulsion using the Rubisco protein concentrate of the invention is particularly useful for mayonnaises, dressings, sauces, condiments, and high-fat fillings.Gels

[0296] In various embodiments, the protein concentrate comprising Rubisco also provides favourable gelation characteristics which provide desired textural characteristics to food products prepared using the protein concentrate of theinvention. For example, using the protein concentrate comprising Rubisco to prepare a food product can improve the textural characteristics of food product, compared to the same food product prepared without the protein concentrate comprising Rubisco.

[0297] Protein gels are three dimensional cross-linked networks of proteins which entrap water and other compounds to create a gel structure.

[0298] Gel strength is an important characteristic of a gel because it measures the gel's resistance to compression. When a gel has a high resistance to compression (or elastic modulus) it means that the formed gel is more likely to hold its shape when force is applied. Gel strength can be measured using a texture analyser using standard methodologies known to those in the art.

[0299] Rubisco protein concentrate forms thermoirreversible gels upon thermal activation. The viscosity (q), storage modulus (G'), and shear stress (a) of Rubisco protein concentrate were recorded on an Anton Paar MCR 302 Rheometer (PP50 geometry) through heating from 25 to 85 °C. At low temperatures (28 °C), the solution is in a liquid state (n = 1500000 mPa s, a = 47 Pa , G'= 7836 Pa) and as temperature increases some weak interactions within the material that do not play a role in gel formation are temporarily destabilized, leading to a slight drop in the rheological properties (at 56 °C, q = 639000 mPa s, a = 20 Pa , G'= 3466 Pa). Upon further heating, the material reaches a critical activation temperature where stronger, irreversible cross-linking interactions occur. This leads to a permanent, strong gel network formation, drastically increasing the storage modulus, viscosity, and shear stress (at 85 °C, q = 3050000 mPa s, a = 96 Pa , G'= 18835 Pa). Since this gelation is thermoirreversible, the gel network remains even upon cooling.

[0300] The initial decrease followed by a strong increase is often seen in materials that need thermal activation to reach their final gelled state.Foaming

[0301] In various embodiments, the protein concentrate comprising Rubisco protein has favourable foaming properties, which make it useful in various food applications, such as: froth, foams, whipped creams, meringues, mousses and volume expanders. For example, using the protein concentrate comprising Rubisco to prepare a food product can improve the textural characteristics of food product by providing creamy texture, visual appeal, and lighter and airy structures textures compared to the same food product prepared without the protein concentrate comprising Rubisco of the invention.

[0302] A functional assay of foaming capacity and stability was performed to test and quantify the ability of the Rubisco protein concentrate to form and hold a foam (Example 17). This was done by hydrating the protein sample and incorporating air using a homogeniser. The foam formed was then measured and left to sit for 30 minutes before being measured again. The foam formed was also heated at 85°C for 15 minutes to assess the foam decay and thermal stability.

[0303] Rubisco protein concentrate of the invention was found to have good foam capability and foam stability, substantially comparable or superior to the foam capability and foam stability of plant and animal protein concentrates analysed concurrently and when compared to reported values (Jakobson, et al. Foods 2023, 12 (14), 2805.Water Holding Capacity (WHC) and Oil Holding Capacity (OHC)

[0304] In various embodiments, the protein concentrate comprising Rubisco protein has favourable oil and water holding properties, which make it useful in various food applications, such as: meat products, bakery product, dairy alternatives, confectionary and snack. For example, using the protein concentrate comprising Rubisco to prepare a food product can improve the textural characteristics of food product by retaining moisture and fat, influencing crispiness and oil content, improving juiciness, and texture, imparting tenderness and mouthfeel, and mimicking creamy textures, smoothness and mouthfeel, compared to the same food product prepared without the protein concentrate comprising Rubisco.

[0305] The inclusion of proteins with high oil holding capacity (OHC) like Rubisco protein concentrate are integral to achieving a visually appealing product with a moist and cohesive texture. The retention of oil by Rubisco protein enhances juiciness, improves mouthfeel, and maintains the form of the product while cooking avoiding excessive surface oiliness. OHC also prevents dryness, ensuring a firm yet tender texture and bite similar to animal meat consistency. High OHC proteins also contribute to flavour retention, allowing for an even distribution of seasoning within the product.

[0306] A functional assay of water holding capacity (WHC) and of oil holding capacity (OHC) was performed to test and quantify the ability of the Rubisco protein concentrate to hold water or to hold oil (Example 18). This was done by hydrating the protein with either water or oil, centrifuging the sample and measuring the weight gain of the sample after the supernatant was removed. The WHC or OHC was calculated as the gained weight by the initial sample weight (g / g).

[0307] Rubisco protein concentrate of the invention was found to have high WHC and high OHC capability, substantially comparable or superior to the WHC and OHC of plant and animal protein concentrates analysed concurrently and when compared to reported values (Jakobson, et al. Foods 2023, 12 (14), 2805).

[0308] In various embodiments, the Rubisco protein concentrate has an oil holding capacity of greater than about 2.5 g / g, for example between about 2.5 and about 6.9g / g, or between about 2.5 and about 6.0 g / g, or between about 2.5 and about 5.0 g / g, or between about 2.5 and about 4.0 g / g, or between about 3.0 and about 6.0 g / g, or between about 3.0 and about 5.0 g / g, or between about 3.0 and about 4.0 g / g, or between about 3.1 and about 3.6 g / g).

[0309] in various embodiments, the Rubisco protein concentrate has a water holding capacity of between about 2.5 and about 3.5 g / g, for example between about 2.7 and about 3.4 g / g.Food product

[0310] The protein concentrate has advantageous functional properties across multiple food applications, delivering emulsification, water binding and gelling, allowing for unique texturisation and stability. Its pale colour, low flavour and aroma volatiles and neutral taste allow its incorporation into sweet and savoury applications. Its unique flavour profile allows for an enrichment of some savoury and proteinaceous notes in certain plant-based applications. The protein concentrate of the present invention may provide improved succulence and texture in food applications, such as burgers, hotdogs and sausages, using alternative non-animal protein sources.

[0311] The invention provides a food product comprising a protein concentrate of the invention. The invention provides a food product comprising a protein concentrate prepared by the process of the invention.

[0312] Consumer food products include but are not limited to emulsified foods such as mayonnaises, dressings, sauces, condiments, and high-fat fillings; dairy alternatives such as plant-based milks, yoghurts, cheeses, spreads, creams, and creamers; frozen and / or chilled whipped items such as fruit ices, ice creams, mousses, custards and desserts; confectionery items such as meringues, candies and prepared and / or instantised beverages such as sports drinks, shakes, juices; plant-based egg alternatives such as egg analogues, replacers, and substitutes; formed, emulsified and / or extruded plant-based meat and fish analogues such as burgers, sausages, hot dogs, salami, cold cuts, cuts, strips and pieces, and frozen portions and meat snacks;bakery items such as breads, cakes, wraps, biscuits, crackers, bars, and pastries; extruded products such as snack foods, pasta, and breakfast cereals.

[0313] The protein concentrate can be used as a base for cultured meat, including fish, extruded products including meat replacements, and fermentation media for cell culture.

[0314] In a further aspect, the invention provides a method for preparing a food product, the method comprising mixing a protein concentrate of the invention, and one or more additional ingredients.[0031S] In another aspect, the invention provides use of a protein concentrate of the invention or made by the process of the invention in the preparation of a food product.

[0316] In another aspect, the invention provides a food product comprising a Rubisco protein concentrate of the invention or a Rubisco protein concentrate prepared by the process of the invention.

[0317] Other steps for preparing a food product will depend on the food product to be produced and will be known to a skilled worker.

[0318] In various embodiments, the one or more additional ingredients may be a lipid, a carbohydrate, a protein, a flavour, a vitamin, a mineral, a milk product, water, a food additive, a colour, a fruit preparation, or any combination of any two or more of these ingredients.

[0319] In various embodiments the lipid may be plant lipid or animal lipid, including but not limited to coconut oil / fat, canola (rapeseed) oil, corn oil, sunflower oil, soybean oil, hydrogenated vegetable oil, cottonseed oil, canola oil, rapeseed oil, olive oil, palm oil, peanut oil, ground nut oil, safflower oil, sesame oil, hazelnut oil, almond oil, cashew oil, macadamia oil, pecan oil, pistachio oil, walnut oil, oils from melon and gourd seeds, pumpkin seed oil, apricot oil, argan oil, avocado oil, flax oil, flax seed oil, grape seed oil, hemp oil, linseed oil, rice bran oil, wheat germ oil, or any combination of any two or more thereof.

[0320] In various embodiments, the carbohydrate may comprise monosaccharides, disaccharides, oligosaccharides and polysaccharides and mixtures thereof, sugar, sucrose, sucralose, starch, modified starch, modified potato starch, modified waxy maize starch, maltodextrin, fructo-oligosaccharides, inulin, galacto-oligosaccharides, glycerol (glycerine), maltitol, erythritol, sorbitol and any combination of any two or more thereof.

[0321] In various embodiments, the protein may be textured vegetable protein, milk, whey, casein, caseinate, egg, egg white, egg yolk, vegetable, plant, alfalfa, clover, pea, bean, kidney bean, soybean, lentil, lupin, cocoa, carob, nut, peanut, rye, cereal, whole wheat, rice, hemp, wheat gluten, fungal, or algal protein, a protein concentrate thereof, a protein isolate thereof, a hydrolysate thereof, or any combination of any two or more thereof. In various embodiments, the protein may be textured vegetable protein, milk, whey, casein, caseinate, egg, egg white, egg yolk, vegetable, plant, alfalfa, clover, pea, bean, kidney bean, soybean, lentil, lupin, cocoa, carob, nut, peanut, rye, cereal, whole wheat, rice, hemp, wheat gluten, fungal, microbial, or algal protein, a protein concentrate thereof, a protein isolate thereof, a hydrolysate thereof, or any combination of any two or more thereof.

[0322] In various embodiments the one or more additional ingredients may be flavours, including but not limited to sweeteners, yeast extract, natural flavours, nature identical flavours, artificial flavours, herbs, and spices.

[0323] In various embodiments the one or more additional ingredients may be vitamins. Vitamins may include fat-soluble or water-soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. The form of the vitamin may include salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of a vitamin, and metabolites of a vitamin.

[0324] In various embodiments the one or more additional ingredients may be minerals, including, but not limited to chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, phosphorus, potassium and chromium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non- reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.

[0325] In various embodiments the one or more additional ingredients may be a food additive, including but not limited to rennet, antifoams, stabilisers, emulsifiers, preservatives, fibre, probiotics, antioxidants such as rosemary extract, flavours, freeze-dried fruit, flavour enhancers, colours, acidity regulators, or emulsifying salts.

[0326] In various embodiments the one or more additional ingredients may be stabilisers or emulsifiers. Useful emulsifiers include lecithins, mono and diglycerides, polyglycerol esters, milk phospholipids, citric acid esters (citrems), polysorbate 60, glyceryl monostearate, and datems. Useful stabilisers include methyl cellulose, carrageenan, gellan gum, pectin, guar gum, locust bean gum, carboxymethyl cellulose, alginates, agar, oat gum, tragacanth gum, acacia gum, xanthan gum, karaya gum, tara gum, starch, and modified starch and microcrystalline cellulose, gelatin, or combinations thereof. In various embodiments the one or more additional ingredients may be stabilisers or emulsifiers. Useful emulsifiers include lecithins, mono and diglycerides, polyglycerol esters, milk phospholipids, citric acid esters (citrems), polysorbate 60, glyceryl monostearate, and datems. Useful stabilisers include methyl cellulose, carrageenan, gellan gum, pectin, guar gum, locust bean gum, carboxymethyl cellulose, alginates, agar, oat gum, tragacanth gum, acacia gum, xanthan gum, karaya gum, konjac gum, tara gum, starch, and modified starch and microcrystalline cellulose, gelatin, or combinations thereof. Useful stabilisers include carrageenan, gellan gum, pectin, guar gum, locust bean gum, carboxymethyl cellulose, alginates, agar, oat gum, tragacanth gum, acacia gum, xanthan gum, karaya gum, tara gum, starch, and modified starch and microcrystalline cellulose or combinations thereof. In some embodiments, use of the Rubisco protein concentrate of the invention allows that additional stabilizers such as methyl cellulose (E461) are not required (see Examples 13 and 14), which is seen as desirable by some consumers.

[0327] In various embodiments, the one or more additional ingredients may be salts or acidity regulators, such as sodium chloride, vinegar, potassium chloride, ethylenediaminetetraacetic (EDTA) salts, lactic acid, acetic acid, citric acid, potassium hydroxide, phosphate salts such as dipotassium phosphate and disodium phosphate, citrate salts such as disodium citrate, dipotassium citrate, or tripotassium citrate.[0032S] In various embodiments, the one or more additional ingredients may be a source of amino acids, amino acid precursors or amino acid metabolites or any combination of any two or more thereof, preferably free amino acids, amino acid precursors or amino acid metabolites.Mayonnaise

[0329] The protein concentrate of the invention is particularly useful in the manufacture of mayonnaise, as described in Example 9. Mayonnaise may be preparedby mixing the protein concentrate of the invention with salt, sugar, oil, and vinegar. Optionally, an antioxidant such as rosemary extract may be added.Burger

[0330] The protein concentrate of the invention is particularly useful in the manufacture of a meat-free burger patty, as described in Example 10. A burger patty may be prepared by mixing the protein concentrate of the invention with water, oil, hydrated textured vegetable protein, methyl cellulose, salt, starch, and coconut fat. The protein concentrate of the invention is also particularly useful in the manufacture of burgers, which use a reduced amount of gums, as described in Example 13.Hot Dogs / Sausages

[0331] The protein concentrate of the invention is particularly useful in the manufacture of hotdogs and sausages, which do not use methyl cellulose, binders or gum, or which do not use methyl cellulose and reduced amount of gums, as described in Examples 13 and 14.Yoghurt

[0332] The protein concentrate of the invention is particularly useful in the manufacture of a yoghurt, as described in Example 11. A yoghurt may be prepared by mixing the protein concentrate of the invention with modified potato starch, modified waxy maize starch, sugar, water, freeze-dried raspberry, hydrogenated coconut oil, natural flavourings, colour, acidity regulator, and preservative.Cheese

[0333] The protein concentrate of the invention is particularly useful in the manufacture of a cheese, as described in Example 12. A cheese may be prepared by mixing the protein concentrate of the invention with modified potato starch, modified waxy maize starch, water, hydrogenated coconut oil, canola oil, salt, yeast extract, maltodextrin, natural flavourings, colour and preservative.Embodiments described herein

[0334] The invention may be seen to provide a Rubisco protein concentrate, that may be used as a food ingredient, having a water content of at least about 50% (w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, and a dry matter content between 5% - 50% w / w, wherein the dry matter comprises Rubisco protein in anamount between about 70% to about 90% w / w. The Rubisco protein concentrate may also comprise one or more of the following characteristics:(a) an emulsion activity of greater than about 70%. Preferably, the Rubisco protein concentrate has an emulsion activity of between about 70% and about 90%, more preferably between about 70% and about 88%, (when measured at 5% protein inclusion rate). When measured at 1% protein inclusion rate, the Rubisco protein concentrate has an emulsion activity of between about 52% and about 60%, preferably between about 53% and 56%, which is very competitive with or superior to alternative non-synthetic emulsifier options when also measured at the 1% protein inclusion rate;(b) an oil holding capacity of greater than about 2.5 g / g, (preferably between about 2.5 and about 6.9g / g, more preferably between about 3.0 and about 5.0 g / g, more preferably between about 3.1 and about 3.6 g / g) and a water holding capacity of between about 2.5 and about 3.5 g / g (preferably between about 2.7 and about 3.4 g / g);(c) less than about 0.5% total saponins and / or less than about 0.5% flavones and polyphenols compounds and / or a lower alcohol content, preferably ethanol, of less than about 0.5%;(d) when the Rubisco protein concentrate has a water content of between about 80% and about 99.2%w / w the Rubisco protein concentrate is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably when the Rubisco protein concentrate has a water content of between about 90 and about 99.2% w / w the Rubisco protein concentrate is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein;(e) a foaming stability after 30 minutes of between about 55% and about 90%, more preferably between about 58% and 86%; a foaming capability of between about 70% and about 115%, more preferably between about 75% and 113%; and a heated foaming stability (at 85C) of between about 95% and about 140%, more preferably between about 100% and about 138%.

[0335] The Rubisco concentrate will preferably comprise (i) CIE colour values of a* > 0; b* > 10; and L* > 50, and / or a water content of between 50% - 95% w / w, preferably between about 65% and about 85%, between about 80% and about 99.2%w / w, or between about 90% and about 99.2%w / w. The Rubisco proteinconcentrate will preferably include between about 5% and about 50% dry matter (when the water content is between 50% - 95% w / w) and the dry matter will preferably comprise Rubisco protein in an amount between 70% - 90% w / w.

[0336] The invention may also be seen to provide a Rubisco protein concentrate that may be used as a food ingredient, having a water content of at least about 50% (w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, an emulsion activity of greater than about 70%, an oil holding capacity of greater than about 2.5 g / g, (preferably between about 2.5 and about 6.9g / g, more preferably between about 3.0 and about 5.0 g / g, more preferably between about 3.1 and about 3.6 g / g) and a water holding capacity of between about 2.5 and about 3.5 g / g (preferably between about 2.7 and about 3.4 g / g). The water content of the Rubisco protein concentrate is preferably between 50% - 95% w / w and more preferably between about 65% and about 85%. The Rubisco protein concentrate will preferably have CIE colour values of a* > 0; b* > 10; and L* > 50. The Rubisco protein concentrate will also preferably have less than about 0.5% total saponins and / or less than about 0.5% flavones and polyphenols compounds and / or a lower alcohol content, preferably ethanol, of less than about 0.5%. In a more preferred option, the inventors have found that when the Rubisco protein concentrate has a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably between about 90 and 99.2% w / w and the Rubisco protein concentrate is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein. Preferably, the Rubisco protein concentrate has an emulsion activity of between about 70% and about 90%, more preferably between about 70% and about 88%, (when measured at 5% protein inclusion rate). When measured at 1% protein inclusion rate, the Rubisco protein concentrate has an emulsion activity of between about 52% and about 60%, preferably between about 53% and 56%, which is very competitive with or superior to alternative non-synthetic emulsifier options when also measured at the 1% protein inclusion rate.

[0337] The Rubisco concentrate may be prepared with a low water content (for example between about 50% and about 75% w / w) and then later diluted to have a higher water content (for example between about 80% and about 99.2%w / w) for use when preparing a food product. This may allow for efficiencies of transportation or storage for example.

[0338] The inventors have also found that Rubisco protein concentrate has a foaming stability after 30 minutes of between about 55% and about 90%, more preferably between about 58% and 86%; a foaming capability of between about 70%and about 115%, more preferably between about 75% and 113%; and a heated foaming stability (at 85°C) of between about 95% and about 140%, more preferably between about 100% and about 138%. Foamability and foam stability are important in a wide range of food products as would be known to skilled person, for example, in bread, ice cream, cakes, mousse, meringues, amongst others (Murray B.S. Recent developments in food foams. Curr. Opin. Colloid Interface Sci. 2020;50: 101394). The Rubisco protein concentrate of the invention as described above therefore offers an alternative, and in some cases an improved alternative, to commonly used foaming options such as egg whites. The ability to use a plant based product having the foaming characteristics shown by the Rubisco protein concentrate of the invention opens options for users in the alternative meat and milk industries and avoids allergen issues that can occur with egg white use. The Rubisco protein concentrate of the invention may in particular be used as a foamer product for the alternative milk coffee market (refer: Example 20), as well as in products such as ice cream, cakes, mousse, meringues, for example.

[0339] The Rubisco protein concentrate of the invention may also be dehydrated into a powder form using known techniques (such as freeze drying, spray drying, or evaporation). The dehydrated Rubisco protein concentrate contains between 70% and 90% Rubisco protein and can be reconstituted with water such that Rubisco protein contains at least 50% water as discussed previously as will retain the Rubisco protein concentrate characteristics discussed above. The powder will also contain less than about 0.5% total saponins and / or less than about 0.5% flavones and polyphenols compounds and CIE colour values of a* > -1; b* > 10; and L* > 50. The powder may also provide one or more of a lower alcohol content, preferably ethanol, of less than about 0.5%, an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate) (preferably, between about 70% and about 90%, more preferably between about 70% and about 88%, (when measured at 5% protein inclusion rate)), an oil holding capacity of greater than about 2.5 g / g, (preferably between about 3 and about 4g / g, more preferably between about 3.1 and about 3.6 g / g) and a water holding capacity of between about 2.5 and about 3.5 g / g (preferably between about 2.7 and about 3.4 g / g) and / or foaming stability after 30 minutes of between about 55% and about 90%, more preferably between about 58% and 86%; a foaming capability of between about 70% and about 115%, more preferably between about 75% and 113%; and a heated foaming stability (at 85C) of between about 95% and about 140%, more preferably between about 100% and about 138%.

[0340] The Rubisco protein concentrate, and therefore the food product that includes the Rubisco protein concentrate, may have a low ethanol (or other loweralcohol) content that is preferably less than about 0.5%, less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds. The water content of the Rubisco protein concentrate is preferably between 50% - 95% w / w and more preferably between about 65% and about 85%. In particular, the Rubisco protein concentrate preferably has a water content of between about 80% and about 99.2%w / w and is able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein, more preferably between about 90% and 99.2% w / w and is able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein. The Rubisco protein concentrate will preferably include between about 5% and about 50% dry matter w / w (when the water content is between 50% - 95% w / w) and the dry matter will preferably comprise Rubisco protein in an amount between 70% - 90% w / w.

[0341] The edible oil in the emulsion formed in the food product may be any suitable edible oil or fat that would be present in a food product as would be known to person skilled in the art. Non-limiting options include one or more of canola, coconut, rice, sunflower, olive, rice bran, soybean, rape, vegetable, palm, and flax oils, as well as animal fats. The aqueous phase is preferably water.

[0342] The food product may include any one or more of the other additional ingredients described above and may be any one or more of the consumer food products described herein (see paragraph

[0307] for example). The inventors have found that the Rubisco protein concentrate of the invention is able to replace or substitute commonly used emulsifiers (such as methyl cellulose and synthetic emulsifying agents such as polyoxyethylene derivatives of sorbitan fatty acid esters, mono- and diglycerides derivatives of sugar alcohol fatty acid esters, and modified emulsifying starches) in food products and, in some products (such as sausages, hotdogs, and burger patties) can replace or substitute such commonly used emulsifiers and also stabilisers (such as gums). As the Rubisco concentrate is a natural, non-synthetic, emulsifying agent, this is particularly desirable for some consumers.

[0343] The invention also provides a food product (that may include one or more of the additional ingredients described above) that comprises an emulsion of a Rubisco protein:oil:aqueous phase in a ratio of about 1 :2.5:5.3 to about 1 :54.5: 108 (w / w). The emulsion is formed in the food product by combining (i) a Rubisco protein concentrate having a water content of at least about 70% (w / w) (and preferably between about 70% and about 99.5% w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, an emulsion activity of greater than about 70% (when measured at 5%protein inclusion rate) (preferably, between about 70% and about 90%, more preferably between about 70% and about 88%, (when measured at 5% protein inclusion rate)), an oil holding capacity of greater than about 2.5 g / g, (preferably between about 2.5 and about 6.9g / g, more preferably between about 3.0 and about 5.0 g / g, more preferably between about 3.1 and about 3.6 g / g) and a water holding capacity of between about 2.5 and about 3.5 g / g (preferably between about 2.7 and about 3.4 g / g), (ii) edible oil, and (iii) water, when preparing the food product.

[0344] The Rubisco protein:oil:aqueous phase emulsion may be formed prior to combining the emulsion with the other additional ingredients when preparing the food product or may be formed incorporating one or more of the additional ingredients prior to combining the emulsion with the remaining other additional ingredients when preparing the food product. Such matters would be known to person skilled in the art.

[0345] The invention also provides a process for producing a Rubisco protein concentrate according to the invention from a biomass material comprising Rubisco protein. The Rubisco protein concentrate is as described above and includes a Rubisco protein concentrate having a water content of at least about 50% (w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, and a dry matter content between about 5% to about 50% w / w, wherein the dry matter comprises Rubisco protein in an amount between about 70% to about 90% w / w, and where the Rubisco protein concentrate optionally comprises one or more of the following characteristics (preferably, the Rubisco protein concentrate comprises one or more of the following characteristics): i. an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate) (preferably about 70% - 90%), ii. an oil holding capacity of greater than about 2.5 g / g (preferably between about 2.5g / g and about 6.9g / g), iii. a water holding capacity of between about 2.5 and about 3.5 g / g (preferably between about 2.7 and about 3.4 g / g), iv. less than about 0.5% total saponins and / or less than about 0.5% flavones and polyphenols compounds, v. a lower alcohol content of less than about 0.5% (preferably between 0.01% and 0.5%), and vi. a foaming stability after 30 minutes of between about 55% and about 90%.

[0346] The process for producing a Rubisco protein concentrate of the invention comprises steps of: i. providing a clarified juice from a biomass material comprising Rubisco protein, wherein the clarified juice is at pH 6.0 to 8.0 and comprises between about 50-95% w / w Rubisco protein, ii. subjecting the clarified juice to acid precipitation at a pH between about 3.0 to about 5.5 to precipitate Rubisco protein from the clarified juice to produce a liquid and a solid protein fraction,Hi. separating the liquid from the solid protein fraction; iv. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol, v. separating the solid protein fraction from the first solvent or solvent mixture, vi. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol, vii. separating the solid protein fraction from the second solvent or solvent mixture, and viii. washing the solid protein fraction with water to remove residual solvent from the solid protein fraction and to produce the Rubisco protein concentrate, wherein the water content of the Rubisco protein in steps (i) to (iii) is above 50% w / w, and wherein the water content Rubisco protein concentrate after water washing in step (viii) is above 50% w / w.

[0347] The biomass material used can be selected from one or more of alfalfa, kudzu, forage peas, oats, duckweed, spinach, kale, cocksfoot, Italian ryegrass, Raphno, clover, Persian clover, rapeseed leaves, carrots, radishes, Jerusalem artichoke, beets, spinach, fodder beet; triticale, white clover, barley, tobacco, chicory, sugar cane, or fava bean leaves, or from an algae selected from the classes Bacillariophyceae, Chloropyceae, Cyptophyceae, Dinophyceae, Euglenophyceae and Rhodophyceae.

[0348] The pH of the clarified juice can be the natural pH of the juice of the biomass material used, for example, when the biomass is alfalfa the pH of the clarified juice may be between about 6.3 to 6.5. If desired, the pH can be modified from the natural pH of the clarified juice but will remain at a substantially neutral pH of between about 6 and about 8. More preferably, the pH may be between about 6 and about 7.5.

[0349] The acid used in the acid precipitation step (ii) may be selected from any one or more of acetic acid, ascorbic acid, citric acid, lactic acid, propionic acid, succinic acid, sorbic acid, tartaric acid, phosphoric acid, sulfuric acid, or hydrochloric acid. Preferably, the acid used is phosphoric acid.

[0350] The solid protein fraction is separated from the liquid fraction and the solid protein fraction is separated from the solvent(s) by any suitable means including a centrifuge, disc stack centrifuge, clarifier, decanter, sedicanter, filter press / coarse filter, settling tank, hydrocyclone, microfiltration, or ultrafiltration. The first and second solvents are preferably both ethanol.

[0351] Steps (iv) to (vii) are repeated until the solid protein fraction comprises CIE colour values of a* > -1; b* > 10; and L* > 50 (preferably a* > 0; b* > 10; and L* > 50) and the water washing step (viii) is repeated until the Rubisco protein concentrate comprises less than about 0.5% residual solvent (preferably between 0.01 and 0.5% residual solvent). There may therefore be more than 2 solvent extraction steps and there may be a plurality of water washing steps. Solvent extraction steps (iv) to (vii) may be performed using a continuous process, in which the use of multiple steps, phases, effects, or loops, is considered to be two (or more) solvent extraction steps to achieve the desired CIE values as described above.EXAMPLESExample 1

[0352] This example shows one process according to the invention on a 40kg scale.

[0353] Method: 40 kg of juice, juiced from alfalfa, was heated to 58 °C using an immersed coil heat exchanger and held at that temperature for one minute, in order to coagulate the green protein fraction. Following the 1-min hold, the juice was cooled to 8 °C. The heat-coagulated juice was then loaded into a centrifuge (Beckman J-20I) and spun at 7,000 xg for 10 min, separating the coagulated green pellet fromthe protein-rich supernatant (clarified juice). The clarified juice was decanted off and collected. The pH of the clarified juice was adjusted to 4.5 using phosphoric acid (85% w / w, Ixom), causing the proteins to precipitate. The precipitated solution was spun in a centrifuge at 5,000 xg for 5 min, producing a creamy / pale light-green pellet. The pellet was mixed with in 95% ethanol (food grade, EcoChem) at a ratio of 3:2 ethanol: pellet, spun in a centrifuge at 5,000 xg for 5 min, and a lighter (less green) pellet was collected and brown supernatant (rich in flavonoids, among other components as measured by UV-vis spectrometry, GC-FID, LC-MS etc.) decanted off. The lighter pellet was resuspended in ethanol a ratio of 3:2 ethanol: pellet, spun in a centrifuge at 5,000 xg for 5 min, resulting in a decoloured pellet and green supernatant, the latter of which was decanted off. The white pellet was suspended in water in a ratio of 3:2 water to pellet to wash out the bulk of ethanol and spun in a centrifuge at 5,000 xg for 5 min. This water wash step was repeated once. The resulting white protein pellet was frozen in a blast freezer. Through this process, 53% w / w of protein from the extracted juice was recovered as protein in the white protein pellet.

[0354] Following this process, the washed pellet would further be washed and / or evaporated as necessary in order to reach the desired ethanol levels in the product.

[0355] Figure 4 shows an image of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel with Coomassie blue staining, illustrating the purification and retention of protein content achieved using Example 1 for purification of the Rubisco proteins. Rubisco large subunit, ~ 55kDa, Rubisco small subunit, ~15 kDa).

[0356] Figure 5 shows an image of a native non-denaturing polyacrylamide gel electrophoresis (Native PAGE) gel with Coomassie blue staining, illustrating the purification protein content achieved using Example 1 for purification of the Rubisco proteins. Rubisco native oligomeric molecule weight ~ 550kDa.The water content and protein content of samples at various stages of the process were measured according to table 1. Water content is measured by AOAC 925.09 and protein content is measured by the Kjeldahl method AOAC 981.10. Dry protein content is calculated from these measurements.Table 1: Mass balance & compositionsExample 2:

[0357] This example shows one process according to the invention on a 120kg scale.

[0358] Method: 120 kg of juice, obtained from alfalfa, was heated to 58 °C using a plate heat exchanger and held at that temperature for one minute in order to coagulate the green protein fraction. Following the 1-min hold, the juice was cooled to 8 °C. The heat-coagulated juice was then pumped through an industrial clarifier to provide a clarified juice. The pH of the clarified juice was adjusted to 4.5 using phosphoric acid, causing the soluble proteins to precipitate. The precipitate and solution were separated on an industrial clarifier (GEA GSC25), and a creamy / pale light-green pellet was obtained. The pellet was suspended in 95% ethanol at a ratio of 3:2 ethanol:pellet, loaded into a centrifuge and spun at 7,000 xg for 10 min, and a lighter (less green) pellet was collected and brown solvent solution as supernatant (rich in flavonoids, among other components) decanted off. The lighter pellet was resuspended in ethanol at a ratio of 3:2 ethanol: pellet), loaded into a centrifuge and spun at 7,000 xg for 10 min, resulting in a decoloured pellet and green solvent solution as supernatant, the latter of which was decanted off. The white pellet was suspended in water in a ratio of 3:2 water to pellet to wash out residual ethanol and spun down - this water wash step was then repeated once. The resulting white protein pellet was frozen in a blast freezer. The water content and protein content of samples at various stages of the process were measured according to table 2.Table 2: Mass balance & compositionsExample 3: Evaporation of residual lower alcohol and water from white pellet

[0359] This example shows preparation of a protein concentrate according to the invention, via two different evaporation methods.Method 2: Static simple, manual agitation evaporator

[0360] 1.14 kg of white pellet obtained according to Example 1 was placed in a tared stainless-steel vessel (200 mm diameter, 200 mm high) with a sealing lid and immersed in a 35 °C water bath (Digisystems Laboratory Instruments). Residual solvent and water were evaporated at 5-20 kPa (RobinAir vacuum pump, RA15501A). The pellet was stirred manually every 30 minutes and evaporated for variable lengths of time until 20-30% of initial weight was removed. The residual solvent in the white pellet was quantified using GC-FID. Water content was measured by loss of water mass on drying. Ethanol content was determined to have been reduced from 5.4% to 1.3% following evaporation, and water content reduced from 80% to 59%.Method 3: Thermomix evaporator

[0361] 1.022 kg of white pellet obtained according to Example 1 was placed in the bowl in a Thermomix TM6 with a sealing lid, heated at 37 °C with stirring. Residual solvent and water were evaporated at 5-20 kPa (RobinAir vacuum pump, RA15501A). The pellet was stirred until 20-30% of initial weight was removed. The residual solvent in the white pellet was quantified using GC-FID measurements. Ethanol content was determined to have been reduced from 1.5% to 0.36%, and water content reduced from 80% to 74%.Example 4 - Batch-mode Counter Current Extraction

[0362] The example shows a batch-mode counter current extraction according to the invention as shown in Figure 2.

[0363] 10 g precipitated protein material obtained according to Example 1 was subjected to a counter current extraction process using ethanol to prepare white protein precipitate.

[0364] Method: 10 kg of juice, obtained from alfalfa was heated to 58 °C using an immersed coil heat exchanger and held at that temperature for one minute, in order to coagulate the green protein fraction. Following the 1-min hold, the juice was cooled to 8 °C. The heat-coagulated juice was then loaded into a centrifuge (Beckman J-20I) and spun at 7,000 xg for 10 min, separating the coagulated green pellet from the protein-rich supernatant (clarified juice). The clarified juice was decanted off and collected. The pH of the clarified juice was adjusted to 4.5 using phosphoric acid, causing the proteins to precipitate. The precipitated solution was spun in a centrifuge at 5,000 xg for 5 min, producing a creamy / pale light-green pellet.

[0365] A batch-mode counter current extraction process was then conducted on 10 g samples of protein material as follows. 100% purity ethanol (grade 1) was used to extract the penultimate precipitate fraction (pellet 1), the result of which was clean white Final pellet and an ethanol fraction 2. This ethanol fraction 2 (78% ethanol) was used to extract a precipitate fraction (pellet 2) resulting in a clean precipitate fraction (pellet 1) and an ethanol fraction 3. This ethanol fraction 3 (61% ethanol) was used to extract the first crude precipitate fraction which had been prepared as described above (pellet 3) resulting in a precipitate fraction (pellet 2) and an ethanol fraction 4 (47% ethanol). The process was continued iteratively until the alcohol extraction liquid fraction had an absorbance at 664nm of less than 0.05 Absorbance units in a 1 cm pathlength, at which point crude pellet prepared as described in the preceding paragraph was introduced to the counter current method. The obtained pellet after the counter-current ethanol extraction cycle was a light tan colour. The protein pellet was then subjected to a counter-current wash cycle using water as a solvent to reduce the ethanol concentration.Example 5: Determination of improvements in colour as measured by UV absorbance and CIELAB measurements

[0366] The extracted protein pellet has a less green colour which can be quantified using UV / visible absorbance spectroscopy of an ethanolic extract of the protein pellet.

[0367] Approximately 1 gram of protein pellet was suspended and mixed in 95% ethanol in a 3:2 ratio of ethanol solvent to protein pellet (w / w). 500 uL of solution phase samples in microfuge tubes were clarified by centrifuge (Eppendorf 5424R) at 10,000 RCF for 5 minutes to sediment the debris. 100 uL was transferred to a 96-well flat bottom plate and an UV / visible absorbance scan performed across the wavelength range 250 - 700 nm on a UV / visible spectrophotometer (SpectraMax iD5). Samples dilutions were performed where necessary to ensure the absorbance value was within the dynamic range. A wavelength of 664nm was used to quantify residual colour.

[0368] The colour of the protein pellet can be quantified and defined using the CIELAB colour space values for perceived colour.Example 6: Comparative Example - Low yielding process using filtration

[0369] 10 kg of clarified alfalfa juice was pumped across a 0.45 pm TAMI ceramic microfiltration membrane to separate the protein from large soluble fats and carbohydrates and obtain a permeate fraction of 5% dry matter, with 30% of the dry matter being protein. The permeate fraction from microfiltration was concentrated and diafiltrated on a Koch 100 kDa ultrafiltration membrane to increase the solids fraction of the liquid, and wash out soluble sugars and minerals from the protein. The resulting retentate was dried on a spray drier, where 4.7 g of protein was obtained. The purity of the protein was determined to be 82% by Bradford assay. Through this process, 3.5% of the soluble protein was recovered as finished product.Example 7: Emulsion composition

[0370] Two emulsion preparations were made: one comprising the unwashed acid precipitated material, and one comprising the acid precipitation material which had gone through the process disclosed in Example 1. Both preparations were made as follows: 1 part protein material was combined with 1 part of water at 3500 rpm for four minutes on a high-shear Silverson mixer. 3 parts of canola oil was slowly addedto the mixture while mixing at 4500 rpm. Mixing was then increased to 7500 rpm until the oil was fully incorporated into the emulsion. This obtained a 1 : 1:3 emulsion for both fractions for protein material:water:oil, or 0.24: 1:3 on a dry-protein basis, demonstrating high emulsification efficiency of the protein. The emulsions were left in the refrigerator at 4°C for 24 hours. On standing, the samples were centrifuged and the top oil layer decanted off and weighed. 82% by weight of the oil separated from the emulsion of the unwashed acid precipitated material, while only 10% by weight of the oil separated from the emulsion of the protein concentrate prepared according to Example 1.Example 8: Emulsion stability

[0371] The stability of the emulsions prepared in Example 7 was determined under acid and alkali conditions.

[0372] For acid stability, the pH of the respective emulsions was lowered to 4.0 using phosphoric acid and centrifuged at 10,000 xg for 10 mins (Sorvall RC6). The oil phase released during centrifugation was measured for the respective emulsions. 70% by weight of the oil was released in the unwashed acid precipitated protein emulsion, while no oil was measured to be released from emulsion of the protein concentrate prepared according to Example 1.

[0373] Alkali stability was conducted in a similar manner, where the pH was raised 1.0 pH unit above the emulsion preparation pH using 10% sodium hydroxide. The released oil from both emulsions was measured. 62% by weight of the oil was released from the unwashed protein emulsion, while no oil was measured to be released from the emulsion of the protein concentrate prepared according to Example 1. The protein concentrate prepared according to Example 1 demonstrated a significantly higher emulsion stability than the unwashed acid precipitated protein.Example 9: Product with stable emulsion - mayonnaise

[0374] 9.8 g of protein concentrate prepared according to Example 1, 1.2 g of salt and 0.8 g of powdered sugar was hydrated in 21.8 mL of water and mixed in a high shear blender (Magimix 5200 XL Food Processor). Canola oil 21.8 g was slowly poured in under high shear mixing whilst simultaneously adding white vinegar 1 g until a pH of 4.0 was reached. The mayonnaise product was transferred into a container and allowed to rest for 12 hours at room temperature and then chilled at 4 °C.

[0375] Comparison was made to a control mayonnaise sample prepared as described above, but with substitution of the protein concentrate of Example 1 with 6 g of liquid egg yolk and 1.5 g of liquid whole yolk, a reduction in the water to 19.1 g and an increase in white vinegar to 6 g.

[0376] The mayonnaise viscosity was measured using a Rheomat RM 100, Lamy Rheology, Spindel ASTM 6 / speed 100 rpm, 60 s. The mayonnaise prepared with protein concentrate was measured to have 63% higher viscosity (1852 mPa.s-1) than the control mayonnaise prepared with egg (1138 mPa.s-1).Example 10: Meat substitute

[0377] A protein concentrate emulsion was prepared in a Magimix bowl by addition of 19 ml of water to 58 g of protein concentrate prepared according to Example 1 with mixing for 2 minutes, followed by the addition of 58 g of canola oil with mixing continued for 2 minutes. Separately, 335 g of hydrated textured vegetable protein (TVP, ADM Arcon® T158-171) was prepared by soaking TVP in 1:2.8 ratio of water for 30 minutes following by chilling to 4 °C. The prepared chilled TVP (335 g) and protein concentrate emulsion (125 g) were combined with mixing, followed by the addition of a mixture of 5g of methyl cellulose (Dupont Methocel MX) combined with 7.5 g of salt. 5 g of starch was added and mixed in, followed by the addition of 22.5 g of finely minced coconut fat with mixing. The mixture was chilled, formed and shaped into patties with a metal patty press.

[0378] Comparison was made to a control burger patty, prepared as described above but with substitution of the protein concentrate emulsion of Example 1 with 50 g of canola oil and 75 g of soy gel (prepared by mixing 80 g of soy protein concentrate (ADM Arcon® SM Cone 70%) with 320 g of water for 2 minutes).

[0379] The prepared burger patties were cooked on a hot plate.

[0380] Burger patties were analysed using a Perten TVT 6700XP texture analyser with a 25 mm cylinder probe, using 50% compression testing, with 2 cycles (two-bite testing) reporting data on hardness, chewiness, springiness, and gumminess.

[0381] The burger patties prepared with protein concentrate were measured to have an improved hardness (8275 g) when compared to the control patties prepared without protein concentrate (5957 g). The burger patties prepared with protein concentrate were measured to have an improved two-bite chewiness (3548) whencompared to the control patties prepared without protein concentrate (1873). This data indicates the favourable rheological modifying properties of the protein concentrate of the current invention to achieve increased hardness and chewiness associated with bite strength and which contribute to succulence and favourable texture in patties.Example 11: Yoghurt

[0382] 24.2 g of modified potato starch (E1420) was combined with 9.08 g of modified waxy maize starch (E1450) and 24.7 g of sugar in a Thermomix bowl with 264 g of water and mixed at ambient temperature on high until well combined. The suspension was heated to 60 degrees Celsius and mixed at high shear for 5 minutes followed by heating to 70 degrees for 2 minutes. The starch pre-gel slurry was allowed to cool to 45 degrees. 176 g of protein concentrate paste prepared according to Example 1 was added, while mixing on high shear, and mixed for 3 minutes at 45 degrees. 11 g of freeze-dried raspberry, 33 g of hydrogenated coconut oil, 3.02 g of combined natural flavourings, 3.30 g of colour 162, 0.55 g of acidity regulator, 0.83 g of preservative (E202) and 0.16 g of Colour 160a was added. The mixture was heated to 60-65 degrees and mixed for 8 minutes, then the hot mixture was spooned into silicon moulds, covered and chilled overnight to set.

[0383] The prepared yoghurt was observed to have a smooth and creamy texture from a strong and stable emulsion.Example 12: Cheese

[0384] 80.0 g of modified potato starch (E1420) was combined with 10.0 g of modified waxy maize starch (E1450) in a Thermomix bowl with 62.5 g of water and mixed at ambient temperature on high until well combined. The suspension was heated to 60 degrees Celsius and mixed at high shear for 5 minutes followed by heating to 70 degrees for 2 minutes. The starch pre-gel slurry was allowed to cool to 45 degrees. 200 g of protein concentrate paste, prepared according to Example 1, was added while mixing on high shear and mixed for 3 minutes at 45 degrees. 120 g of hydrogenated coconut oil, 5 g of canola oil, 11.25 of salt, 2 g of yeast extract, 1.88 g of maltodextrin, 4.65 g of combined natural flavourings, 1.63 g of colour 162, 0.3 g of colour 160a, 0.3 g of colour 160b and 0.5 g of preservative (202) were added and mixed on high shear until well emulsified. The mixture was heated to 60-65 degrees and mixed for 8 minutes, then the hot mixture was spooned into silicon moulds, covered and chilled overnight to set.

[0385] Comparison was made to a control cheese, prepared as described above but without the protein concentrate emulsion of the current invention.

[0386] Texture was measured using a Perten TVT 6700XP texture analyser with a 75 mm cylinder probe using 15% compression with a 3 second hold time, and a cutting wire (0.4 mm gauge, 90 mm).

[0387] The cheeses prepared with protein concentrate were measured to have a hardness of 6.24 g compared to a hardness of 29.6 g for the control cheese prepared without protein concentrate indicating an improved softness and texture in the current Example.Example 13: Rubisco protein concentrate used as a cold binder solution in plant-based meat productsBurger

[0388] Method:1. According to Table 3, add seasoning to soy TVP flakes. Hydrate TVP with water, store at 4°C for 45 minutes.2. Using Magimix food processor, add Rubisco protein concentrate (made generally as described in Example 1) to water for mixing, and mix on high shear until a smooth solution is formed (3 minutes). (Skip this step on the control sample without Rubisco protein concentrate)3. Add gellan gum and mix on highest shear setting in the Magimix for 30 sec.4. Slowly pour in oil while mixing on high shear. Emulsify fat into the hydrated mix solution until a smooth, glossy mix is obtained (2-3 minutes).5. Transfer the mix to a planetary mixer and mix with the flat beater attachment for 1 minute at lowest speed.6. Add minced coconut fat, keep mixing for 1 more minute until combined.7. Add starch and mix for 30 s until combined, scraping down the bowl walls in between. This will be the burger mixture.8. Weigh out burger mixture into 100g portions.9. Form each 100g portion into a patty shape using a patty press.10. Place chilled burgers on a tray and chill at 4°C or freeze at -20°C.11. Cook patties in a frying pan flipping every minute until internal temperature reaches 70°C (68-74°C range).Table 3: Burger products* Gellan gum in burger patty with Rubisco protein concentrate can be adjusted to achieve desired texture (0.5-1%).

[0389] The binding agents used were 1% of gellan gum (control sample) or 1% Rubisco protein concentrate + 0.5% gellan gum (see Table 3). Rubisco protein concentrate was able to reduce the amount of gellan gum needed to achieve the same cold binding effect to provide a firm soy TVP patty that held it shape when handled raw. After cooking, Rubisco protein concentrate provided browning and umami flavour (not perceived in the control sample), chewier texture, and a bite sensation closer to animal meat compared to the control sample, which was considered mushy.Hotdog

[0390] A plant-based hotdog was created with the addition of Rubisco protein concentrate as the sole protein binder. 7% of Rubisco protein concentrate (2% total protein) was able to replace soy protein concentrate (2%), methylcellulose (1.5%),and gellan gum (0.5%), which is the typical binder solution found in the market for plant-based hotdogs. Rubisco protein concentrate is a clean label, non-allergenic binder alternative to non-natural ingredients in the typical binder mix (E-461 methylcellulose) and allergens (soy protein concentrate).

[0391] Method:1. According to Table 4, using a Magimix food processor or homogeniser, add soy protein concentrate or Rubisco protein concentrate (made generally as described in Example 1) to one third of the water (18g) and mix on high shear until smooth (3 minutes).2. Slowly pour in canola oil while mixing on high shear into hydrated protein until a smooth, glossy mix is obtained (2-5 minutes). Save this mix3. In a clean food processor, combine remaining water (36g), wheat gluten and seasoning. Blend for 1 minute to hydrate gluten. For the control sample, add the methyl cellulose and gellan gum, blend until homogenous (30s-lmin).4. Add soy or Rubisco pre-emulsion from step 2 and mix well (30s).5. Add starch and mix until combined (lmin), scraping down in between.6. Transfer mix to a planetary mixer and beat to activate and work gluten at medium speed for 5-10 minutes until a homogeneous mixture is achieved.7. Stuff mixture from control or Rubisco concentrate into sausage casings of choice and steam cook hotdogs at 80 °C (range 80-85°C) for 20 minutes (range is 20-40 minutes).8. Chill hotdogs well at 4 °C (min. 6-8 hours).9. Remove peelable sausage casings. Fry hot dogs in a pan (optional).Table 4: Hotdog products

[0392] It is notable that the Rubisco concentrate of the invention can replace all of methylcellulose, gellan gum, and soy protein concentrate to provide a plantbased hotdog with similar protein, moisture, and fat as the control sample (see Table 4).

[0393] The hotdog prepared with Rubisco protein concentrate was observed with be more uniform, shinier, and a bit softer than the control hotdog. The Rubisco protein concentrate hotdog was more homogeneous and the cross-section cut was observed to be cleaner. The control hotdog stuck to the knife when cut and it looked more heterogeneous and "bready".

[0394] During cooking, the Rubisco protein concentrate hotdog browned more than the control hotdog. The aroma of the Rubisco protein concentrate hotdog was described as umami and a bit green, pleasant.

[0395] When eaten, the control was stickier and softer at bite and had a glutinous aftertaste compared to the Rubisco protein concentrate hotdog.Example 14: Rubisco Protein concentrate as a hot gelling agent

[0396] Burger patties were prepared with either Methylcellulose (control) or Rubisco protein concentrate (sample, made generally as described in Example 1) as a hot gelling agent. These samples do not contain any additional gelling agents such as cold-binding polysaccharide.

[0397] First, hydrated textured vegetable protein (TVP, ADM Arcon® T158- 171) was prepared by soaking TVP in 1:2.5 ratio of water for 30 minutes at 4°C. Then the binding solutions were prepared by mixing either methylcellulose or Rubisco protein concentrate with ice cold water (<10 °C) with a high shear homogeniser to mix thoroughly. Then the chilled hydrated TVP was mixed into chilled binding solution using a Magimix blender (1 minute per sample in small mixing bowl). The mix was transferred into large vacuum bags and vacuum sealed to remove whipped-in air bubbles (vac setting >98). The mix was spooned into silicon muffin moulds, pressed down well with a spatula and covered with cling film followed by chilling for 2 hours at 4 °C. The patties were steamed cooked in the silicon mould at bain-marie (150 °C fan forced oven for 1 hr). The cooked patties were cooled at 4 °C overnight and unmoulded to perform the texture profile analysis (analysis was performed at 12 C).

[0398] The final weight of each patty was 115g composed of 80% hydrated TVP, 1% of Methylcellulose, 3% or 4.7% Rubisco protein (total protein content), and the rest was water for mixing or water included in the Rubisco protein concentrate.

[0399] For the control patties, three different grades of methylcellulose were used at 1% of the total weight (Methocel MX (strong binding), Benecel MX (medium binding), and Methocel A4C (weak binding). Two samples with Rubisco protein concentrate were prepared, at 3% and 4.7% protein content of the total weight.

[0400] Texture profile analysis was performed with a TVT 6700 texture analyser (Perten Instruments). A multiple cycle compression test with 50% compression was performed. The instrument's load cell was 5kg and 16 cm x 15 cm in size. The measurements were done with probe code 673075 (7.5cm diameter).

[0401] The sample size was 35mm diameter, about 15mm height, 16g each. 6 replicates were measured for each sample and an average profile curve was obtained for each sample where the values for hardness and chewiness were obtained and both attributes were improved in the samples with Rubisco Protein. Hardness (Figure 6) refers to the maximum load reached during the first compression cycle. It is related to the stiffness of the material. Chewiness (Figure 7) is the energy required to chew a product until is ready to be swallowed, how easy it can be bitten. It is related to the recovery of the material and its viscoelastic properties. As shown in Figure 6 and 7, the Rubisco protein concentrate provided increased hardness and chewiness compared to patties prepared with methylcellulose.

[0402] The inventors have found gelling agents, such as methyl cellulose, and gums and polysaccharides usually used in food preparation, do not need to beincluded in meat replacements using the Rubisco protein concentrate of the invention when the meat replacement product stays in a mould or casing during cooking. For example, hotdogs (pre-cooked product by definition), sausages (enclosed in a casing during cooking), or burger patties that are cooked before unmoulding are non-limiting examples of such products. After cooking, the product will retain its form just due to Rubisco gelling ability. The Rubisco protein concentrate of the invention can be used with such other gelling agents if desired, however.Example 15:

[0403] This example shows one process according to the invention on a 190 kg scale using membrane filtration to increase the content of protein in the clarified juice.

[0404] Clarified juice was obtained following the method in Example 2. The pH of the clarified juice was adjusted to 4.5 using phosphoric acid (85% w / w, Ixom) causing the soluble proteins to precipitate. 190 kg of the acid-precipitated material was loaded onto a 500 kDa UF hollow fibre membrane (Kovalus Separation Solutions) and diafiltrated with 70 of kg water to remove minor impurities and then increase the total solids concentration in the solution. 80 kg of retentate (UFR) was collected at 16.6% TS. 145 kg of ethanol at 55% w / w was added to the UFR in a feed tank and mixed with a paddle. The ethanol / UFR mixture was then loaded onto a microfiltration membrane (MF) (0.1 micron, TiC tubular, Graver Technologies) and diafiltrated with a further 150L of ethanol at 60% w / w. Once 300 kg permeate (MFP) was collected, the material was diafiltrated with two volumes of 150 kg water. 173 kg of water-washed material was collected and measured to be 4.3% TS. This material was then loaded into a scraped-surface evaporator which was run at 35 °C under vacuum (4 kPa) to remove residual ethanol and some water. 38 kg of final product (Rubisco protein concentrate) was collected and determined to be 19.4% TS and 85.1% protein content, with 0.15% w / w ethanol (measured by GC-FID).Example 16: Emulsion composition

[0405] Emulsions were prepared at 1% total solids by dispersing in solution 1.72 g of protein concentrate prepared according to Example 1 (at 23.3% total solid) into a beaker with 18.3 mL of water and 20 mL of canola oil. The sample was homogenised with a benchtop homogeniser (HG-15A, Daihan Scientific, attachment B) at speed 30 for 1 minute at room temperature. The sample was transferred to a 50 mL falcon tube and the emulsion activity (EA) for samples was determined by centrifuging at 1100 RCF for 5 mins at 20 °C. EA was calculated by measuring the height of the emulsified layer. EA was calculated by measuring the height of theemulsified layer (Hi) and the total height of the liquid ( HT) and reported as EA (%) = HI / HT x 100. For determination of emulsion stability after heat treatment (ES) samples were first heated in a water bath at 80 °C for 30 min, then cooled in an ice-water bath for 15 min, and finally centrifuged for 5 min at 1100 RCF at 20 °C. For the calculation of ES, the height of the emulsified layer (H2) was recorded. Emulsion stability was reported as ES [%] = H2 / HT x 100 (Table 5).

[0406] The emulsion activity and emulsion stability of rubisco protein concentrate was higher than soy, casein, pea, and similar to egg white protein for EA. Rubisco protein concentrate was able to hold emulsions at 80 °C for 30 mins indicating a high emulsion stability.Table 5: Emulsion activity and emulsion stability at 1% TS* reported in Jakobson, et al. Techno-Functional and Sensory Characterization of Commercial Plant Protein Powders. Foods 2023, 12 (14), 2805.

[0407] Emulsions were prepared at 5% total solids (TS) by dispersing in solution 6.88 g of protein concentrate prepared according to Example 1 (at 21.8% total solid) with 8.12 mL of water and 15 mL of canola oil in a 50 centrifuge tube. For powders, 1.5g of protein powder was dispersed in 15 mL of water and 15 mL of canola oil in a 50 mL centrifuge tube. The samples were homogenised with a benchtophomogeniser (HG-15A, Daihan Scientific, attachment B) at speed 30 for 1 minute at room temperature, and then centrifuging at 1100 RCF for 5 mins at 20 °C. Emulsion activity was calculated by measuring the height of the emulsified layer (Hl) and the total height of the liquid (HT) and reported as EA (%) = Hl / HT x 100.

[0408] The emulsion activity of the Rubisco protein concentrate prepared according to Example 1, or as subsequently freeze-dried and then rehydrated for emulsion formation, were higher than soy, casein, pea and egg white protein.Table 6: Emulsion activity and emulsion stability at 5% TSExample 17: Foaming formation and foam stability

[0409] Foaming Capability Method: A protein concentrate solution was prepared at 5% total solids in a 100 mL beaker, for example 4.30 g of protein concentrate prepared according to Example 1 (at 23.3% total solid) was combined with 15.7 g of water to provide a 5% TS solution. The sample was homogenised with a benchtop homogeniser (HG-15A, Daihan Scientific, attachment B) at speed 30 for 30 seconds and then allowed to hydrate in the water by stirring at 350 RPM for 2h until homogenous. The sample pH was adjusted to 7 with 10% NaOH or 5% phosphoric acid and transferred to a 50 mL falcon tube and the initial height of the sample measured (Vol / ). The sample was homogenised for 30 seconds at speed 30 and the resultant foam height measured (Voir). Foaming capacity (%) was calculated as [(Voir - Vol,) / Vol,] x 100.

[0410] Heated Foaming Stability Method. A protein foam prepared as described above in a 50 mL falcon tube was placed in a water bath and heated at 85 °C for 15 mins, then allowed to cool to room temperature for 15 mins. The final height of the foam in the sample was measured (Vohr), and the foam stability (%) calculated as(Volw7 Volf) x 100. A non-heated foaming stability was measured as described for the heated foaming stability except with omission of the heating step with foam stability recorded at 30 mins.

[0411] Foaming capacity and stability of Rubisco protein concentrate was measured and compared to soy, egg white, and whey proteins (Table 7).

[0412] The foam capability of Rubisco protein concentrate was higher than soy and pea, comparable to casein, but less than egg white. After 30min, the foam remaining in the Rubisco protein concentrate sample was higher than any other protein measured and comparable to egg, meaning that the foam formed is highly stable.Table 7: Foaming capability, foaming stability and heated foaming stability* reported in Jakobson, et al. Techno-Functional and Sensory Characterization of Commercial Plant Protein Powders. Foods 2023, 12 (14), 2805.Example 18: Oil and Water Holding Capacity

[0413] Oil Holding Capacity Method : A protein concentrate and oil mixture was prepared at 5% total solids by weighing 1.5 g of dried protein concentrate powder(<1% moisture) into a 100 mL beaker and mixing with 30 mL of canola oil. The sample was homogenised with a benchtop homogeniser (HG-15A, Daihan Scientific) at speed 30 for 30 seconds, and then allowed to absorb oil by stirring at 350 RPM for 2h. The sample was transferred to a 50 mL falcon tube and centrifuged at 10,000 RCF for 10 minutes at 15 °C. The supernatant was decanted, and the tubes placed upside down to drain on absorbent paper for 5 minutes before weight the residual pellet. The OHC was calculated as follows: (pellet weight after centrifugation - ingoing protein weight) / ingoing solids weight.

[0414] Water Holding Capacity Method: A protein concentrate and water mixture was prepared at 5% total solids by accurately weighing approximately 6.45 g of protein concentrate (determined to be 23.3% total solid w / w) into a 50 mL falcon tube and adding sufficient addition water to provide a 5% total solids solution (23.55 g).The sample was homogenised with a benchtop homogeniser (HG-15A, Daihan Scientific) at speed 30 for 30 seconds, and then allowed to hydrate in the water by stirring at 350 RPM for 2h. The sample pH was adjusted to 7 with 10% NaOH or 5% phosphoric acid, and then centrifuged at 10,000 RCF for 10 minutes at 15 °C. The supernatant was decanted, and the tubes placed upside down to drain on absorbent paper for 5 minutes before weight the residual pellet. The WHC was calculated as the weight gained by the initial sample solids on a g / g basis (Table 8).Table 8: Oil holding capacity (OHC) and water holding capacity (WHC) of Rubisco protein concentrate* reported in Jakobson, et al. Techno-Functional and Sensory Characterization of Commercial Plant Protein Powders. Foods 2023, 12 (14), 2805.

[0415] Rubisco protein concentrate shows superior OHC when compared to other plant or animal derived proteins, as shown in Table 7 where Rubisco protein concentrate has an average OHC of 3.69 compared to 2.77 for canola, 2.09 for potato, and other protein isolates in the range 0.87-1.7. This high OHC capacity provides for a high retention of oil in products which incorporate Rubisco protein concentrate, enabling superior texture and food attributes.

[0416] Rubisco protein concentrate also shows comparable or superior WHC compared to other common plant or animal derived proteins, with hydration providing a smooth homogenous paste.Example 19: Rubisco Protein concentrate in coarse cut sausages

[0417] A plant-based coarse cut sausage was created with the addition of Rubisco protein concentrate to replace methyl cellulose.

[0418] Method:Ingredients Preparation• According to Table 9, add seasoning to TVP flakes. Hydrate TVP flakes with water, chill well.• Mince coconut fat through 3mm hole plate.Leaf protein and binding agent pre-emulsification• Using a Magimix food processor or homogeniser add rubisco protein concentrate (made generally as described in Example 1) to water for mixing, and mix on high shear until smooth (3 mins).• Add binding agents (gellan gum and / or MCE ) and mix on high shear.• Slowly pour in oil while mixing on high shear. Emulsify fat into hydrated binder solution until a smooth, glossy mix is obtained.Sausage mixture preparation• In planetary mixer, combine hydrated TVP and binding agent pre-emulsion, mix well• Add finely minced coconut fat, mix until combined.• Add starch and mix until combined, scraping down in between.• Mince sausage mixture through 4.5 or 6mm hole plate, as desired.Filling and packing sausages• Stuff mixture into sausage casings of choice• Chill sausages well (min. 6-8 hours)• Pack chilled sausages into vacuum bag or tray, seal and chill (or freeze).Cook sausages in a frying pan rotating every minute until internal temperature reaches 70 °C (68-74 °C range)Table 9Table 10

[0419] Three variations of coarse-ground sausage were prepared using different binding agents: Rubisco protein concentrate with gellan gum, only gellan gum, and methylcellulose (see Table 8). The sausage with Rubisco protein concentrate was glossier and darker, closer to traditional animal-based sausages. During pan frying, this sausage browned more (similar to a grilled sausage) and released minimal oil, in contrast to the control sausages, which released a noticeable amount of oil. Without wishing to be bound by theory, the inventors believe this difference is due to Rubisco's higher oil-holding capacity, resulting in a chewier, juicier texture. The Rubisco sausage also had a more heterogeneous, meat-like texture when cut, whereas the control sausages appeared and tasted mushier. In terms of flavour, the control had a beany, mushy profile, while the Rubisco sausage delivered an umami, salty, and chewy experience without any beany notes. The raw sausage mixture is placed in a casing and cooked within that casing. Rubisco protein concentrate gels at cooking temperatures and it effectively binding the TVP particles together when pan frying. Since the sausages are cooked within a casing, gellan gum is not required, making Rubisco protein concentrate a viable full replacement for methylcellulose in this application.Example 20: Barista milk foam

[0420] The ability of Rubisco protein concentrate to improve the foaming capability of oat milk was assessed by adding 0%, 0.5%, 1%, or 5% Rubisco protein concentrate to non-barista oat milk followed by frothing with an espresso machine steam wand to 65°C. First, all of the samples were heated to 85 °C for 5 minutes under slow mixing (setting 1 on Thermomix and setting 10 on overhead homogenizer) to mimic pasteurisation and demonstrate that the addition of Rubisco protein does not alter the structure of the oat milk. After frothing, the 0% sample exhibited a small foam layer that was stable for at least 15 minutes. The 0.5% sample displayed a foamlayer 50% higher than 0% inclusion sample and also stable for at least 15 minutes. The 1% sample had similar foam height to 0.5% but the solution was less stable, and some precipitated particles were observed. The 5% sample produced a very dense foam but the solution started to gel and separated in two phases (Rubisco protein gel and oat milk). This shows that an inclusion of Rubisco protein concentrate between 0.5% and 1% improves the foaming capacity of oat milk.Example 21

[0421] Emulsion preparations were made by combining rubisco protein concentrate prepared by the process disclosed in Example 1 (1 part, 1 g) with sufficient parts water (grams of water) to provide a calculated target hydration value, with mixing on a high-shear Silverson mixer at 3500 rpm for four minutes. Canola oil was added slowly in parts to the mixture with mixing at 7500 rpm for incorporation until the oil was fully incorporated into the emulsion. Additional oil was added until it was observed that the emulsion split, which was recorded as the emulsifying capacity at the target protein hydration as oil I gram of protein (Table 11).Table 11

[0422] It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention as set out in the appended claims.

Claims

What we claim is:

1. A Rubisco protein concentrate, that may be used as a food ingredient, having a water content of at least about 50% (w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, and a dry matter content between 5% - 50% w / w, wherein the dry matter comprises Rubisco protein in an amount between 70% - 90% w / w, wherein the Rubisco protein concentrate also comprises one or more of the following characteristics: i. an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate); ii. an oil holding capacity of greater than about 2.5 g / g;Hi. a water holding capacity of between about 2.5 and about 3.5 g / g; iv. less than about 0.5% total saponins; v. less than about 0.5% flavones and polyphenols compounds; vi. a lower alcohol content of less than about 0.5%; and vii. a foaming stability after 30 minutes of between about 55% and about 90%.

2. The Rubisco protein concentrate of claim 1, wherein the Rubisco protein concentrate has a water content of between about 80% and about 99.2%w / w and the Rubisco protein concentrate is able to emulsify between about 2.5g and about 60 g oil per gram of Rubisco protein.

3. The Rubisco protein concentrate of claim 1 or claim 2, wherein the Rubisco protein concentrate has a water content of between about 90 and about 99.2% w / w the Rubisco protein concentrate is able to emulsify between about 25g and about 60 g oil per gram of Rubisco protein.

4. A Rubisco protein concentrate having a water content of at least about 50% (w / w), CIE colour values of a* > -1; b* > 10; and L* > 50, an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate), an oil holding capacity of between about 2.5 and about 6.9g / g, and a water holding capacity of between about 2.5 and about 3.5 g / g.

5. The Rubisco protein concentrate of claim 1 or claim 4, wherein the water content is between about 50% and 75% w / w or between 80% and 99.5% w / w.

6. The Rubisco protein concentrate of any one of the previous claims, comprising CIE colour values of a* > 0; b* > 10; and L* > 50.

7. The Rubisco protein concentrate of any one of the preceding claims, comprising an oil holding capacity of between about 3.0 and about 5.0 g / g.

8. The Rubisco protein concentrate of any one of the preceding claims, comprising a water holding capacity of between about 2.7 and about 3.4g / g.

9. The Rubisco protein concentrate of any one of the preceding claims, comprising an ethanol content of less than about 0.5%.

10. The Rubisco protein concentrate of any one of claims 4 to 9, comprising less than about 0.5 % total saponins and / or less than about 0.5% flavones and polyphenols compounds.

11. The Rubisco protein concentrate of any one of claims 4 to 10, comprising a water content between about 50% and about 95% w / w and a dry matter content between about 5% to about 50% w / w, wherein the dry matter comprises Rubisco protein in an amount between about 70% to about 90% w / w.

12. The Rubisco protein concentrate of any one of claims 4 to 11, comprising an emulsion activity of between about 70% and about 90% (when measured at 5% protein inclusion rate).

13. The Rubisco protein concentrate of any one of claims 4 to 12, comprising a water content of between about 80% and about 99.2%w / w and being able to emulsify between about 2.5g and about 60 grams oil per gram of Rubisco protein14. The Rubisco protein concentrate of any one of claims 4 to 13, comprising a water content of between about 90% and 99.2% w / w and being able to emulsify between about 25g and about 60 grams oil per gram of Rubisco protein.

15. The Rubisco protein concentrate of any one of claims 4 to 14, comprising a foaming stability after 30 minutes of between about 55% and about 90%.

16. A food product comprising the Rubisco protein concentrate of any one of the preceding claims.

17. The food product of claim 16, comprising one or more additional ingredients selected from a lipid, a carbohydrate, a protein, a flavour, a vitamin, a mineral, a milk product, water, a food additive, a colour, a fruit preparation, or any combination of any two or more of these ingredients.

18. The food product of claim 16 or claim 17, wherein the food product is selected from(i) emulsified foods selected from mayonnaises, dressings, sauces, condiments, and high-fat fillings; dairy alternatives such as plant-based milks, yoghurts, cheeses, spreads, creams, and creamers;(ii) frozen and / or chilled whipped items selected from fruit ices, ice creams, mousses, custards and desserts; confectionery items such as meringues, candies;(iii) prepared and / or instantised beverages selected from sports drinks, shakes, juices;(iv) plant-based egg alternatives selected from egg analogues, replacers, and substitutes;(v) formed, emulsified and / or extruded plant-based meat and fish analogues selected from burgers, sausages, hot dogs, salami, cold cuts, cuts, strips and pieces, and frozen portions and meat snacks;(vi) bakery items selected from breads, cakes, wraps, biscuits, crackers, bars, and pastries;(vii) extruded products selected from snack foods, pasta, and breakfast cereals.

19. A food product comprising an emulsion of Rubisco protein:oil:aqueous phase in a ratio of about 1 :2.5:5.3 to about 1 :54.5: 108 (w / w), wherein the emulsion is formed by combining: i. a Rubisco protein concentrate according to any one of claims 1 to 16; ii. an edible oil; andiii. water.

20. The food product of claim 19, wherein the edible oil comprises an oil or fat selected from one or more of canola, coconut, rice, sunflower, olive, rice bran, soybean, rape, vegetable, palm, and flax oil, and animal fats.

21. An emulsifying extender composition comprising a Rubisco protein concentrate having a water content between 50% - 99.5%; CIE colour values of a* > -1; b* > 10, and L* > 50; an emulsion activity of greater than about 70% (when measured at 5% protein inclusion rate); an oil holding capacity of between about 2.5 and about 6.9g / g; a water holding capacity of between about 2.5 and about 3.5 g / g; a dry matter content between about 5% to about 50% w / w, and ethanol; wherein the dry matter comprises protein in an amount between about 70% to about 90% w / w, and wherein the ethanol is present at a concentration of less than about 0.5%.

22. A process for producing a Rubisco protein concentrate according to any one of claims 1 to 14 from a biomass material comprising Rubisco protein, the process comprising steps of: i. providing a clarified juice from a biomass material comprising Rubisco protein, wherein the clarified juice is at pH 6.0 to 8.0 and comprises between about 50 to about 95% w / w Rubisco protein, ii. subjecting the clarified juice to acid precipitation at a pH between about 3.0 to about 5.5 to precipitate Rubisco protein from the clarified juice to produce a liquid and a solid protein fraction, iii. separating the liquid from the solid protein fraction, iv. contacting the solid protein fraction with a first solvent or solvent mixture comprising a lower alcohol, v. separating the solid protein fraction from the first solvent or solvent mixture, vi. contacting the solid protein fraction with a second solvent or solvent mixture comprising a lower alcohol, vii. separating the solid protein fraction from the second solvent or solvent mixture, andviii. washing the solid protein fraction with water to remove residual solvent from the solid protein fraction and to produce the Rubisco protein concentrate, wherein the water content of the Rubisco protein in steps (i) to (iii) is above 50% w / w, and wherein the water content Rubisco protein concentrate after water washing in step (viii) is above 50% w / w.

23. The process of claim 22, wherein the biomass material is selected from alfalfa, kudzu, forage peas, oats, duckweed, spinach, kale, cocksfoot, Italian ryegrass, Raphno, clover, Persian clover, rapeseed leaves, carrots, radishes, Jerusalem artichoke, beets, spinach, fodder beet; triticale, white clover, barley, tobacco, chicory, sugar cane, or fava bean leaves, or from an algae selected from the classes Bacillariophyceae, Chloropyceae, Cyptophyceae, Dinophyceae, Euglenophyceae, and Rhodophyceae.

24. The process of claim 23, wherein the biomass material is alfalfa and pH of the clarified juice is between about 6.3 to 6.5.

25. The process of any one of claims 22 to 24, wherein the acid used in the acid precipitation step (ii) is acetic acid, ascorbic acid, citric acid, lactic acid, propionic acid, succinic acid, sorbic acid, tartaric acid, phosphoric acid, sulfuric acid, or hydrochloric acid.

26. The process of any one of claims 22 to 25, wherein the solid protein fraction is separated from the liquid fraction in step (iii) using a centrifuge, disc stack centrifuge, clarifier, decanter, sedicanter, filter press / coarse filter, settling tank, hydrocyclone, microfiltration, or ultrafiltration.

27. The process of any one of claims 22 to 26, wherein the first solvent or solvent mixture and the second solvent or solvent mixture comprise a lower alcohol solvent or a mixture of lower alcohol solvents.

28. The process of claim 27, wherein the first and second solvents are both ethanol.

29. The process of any one of claims 22 to 28, wherein steps (iv) to (vii) are repeated until the solid protein fraction comprises CIE colour values of a* > - 1; b* > 10; and L* > 50.

30. The process of any one of claims 22 to 29, wherein the water washing step(viii) is repeated until the Rubisco protein concentrate comprises less than about 0.5% residual solvent.

31. The process of any one of claims 22 to 30 wherein extraction steps (iv) to (vii) are performed using a continuous process.

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