Extraction process

Abstract

The invention relates to an extraction process for the recovery of valuable nutritional components from traditional so-called food waste streams and related feedstocks. In particular, the present invention relates to a fractionation process for the recovery of proteins and soluble dietary fibres, insoluble fibres and sugars from feedstock such as barley-derived brewer's spent grain and the like. However, it will be appreciated that the invention is not limited to these particular fields of use.

Description

EXTRACTION PROCESSField of the Invention

[0001] The present invention relates to an extraction process for the recovery of valuable nutritional components from a feedstock. In particular, the present invention relates to a fractionation process for the recovery of proteins and carbohydrates from cereal grain derived feedstocks such as barley, wheat, oats, malted barley, and brewer’s spent grain (BSG) and the like.

[0002] The process of the present invention can produce food-grade products from low and high-starch containing feedstock such as brewer’s spent grain (BSG) by recovering proteins and carbohydrate fractions which would otherwise be disposed of as waste, fertiliser or used as low value livestock feed. However, it will be appreciated that the invention is not limited to these particular fields of use.Background of the Invention

[0003] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0004] Globally, significant amounts of food and beverage waste are produced each year (over 1 billion tonnes of food waste / year), whilst simultaneously over 700 million people are undernourished. One source of significant waste produced in the food and beverage industry is from beer production. Beer production involves many processes and different ingredients which generates a variety of waste or byproducts.

[0005] Beer is the fifth most consumed beverage in the world apart from tea, carbonates, milk and coffee with an estimated annual world production exceeding 1.3 billion hectolitres per year. Various byproducts are generated during the production of beer. The most common byproducts are spent grains (brewer’s spent grain), spent hops and surplus yeast.

[0006] Brewer’s spent grains is a byproduct of the mashing and lautering process which is one of the initial treatment steps in a brewery to solubilise starch malt and cereal grains to ensure adequate extraction of the wort (water with extracted matter). Typically, the amount of brewers’ spent grain produced in the brewing process can be about 85% of the total byproducts generated. Globally over 40 million tonnes of wet brewer spent grain waste is produced each year. Australia produces over 300,000 tonnes of wet brewer spent grain waste is producedeach year, mainly derived from barley. On that, Australia is considered a leading global producer of barley and other cereal grains in terms of quality, quantity, affordability and efficiency thanks to its favourable environment, soils, political stability, transport infrastructure and agriculture industry. In most recent years the country has been producing over 10 million tonnes of barley grain per annum and over 20million tonnes of wheat. Biorefining these types of feedstocks as geographically close to the source as possible provides process efficiency and fundamental unit cost advantages.

[0007] There is a significant amount of barley-derived brewers spent grain feedstock which is usually sent to landfill or used by farmers as a low-value livestock feed.

[0008] Owing to the amount produced annually globally, current low market value, increasing environmental awareness, and the recognition that brewers spent grain (BSG) may represent a nutritionally valuable co-product, developments have been made for the valorisation of this byproduct. Developments have been made to valorise BSG for applications in food and beverage products, pharmaceutical applications and cosmetics.

[0009] The present inventors have developed a fractionation process to extract valuable components from a feedstock such as BSG for use in the food, beverage and other industry.

[0010] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0011] Other prior art has one or more disadvantages, from the list of, but not limited to; lower protein yields, lower protein purity, lack of process synergies, lack of synergistic protein recovery, lack of synergistic soluble dietary fibre recovery, lack of synergistic sugar recovery, more expensive process, more complex processing, more energy intensity per unit of output, environmentally damaging chemical usage, lower protein sensory qualities.

[0012] Although the invention will be described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.Summary of the Invention

[0013] Significant wastage of feedstock which can end up in landfill or as feed for animals has driven the desire to develop extraction processes to recover valuable food-grade components which can have both commercial and environmental benefits. In particular, the present inventors have developed a process to recover valuable food-grade components such as protein and carbohydrates (including soluble dietary fibres) from barley-derived brewer’s spent grain in an efficient and cost-effective manner.

[0014] According to one aspect, the present invention provides a process for the fractionation of a feedstock, comprising the steps of: a) providing a feedstock comprising a bran;b) treating the feedstock with a protein solubilisation agent to provide a liquid fraction comprising protein and a solid fraction; and c) precipitating the protein from the liquid fraction to provide a protein fraction.

[0015] In some embodiments, the feedstock is treated to improve the quality and quantity of fractionated products. In some embodiments, the process further comprises d) treating the solid fraction with a carbohydrate solubilisation agent to provide a liquid carbohydrate fraction and a second solid fraction. In some embodiments, the process comprises e) precipitating a carbohydrate from the liquid carbohydrate fractions with a precipitating agent.

[0016] In certain embodiments, wherein in step c), the process further comprises precipitating the liquid fraction with a precipitating agent to provide a carbohydrate fraction. In certain embodiments, wherein in step d), the process further comprises precipitating a protein from the liquid carbohydrate fraction to provide a protein fraction. In certain embodiments, wherein in step d), the process further comprises treating the second solid fraction with a carbohydrate solubilisation agent to provide a second liquid carbohydrate fraction. In certain embodiments, the process further comprises precipitating a protein from the second liquid carbohydrate fraction to provide a protein fraction. In some embodiments, the process further comprises precipitating a carbohydrate from the second liquid carbohydrate fraction with a precipitating agent to provide a carbohydrate fraction.

[0017] As discussed above, the process of the present invention can provide a solid and a liquid fraction comprising at least one of a protein and a carbohydrate, in each fraction respectively. This can provide recovery of at least one of a protein and a carbohydrate from a single fraction (liquid or solid or both). Each solid or liquid fraction can be further treated or processed sequentially using the process of the invention to generate further downstream fractions (i.e., second, third, fourth and so on liquid and / or solid fractions) for further recovery of the valuable food-grade components. Advantageously, in these embodiments, a higher total yield of protein and / or carbohydrate can be recovered from the feedstock which can reduce wastage.

[0018] In certain embodiments, the process further comprises treating the feedstock with an amylase and, or beta glucanase to substantially convert starch and, or beta glucans to smaller sacharrides, to help extract bound protein, and to enable production of a more pure soluble dietary fibre extract.

[0019] In certain embodiments, the process comprises treating a solid fraction (including or in the alternative a downstream solid fraction) with a cellulase. In these embodiments, the cellulase can catalyse cellulolysis to break down cellulose into monosaccharides, oligosaccharides or polysaccharides. In certain embodiments, the cellulase breaks downcellulose into monosaccharides (simple sugars). Advantageously, this can further provide an improved or additional value product stream.

[0020] As used herein, the term “bran” refers to the hard outer layers of a grain such as barley malt grain or wheat grain, and optionally the germ, but excludes the majority of the starch rich endosperm which will have previously been predominantly removed via fractionation and / or fermentation. Bran typically comprises the combined aleurone and pericarp and optionally the germ. Bran is often produced as a byproduct of milling in the production of refined grains, however, can also include byproducts from other processes such as brewing beer and fermentation. Where fermentation byproducts (i.e. spent yeast) are comingled with the bran (i.e. spent grain) this would all be considered Bran under this definition.

[0021] As used herein, the term “carbohydrate” and “protein” recovered by the process of the present invention comprises isolation of the carbohydrate and / or protein from the initial solid and / or liquid fractions as well as further downstream solid and / or liquid fractions.

[0022] In some embodiments, the feedstock has a protein content of between about 5% to about 50%, preferably between about 15% to 30%. In some embodiments, the feedstock is selected from the group consisting of brewers spent grain, oat bran, distillers spent grain, wheat bran, barley bran, yeast, reduced carbohydrate barley, reduced carbohydrate wheat, whole wheat, whole barley, whole oats and combinations thereof. In some embodiments, the feedstock is malted. In some embodiments, the feedstock is unmalted.

[0023] In another aspect, the present invention provides a protein and / or a carbohydrate produced by the process as described herein.Definitions

[0024] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0025] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0026] As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” (or variations thereof) appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only theelement set forth in that clause; other elements are not excluded from the claim as a whole. As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified elements or method steps, plus those that do not materially affect the basis and novel characteristic(s) of the claimed subject matter.

[0027] With respect to the terms “comprising”, “consisting of’, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of’ or, alternatively, by “consisting essentially of’.

[0028] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about”. The examples are not intended to limit the scope of the invention. In what follows, or where otherwise indicated, “%” will mean “weight %”, “ratio” will mean “weight ratio” and “parts” will mean “weight parts”.

[0029] The term “substantially” as used herein shall mean comprising more than 50% by weight, where relevant, unless otherwise indicated.

[0030] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0031] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0032] It must also be noted that, as used in the specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0033] The prior art referred to herein is fully incorporated herein by reference.

[0034] Although exemplary embodiments of the disclosed technology are explained in detail herein, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosed technology be limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosed technology is capable of other embodiments and of being practiced or carried out in various ways.Brief Description of the Drawings

[0035] Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0036] Figure 1 shows a representative flowchart of an embodiment of the fractionation process of the present invention.Detailed Description of the Invention

[0037] The skilled addressee will understand that the invention comprises the embodiments and features disclosed herein as well as all combinations and / or permutations of the disclosed embodiments and features.

[0038] According to one aspect, the present invention provides a process for the fractionation of a feedstock, comprising the steps of: a) providing a feedstock comprising a bran; b) treating the feedstock with a protein solubilisation agent to provide a liquid fraction comprising protein and a solid fraction; and c) precipitating the protein from the liquid fraction to provide a protein fraction.Fractionation

[0039] In some embodiments, the feedstock is a cereal grain with a reduced starch fraction. In some embodiments, the process further comprises d) treating the solid fraction with a carbohydrate solubilisation agent to provide a liquid carbohydrate fraction and a second solid fraction. In some embodiments, the process comprises e) precipitating a carbohydrate from the liquid carbohydrate fraction with a precipitating agent.

[0040] In certain embodiments, wherein in step c), the process further comprises precipitating the liquid fraction with a precipitating agent to provide a carbohydrate fraction. In certain embodiments, wherein in step d), the process further comprises precipitating a protein from the liquid carbohydrate fraction to provide a protein fraction. In certain embodiments, wherein in step d), the process further comprises treating the second solid fraction with a carbohydrate solubilisation agent to provide a second liquid carbohydrate fraction. In certain embodiments, the process further comprises precipitating a protein from the second liquid carbohydrate fraction to provide a protein fraction. In some embodiments, the process further comprises precipitating a carbohydrate from the second liquid carbohydrate fraction with a precipitating agent to provide a carbohydrate fraction.

[0041] The process of the present invention can provide a solid and a liquid fraction comprising at least one of a protein and a carbohydrate, in each fraction. This can provide recovery of at least one of a protein and a carbohydrate from a single fraction (liquid or solid or both). Each solid or liquid fraction can be further treated or processed sequentially using theprocess of the invention to generate further downstream fractions (i.e., second, third, fourth and so on liquid and / or solid fractions) for further recovery of the valuable food-grade components. Advantageously, in these embodiments, a higher total yield of protein and / or carbohydrate can be recovered from the feedstock which can reduce wastage either by recovering both a protein and a carbohydrate from a single fraction (either solid or liquid) or by performing the process of the invention sequentially. Doing so can also allow less intense treatment conditions than would otherwise need to be used, improving the quality of the recovered proteins. In some embodiments the second or third pass provides higher yields than previous passes.

[0042] In some embodiments, the process is a batch process. In some embodiments, the process is a continuous process.

[0043] In certain embodiments, the carbohydrate is a dietary fibre.Feedstock

[0044] As would be appreciated by a skilled addressee, any suitable feedstock comprising a bran can be used in the present invention. In some embodiments, the feedstock is derived from a cereal grain. In some embodiments, the feedstock is derived from the group selected from at least one of a rice, corn (also known as maize), wheat, oats, barley, rye, millet and combinations thereof. In some embodiments, the feedstock is selected from the group consisting of brewers spent grain (BSG), oat bran, wheat bran, barley bran, distillers spent grain, yeast, reduced carbohydrate wheat, reduced carbohydrate barley and combinations thereof. In preferred embodiments, the feedstock is BSG, also known as barley with a reduced starch fraction.

[0045] BSG is a lignocellulosic material typically rich in carbohydrates (such as cellulose, hemicelluloses, fibre), lignin and protein. The composition of BSG can vary due to a number of factors such as climate, growth conditions, the malting process used, the brewing / starch fractionation process used and the type of barley used. In a typical BSG, about up to 70% of the composition by dry weight comprises carbohydrates in the form of fibre (mainly hemicellulose and cellulose), and up to 30% protein by dry weight as well as other components such as lipids and phenols.

[0046] In some embodiments, the feedstock has a protein content of between about 5% to about 60% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about 5% to about 40% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about 5% to about 30% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about 5% to about 15% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about5% to about 10% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about 10% to about 40% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about 15% to about 35% on a dry weight basis. In some embodiments, the feedstock has a protein content of between about 20% to about 30% on a dry weight basis. In preferred embodiments, the feedstock has a protein content of between about 20% to about 25% on a dry weight basis.

[0047] In some embodiments, the feedstock has a carbohydrate content of between about 10% to about 90% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 10% to about 80% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 10% to about 70% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 20% to about 70% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 30% to about 70% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 30% to about 60% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 30% to about 50% on a dry weight basis. In some embodiments, the feedstock has a carbohydrate content of between about 35% to about 45% on a dry weight basis.

[0048] In some embodiments, the feedstock has a starch content of between about 0.5% to about 60% on a dry weight basis. In some embodiments, the feedstock has a starch content of between about 0.5% to about 40% on a dry weight basis. In some embodiments, the feedstock has a starch content of between about 0.5% to about 30% on a dry weight basis. In some embodiments, the feedstock has a starch content of between about 0.5% to about 20% on a dry weight basis. In some embodiments, the feedstock has a starch content of between about 0.5% to about 10% on a dry weight basis. In some embodiments, the feedstock has a starch content of between about 3% to about 10% on a dry weight basis. In some embodiments, the feedstock has a starch content of between about 3% to about 8% on a dry weight basis.

[0049] As would be understood by a skilled addressee, the feedstock can be used directly in its original form or can be comminuted prior to be used in the process of the present invention. Any suitable comminution method can be used. In some embodiments, comminution can be performed by milling. In this embodiment, the milling step is performed typically using wet milling with high shear such as colloid mill, comitrol mill or in-line high shear pump milling. However, as would be appreciated by a skilled addressee, other mills can be used such as high pressure grinding rolls or dry milling such as with a hammer mill, air classifiermill, jet mill or pin mill. Wet milling comminutes (crushes, grinds, shears, impacts or beats) a material (product material, in this case feedstock to be reduced to smaller dimensions.

[0050] In some embodiments, the milling step can be performed dry. In preferred embodiments, the milling step is performed wet. In preferred embodiments, the milling step is performed in the presence of an aqueous solution or water. Typically, the feedstock is mixed with a water or an aqueous solution and retaining the mixture as a slurry during the milling step.

[0051] As would be appreciated by a skilled addressee, any suitable amount of feedstock by weight of water or aqueous solution can be used during the milling step to provide the desired comminuted particle size. In some embodiments the minimum amount of aqueous solution or water is added to maximise the viscosity of the slurry increasing the impact and shearing forces imparted on particles, to the optimum extent without making handling and pumpability of the slurry unviable.

[0052] In preferred embodiments, the feedstock is comminuted using one or more rotating metal blades. In further preferred embodiments, the feedstock is comminuted using a blender or similar high shear wet mill. In preferred embodiments, the feedstock is comminuted in the presence of water or an aqueous solution.

[0053] In some embodiments, the feedstock comprises greater than about 5%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50% by weight of the water or aqueous solution during comminution. In certain embodiments, the feedstock comprises between about 5% to about 70% by weight of the water or aqueous solution, between about 5% to about 60% by weight of the water or aqueous solution, between about 5% to about 50% by weight of the water or aqueous solution, between about 5% to about 40% by weight of the water or aqueous solution, between about 5% to about 30% by weight of the water or aqueous solution, between about 5% to about 20% by weight of the water or aqueous solution, between about 10% to about 20% by weight of the water or aqueous solution or about 15% by weight of the water or aqueous solution during comminution.

[0054] The comminuted particles of the feedstock can be any suitable size for use in the present invention. In some embodiments, the average diameter (dso) of the comminuted particles is less than about 10 mm, less than about 9 mm, less than about 8 mm, less than about 7 mm, less than about 6 mm, less than about 5 mm, less than about 4 mm, less than about 3 mm, less than about 2 mm, less than about 1 mm, less than about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm or less than about 0.25 mm. In some embodiments, the average diameter (dso) of the comminuted particles is between about 0.1 mm to about 1 mm, between about 0.01 mm to about 1 mm, between about 0.01 mm to about 0.8 mm,between about 0.25 mm to about 1 mm, between about 0.25 mm to about 1 mm or between about 0.05 mm to about 0.7 mm.

[0055] The particle size of the comminuted particles can be characterised using conventional techniques such as transmission electron microscopy or scanning electron microscopy, optical microscopy, dynamic light scattering and the like.

[0056] As would be appreciated by a skilled addressee, the comminution step can be performed for any suitable duration. In certain embodiments, the duration of the milling step is less than about 48 hours, less than about 36 hours, less than about 24 hours, less than about18 hours, less than about 12 hours, less than about 6 hours, less than about 3 hours, less than about 2 hours, less than about 1 hour, less than about 30 minutes, less than about 15 minutes or less than about 5 minutes. In certain embodiments, the duration of the comminution step is between about 0.01 hours to about 48 hours, between about 0.01 hours to about 36 hours, between about 0.01 hours to about 36 hours, between about 0.01 hours to about 24 hours, between about 0.01 hours to about 18 hours, between about 0.01 hours to about 18 hours, between about 0.01 to about 48 hours, about 1 hour.

[0057] In an embodiment, the method further comprises pre-treating the comminuted wet particles with a pulsed electric field. This pre-treatment step may advantageously reduce microbial growth and / or open the cell structure and / or further increase the surface area. In one form of this embodiment, the pulsed electric field pre-treatment is between about 1 to about 10 kV / cm, with about a 1 to 5 cm electrode gap, with about 10 to 15 pulses per volume element, with an energy input of about 5 kJ / kg to 50kJ / kg.

[0058] In some embodiments, the process of the present invention further comprises a high- shear force aggressive redispersion of the protein fraction followed by a water wash under acidic conditions followed by secondary centrifugation to assist with removing sugars, salts and similar impurities to improve protein purity.

[0059] In an embodiment, the method further comprises pre-treating the comminuted wet particles with hydrodynamic cavitation. This pre-treatment step may advantageously reduce microbial growth and / or open the cell structure and / or further increase the surface area and / or efficiently heat the wet feedstock. In one form of this embodiment, the hydrodynamic cavitation conditions occur in the pressure range of 100-5000bar and temperature range of 727 Celsius to 9727 Celsius.

[0060] In an embodiment, the method further comprises pre-treating the comminuted wet particles with an aquas alcohol (ethanol). This pre-treatment step may advantageously reduce microbial growth and / or open the cell structure and / or further increase the surface area, and / or assist with the release of alcohol soluble proteins. In one form of this embodiment, the aquasalcohol conditions are 0.01 %-30% alcohol v / v, 0.1 %-25% alcohol v / v, 1 %-20% alcohol v / v, 1 .5- 18% or 2-15%.

[0061] In an embodiment, the method further comprises pre-treating the comminuted wet particles with an amylase or beta glucanase enzyme to convert (residual) starch or beta glucan polysaccharides to simple sugars. In certain embodiments, the amylase and, or beta glucanase, is selected from the group consisting of a-amylase, p-amylase, y-amylase (also known as glucoamylase) beta glucanase and combinations thereof.

[0062] In certain embodiments, the concentration of the amylase and, or the beta glucanase is less than about 5 mg / mL, less than about 2 mg / mL less than about 1 .5 mg / mL, less than 1 .2 mg / mL, less than 1 mg / mL, less than 0.8 mg / mL or less than 0.6 mg / mL. In certain embodiments, the concentration of the amylase is between 0.1 mg / mL to about 2 mg / mL, between 0.1 mg / mL to about 1 .5 mg / mL, between 0.1 mg / mL to about 1 .2 mg / mL, between 0.1 mg / mL to about 1 mg / mL, between 0.1 mg / mL to about 0.8 mg / mL, between 0.2 mg / mL to about 0.8 mg / mL, between 0.3 mg / mL to about 0.8 mg / mL, between 0.3 mg / mL to about 0.6 mg / mL, between 0.4 mg / mL to about 0.6 mg / mL or about 0.5 mg / mL.

[0063] As would be appreciated by a skilled addressee, any suitable duration can be used for the reduction of starch and, or beta glucan polysacharise in the process of the present invention. In some embodiments, the starch and, or beta glucan polysaccharide reduction step using an amylase or beta glucanase is performed for a duration between about 5 minutes to about 30 minutes, between about 5 minutes to about 20 minutes, between about 5 minutes to about 15 minutes, between about 10 minutes to about 5 hours, between about 10 minutes to about 3 hours, between about 10 minutes to about 2.5 hours, between about 30 minutes to about 2.5 hours, between about 30 minutes to about 2 hours, between about 20 minutes to about 40 minutes, between about 1 hour to about 2.5 hours, about 10 minutes, about 30 minutes or about 2 hours.

[0064] As would be appreciated by a skilled addressee, any suitable temperature can be used for the reduction of starch and, or beta glucan polysaccharide using an amylase and, or beta glucanase in the process of the present invention. In some embodiments, the starch, and, or beta glucan polysaccharide removal step using an amylase and, or beta glucanase is performed at a temperature greater than about 20°C, greater than about 30°C, greater than about 40°C, greater than about 50°C or greater than about 60°C. In some embodiments, the starch, and, or beta glucan polysaccharide removal step using an amylase and, or beta glucanase is performed at a temperature between about 20°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about 40°C to about 90°C, between about 40°C to about 80°C, between about 40°C to about 70°C, between about 40°C to about 60°C, between about 50°C to about 60°C or about 55°C.

[0065] As would be appreciated by a skilled addressee the starch and, or beta glucan polysaccharide fraction can be partially to majority removed from the feedstock during pretreatment by options including; draining the liquefied and / or saccharified starch and, or saccharified beta glucan polysacharide fraction from the bran via a suitable sieve as in a traditional beer production process; by centrifuging the feedstock, by dry fractionating the feedstock as a pre-treatment using air classification and / or triboelectric effect; by in-situ wet fermentation conversion of the starch fraction into yeasts and, or ethanol, or other yeast expressed product, or by letting the liquified and saccharified fraction carry through the process to end up in the aqueous by-product stream.Protein solubilisation and precipitation

[0066] As described herein, the process of the present invention comprises use of a protein solubilisation agent to provide a liquid fraction or downstream liquid fraction for the recovery of a protein. In these embodiments, the protein solubilisation agent is selected from the group consisting of a base, alcohol, protease and combinations thereof. In preferred embodiments, the solubilisation agent is a base.

[0067] As would be appreciated by a skilled addressee, any suitable base can be used in the present invention to solubilise protein. In certain embodiments, the base is selected from the group consisting of a metal oxide, hydroxide, alkoxide, conjugate base of a weak acid, amine, ammonia and combinations thereof.

[0068] In certain embodiments, the metal oxide is selected from the group consisting of lithium oxide, sodium oxide, potassium oxide, rubidium oxide, cesium oxide, magnesium oxide, calcium oxide, strontium oxide, barium oxide, thallium(l) oxide, bismuth(ll) oxide and combinations thereof.

[0069] In certain embodiments, the hydroxide is selected from the group lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, thallium(l) hydroxide, bismuth(ll) hydroxide, tetramethylammonium hydroxide and combinations thereof. In preferred embodiments, the hydroxide is selected from the group consisting of sodium hydroxide, potassium hydroxide, magnesium hydroxide and combinations thereof.

[0070] In certain embodiments, the alkoxide is selected from the group consisting of methoxide, ethoxide, propoxide, butoxide and combinations thereof.

[0071] In certain embodiments, the amine is selected from the group consisting of methylamine, dimethylamine, trimethylamine, ethylamine, aniline, 4-methoxyaniline, N,N- dimethylaniline, 3-nitroaniline, 4-nitroaniline, 4-trifluoromethylaniline, N,N- diisopropylethylamine and combinations thereof.

[0072] In certain embodiments, the protein solubilisation agent is an alcohol. In some embodiments, the alcohol is selected from the group consisting of methanol, ethanol, 1- propanol, isopropyl alcohol, butanol (n-butanol, sec-butanol, isobutanol and tert-butanol) and combinations thereof.

[0073] In certain embodiments, the protein solubilisation agent is a peptidase. In some embodiments, the peptidase is an exopeptidase. In some embodiments, the peptidase is selected from the group consisting of a aminopeptidase, a carboxypeptidase and combinations thereof.

[0074] As would be appreciated by a skilled addressee, any suitable amount or concentration of the protein solubilisation agent can be used in the process of the present invention. In certain embodiments, the concentration of the base used to solubilise protein is less than about 0.5 M, less than about 0.4 M, less than about 0.3 M, less than about 0.25 M, less than about 0.2 M, less than about 0.15 M, less than about 0.1 M or less than about 0.05 M. In certain embodiments, the concentration of the base is between about 0.01 M to about 0.5 M, between about 0.01 M to about 0.4 M, between about 0.01 M to about 0.3 M, between about 0.01 M to about 0.2 M, between about 0.01 M to about 0.15 M, between about 0.01 M to about 0.1 M, between about 0.05 M to about 0.1 M, between about 0.03 M to about 0.08 M, between about 0.03 M to about 0.06 M or about 0.05 M.

[0075] In certain embodiments, the concentration of the alcohol is greater than about 5% v / v, greater than about 10%v / v, greater than about 20%v / v, greater than about 30% v / v, greater than about 40% v / v, greater than about 50%v / v or greater than about 60%v / v. In certain embodiments, the concentration of the alcohol is between about 10%v / v to about 80%v / v, between about 10%v / v to about 70%v / v, between about 20%v / v to about 70%v / v, between about 20%v / v to about 65%v / v, between about 30%v / v to about 65%v / v, between about 40%v / v to about 65%v / v, between about 50%v / v to about 65%v / v, between about 55%v / v to about 65%v / v or about 60%v / v.

[0076] In certain embodiments, the concentration of the protease is less than about 150 pL / g by dry weight (such as dry weight of feedstock). In certain embodiments, the concentration of the protease is less than about 130 pL / g by dry weight, less than about 100 pL / g by dry weight, less than about 80 pL / g by dry weight, less than about 60 pL / g by dry weight, less than about 50 pL / g by dry weight, less than about 40 pL / g by dry weight, less than about 30 pL / g by dry weight or less than about 20 pL / g by dry weight. In certain embodiments, the concentration of the protease is between about 1 pL / g to about 130 pL / g by dry weight, between about 1 pL / g to about 100 pL / g by dry weight, between about 1 pL / g to about 80 pL / g by dry weight, between about 1 pL / g to about 60 pL / g by dry weight, between about 1 pL / g to about 50 pL / g by dry weight, between about 1 pL / g to about 40 pL / g by dry weight, betweenabout 1 pL / g to about 30 pL / g by dry weight, between about 1 pL / g to about 25 pL / g by dry weight, between about 2 pL / g to about 25 pL / g by dry weight or between about 5 pL / g to about 20 pL / g by dry weight.

[0077] As would be appreciated by a skilled addressee, any suitable pH can be used for the protein solubilisation step using a protein solubilisation agent in the process of the present invention. In some embodiments, the protein solubilisation step comprising treatment with the protein solubilisation agent is performed at a pH about 7.8 to about 11 .5, pH about 7.8 to about 10, pH about 7.8 to about 9, pH about 8 to about 11 , pH about 9 to about 11 , pH about 10 to about 11 or about pH 10.5.

[0078] As would be appreciated by a skilled addressee, any suitable duration can be used for the protein solubilisation step using a protein solubilisation agent in the process of the present invention. In some embodiments, the protein solubilisation step comprising treatment with the protein solubilisation agent is performed for a duration between about 5 minutes to about 30 minutes, between about 5 minutes to about 20 minutes, between about 5 minutes to about 15 minutes, between about 10 minutes to about 5 hours, between about 10 minutes to about 3 hours, between about 10 minutes to about 2.5 hours, between about 30 minutes to about 2.5 hours, between about 30 minutes to about 2 hours, between about 20 minutes to about 40 minutes, between about 1 hour to about 2.5 hours, about 10 minutes, about 30 minutes or about 2 hours.

[0079] As would be appreciated by a skilled addressee, any suitable temperature can be used for the protein solubilisation step using a protein solubilisation agent in the process of the present invention. In some embodiments, the protein solubilisation step comprising treatment with the protein solubilisation agent is performed at a temperature greater than about 20°C, greater than about 30°C, greater than about 40°C, greater than about 50°C, greater than about 60°C, greater than about 70°C, greater than about 80°C or greater than about 90°C. In some embodiments, the protein solubilisation step comprising treatment with the protein solubilisation agent is performed at a temperature between about 20°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about 50°C to about 100°C, between about 60°C to about 100°C, between about 70°C to about 100°C, between about 70°C to about 90°C, between about 80°C to about 100°C between about 90°C to about 100°C, between about 20°C to about 90°C, between about 20°C to about 80°C, between about 30°C to about 80°C, between about 40°C to about 80°C, between about 50°C to about 80°C, between about 60°C to about 80°C, between about 65°C to about 75°C, about 95°C or about 70°C.

[0080] In an embodiment, the method further comprises adding to the protein solubilisation step a simultaneous ultrasonic treatment. This treatment may advantageously increase therelease of proteins, including peptides and amino acids, from the matrix structure by further opening the cells, dislodging proteins and, or, further increasing the surface area. In one form of this embodiment, the ultrasonic treatment is between 1 to 5 watts per gram of dry matter feedstock at 15 to 45 KHz, for 1 minute to 45 minutes.

[0081] Once a protein solubilisation step has been performed, an initial separation of protein rich liquid and the fibrous solids can also be performed. In the preferred embodiment this initial separation may involve a centrifugation step using a continuous-flow high-speed decanter. In some embodiments this may involve a lipid separator device, such as a dairy fat centrifuge separator, disc stack separator, additional decanter or tricanter. The liquid fraction or downstream liquid fraction comprising protein, can then be treated to concentrated protein via by protein precipitation and centrifugation.

[0082] As would be appreciated by a skilled addressee the temperature of the initial centrifugation can influence the yield and purity of the protein to be recovered. The temperature should be set according to the specifications of the desired output protein product. In some embodiments, the initial centrifugation step is performed with a feedstock at a temperature greater than about 0°C, greater than about 10°C, greater than about 20°C, greater than about 30°C, greater than about 40°C, greater than about 50°C, greater than about 60°C, greater than about 70°C, greater than about 80°C or greater than about 90°C. In some embodiments, the initial centrifugation step is performed with a feedstock at a temperature between about 0°C to about 100°C, between about 0°C to about 80°C, between about 0°C to about 70°C, between about 50°C to about 100°C, between about 60°C to about 100°C, between about 70°C to about 100°C, between about 70°C to about 90°C, between about 80°C to about 100°C between about 90°C to about 100°C, between about 20°C to about 90°C, between about 20°C to about 80°C, between about 30°C to about 80°C, between about 40°C to about 80°C, between about 50°C to about 80°C, between about 60°C to about 80°C, between about 65°C to about 75°C, about 0°C, about 5°C, about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, about 100°C.

[0083] In some embodiments it is desirable to treat the protein rich liquid with an enzyme after the fibrous solids have been separated to improve the sensory characteristics, for example to reduce bitterness and create an umami flavour in the final protein concentrate. In some embodiments this may be a flavourzyme enzyme and / or a laccase enzyme. Alternative flavour improvements techniques include treating with a food-grade resin, such as polyvinylpolypyrrolidone (PVPP) or passing the protein rich liquid through an activated carbon filter.

[0084] In an embodiment, a suitable pH can be used for this sensory improvement treatment step using in the process of the present invention. In some embodiments, the treatment isperformed at a pH about 10.5 to 3.5, about 5 to about 8, pH about 5.5 to about 7.5, pH about 6.5 to about 7.5 or about pH 7.0.

[0085] In certain embodiments, the concentration of the enzyme where used in the sensory improvement treatment step is less than about 150 pL / g by dry weight (such as dry weight of feedstock). In certain embodiments, the concentration of the enzyme is less than about 130 pL / g by dry weight, less than about 100 pL / g by dry weight, less than about 80 pL / g by dry weight, less than about 60 pL / g by dry weight, less than about 50 pL / g by dry weight, less than about 40 pL / g by dry weight, less than about 30 pL / g by dry weight or less than about 20 pL / g by dry weight. In certain embodiments, the concentration of the enzyme is between about 1 pL / g to about 130 pL / g by dry weight, between about 1 pL / g to about 100 pL / g by dry weight, between about 1 pL / g to about 80 pL / g by dry weight, between about 1 pL / g to about 60 pL / g by dry weight, between about 1 pL / g to about 50 pL / g by dry weight, between about 1 pL / g to about 40 pL / g by dry weight, between about 1 pL / g to about 30 pL / g by dry weight, between about 1 pL / g to about 25 pL / g by dry weight, between about 2 pL / g to about 25 pL / g by dry weight or between about 5 pL / g to about 20 pL / g by dry weight.

[0086] As would be appreciated by a skilled addressee, any suitable temperature can be used for the sensory improvement treatment step in the process of the present invention. In some embodiments, the enzyme treatment step is performed at a temperature greater than about 20°C, greater than about 30°C, greater than about 40°C, greater than about 50°C, greater than about 60°C, greater than about 70°C, greater than about 80°C or greater than about 90°C. In some embodiments, the enzyme treatment step is performed at a temperature between about 20°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about 50°C to about 100°C, between about 60°C to about 100°C, between about 70°C to about 100°C, between about 70°C to about 90°C, between about 80°C to about 100°C between about 90°C to about 100°C, between about 20°C to about 90°C, between about 20°C to about 80°C, between about 30°C to about 80°C, between about 40°C to about 80°C, between about 50°C to about 80°C, between about 60°C to about 80°C, between about 50°C to about 63°C, about 60°C or about 70°C.

[0087] As would be appreciated by a skilled addressee, any suitable duration can be used for the sensory improvement treatment step in the process of the present invention. In some embodiments, the enzyme treatment step is performed for a duration between about 5 minutes to about 30 minutes, between about 5 minutes to about 20 minutes, between about 5 minutes to about 15 minutes, between about 10 minutes to about 5 hours, between about 10 minutes to about 3 hours, between about 10 minutes to about 2.5 hours, between about 30 minutes to about 2.5 hours, between about 30 minutes to about 2 hours, between about 20 minutes toabout 40 minutes, between about 1 hour to about 2.5 hours, about 10 minutes, about 30 minutes or about 1 hour.

[0088] As would be appreciated by a skilled addressee, the protein can be precipitated using any suitable method. In certain embodiments, the protein is precipitated by isoelectric precipitation. The isoelectric point (pl) is the pH of a solution at which the net primary charge of a protein becomes zero. In these embodiments, the solubility of the protein to be recovered by the present invention is at its lowest. In certain embodiments, protein is precipitated at a pH between about 1 .5 to about 6, between about 2 to about 5.5, between about 2 to about 5, between about 3 to about 5, between about 1 .8 to 3, about 3, about 3.5 about 4, or about 4.5 or about 5.

[0089] In certain embodiments, the protein can be sequentially precipitated at different pH levels. In these embodiments, each portion of recovered protein is isolated from the liquid before adjusting to a different pH for further recovery of protein. The recovered protein from each adjusted pH can be combined for each liquid fraction. In preferred embodiments, the protein can be sequentially precipitated at a pH of around 3.0, around 4.0 and around 5.0. In these embodiments, the pH can be adjusted using an acid or a base depending on the starting pH of the liquid fraction or downstream liquid fraction.

[0090] In some embodiments, the protein is precipitated by addition of an acid. Suitable acids can be selected from the group consisting of citric acid, formic acid, acetic acid, trifluoroacetic acid, (neat) sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, hydroiodic acid and combinations thereof. In preferred embodiments, the protein is precipitated by addition of citric acid, hydrochloric acid or sulfuric acid. In preferred embodiments, the acid is saturated citric acid, saturated hydrochloric acid, saturated sulfuric acid and combinations thereof.

[0091] For liquid fractions or downstream liquid fractions with acidic pH less than about pH 3, the protein can be precipitated by addition of a base. As would be appreciated by a skilled addressee, any suitable base can be used including bases described herein. In preferred embodiments, the base is sodium hydroxide, potassium hydroxide and combinations thereof.

[0092] As would be appreciated by a skilled addressee, the protein can be precipitated at any suitable temperature. In some embodiments, the protein is precipitated at a temperature greater than about 0°C, greater than about 10°C, greater than about 20°C, greater than about 30°C, greater than about 40°C, greater than about 50°C, greater than about 60°C, greater than about 70°C, greater than about 80°C, greater than about 90°C, greater than about 100°C, greater than about 110°C, greater than about 120°C or greater than about 130°C. In some embodiments, the protein is precipitated at a temperature between about -3°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about50°C to about 100°C, between about 60°C to about 10°C, between about 70°C to about 100°C, between about 80°C to about 100°C, between about 90°C to about 100°C. In the preferred embodiment the protein is precipitated between -3°C to 20°C.

[0093] In some embodiments a water wash of the acidic protein will reduce the amount of sugars, salts, minerals and other impurities increasing the final protein purity. In some embodiments this wash treatment will involve an aggressive redispersion of the protein in water under acidic conditions followed by a centrifugation. In some embodiments this aggressive redispersion with involve high shear forces generated by a wet mill, high shear pump, hydrodynamic cavitation pump, pulse electric field generator and / or ultrasonic or megasonic generator.

[0094] In some embodiments, the recovered carbohydrate yield of one fraction comprising carbohydrate is at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 8%, at least about 10%, at least about 15% or at least about 20% relative to the amount of carbohydrate in the feedstock. As would be appreciated by a skilled addressee, the recovered fractions of carbohydrate can be combined to provide a total yield. In some embodiments, the total recovered carbohydrate yield is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25% or at least about 30% relative to the amount of carbohydrate in the feedstock.

[0095] In some embodiments, the recovered dietary fibre yield is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10% or at least about 12% relative to the amount of carbohydrate in the feedstock .

[0096] In some embodiments, the protein can be treated with a bleaching agent. As would be appreciated by a skilled addressee, treatment with a bleaching agent may be preferred in applications where a lighter coloured protein is desirable. In certain embodiments, the bleaching agent is an oxidising agent. In certain embodiments, the oxidising agent is selected from the group consisting of fluorine, peroxide (such as hydrogen peroxide), chlorine, ozone, oxygen, bromide, iodide, hypochlorite (such as sodium hypochlorite), chlorate, chromate, dichromate, chromium trioxide, permanganate, manganate and combinations thereof.

[0097] In some embodiments, the protein can be treated with activated carbon filtration to remove some polyphenolics including tannins to better neutralise off-flavours or where a marginally lighter colour is desired. Typically a resin, such as polyvinylpolypyrrolidone (PVPP) may be expected to produce a similar or better effect than activated carbon but this was unexpectedly not the case. As may be appreciated by the skilled addressee, proteins are amphoteric and can have net positive or negative charge. By modifying the surface charge of the activated carbon (through oxidation or anime functionalisation) it is possible to better repel proteins while attracting polyphenols. As would be appreciated by a skilled addressee, somepolyphenols have an antioxidant benefit for human and animal health but are also often bitter so optimising the level of removal would be dependent of the final food, health supplement or feed application. This optimisation can be influenced by pH, temperature of the liquid, modifications to the characteristics of the activated carbon including particle size, duration of the exposure to activated carbon, hydraulic pressure and nature of any bonding between protein and polyphenols. As would be appreciated by the skilled address the activated carbon will need to be replaced or recharged intermittently to maintain the optimum effect. As would be appreciated the recovered phenolics may find valuable use in other applications. In some embodiments, the recovered activated carbon is granulated activated carbon rather than powder activated carbon. In some embodiments the activated carbon can be recharged using hot water or an alcohol, or combination thereof. In some embodiments, the alcohol is selected from the group consisting of methanol, ethanol, glycerol, 1-propanol, isopropyl alcohol (2- propanol), butanol (n-butanol, sec-butanol, isobutanol and tert-butanol) and combinations thereof. In preferred embodiments, the precipitating agent is ethanol. In some embodiments the recovered phenolics are high in ferulic acid.

[0098] In some embodiments the liquid protein streams from separate passes can be combined in storage vessels to improve process efficiency by reducing the total number of centrifugation devices otherwise required. In some embodiments this combining of stream is done prior to, or after, the addition of a protein precipitating agent but prior to the centrifugation of an acidified protein precipitate.

[0099] In some embodiments, final protein can be blended with a protein from a feedstock that is lighter in colour, to produce a blended protein that is lighter in colour. This blending protein sourced from one or more feedstocks that includes lupins, peas, wheat, rice, whey, hemp, meat. As would be appreciated by a skilled addressee, any suitable ratio could be used that is acceptable to meet the desired colour.Starch treatment and sugar production

[0100] Depending on the residual starch or beta glucan content of the feedstock used in the present invention at this point, the feedstock can be treated with an amylase to substantially convert starch to smaller molecules of carbohydrates and sugars. In certain embodiments, the carbohydrate recovered by the process of the present invention is treated with an amylase to increase the purity of a soluble dietary fibre. In certain embodiments, the amylase is selected from the group consisting of a-amylase, p-amylase, y-amylase (also known as glucoamylase) and combinations thereof. In certain embodiments, the carbohydrate fraction obtained by the process of the present invention is treated with an amylase to substantially remove starch to provide a soluble dietary fibre. In these embodiments, treatment with an amylase can dependon the feedstock used in the process of the present invention. For example, a high starch containing feedstock (greater than about 5 dry weight%) in starch can be treated with the amylase.

[0101] It will be understood by a skilled addressee that dietary fibre is the part of plantbased food that mostly passes through the digestive system without breaking down into sugars or being digested and comprises soluble and insoluble fibre. Soluble fibre can dissolve in water and typically comprises pectin and gums while insoluble fibre is insoluble in water and typically comprises cellulose, hemicellulose and lignin. The three main types of carbohydrates include sugars, starches and dietary fibre.

[0102] In certain embodiments, the concentration of the amylase is less than about 2 mg / mL, less than about 1.5 mg / mL, less than 1.2 mg / mL, less than 1 mg / mL, less than 0.8 mg / mL or less than 0.6 mg / mL. In certain embodiments, the concentration of the amylase is between 0.1 mg / mL to about 2 mg / mL, between 0.1 mg / mL to about 1.5 mg / mL, between 0.1 mg / mL to about 1.2 mg / mL, between 0.1 mg / mL to about 1 mg / mL, between 0.1 mg / mL to about 0.8 mg / mL, between 0.2 mg / mL to about 0.8 mg / mL, between 0.3 mg / mL to about 0.8 mg / mL, between 0.3 mg / mL to about 0.6 mg / mL, between 0.4 mg / mL to about 0.6 mg / mL or about 0.5 mg / mL.

[0103] As would be appreciated by a skilled addressee, any suitable duration can be used for the reduction of starch in the process of the present invention. In some embodiments, the starch reduction step using an amylase is performed for a duration between about 5 minutes to about 30 minutes, between about 5 minutes to about 20 minutes, between about 5 minutes to about 15 minutes, between about 10 minutes to about 5 hours, between about 10 minutes to about 3 hours, between about 10 minutes to about 2.5 hours, between about 30 minutes to about 2.5 hours, between about 30 minutes to about 2 hours, between about 20 minutes to about 40 minutes, between about 1 hour to about 2.5 hours, about 10 minutes, about 30 minutes or about 2 hours.

[0104] As would be appreciated by a skilled addressee, any suitable temperature can be used for the reduction of starch using an amylase in the process of the present invention. In some embodiments, the starch removal step using an amylase is performed at a temperature greater than about 20°C, greater than about 30°C, greater than about 40°C or greater than about 50°C. In some embodiments, the starch removal step using an amylase is performed at a temperature between about 20°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about 40°C to about 90°C, between about 40°C to about 80°C, between about 40°C to about 70°C, between about 40°C to about 60°C, between about 50°C to about 60°C or about 55°C.

[0105] In some embodiments, the process of the present invention further comprises treating at least one of the solid fraction and second solid fraction with a cellulase. In these embodiments, the carbohydrates can be broken down further into simple sugars, oligosaccharides or polysaccharides to be used as a further valuable product whilst releasing more protein from the feedstock matrix. In certain embodiments, the cellulase is selected from the group consisting of endocellulase, exocellulase, cellobiase, oxidative cellulase, cellulose phosphorylase and combinations thereof.

[0106] In certain embodiments, the concentration of the cellulase is greater than about 1 FPU (filter paper unit) / g solids, greater than about 2 FPU / g solids, greater than about 3 FPU / g solids, greater than about 4 FPU / g solids, greater than about 5 FPU / g solids, greater than about 6 FPU / g solids, greater than about 7 FPU / g solids, greater than about 8 FPU / g solids, greater than about 9 FPU / g solids or greater than about 10 FPU / g solids. In certain embodiments, the concentration of the cellulase is between about 1 to about 20 FPU / g solids, between about 1 to about 15 FPU / g solids, between about 2 to about 15 FPU / g solids, between about 3 to about 15 FPU / g solids, between about 4 to about 15 FPU / g solids, between about 5 to about 15 FPU / g solids, between about 6 to about 15 FPU / g solids, between about 7 to about 15 FPU / g solids, between about 8 to about 15 FPU / g solids, between about 8 to about 13 FPU / g solids, between about 8 to about 12 FPU / g solids or about 10 FPU / g solids.

[0107] As would be appreciated by a skilled addressee, any suitable duration can be used using cellulase in the process of the present invention. In some embodiments, the step of using a cellulase is performed for a duration between about 0.25 hours to about 72 hours, between about 0.5 hours to about 48 hours, between about 0.5 hours to about 24 hours, between about 0.5 hours to about 12 hours, between about 0.25 hours to about 12 hours, between about 0.5 hours to about 6 hours, between about 0.5 hours to about 3 hours, between about 0.25 hours to about 3 hours or about 3 hours.

[0108] As would be appreciated by a skilled addressee, any suitable temperature can be used using cellulase in the process of the present invention. In some embodiments, the step of using cellulase is performed at a temperature greater than about 20°C, greater than about 30°C, greater than about 40°C or greater than about 45°C. In some embodiments, the step of using a cellulase is performed at a temperature between about 20°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about 40°C to about 90°C, between about 40°C to about 80°C, between about 40°C to about 70°C, between about 40°C to about 60°C, between about 45°C to about 55°C or about 50°C.

[0109] As would be appreciated by a skilled addressee smaller polysaccharides and simple sugars will be released into the aqueous streams during the treatment whilst they are not being targeted. In some embodiments these smaller polysaccharides and simple sugars can berecovered from the wastewater streams via membrane filtration to provide a useful product. In some embodiments this membrane filtration has an average pore size between about 10 kilodaltons to 10 daltons, between about 5 kilodaltons to 50 daltons, between about 1 kilodalton to 100 daltons, about 100daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons.Carbohydrate solubilisation and precipitation

[0110] As would be appreciated by a skilled addressee, any suitable method can be used to assist with solubilising carbohydrate in the present invention. In certain embodiments, the solid fraction or downstream combined solid plus liquid fraction is acidified prior to precipitation with a precipitating agent. Suitable acids for solubilisation of carbohydrate can be selected from the group consisting of citric acid, formic acid, acetic acid, trifluoroacetic acid, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, hydroiodic acid and combinations thereof. In preferred embodiments, the acid for acidification is hydrochloric acid.

[0111] In certain embodiments, the pH of the acidified solid fraction or downstream combined solid plus liquid fraction is less than about pH7, is less than about pH 6, less than about pH 5.5, less than about pH 5, less than about pH 4, less than about pH 3 or less than about pH 2. In some embodiments, the pH of the acidified solid fraction or downstream combined solid plus liquid fraction is between about pH 1 to about pH 6, between about pH 1 .5 to about pH 6, between about pH 1 to about pH 5.5, between about pH 1 to about pH 4, between about pH 1 .8 to about pH 3, between about pH 1 to about pH 3, between about pH 1 .5 to about pH 2 or about pH 1.8.

[0112] In some embodiments, the precipitating agent is selected from the group consisting of a salt, a lactone, an alcohol and combinations thereof.

[0113] In some embodiments, the salt is selected from the group consisting of a chloride, sulfate, nitrate, chlorate and combinations thereof. In some embodiments, the salt is a neutral salt. In some embodiments, the salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, choline chloride, potassium sulfate, magnesium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, magnesium nitrate, sodium chlorate, potassium chlorate, magnesium chlorate and combinations thereof.

[0114] In some embodiments, the lactone is selected from the group consisting a- acetolactone, p-propiolactone, y-butyrolactone, d-glucono-6-lactone, E-caprolactone, y- valerolactone and combinations thereof.

[0115] In some embodiments, the alcohol is selected from the group consisting of methanol, ethanol, glycerol, 1 -propanol, isopropyl alcohol (2-propanol), butanol (n-butanol, sec-butanol,isobutanol and tert-butanol) and combinations thereof. In preferred embodiments, the precipitating agent is ethanol.

[0116] As would be appreciated by a skilled addressee, in some embodiments it may be preferable to utilise membrane filtration in lieu of, or in addition to, precipitation to recover a target carbohydrate. In some embodiments this membrane filtration has an average pore size between about 300 kilodaltons to 1 kilodaltons, between about 5 kilodaltons to 200 kilodaltons, between about 10 kilodaltons to 100 kilodaltons, about 1 kilotaltons, about 10 kilodaltons, about 10 kilodaltons.

[0117] As would be appreciated by a skilled addressee, any suitable amount of precipitating agent can be used to precipitate carbohydrate in the present invention. In some embodiments, the amount of precipitating agent is at least about 10%v / v, at least about 20%v / v, at least about 30%v / v, at least about 40%v / v, at least about 50%v / v, at least about 60%v / v, at least about 70%v / v by total volume of the precipitating agent and liquid fraction or downstream liquid fraction. In some embodiments, the amount of precipitating agent is between about 10%v / v to about 90% v / v, between about 20%v / v to about 90% v / v, between about 30%v / v to about 90% v / v, between about 40%v / v to about 90% v / v, between about 50%v / v to about 90% v / v, between about 60%v / v to about 90% v / v, between about 60%v / v to about 80% v / v, between about 65%v / v to about 75% v / v or about 70% v / v by total volume of the precipitating agent and liquid fraction or downstream liquid fraction after centrifugation of the solid fraction or downstream combined solid plus liquid fraction.

[0118] As would be appreciated by a skilled addressee, any suitable duration can be used for the precipitation of carbohydrate using a precipitating agent in the process of the present invention. In some embodiments, the precipitation step is performed for a duration between about 10 seconds to about 30 minutes, between about 10 seconds to about 25 minutes, between about 10 seconds to about 20 minutes, between about 10 seconds to about 15 minutes, between about 10 seconds to about 10 minutes, between about 10 seconds to about 5 minutes, between about 20 seconds to about 5 minutes, between about 30 seconds to about 5 minutes, between about 1 minute to about 5 minutes, between about 2 minutes to about 5 minutes or about 3 minutes.

[0119] In some embodiments, the recovered protein yield of one fraction comprising protein is at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% or at least about 65% relative to the amount of protein in the feedstock. As would be appreciated by a skilled addressee, the recovered fractions of protein can be combined to provide a total yield. In some embodiments, the total recovered protein yield is at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% or at least about 70% relative to the amount of protein in the feedstock.EXAMPLES

[0120] Materials

[0121] A Brewer’s spent grain (BSG) sample labelled with “MG” was used, provided as part of a selection of fresh frozen BSG samples. This BSG sample had a moisture content of 76.6%. Based on its dry mass, the BSG contained 26.2% crude protein, 7.1 % lipids, 7.3% starch, 13.2% cellulose, 13.5% xylan, 7.2% arabinan and 3.3% ash. In addition, the BSG sample labelled with “CV” was used to verify the conditions developed with MG sample. CV contained 28.1 % protein, 0.7% starch, 9.1 % lipid, 13.2% cellulose, 14.9% xylan, 7.9% arabinan and 4.0% ash.

[0122] To test the process of the present invention with other potential feedstock; an oat bran and a wheat bran was purchased from an Australian supermarket (home brand, Woolworths) as comparative samples. According to the product specification, the oat bran and wheat bran samples contained 16.6% and 16.3% protein, respectively.

[0123] Alcalase (a commercial protease) and Accellerase 1500 (a commercial cellulase) was purchased from Novozymes (Denmark) and DuPont (USA), respectively. Distiller’s glucoamylase was purchased from Still Spirits (New Zealand).

[0124] Methods

[0125] A sequential fractionation approach was used to extract crude protein and soluble dietary fibres from the BSG sample in the process of the present invention. The sequential treatment process included (1 ) sample crushing, (2) treatment for protein solubilisation, (3) hydrochloric acid treatment for dietary fibre solubilisation.

[0126] BSG sample preparation

[0127] A large portion of the BSG sample was crushed for 5 min using a commercial blender (908™, Hamilton Beach, Australia). Prior to crushing, the solid content of the BSG sample was adjusted to 15% (weight / weight) with the addition of water. For each batch of crushing, 500 - 600 g of the BSG sample (containing 15% solids) was used.

[0128] A small portion of the BSG sample was placed at a shallow tray and dried at 45 °C for 24 h. All the dried and milled sample were passed through a sieve with an aperture size of 2.0 mm. A sub-portion of the sieved sample was further milled and passed through a sieve with an aperture size of 0.25 mm.

[0129] Treatment for protein solubilisation

[0130] Treatment for protein solubilisation was performed using the starch-containing MG sample unless it is stated otherwise.

[0131] Comparison of different treatment for protein solubilisation

[0132] NaOH, ethanol and protease-assisted treatments for protein solubilisation were compared. The treatment trials were undertaken at 60 °C and 200 rpm for 2 h in 250 mL shake flasks containing 50 g of BSG samples (5.0 g dry BSG). The concentrations of NaOH and ethanol in the BSG solutions were 0.05 M and 60% (volume / volume).

[0133] For the enzyme-assisted treatment, the Alcalase was used with enzyme loadings of 0, 5, 10 and 20 pL / g dry BSG, respectively at pH 8.0. At the end of enzyme treatment, the BSG mixtures were incubated at 95 °C for 10 min to denature the protease.Following treatment, the solid and liquid fractions were neutralised by 5 M HCI solution to pH 7.0. The solid and liquid fractions were separated by filtration using Miracloth (pore size of 22 - 25 pm, Merck Millipore, USA). The solid samples were washed with water, collected and freeze-dried for compositional analysis. The liquid samples were collected and stored at 4 °C prior to further processing and analysis.

[0134] Effects of operational parameters on protein solubilisation by NaOH treatment

[0135] Effect of drying, particle size and temperature: The effects of drying, particle size and temperature on protein solubilisation were also investigated. The dried BSG samples with two different particle size ranges (0.25-2.0 mm and < 0.25 mm) were treated with 0.05 M NaOH at 60 °C, 70 °C and 80 °C for 2 h, respectively. The treatments of dried and milled samples by NaOH were also compared with the wet and crushed sample under the same conditions. One NaOH treatment at 60 °C with the wet and uncrushed sample was used as a control. The other conditions were the same as those used for the treatment comparison trial.

[0136] Effect of treatment time: The effect of time (30 min, 60 min and 120 min) on protein solubilisation by 0.05 M NaOH treatment was studied at 70 °C with the use of wet and crushed BSG samples. All the other conditions were the same as those used in the treatment comparison trials.

[0137] Following treatment, the samples were processed as described in the treatment comparison trial.

[0138] Protein precipitation from NaOH treatment solution

[0139] A NaOH solution (0.05 M), at a temperature of 70 °C and a treatment time of 2 h were selected for processing large quantities of BSG. Under this condition, a total of about 13.0 kg of wet and crushed BSG (about 1 .3 kg dry mass) were treated in a 10 L bucket (two batches at 5 kg wet BSG and one batch at 3 kg wet BSG). The treatment was conducted in a water bath with an agitation of 50 rpm using an overhead stirrer. Following treatment, the solid and liquid fractions were neutralised by 5 M HCI solution to pH 7.0 and separated as describedabove. The solid fraction was water-washed. Both solid and liquid fractions were collected and stored at 4 °C for further processing and analysis.

[0140] 1st citric acid treatment for carbohydrate solubilisation

[0141] The wet MG BSG solid residues after NaOH treatment (70 °C for 2 h) were used for the first citric acid treatment for dietary fibre solubilisation. Prior to treatment, BSG residues were firstly transferred to 250 mL flasks and each flask contained 55 g of the BSG residue mixture (5.0 g dry mass).

[0142] Effect of pH: The effect of pH (2.0, 3.0 and 4.0 adjusted by saturated citric acid; 5.5 - in water and pH adjustment) on solubilisation of solid residues was performed at 110 °C and 130 °C, respectively, with a treatment time of 15 min. The treatment was performed in autoclave.

[0143] Effect of treatment time: The effect of treatment time (15 min, 30 min and 60 min) on solubilisation of solid residues was investigated at 90 °C and 110 °C with an initial treatment pH of 2.0. The treatment at 90 °C and 110 °C, was performed in a water bath and an autoclave, respectively.

[0144] After treatment, the BSG mixtures were filtered through the Miracloth. The solids were water-washed. Both solid and liquid fractions were collected and stored at 4 °C forfurther processing and analysis.

[0145] 2ndcitric acid treatment for dietary fibre solubilisation

[0146] After studying the effects of operational parameters on dietary fibre solubilisation by citric acid treatment, the operational conditions of pH 2.0 and 110 °C for 15 min were selected for the second citric acid treatment of the BSG solid residues from the first citric acid treatment. After treatment, the BSG mixtures were filtered through the Miracloth. The solids were water- washed. Both solid and liquid fractions were collected and stored at 4 °C forfurther processing and analysis.

[0147] Protein recovery from treatment solutions

[0148] Protein recovery from NaOH treatment solution

[0149] Proteins dissolved in the NaOH treatment solutions were recovered by acid precipitation. For the fresh treatment solution, saturated citric acid was added dropwise until the pH reached about 3.0 under continuous gentle stirring. At pH 3.0, the solution was stirred for 3 min, followed by centrifugation at 3900 rpm (about 3200 x g). The solids were washed by water and freeze-dried. The dried solids were labelled as Crude protein 1. The supernatant was collected for the recovery of dietary fibres.

[0150] For the treatment solution stored at 4 °C, it was observed that storage of the solution led to precipitation. Therefore, for the stored solution, the precipitates were firstly separated by centrifugation and the supernatant was acidified using the above protein precipitationprocedure. The two proteins were labelled as Crude protein 1 -1 (storage precipitation) and Crude protein 1-2 (acid precipitation), respectively.

[0151] Protein recovery from 1stand 2ndcitric acid treatment solutions

[0152] Proteins dissolved in the first citric acid treatment solutions were recovered by a sequential precipitation approach. The solution pH was firstly increased to 3.0 by 5 M NaOH under continuous gentle stirring. At pH 3.0, the solution was stirred for 3 min, followed by centrifugation at 3900 rpm (about 3200 x g) for 10 min. The pH of the supernatant was further increased to 4.0 by 5 M NaOH under continuous gentle stirring. At pH 4.0, the solution was stirred for 3 min, followed by centrifugation at 3900 rpm (-3200 x g) for 10 min. The pH of the supernatant was finally increased to 5.0. At pH 5.0, the solution was stirred for 3 min, followed by centrifugation at 3900 rpm (-3200 x g) for 10 min. The solids from the sequential precipitation were combined and washed by deionised water and freeze-dried. The dried solids were labelled as Crude Protein 2 forthose recovered from the first citric acid treatment solution and as Crude Protein 3 from the second citric acid solution. The supernatants from the final steps were collected for the recovery of dietary fibres.

[0153] Carbohydrate recovery from treatment solutions

[0154] Following protein recovery, the pH of the collected supernatants were reduced to about pH 1.8 with 32% (weight / weight) HCI solution. Following pH adjustment, absolute ethanol was added to the acidified solution to a final ethanol / supernatant solution volume ratio of 7:3 (70% ethanol) to precipitate dietary fibres under continuous gentle stirring. After ethanol addition, the mixture was stirred for 3 min, followed by centrifugation to collect the solids. The solids were washed by 70% ethanol and freeze-dried. The dried solids were labelled as Carbohydrate 1 , Carbohydrate 2 and Carbohydrate 3 for the solids precipitated from the NaOH treatment solution, 1st citric acid treatment solution and 2nd citric acid treatment solution, respectively.

[0155] Preliminary tests to remove protein colour

[0156] To remove the colour of crude protein, different approaches were tested.

[0157] Adsorption by activated carbon: 2 mL of protein solution after the first NaOH treatment was mixed with 2 mg activated carbon for 60 min. The mixture was centrifuged to separate the solid and liquid. The colour of the liquid was monitored.

[0158] Solvent extraction: three solvents - ethyl acetate, hexane, and petroleum ether, were tested to extract the colourant of the protein solution after the first NaOH treatment. 4 mL of a solvent was mixed with 20 mL of the NaOH treatment solution for at least two minutes. The mixture was settled to observe the extraction of colourant by the solvent.

[0159] Dialysis: a dialysis method was tried to remove the colour of the protein solution. 50 mL of the protein solution were placed in a dialysis bag (molecular weight cut-off = about3.5kDa, Spectra / Por). The dialysis was performed for 48 h. At the end of dialysis, the colour of the solution was observed and compared to that without dialysis.

[0160] Bleaching: Crude protein 1 recovered from NaOH treatment solution was used in the bleaching test. In one trial, 0.5 g of crude protein was firstly suspended into 5.5 mL water (~pH 3.0), followed by the addition of 3 mL 30% (w / w) H2O2. The solution was mixed well and poured into a beaker. The beaker was incubated at 60 C with ventilation for 24 h for bleaching and drying the sample. After 24 h, the bleached and dried sample was collected. In another trial, 0.5 g of crude protein was suspended into 5.0 mL water, followed by the addition of 3 mL of 30% H2O2. The mixture was added with 0.5 mL of 5 M NaOH to increase the pH to 10.5. It was observed that the addition of NaOH solution caused vigorous foaming. The foam and the solution were transferred to a beaker, followed by bleaching and drying using the same method as above.

[0161] Preliminary tests to precipitate carbohydrates using alternative solvents

[0162] High boiling point solvents including n-butanol, y-valerolactone (GVL) and choline chloride were used for precipitation of carbohydrates as an alternative to ethanol above. Due to solubility, n-butanol, 70% GVL and choline chloride were mixed with the carbohydrate solution at -20%, 50% and 50%, respectively. After mixing for a couple minutes, the mixtures were settled to observe the carbohydrate precipitates.

[0163] Extraction of protein and dietary fibres from bran samples

[0164] Sequential extractions of protein and dietary fibres from the two bran samples were undertaken using the procedure and conditions determined for the BSG. 5 g of each bran sample was first treated with 0.05 M NaOH solution, followed by separation of the solid and liquid fractions by filtration using the Miracloth.

[0165] Cellulose digestibility of solid residues after each extraction

[0166] Enzymatic hydrolysis of solid residues after each treatment was carried out in 20 ml vials at 50 °C and 150 rpm for 48 h. Each vial contained 5ml of reaction mixtures including the solids (1 %), buffer (0.05 mM citrate at pH 4.8), and Accellerase 1500 (10 FPU / g solids). At the end of enzymatic hydrolysis, liquid samples were collected and were filtered through 0.45 pm disc membrane, followed by sugar analysis by the high-performance liquid chromatography (HPLC) method.

[0167] Starch removal from recovered carbohydrates

[0168] The recovered and freeze-dried carbohydrate samples of 100 mg and 500 mg were resuspended in 5 mL and 10 mL of aqueous solutions containing 0.5 mg / mL glucoamylase, which corresponded to carbohydrate loadings of 2% and 5%, respectively. The enzymatic hydrolysis was undertaken at 55 °C and 160 rpm for 2 h. At the end of cultivation, the solutions were filtered through 0.8 pm disc membranes, followed by precipitation of carbohydrates byethanol as described herein. The precipitated carbohydrates were freeze-dried and acid- hydrolysed to determine the composition.

[0169] Treatment of low starch-containing CV sample for protein solubilisation

[0170] To verify the protein solubilisation method developed based on the high starch- containing MG sample, the low starch-containing CV sample was also treated. Prior to treatment, CV sample was crushed using the method same as that for the MG sample. The treatment was undertaken with 0.05 M NaOH at 70 °C for 2 h, 0.1 M NaOH at 70 °C for 2 h and 0.1 M NaOH at 90 °C for 2 h, respectively.

[0171] Following treatment by NaOH solutions, recovery of protein and carbohydrate was performed using the methods same as those for the MG sample as described herein.

[0172] Characterisation

[0173] Biomass compositional analysis

[0174] Water and ethanol extractions: the water and ethanol extracts of two bran samples were determined using the Soxhlet extraction approach.

[0175] Cellulose, hemicellulose and lignin: the solid residues after each treatment step, recovered crude protein samples, recovered carbohydrate samples and the two bran samples (after water and ethanol extractions) were acid-hydrolysed to determine the contents of cellulose, hemicellulose and lignin. Briefly, the samples were first solubilised in 72% sulfuric acid at 30 °C, followed by acid-hydrolysis in 4% sulfuric acid solution at 121 °C for 1 hour. Following the acid-hydrolysis, the liquid and solid residue were separated by vacuum filtration. The liquid samples (hydrolysates) were analysed by the HPLC method to determine sugars generated from the hydrolysates. The amounts of sugars were converted back to cellulose (glucan) and hemicellulose (mainly xylan and arabinan) based on stoichiometry.

[0176] Protein analysis

[0177] Crude protein: The total nitrogen concentrations / contents in the treatment solutions, solid residues after each treatment, recovered crude protein samples, recovered carbohydrate samples and the two bran samples were determined using a CNS (carbon, nitrogen and sulfur) analyser (LECO TruMac™ Analyzer, USA). For the liquid samples, prior to nitrogen analysis, 1 mL of liquid sample was diluted to 50 mL, followed by centrifugation at 3900 rpm (about 3200 x g) 10 min. The supernatant was withdrawn for nitrogen analysis using a Shimadzu TOC analyser (TOC-VCSH) coupled with a chemiluminescence detector (TNM-L TN Unit). Crude protein content was estimated by multiplying the nitrogen content with 6.25.

[0178] True protein: Soluble protein concentrations in the treatment solutions were also determined using the Bradford method (Bradford protein assay) with the use of bovine serum albumin (BSA) as a standard.

[0179] Amino acid composition: amino acid compositions of crude protein samples were determined according to the following procedure. 50 mg of protein samples were firstly transferred to 50 mL glass pressure tubes, followed by the addition of 5 mL of 6 M hydrochloric acid containing 20 mM of dithiothreitol. The glass tubes were purged with nitrogen for 10-15 seconds to remove oxygen. The tubes were sealed with PTFE screw caps and digested at 110 °C in an oil bath overnight. At the end of digestion, the tubes were cooled to room temperature, followed by transferring digested protein solution into a round bottom flask. The acid was removed by rotary evaporation at 60°C. The dried residue was dissolved in 5 mL of 0.1 % (weight / volume) formic acid and filtered through 0.22 pm filters. The filtered samples were frozen at -20 °C until analysis by a liquid chromatography-mass spectrometry (LC-MS) method.

[0180] Determination of sugars, citric acid and amino acids

[0181] Sugars generated from the acid hydrolysis were determined by an HPLC method. In brief, a Waters HPLC system equipped with an Aminex HPX-87P column and a Waters refractive index detector was used to analyse sugars. The temperature for the column was set up at 85 °C and water was used as the mobile phase at a flow rate of 0.5 ml / min.

[0182] The contents of citric acid in the dietary fibre (DF) samples were determined by another HPLC method. In brief, the DF samples were firstly dissolved in water, followed by analysis of the citric acid concentration in the aqueous solution. A Waters HPLC system equipment with Aminex HPX-87H column and a Waters refractive index detector. The temperature for the column was set up at 60 °C and 5 mM H2SO4 was used as the mobile phase at a flow rate of 0.5 mL / min.

[0183] Amino acids were analysed using a Shimadzu LCMS 8050 system (Japan) equipped with an Ascentis Express F5 column (Sigma-Aldrich, USA). The samples were diluted in 0.1 % formic acid in MS-grade water prior to injection. Ions were detected in positive mode and monitored using a targeted multiple reaction monitoring (MRM) approach. Spectra results observed from the amino acid analysis were analysed using the Skyline software package (Sciex, USA). The amount of each amino acid was quantified using a standard curve generated from a set of known standards.EXAMPLE 1 - Comparison of different treatment methods on protein solubilisation

[0184] Table 1 shows solid recoveries and protein distributions after treatment with different methods. The use of protease led to lower solid recoveries than others, indicating higher protein solubilisation. The protein distribution results based on the total nitrogen assay confirmed that the use of protease solubilised more protein into the liquids than the other treatments. Increasing enzyme loading from 5 to 20 pL / g dry BSG led to a slight increase in protein solubilisation.

[0185] Although the use of protease solubilised more BSG protein based on the total nitrogen assay, the Bradford assay results showed that most of the solubilised protein were hydrolysed to amino acids. In contrast, although the NaOH treatment only solubilised -30% of the total protein (based on the total nitrogen assay), the Bradford assay result showed that the majority of the solubilised protein still existed as protein instead of amino acids.Table 1 : Solid recovery and protein distribution using different treatment approaches1. Control - treatment of crushed wet BSG in water without chemical and enzyme.2. Protein solubilisation is based on the comparison of crude protein (estimated by total nitrogen assay) in initial BSG.EXAMPLE 2 - Treatment optimisation for protein solubilisation

[0186] Table 2 shows the effects of sample conditions and temperature on protein solubilisation. The results from treatment of dried and milled samples with particle sizes of < 0.25 mm and 0.25 - 2.0 mm by 0.05 M NaOH indicated that the particle size did not seem to have a significant effect on protein solubilisation. However, treatment of wet and crushed samples by 0.05 M NaOH solubilised more proteins than treatment of dried and milled samples three tested temperatures indicating it is better to keep the BSG wet. For NaOH treatment, it appears that 70 °C was the preferred treatment temperature. Further, for NaOH treatment, the percentages of solubilised protein based on the total nitrogen assay were in line with those based on the Bradford assay. These results confirm that treatment with 0.05 M NaOH did not lead to the severe hydrolysis of protein.

[0187] Enzyme treatment was also performed at two different temperatures and the results showed that higher treatment temperature led to lower solid recovery, although the difference in protein solubilisation based on the total nitrogen assay was not significant. The results fromthe Bradford assay indicated that the majority of the solubilised protein by the enzyme were hydrolysed to amino acids.

[0188] Table 2 also shows that the extent of protein solubilisation was relatively low at all the tested temperatures without the use of enzyme and NaOH.Table 2: Effect of sample conditions and treatment temperature on protein solubilisation

[0189] Table 3 shows the effect of time on protein solubilisation by NaOH treatment. The increase of treatment time from 30 min to 120 min did not appear to lead to greater solubilisation of protein compared to the shorter duration.Table 3: Effect of NaOH treatment time on solid recovery and protein solubilisation** Treatment was performed at 70 °C with the use of wet and crushed BSG and 0.05 M NaOH.

[0190] Based on the above results, 0.05 M NaOH, 70 °C and 120 min with the use of wet and crushed BSG were preferred for protein solubilisation. It should be noted that a shorter treatment time, e.g., 30 min or shorter, is feasible given that longer treatment time did not appear to lead to the solubilisation of significantly more protein. This may be adapted for larger scales using an extruder.EXAMPLE 3 - Recovery of protein and carbohydrates from NaOH treatment solution

[0191] NaOH treatment solution from the treatment of about 13 kg of wet and crushed BSG with 0.05 M NaOH at 70 °C for 2 h was used for recovery of crude protein and carbohydrates. From 1 .0 kg of dry BSG, approximately 83 g Crude protein 1 and 30 g Carbohydrate 1 was recovered by acid precipitation and ethanol precipitation methods, respectively with the use of fresh treatment solution.

[0192] It was observed that precipitation occurred during the storage of the treatment solution. With the use of storage solution, precipitates (Crude protein 1 -1) during storage were firstly separated by centrifugation and the supernatant was used to recover Crude protein 1-2 and Carbohydrate 1.

[0193] From 1.0 kg of dry BSG, approximately 37 g Crude protein 1-1 and 46 g Crude protein 1 -2 was recovered. The recovery of Carbohydrate 1 did not appear to be affected by the storage and the yield was similar to that with the use of fresh treatment solution.

[0194] The recovered crude proteins were brownish, likely due to the presence of phenolics and lignin. Three methods (activated carbon adsorption, solvent extraction and dialysis) were used to reduce the colour of NaOH treatment solution prior to protein recovery by precipitation.

[0195] A H2O2-assisted bleaching method was applied to Crude protein 1 for colour reduction. The crude protein (brown) became light (yellowish) after bleaching. A darker brown protein may suit alternative meat manufactured food products whilst the lighter yellowish protein may suit other products.EXAMPLE 4 - Effect of 1stcitric acid treatment on solubilisation of protein and carbohydrates

[0196] The solid residue after NaOH treatment of BSG was used for the first citric acid treatment.

[0197] Effects of pH and temperature: Table 4 shows that increasing pH from 2.0 to 5.5 at both treatment temperatures led to the recovery of more solids (lower level of solid solubilisation). The present inventors surprisingly found that the amounts of protein solubilised into the treatment solutions were the highest at both the lowest and highest pHs, supportive ofa sequential precipitation technique. These results indicate that the average isoelectric point of the protein is likely in the range of 3 - 4, at which the protein is least soluble. This is also in line with the selection of pH 3 for protein recovery from treatment solution by acid-assisted precipitation.

[0198] Further, the protein and carbohydrates were precipitated from the solutions after treatment at pH 2.0 with 110 °C and 130 °C, respectively. Protein was recovered by a sequential precipitation approach (increasing solution pH to 3.0, 4.0 and 5.0) as described herein. The proteins recovered at pH 3.0, 4.0 and 5.0 were combined. It should be noted that more proteins were precipitated at pH 3.0 and increasing pH from 3.0 to 5.0 led to the reduced protein recovery. These observations indicate that the average isoelectric point of the protein is around 3.0-3.5 (protein is least soluble at its isoelectric point).

[0199] After protein recovery, the pH of the protein-free solution was dropped back to about pH 1 .8, followed by precipitation of carbohydrates by 70% (v / v) ethanol according to the method described as described herein. In addition, other solvent-assisted precipitation methods were used to precipitate carbohydrates.

[0200] Table 4 shows the results on solid recovery, protein solubilisation and compound recovery at different pH values and the two treatment temperatures. Treatment at 130 °C led to the recovery of slightly more crude protein but much less carbohydrates, indicating greater hydrolysis / degradation of carbohydrates at 130 °C. In addition, it was observed that the crude protein recovered from 130 °C treatment was darker than that from 110 °C.Table 4: Solid recovery, protein solubilisation and compound recovery at different pH values#. Treatment time, 15 min,§. Not recovered

[0201] Effect of treatment time: effect of treatment time by citric acid at pH 2.0 on extraction of protein and dietary fibres was investigated at 90 °C and 110 °C. As shown in Table 5, at 110 °C increasing treatment time from 15 minutes to 60 minutes solubilised more solids. Increasing treatment time from 15 minutes to 30 minutes led to the solubilisation of more protein in the solution. However, an increased treatment time did not lead to the solubilisation of more protein, possibly due to the hydrolysis of solubilised protein. Treatment time did not significantly affect protein recovery, however, increasing treatment time reduced carbohydrate recovery. The reduced carbohydrate recovery was possibly due to the hydrolysis / degradation of carbohydrate with the prolonged treatment time.

[0202] Treatment at 90 °C was less effective compared to that 110 °C, however, increasing treatment time from 15 minutes to 90 minutes solubilised more solids and more protein. Citric acid treatment at pH 2.0 and 110 °C for 15 min was also directly applied to the wet and crushed BSG (without NaOH treatment). As shown in Table 5, the percentage of solubilised protein accounted for about 18% of the total protein in BSG, lower than that of 0.05 M NaOH treatment at 70 °C for 30 - 120 min (Table 3). These results indicate that direct treatment by citric acid is less preferable for protein solubilisation.Table 5: Solid recovery, protein solubilisation and compound recovery after treatment for different times at 90 °C and 110 °C*. Treatment pH 2.0,f. Not determined.

[0203] Based on the above results, citric acid treatment at pH 2.0 and 110 °C for 15 minutes was suitable for solubilisation of carbohydrates. A repeated citric acid treatment (second citric acid treatment) at 110 °C is preferable instead of using a single citric acid treatment at 110 °C for a longer time as prolonged treatment time can lead to the hydrolysis of carbohydrates.

[0204] Carbohydrate recovery by other solvent-assisted precipitation: In addition to the ethanol-assisted precipitation, other solvents, such as n-butanol, GVL and petroleum ether were also tried to precipitate carbohydrates.

[0205] From 1.0 kg of initial dry BSG, approximately 59 g protein (Crude protein 2) and 92 g carbohydrates were recovered by acid-precipitation after first citric acid treatment at pH 2.0 and 110 °C for 15 min.EXAMPLE 5 - Protein and carbohydrate recovery from 2ndcitric acid treatment solution

[0206] The solid residues from the first citric acid treatment at pH 2.0 and 110 °C for 15 minutes was subjected to a second citric acid treatment under the same conditions for carbohydrate solubilisation. The treatment solution was used for recovery of protein and carbohydrates by precipitation.

[0207] From 1.0 kg of initial dry BSG, approximately 26 g protein (Crude protein 3) and 20 g carbohydrates were recovered, which accounted for 10% of the total protein and 4% of the dietary fibres in the initial BSG.EXAMPLE 6 - Sugar production from treatment residues

[0208] The BSG solid residues from this fractionation process have further commercial product potential. Simple sugar production (glucose and xylose) was investigated which could be a useful range of applications. These could include: feeding yeast for enhanced beer or ethanol production, feeding other yeast for industrial ethanol production, feeding yeast to produce single cell proteins for human or animal feed, or feeding other microorganisms to produce lactic acid and poly-3-hydroxybutyrate (precursors for some bioplastics). The remaining biomass could potentially be used in products including but not limited to livestock feed, feedstock for cellulosic bioplastic, nanocellulose and lignin based hard carbon.

[0209] The BSG solid residues after each treatment were enzymatically hydrolysed to provide at least a portion of monosaccharide such as glucose. Prior to enzymatic hydrolysis, the compositions of the solid residues were firstly characterised, followed by determination of the amounts of glucose produced from these residues by enzymatic hydrolysis. As shown inTable 6, the untreated BSG sample had the lowest total content of glucan and hemicellulose. NaOH treatment improved the carbohydrate content in solid residue due to the removal of both lignin and protein. Citric acid treatment improved glucan content, possibly due to the higher cellulose stability compared to other biomass components such as protein and hemicellulose under acidic conditions.Table 6: Compositions of BSG solid residuesincluding starch, -glucan and cellulose

[0210] Table 7 shows glucan digestion and glucose yield. It should be noted that glucan in untreated samples (raw BSG and crushed BSG) also included starch. The three treatments led to similar levels of glucan digestion. Based on the solid yields, glucan content and glucan digestion data, the amounts of glucose from solid residues after treatment and hydrolysis were calculated. After the second citric acid treatment and enzymatic hydrolysis of the solid residue, a total of 103 g sugars (75 g glucose and 28 g xylose) can be produced from 1 .0 kg dry BSG.Table 7: Solid recovery and sugar production from the solid residue after each treatment.aGlucan including starch, -glucan and cellulose.

[0211] Although glucose and xylose can be produced from BSG and treated BSG, separation and concentration of these sugars as carbon sources for fermentation can in some embodiments be costly.EXAMPLE 7 - Purity and amino acid profile of crude protein

[0212] Protein purity

[0213] Table 8 shows the contents of protein and carbohydrates in crude proteins. Food Nutrition Information Panels (NIPs) to Australian standards consider protein in the form of crude protein. The crude protein content of Crude protein 1 was 57% (prior to optimisation). Carbohydrates accounted for about 20% of the total crude protein. Due to the darker tan colour of the crude protein, the crude protein likely contains phenolics and lignin (lignin could not be determined due to the interference by protein) and as a result of Maillard reactions. Further, it was found that the weight ratio of Crude protein 1-1 to Crude protein 1-2 was approximately 45:55 and the protein content of Crude protein 1-2 was more than double of that of Crude protein 1-1. These results indicate that crude protein fractions have different particle size and / or average isoelectric points and may be separated with the recovery ofprotein with a high purity (for example 70% purity or above) through for example membrane filtration and / or sequential precipitation or other process optimisation techniques.Table 8: Crude protein purity’Calculated contents based on the data of Crude protein 1-1 and Crude protein 1-2;2Not determined due to insufficient sample

[0214] Crude protein 2 had a similar level of protein content compared to Crude protein 1. The glucan content was lower and the total content of hemicellulose (xylan, arabinan and galactan) was higher than that of Crude protein 1 , respectively. Crude protein 2 may also be further fractionated to higher purity protein.

[0215] Crude protein 3 had the lowest protein content of 51% and the contents of carbohydrates were not determined due to the insufficient amount of sample.EXAMPLE 8 - Amino acid profiles

[0216] Table 9 shows the amino acid profiles of crude proteins recovered from NaOH treatment solutions and citric acid treatment solutions. Glutamic acid was the most abundant amino acids in all the protein.Table 9: Amino acid profiles of recovered crude proteins‘The 9 essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.EXAMPLE 9 - Soluble carbohydrate composition

[0217] The composition of the Carbohydrate 1 and Carbohydrate 2 samples were determined. As shown in Table 10, Carbohydrate 1 and Carbohydrate 2 had total carbohydrate contents of 83% and 76%, respectively. Surprisingly, both carbohydrate samples containedrelatively high content of glucan. In addition, the two carbohydrate samples also contained 1 - 3% crude proteins and citric acid. For Carbohydrate 3, only crude protein content was determined as the amount of sample was not sufficient for compositional analysis.Table 10: Solub e carbohydrate sample composition and purity1not determined due to insufficient sample.

[0218] The glucan in the recovered carbohydrate samples was likely starch considering the high starch content (7.3%) in the MG sample. Starch can be removed from the dietary fibers by enzymatic hydrolysis using amylase and / or glucoamylase. When the recovered and freeze- dried carbohydrate sample was mixed with the glucoamylase solutions at a solid loading of 20%, the solution became jam-like, and mixing and solubilisation is less preferable. With 2% and 5% solid loadings, the solutions could be mixed readily. Therefore, the carbohydrate samples were only hydrolysed at 2% and 5% solid loadings. Table 11 shows the yield and composition of hydrolysed carbohydrates. After hydrolysis and precipitation, about 45%-50% of carbohydrates were recovered. Hydrolysis by glucoamylase reduced the content of glucan and the use of a lower carbohydrate loading was associated with a lower glucan content. With 2% solid loading, the glucan content was reduced to less than 4% compared to 15.7% with 5% solid loading. The relative abundances of xylan and arabinan increased after hydrolysis by glucoamylase compared to those in the unhydrolysed carbohydrate sample.Table 11 : Sample composition in recovered carbohydrates after hydrolysis by glucoamylase

[0219] The results indicate that the majority of the glucan (if not all) in the unhydrolysed carbohydrate samples were starch and post-treatment by glucoamylase could remove the starch component.EXAMPLE 10 - Verification of NaOH treatment for protein solubilisation with low starch-containing BSG sample

[0220] Table 12 shows protein solubilisation, protein recovery and carbohydrate recovery after NaOH treatment of the low-starch containing CV sample. If the treatment conditions developed for the high-starch containing MG sample were used, only 10.3% of the total proteins were solubilised in the liquid. Increasing NaOH concentration from 0.05 M to 0.1 M and treatment temperature from 70 °C to 90 °C increased protein solubilisation. With 0.1 M NaOH and treatment temperature of 90 °C for 2 h, the yields of recovered protein and carbohydrates were higher than those with the high starch-containing BSG sample.

[0221] Although the composition of the recovered carbohydrates was not determined at this time, based on the composition of initial CV-labelled sample, it is likely that the recovered carbohydrates are dominant by dietary fiber components, namely xylan and arabinan instead of starch-derived glucan.Table 12: Protein solubilisation, protein recovery and carbohydrate recovery after NaOH treatment of CV-labelled sample* Treatment time: 2hEXAMPLE 11 - Estimated mass balance and yields of protein and carbohydrates based on the high starch-containing BSG

[0222] Table 13 shows the estimated mass balance and yields (based on the mass of BSG feedstock byproduct) of protein and carbohydrate components (glucan and xylan+arabinan) based on the NaOH treatment and two sequential citric acid treatments. The missing amounts of protein, glucan and xylan is likely present in the process liquid residue solutions.Table 13: Estimated mass balances and yields of protein, glucan and xylan+arabinan.1The amounts of glucan and xylan were estimated based on the composition of Crude protein 2;2The amounts of glucan and xylan were estimated based on the composition of Carbohydrate 2; and3The amount of protein was assumed the same as that in the final treatment residue.EXAMPLE 12 - Processing of bran samples

[0223] The biomass composition of the oat bran and wheat bran samples were analysed using the standard method as described herein. In certain embodiments, it is preferable to provide a pre-hydrolysis step using an amylase and glucoamylase to remove starch in a feedstock byproduct (such as an oat bran) and reduce the viscosity prior to protein and dietary fibre extraction using the process of the present invention. A wheat bran sample was treated with 0.05 M NaOH at 70 °C for 2 h, followed by liquid / solid separation by filtration and recovery of protein and carbohydrates from the treatment solution.

[0224] The compositional results showed that wheat bran contained 23.0% cellulose, 11 .5% xylan, 5.9% arabinan and 1.5% galactan. The wheat bran also contained 16.3% protein as stated on the product packaging. The glucan from hot water extraction (at 100 °C) was not determined but the total water extracts accounted for 20% of the total wheat bran weight.

[0225] From 1 kg wheat bran dry matter, approximately 110 g Crude protein 1 and 29 g Carbohydrate 1 were precipitated from NaOH treatment solution. These results indicate that the method developed for the fractionation of BSG is also applicable for the fractionation of wheat bran.

[0226] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.

[0227] Future patent applications may be filed in Australia or overseas on the basis of or claiming priority from the present application. It is to be understood that the following provisional claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Features may be added to or omitted from the provisional claims at a later date so as to further define or re-define the invention or inventions.

Claims

CLAIMS1. A process for fractionating a feedstock, comprising the steps of: a) providing a protein-containing feedstock comprising a bran; b) treating the feedstock with a protein solubilisation agent to provide a liquid fraction comprising protein and a solid fraction; and c) precipitating the protein from the liquid fraction to provide a protein fraction and a liquid carbohydrate stream.

2. A process according to claim 1 , further comprising the step of wet milling the feedstock.

3. A process according to claim 1 or 2, further comprising the step of treating the feedstock with a pulse electric field.

4. A process according to claim 1 or 2, further comprising the step of treating the feedstock with ultrasound.

5. A process according to claim 1 or 2, further comprising the step of treating the feedstock with hydrodynamic cavitation.

6. A process according to claim 1 or 2, further comprising the step of treating the feedstock with an alcohol.

7. A process according to claim 1 or 2, wherein the solubilised protein liquid fraction b) is separated from solids using a centrifugation device.

8. A process according to claim 7, wherein the centrifugation device is a three-phase centrifugation device to provide an additional lipid rich fraction.

9. A process according to claim 1 or 2, wherein the protein precipitation c) uses a precipitating agent followed by separating the solids fractions via centrifugation to recover a protein rich fraction.

10. A process according to any one of claims 1 to 9, wherein the process further comprises a high-shear force mechanical redispersion of the protein fraction followed by a water wash under acidic conditions followed by additional centrifugation to recover a higher purity protein rich fraction.

11. A process according to any one of claims 1 to 9, comprising d) treating the solid fraction with a carbohydrate solubilisation agent to provide a liquid carbohydrate fraction and a second solid fraction.

12. A process according to claim 10, comprising e) precipitating a carbohydrate from the liquid carbohydrate fraction with a precipitating agent.

13. A process according to claim 11 or claim 12, wherein step c) further comprises precipitating the liquid fraction with a precipitating agent to provide a carbohydrate fraction.

14. A process according to any one of claims 11 to 13, wherein step d) further comprises precipitating a protein from the liquid carbohydrate fraction to provide a protein fraction.

15. A process according to any one of claims 11 to 14, wherein step d) further comprises treating the second solid fraction with a carbohydrate solubilisation agent to provide a second liquid carbohydrate fraction.

16. A process according to claim 15, further comprising precipitating a protein from the second liquid carbohydrate fraction to provide a protein fraction.

17. A process according to claim 15 or 16, further comprising precipitating a carbohydrate from the second liquid carbohydrate fraction with a precipitating agent to provide a carbohydrate fraction.

18. A process according to any one of claims 11 to 16, wherein the carbohydrate is treated with an amylase to substantially reduce residual starch to provide a more purified soluble dietary fibre.

19. A process according to any one of claims 11 to 17, wherein the carbohydrate is a dietary fibre.

20. A process according to any one of claims 1 to 18, wherein the feedstock has a protein content of between about 5% and about 50% on a dry weight basis, preferably between about 15% and 30% on a dry weight basis.21 . A process according to any one of claims 1 to 20, wherein the feedstock bran is selected from the group consisting of brewers spent grain, oat bran, distillers spent grain, reduced-carbohydrate barley, reduced-carbohydrate wheat, whole barley, whole wheat, whole oat, wheat bran and combinations thereof.

22. A process according to any one of claims 1 to 21 , wherein the protein solubilisation agent is a base, a protease, an alcohol blend and combinations thereof.

23. A process according to claim 22, wherein the base is sodium hydroxide, potassium hydroxide and combinations thereof.

24. A process according to any one of claims 1 to 23, wherein the protein solubilisation step comprising treatment with the protein solubilisation agent is performed at a temperature greater than about 50°C, preferably about 70°C.

25. A process according to any one of claims 1 to 24, wherein the protein solubilisation step comprising treatment with the protein solubilisation agent is performed at a pH of about 7.8 to about 11 .5, preferably about pH 10.5.

26. A process according to any one of claims 1 to 25, wherein the protein is precipitated by isoelectric precipitation.

27. A process according to any one of claims 1 to 26, wherein the protein liquid stream is filtered using activated carbon.

28. A process according to any one of claims 1 to 27, wherein the protein is precipitated by addition of an acid, preferably citric acid, hydrochloric acid, sulphuric acid or a combination thereof.

29. A process according to any one of claims 1 to 27, wherein carbohydrates are solubilised at a pH between about 1.5 and about 11 , preferably between about 1.8 and about 5.

30. A process according to any one of claims 11 to 29, wherein the solubilised carbohydrates are precipitated and the precipitating agent is selected from the group consisting of a salt, a lactone, an alcohol and combinations thereof.

31. A process according to claim 30, wherein the alcohol is selected from the group consisting of ethanol, glycerol, 2-propanol and combinations thereof.

32. A process according to any one of claims 1 to 30, wherein the recovered protein yield is at least 20% relative to amount of protein in the feedstock.

33. A process according to any one of claims 1 to 30, wherein the recovered protein purity is at least 50% on a dry matter basis.

34. A process according to any one of claims 1 to 30, wherein the recovered carbohydrate yield is at least 5% relative to amount of carbohydrate in the feedstock

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