WO2025238352A1

WO2025238352A1 - A protein-based material

Info

Publication number: WO2025238352A1
Application number: PCT/GB2025/051031
Authority: WO
Inventors: Edward Mitchell; Daniel Whitaker
Original assignee: Arda Biomaterials Ltd
Current assignee: Arda Biomaterials Ltd
Priority date: 2024-05-15
Filing date: 2025-05-14
Publication date: 2025-11-20
Anticipated expiration: 2026-11-15

Abstract

The present invention is directed towards a method of forming a protein-based plastic substitute material, the method comprising: (a) providing a protein composition comprising at least 30 wt.% of protein, based on the total solids content of the protein composition, wherein the protein includes one or more seed storage proteins selected from albumins, globulins, prolamins, and/or glutelins; (b) forming the protein composition into a solid fused protein-containing structure; and (c) heating the solid fused protein-containing structure to a temperature of more than 60 °C for at least 5 minutes to provide the protein-based plastic substitute material. A protein-based plastic substitute material formed by said method is also encompassed.

Description

A PROTEIN-BASED MATERIAL

FIELD OF THE INVENTION

The present invention relates to methods for the production of a protein-based plastic substitute material that provides a sustainable non-synthetic alternative to plastic materials.

BACKGROUND

Many materials, including synthetic leather, are typically composed of, or predominantly comprise, plastics. These materials are generally derived from non-sustainable fossil fuel resources, and they are typically non-biodegradable and contribute to plastic pollution. There is a desire to replace fossil-fuel derived plastics with other materials from sustainable sources and which avoid the issues associated with the recycling of plastics.

Various plastic alternatives have been considered. However, such materials often require intensive growth of feedstocks, for example cultivated fungi, and thus present significant difficulties with respect to scaling.

It is therefore desirable to provide a sustainable plastic substitute material that mimics the behaviour of fossil fuel-based plastic materials and that can be manufactured via an easily scalable production process.

SUMMARY OF INVENTION

According to a first aspect of the present invention, there is provided a method of forming a protein-based plastic substitute material, the method comprising:

(a) providing a protein composition comprising at least 30 wt.% of protein based on the total solids content of the protein composition, wherein the protein includes one or more seed storage proteins selected from albumins, globulins, prolamins, and/or glutelins;

(b) forming the protein composition into a solid fused protein-containing structure; and (c) heating the solid fused protein-containing structure to a temperature of more than 60 °C for at least 5 minutes to provide the protein-based plastic substitute material.

According to a second aspect of the present invention, there is provided a method of forming a protein-based plastic substitute material, the method comprising: heating a solid fused protein-containing structure to a temperature of more than 60 °C for at least 5 minutes to provide the protein-based plastic substitute material; wherein the solid fused protein-containing structure comprises one or more seed storage proteins selected from albumins, globulins, prolamins and/or glutelins.

According to a third aspect of the present invention, there is provided a proteinbased plastic substitute material obtainable by the method according to the first or second aspect of the present invention.

DETAILED DESCRIPTION

It has been found that the present invention provides a sustainable, non-synthetic alternative to plastic materials, for example, a sustainable alternative to synthetic leather. This novel material is formed utilising a protein composition obtained from plants, such as cereals and legumes. The material can be produced sustainably from a natural source.

The protein-based plastic substitute material formed by the present invention is plastic-free, fully biodegradable, and capable of mimicking properties of plastic materials such as synthetic leather with respect to flexibility and hand feel. It can therefore be used as a non-synthetic alternative to plastic-based materials in, for example, the fashion, textile, packaging, and furniture industries.

It has also been found that the present invention produces a protein-based plastic substitute material having advantageous water stability. The protein-based plastic substitute material does not lose its shape or structure (e.g. does not disintegrate or fall apart) when completely submerged in water, partially submerged in water, or when directly contacted with water, preferably for at least 1 day (24 hours), or for at least 2 days, or 4 days, or for at least 7 days, such as for at least 14 days, or even at least 21 days.

By ‘plastic substitute material’ is meant an alternative material that can be used to replace plastic or synthetic materials, such as synthetic leather. By ‘protein-based’ or ‘protein-containing’ is meant containing a protein composition obtained from plants, i.e. a plant source(s), as defined herein, as opposed to non-renewable materials such as petroleum. The protein-based plastic substitute material of the present invention is a protein-based material that is suitable for use as a plastic substitute material.

The protein of the protein composition is obtained from a plant source(s) containing the one or more seed storage proteins. The protein composition may optionally comprise proteins other than the one or more seed storage proteins. The protein composition may comprise a mixture of proteins in proportions and with chemical structures (i.e. amino acid sequences) that are unique to the plant source(s) used. This means that the protein-based plastic substitute material formed from the protein composition is uniquely suitable for the formation of a sustainable, plastic-free, and biodegradable material. The protein composition may comprise a mixture of proteins obtained from multiple plant sources, or from the same plant source using different extraction processes.

The protein composition may be provided in a solid form, such as a powder form, or may comprise a liquid carrier such as that defined herein.

The plant source may be a plant or crop, such as a cereal or legume, a by-product of oil extraction from plants such as soybean meal or rapeseed meal, a crop residue such as leguminous crop residue, or a by-product of manufacturing or production processes using plants, such as cereals, for example Brewer’s Spent Grain and/or Distiller’s Spent Grain. Any plant source containing one or more seed storage proteins may be used. More than one plant source may be used.

The plant source may be a plant or crop, such as a cereal or legume, for example barley, oat, rye, corn, sorghum, wheat, pea, or soybean, or combinations thereof. The plant source may be a by-product of oil extraction from plants such as soybean meal or rapeseed meal. Rapeseed meal is a by-product of the production of rapeseed oil, whilst soybean meal is by-product of soybean oil extraction. The plant source may be a crop residue, typically process residues left after the crop has been processed into a usable resource. Cereal crop residues include wheat, corn, sorghum, barley, oat, and rye crop residues. Leguminous crop residues include pea or soybean crop residue. The plant source may be a by-product of a manufacturing or production processes using plants such as cereals, for example Brewer’s Spent Grain and/or Distiller’s Spent Grain.

As used herein, the term ‘Brewer’s Spent Grain and/or Distiller’s Spent Grain’ refers to the protein-containing grain by-product of beer (Brewer’s) or spirit (Distiller’s) production, such as beer or whiskey production, in which a grain is utilised. Distiller’s Spent Grain may also refer to the protein-containing grain byproduct of ethanol biofuel production. Brewer’s Spent Grain typically comprises barley grain components, typically comprising protein, lignin, lipid, and cellulose, and optionally other grain components introduced during the brewing process, such as oats, wheat and/or rye. The Brewer’s Spent Grain may further comprise additional components as a result of the processes to which it has been subjected, for example, excess sugars that invite decomposition via bacteria and/or fungal growth within the Brewer’s Spent Grain, and/or enzymes such as amylase utilised in the brewing process and capable of producing more sugars. Distiller’s Spent Grain typically comprises barley, rice, wheat, rye and/or corn grain components, preferably barley grain components, typically comprising protein, lignin, lipid and cellulose, and optionally other grain components introduced during the fermentation process. The Distiller’s Spent Grain may further comprise additional components as a result of the processes to which it has been subjected, for example, insoluble fibrous components and/or excess sugars that invite decomposition via bacteria and/or fungal growth within the Distiller’s Spent Grain and/or enzymes such as amylase utilised in the distillation process and capable of producing more sugars. Typically, Brewer’s Spent Grain and/or Distiller’s Spent Grain may comprise 5 to 40 wt.% protein, preferably 5 to 35 wt.% protein, or 10 to 30 wt.% protein, or 12 to 30 wt.% protein, based on the dry weight of Brewer’s Spent Grain and/or Distiller’s Spent Grain. Typically, Brewer’s Spent Grain and/or Distiller’s Spent Grain may comprise 5 to 40 wt.% lignin, preferably 11 to 32 wt.% lignin, based on the dry weight of Brewer’s Spent Grain and/or Distiller’s Spent Grain. Typically, Brewer’s Spent Grain and/or Distiller’s Spent Grain may comprise 1 to 15 wt.% lipid, based on the dry weight of Brewer’s Spent Grain and/or Distiller’s Spent Grain. Typically, Brewer’s Spent Grain and/or Distiller’s Spent Grain may comprise 10 to 80 wt.% cellulose, such as 20 to 60 wt.% cellulose, preferably 23 to 60 wt.% cellulose, based on dry weight of the Brewer’s Spent Grain and/or Distiller’s Spent Grain. As referred to herein, the ‘cellulose’ of the Brewer’s Spent Grain and/or Distiller’s Spent Grain also encompasses any hemi-cellulose and starch present in the Brewer’s Spent Grain and/or Distiller’s Spent Grain, e.g. a cellulosic fraction. Preferably, at least 90% of the Brewer’s Spent Grain is a barley grain by-product. Brewer’s Spent Grain may be obtained directly from breweries. Distiller’s Spent Grain may be obtained directly from distilleries or ethanol biofuel plants. Brewer’s Spent Grain and/or Distiller’s Spent Grain may be provided in a wet or dry form. Distiller’s Spent Grain encompasses wet distiller’s grains (WDG) and dried distiller’s grains with solubles (DDGS). Preferably, Brewer’s Spent Grain and/or Distiller’s Spent Grain comprises barley grain. Brewer’s Spent Grain and/or Distiller’s Spent Grain may consist of barley grains as the grain source.

Preferably, the plant source from which the protein composition is obtained is Brewer’s Spent Grain and/or Distiller’s Spent Grain, more preferably Brewer’s Spent Grain.

When the seed storage proteins of the protein composition are obtained from Brewer’s Spent Grain and/or Distiller’s Spent Grain, the resulting protein-based plastic substitute material enables the formation of a strong and supple material having increased abrasion resistance, elasticity, and hand feel.

It is particularly advantageous and surprising that Brewer’s Spent Grain and/or Distiller’s Spent Grain can be utilised as the plant source to produce the proteinbased plastic substitute material of the invention. Brewer’s Spent Grain or Distiller’ Spent Grain is not a pure grain, but a waste by-product of beer or ethanol production. Prior to use in the present invention, the grains have already been subjected to highly intensive brewing or distillation processes. It was unexpected that Brewer’s Spent Grain or Distiller’ Spent Grain could provide a suitable feedstock for the production of a protein-based plastic substitute material, since it would have been expected that the intensive processing would cause unpredictable protein degradation, such as hydrolysis and/or denaturing of the proteins. The fact proteins derived from Brewer’s Spent Grain or Distiller’ Spent Grain can be used to form plastic-substitute materials having desirable mechanical properties including strength and flexibility could not have been predicted.

The protein composition and thus one or more seed storage proteins are obtained from a plant source(s) containing the one or more seed storage proteins, as defined herein. Proteins obtained from a plant source differ from those obtained from any non-plant source. The protein composition preferably does not include proteins from non-plant sources. The protein composition preferably does not include proteins from animal sources, for example albumins from human sources. These differ in structure and composition to albumins from plant sources.

Preferably, the one or more seed storage proteins of the protein composition comprise or consist of prolamins. Preferably, the one or more seed storage proteins of the protein composition are prolamins, in addition to one or more of albumins, globulins, and/or glutelins. Preferably, the one or more seed storage proteins of the protein composition comprise or consist of prolamins and glutelins. Preferably, the one or more seed storage proteins of the protein composition comprise 10 wt% or less of albumins.

Preferably, the protein composition comprises prolamins. Preferably, the protein composition comprises prolamins and one or more of albumins, globulins and/or glutelins. Preferably, the protein composition comprises prolamins and glutelins.

Prolamins include gliadins, hordeins, secalins, zein, kafirins and avenins. Gliadins may be obtained from wheat. Hordeins may be obtained from barley. Secalins may be obtained from rye. Zein may be obtained from corn. Kafirin may be obtained from sorghum and avenin may be obtained from oats.

Preferably, the prolamins are hordeins. Preferably, the one or more seed storage proteins of the protein composition comprise or consist of hordeins. Preferably, the one or more seed storage proteins of the protein composition are hordeins, in addition to one or more of albumins, globulins, and/or glutelins. Preferably, the one or more seed storage proteins of the protein composition comprise or consist of hordeins and glutelins.

Preferably, the protein composition comprises hordeins. Preferably, the protein composition comprises hordeins, in addition to one or more of albumins, globulins and/or glutelins. Preferably, the protein composition comprises hordeins and glutelins.

Brewer’s Spent Grain and/or Distiller’s Spent Grain typically comprises barley grain. Hordeins are prolamins of barley.

The protein composition may comprise proteins having a molecular weight falling within the range of 10 to 100 kDa, such as 15 to 100 kDa, or 15 to 90 kDa, or 17 to 70 kDa. The protein composition may comprise proteins having a molecular weight falling within the range of 17 to 100 kDa, or 20 to 90 kDa, or 30 to 80 kDa. The molecular weights of the proteins may be distributed across this range.

The presence of hordeins may be confirmed by SDS-page (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a technique known for the separation of proteins by molecular weight from samples and mixtures. In particular, in the context of the present invention, protein, preferably protein obtained from Brewer’s Spent Grain and/or Distiller’s Spent Grain (obtained as described herein) was boiled in a laemmli buffer (0.125M tris-HCI, pH 6.8, 4% w/v sodium dodecyl sulfate, 20% v/v glycerol, 1 % w/v bromophenol blue and 0.5% 2-mercaptoethanol) and loaded onto a pre-cast 4-15% v/v polyacrylamide gel (Mini-PROTEAN® TGX™ Precast Protein Gels, 15-well, 15 pl #4561086). Samples were subjected to electrophoresis at a constant voltage of 180V. Protein bands were visualised using coomassie blue stain and de-stained in dH^O. Samples were compared to the Precision Plus Protein All Blue (supplied by BioRad) standard protein ladder simultaneously run to quantify the molecular weight.

The protein composition may comprise at least 40 wt.%, preferably at least 50 wt.%, and more preferably at least 60 wt.%, of protein, based on the total solids content of the protein composition.

The protein composition may comprise 30 to 80 wt.%, preferably 30 to 70 wt.%, preferably 40 to 60 wt.% of protein, based on the total solids content of the protein composition.

The protein of the protein composition preferably comprises at least 70 wt.%, preferably at least 80 wt.%, preferably at least 90 wt.%, preferably at least 95 wt.%, preferably at least 98 wt.%, preferably at least 99 wt.%, of said one or more seed storage proteins selected from albumins, globulins, prolamins, and/or glutelins. Optionally, the protein of the protein composition may consist of said one or more seed storage proteins selected from albumins, globulins, prolamins, and/or glutelins.

The protein of the protein composition may be present in the protein-based plastic substitute material in any suitable amount. The protein-based plastic substitute material may comprise from 6 to 90 wt.% of protein, such as from 12 to 80 wt.% of protein. The protein-based plastic substitute material may comprise from 55 to 90 wt.% of protein, such as from 55 to 80 wt.% of protein. The protein-based plastic substitute material may comprise 6 to 60 wt.% of protein, or 12 to 50 wt.% of protein, or 12 to 30 wt.% of protein.

The protein composition, solid fused protein-containing structure and the proteinbased plastic substitute material may further comprise at least one crosslinker. A crosslinker reacts with the proteins of the protein composition. In such cases, the protein composition, the solid fused protein-containing structure, and the proteinbased plastic substitute material may comprise a reaction product of the proteins and the crosslinker. Preferably, the protein composition further comprises a crosslinker, as described herein. Preferably, the solid fused protein-containing structure and/or the protein-based plastic substitute material further comprises a crosslinker. Preferably, the protein composition further comprises a crosslinker and a liquid carrier, as further described herein.

The protein composition may comprise from 1 to 40 wt.% of one or more crosslinkers, preferably from 5 to 35 wt.% of one or more crosslinkers, such as from 5 to 30 wt.%, or from 10 to 25 wt.%, or 5 to 20 wt.%, such as 5 to 15 wt.% or even 7.5 wt.% to 12.5 wt.%, such as 10 wt.%, based on the total solids content of the protein composition.

The solid-fused protein-containing structure and/or the protein-based plastic substrate may comprise from 1 to 40 wt.% of one or more crosslinkers, such as from 5 to 35 wt.%, or from 5 to 30 wt.%, or from 10 to 25 wt%, or 5 to 20 wt.%, such as 5 to 15 wt.%, or even 7.5 wt.% to 12.5 wt.%, such as 10 wt.%.

In the context of the present invention, one or more crosslinkers may be used. Two different crosslinkers may be used. Each crosslinker may be a bridging reagent or a crosslinking catalyst. As used herein, a bridging reagent refers to a chemical reagent comprising a bridging moiety that is incorporated into a crosslink between two or more amino acid residues of the proteins. A bridging reagent comprises reactive groups, the reactive groups interacting with the proteins such that the bridging moiety is incorporated into and forms a link between proteins, for example, between two or more amino acid residues of the proteins. The bridging moiety forms part of the crosslinked structure. It is retained in the crosslinked structure, for example, once the reactive moieties of the bridging reagent have interacted with the protein. With bridging reagents, the proteins may be linked via the bridging reagent or the bridging moiety of the bridging reagent, dependent on the type of crosslinking that occurs. The bridging reagent or bridging moiety forms part of the crosslinked protein structure. The bridging reagent may have reactive groups which interact with the protein. The bridging moiety of the bridging reagent is the moiety that forms part of the crosslinked protein structure. It is a moiety retained in the crosslinked protein structure after the reaction between the protein and bridging reagent, for example once the reactive groups of the bridging reagent have interacted with the protein extract. A crosslinking catalyst refers to a reagent that promotes crosslinking reactions between the existing functional groups of the protein but is not itself incorporated into the crosslinked protein structure.

Preferably, the crosslinker is a bridging reagent.

A bridging reagent and a crosslinking catalyst may be utilised. The bridging reagent and crosslinking catalyst may be used together or sequentially, preferably in step (a).

The protein composition, solid fused protein-containing structure, or protein-based plastic substitute material, may comprise a reaction product of a sequential reaction of the proteins of the protein composition with a bridging reagent and a crosslinking catalyst. For example, the protein composition, solid fused proteincontaining structure, or the protein-based plastic substitute material may comprise a reaction product of a sequential reaction of the proteins of the protein composition with a bridging reagent and then a crosslinking catalyst. Alternatively, the protein composition, solid fused protein-containing structure, or the proteinbased plastic substitute material may comprise a reaction product of a sequential reaction of the proteins of the protein composition with a crosslinking catalyst and then a bridging reagent.

The protein composition, solid fused protein-containing structure, or protein-based plastic substitute material may comprise a reaction product of a sequential reaction of the proteins of the protein composition with a bridging reagent and then a crosslinking catalyst. In this case, the first reaction forms crosslinked proteins linked by bridging moieties. The crosslinked protein composition, and any other proteins, are then induced to further crosslink in a second reaction promoted by the crosslinking catalyst. In the context of the present invention, the term ‘sequential reaction’ includes the reaction of the proteins of the protein composition with a bridging reagent followed directly by the reaction of the resulting product with a crosslinking catalyst. The term also includes the reaction of the proteins of the protein composition with a bridging reagent, followed by other processing steps before the reaction with the crosslinking catalyst. The use of both a crosslinking catalyst and a bridging reagent in the formation of the proteinbased plastic substitute material enables the production of a material having increased tensile strength, tear strength, abrasion resistance, and/or flexibility. This leads to a protein-based plastic substitute material of improved texture and/or hand feel.

Suitable bridging reagents include, but are not limited to: citric acid, sebacic acid, formaldehyde, glutaraldehyde, benzaldehyde, oxalic acid, phosphoric acid, glucuronic acid, fumaric acid, ascorbic acid, tartaric acid, maleic acid, tyrosine, riboflavin, bis(sulfosuccinimidyl)suberate, calcium hydroxide Ca(OH)2, N- hydroxysulfosuccinimide, urea, genipin, azetidinium, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, feruic acid, caffeic acid, and vanillin, or combinations thereof.

Suitable bridging agents may also be one or more poly-epoxy functionalised compounds, or one or more poly-aldehyde functionalised compounds, or combinations thereof. The poly-epoxy functionalised compounds contain two or more epoxide groups. The poly-aldehyde functionalised compounds contain two or more aldehyde groups.

The crosslinker may be one or more poly-epoxy functionalised compounds, or one or more poly-aldehyde functionalised compounds, or combinations thereof.

The one or more poly-epoxy functionalised compounds and/or one or more polyaldehyde functionalised compounds are preferably naturally obtained compounds, i.e. obtained or derived from natural sources. Naturally obtained compounds include bio-based or plant-based compounds. The one or more poly-epoxy functionalised compounds and/or one or more poly-aldehyde functionalised compounds are preferably bio-based, such as plant-based compounds. Plantbased compounds are derived or obtained from plant source(s).

The one or more poly-epoxy functionalised compounds may be formed through the epoxidation of two or more alkene moieties in a single compound, for instance, the epoxidation of two or more carbon-carbon double bond(s) in an unsaturated fatty acid, monoglyceride, diglyceride, or triglyceride, leading to an epoxidised fatty acid, epoxidised monoglyceride, epoxidised diglyceride or epoxidised triglyceride. For example, epoxidised soybean oil can be produced from soybean oil.

The one or more poly-epoxy functionalised compounds may be an epoxidised fatty acid, an epoxidised monoglyceride comprising an epoxidised fatty acid, an epoxidised diglyceride comprising at least one epoxidised fatty acid, or an epoxidised triglyceride comprising at least one epoxidised fatty acid. The epoxidised fatty acid comprises two or more epoxide groups. The epoxidised fatty acid of the epoxidised monoglyceride comprises two or more epoxide groups. The epoxidised diglyceride or epoxidised triglyceride may comprise one epoxidised fatty acid having two or more epoxide groups. Alternatively, the epoxidised diglyceride or epoxidised triglyceride may comprise two or more epoxidised fatty acids with at least one epoxide group on each fatty acid. The epoxide groups may be formed as described above through epoxidation of a carbon-carbon double bond(s) in an unsaturated fatty acid. The fatty acids may have aliphatic carbon chains of at least 6 carbon atoms, such as from 6 to 20 carbon atoms. Suitable unsaturated fatty acids include, but are not limited to: a-linolenic acid, linoleic acid, oleic acid, or combinations thereof. The epoxidised diglyceride or epoxidised triglyceride may further comprise one or more saturated fatty acids.

The epoxidised monoglyceride, epoxidised diglyceride, or epoxidised triglyceride may have the following formula (A): wherein one of -ORi to -OR3 is an epoxidised fatty acid residue comprising two or more epoxide groups, and the remaining two of -OR1 to -OR3 are independently selected from H and a non-epoxidised fatty acid residue; or two or more of -OR1 to -OR3 are independently selected to be an epoxidised fatty acid residue comprising one or more epoxide group, and any remaining R1 to R3 is selected from H and a non-epoxidised fatty acid residue.

Preferably, two or more of -OR1 to -OR3 are independently selected to be an epoxidised fatty acid residue comprising one or more epoxide group, and any remaining -OR1 to -OR3 is selected from H and a non-epoxidised fatty acid residue.

More preferably, two or more of -OR1 to -OR3 are independently selected to be an epoxidised fatty acid residue comprising one or more epoxide group, and any remaining -OR1 to -OR3 is selected to be a non-epoxidised fatty acid residue.

The ‘non-epoxidised fatty acid residue’ may be an unsaturated or saturated fatty acid residue.

The one or more poly-epoxy functionalised compounds may be an epoxidised oil, wherein the epoxidised oil is plant-based. The poly-epoxy functionalised compound may be formed from plant-based oils. A plant-based oil is derived or obtained from a plant source(s).

Suitable epoxidised oils include, but are not limited to: epoxidised canola oil, epoxidised corn oil, epoxidised linseed oil, epoxidised grape seed oil, epoxidised hemp seed oil, epoxidised olive oil, epoxidised peanut oil, epoxidised sesame oil, epoxidised soybean oil, epoxidised walnut oil, epoxidised sunflower oil, epoxidised high oleic canola oil, epoxidised high oleic sunflower oil, and epoxidised high oleic soybean oil.

The one or more poly-epoxy functionalised compounds and/or one or more polyaldehyde functionalised compounds are preferably water-soluble compounds. Preferably, the one or more poly-epoxy functionalised compounds comprise epoxide end groups, i.e. an epoxide group at each end of the compound. Preferably, the one or more poly-aldehyde functionalised compounds comprise aldehyde end groups, i.e. an aldehyde groups at each end of the compound. The end groups may be those at the ends of the length of the main chain of the compound. The main chain is a consecutive chain of atoms that can be considered as the ‘backbone’ of the compound (similar to the ‘backbone’ of a polymer being the main chain of a polymer).

The one or more poly-epoxy functionalised compounds may be poly-epoxy functionalised polyols, such as poly-epoxy functionalised bio-based polyols. The one or more poly-aldehyde functionalised compounds may be poly-aldehyde functionalised polyols, such as poly-aldehyde functionalised bio-based polyols.

The one or more poly-epoxy functionalised compounds may be poly-glycidyl ether epoxy compounds.

The one or more poly-epoxy functionalised compounds, in particular poly-glycidyl ether epoxy compounds, may be formed by reacting a polyol with epichlorohydrin. A base, for example NaOH, may be used as a catalyst. For example, reacting glycerol with epichlorohydrin to make glycerol diglycidyl ether. Preferably, the polyols are bio-based, such as plant-based polyols. Preferably, the epichlorohydrin is bio-based epichlorohydrin. Bio-based epichlorohydrin is typically formed from glycerol, such as glycerol derived or obtained from a plant source. Reagents such as HCI and/or NaOH may be used.

Suitable polyols include bio-based polyols, such as plant-based polyols. Suitable polyols include sugar alcohols and reduced dimer-acid di-alcohols. Suitable polyols include glycerol, sorbitol, isosorbide, propanediol, xylitol, tannic acid, or combinations thereof. Suitable reduced dimer-acid di-alcohols are dimerised fatty acids, including hydrogenated dimerised fatty acids, in which the carboxylic acid groups are, or have been, reduced to alcohols. For example, a reduced dimer of oleic acid. The one or more poly-epoxy functionalised compounds may be poly-glycidyl ether epoxy compounds, in particular naturally derived or obtained poly-glycidyl ether epoxy compounds formed by reacting naturally derived or obtained polyols with bio-based epichlorohydrin, the polyol preferably selected from sugar alcohols and reduced dimer-acid di-alcohols, more preferably from glycerol, sorbitol, isosorbide, propanediol, xylitol, tannic acid, or combinations thereof, and more preferably selected from glycerol, sorbitol or xylitol.

The one or more poly-epoxy functionalised compounds may be poly-epoxy functionalised polycarboxylates, such as poly-epoxy functionalised bio-based polycarboxylates. The one or more poly-aldehyde functionalised compounds may be poly-aldehyde functionalised polycarboxylates, such as poly-aldehyde functionalised bio-based polycarboxylates. A polycarboxylate is a compound comprising two or more carboxylic acid groups.

The one or more poly-epoxy functionalised compounds may be poly-glycidyl ester epoxy compounds.

The one or more poly-epoxy functionalised compounds, in particular poly-glycidyl ester epoxy compounds, may be formed by reacting a polycarboxylate with epichlorohydrin. Preferably, the polycarboxylates are bio-based, such as plantbased polycarboxylates. Preferably, the epichlorohydrin is bio-based epichlorohydrin. Bio-based epichlorohydrin is typically formed from glycerol, such as glycerol derived or obtained from a plant source. Reagents such as HCI and/or NaOH may be used.

Suitable polycarboxylates include dimer acids. Dimer acids are dimerised fatty acids. Suitable polycarboxylates include dicarboxylic acids such as azelaic acid, sebacic acid, aconitic acid, a-keto glutaric acid, tartaric acid, fumaric acid, malic acid, citric acid, dodecanedioic acid, dimerised fatty acids, and hydrogenated dimerised fatty acids. Suitable polycarboxylates include dicarboxylic acids such as polyester dicarboxylic acids, polyamide dicarboxylic acids, polyether dicarboxylic acids, and polycarbonate dicarboxylic acids. Suitable polycarboxylates include sebacic acid. Suitable polycarboxylates include polyester dicarboxylic acid formed by reacting a polyester polyol with one or more dicarboxylic acid monomers. Suitable polycarboxylates include polyamide dicarboxylic acid formed from diamines generated by a bio-fermentation process. Suitable polycarboxylates include dicarboxylic acid formed from diamines generated by a bio-industrial process. Suitable polycarboxylates include branched polycarboxylic acids or hyperbranched polycarboxylic acids. Suitable polycarboxylates include branched polycarboxylic acids or hyperbranched polycarboxylic acids prepared by reacting multifunctional carboxylic acids monomers, or multifunctional alcohols monomers, or multifunctional amines monomers in the polymerization. Suitable polycarboxylates include naturally derived polyfunctional carboxylic acids.

The one or more poly-epoxy functionalised compounds may be poly-glycidyl ester epoxy compounds, in particular naturally derived or obtained poly-glycidyl ester epoxy compounds formed by reacting naturally derived or obtained carboxylates with bio-based epichlorohydrin, the carboxylates being as defined herein.

The one or more poly-epoxy functionalised compounds may have the following formula (la), (lb), or (Ic):

wherein n is from 1 to 12; each occurrence of L is a direct bond or -C(=O)-; each occurrence of R4 is independently selected from a divalent linking moiety; and each occurrence of R5 is independently selected from hydrogen, Ci-C4alkyl,

Optionally, each occurrence of R4 may be independently selected from Ce-C aryl and C2-C7alkylene, preferably C2-C7alkylene. C2-C7alkylene may be selected from C₂-C7alkylene, or C2-C5alkylene, or C^alkylene. Each occurrence of R4 may optionally be independently substituted with one or more of -C(= e Re is independently selected from hydrogen an , preferably -ORe.

Optionally, each occurrence of R₄ may be independently based on a sugar alcohol or reduced dimer-acid di-alcohols, more preferably independently based on glycerol, sorbitol, isosorbide, propanediol, xylitol, tannic acid, or combinations thereof, and more preferably glycerol, sorbitol or xylitol.

Optionally, each occurrence of R4 is the same.

Optionally R4 may be a divalent oligomeric or polymeric moiety, such as an oligosaccharide, polysaccharide, oligopeptide or polypeptide. Optionally R4 may be a monosaccharide or peptide.

Optionally, R5 is independently selected from Ci-C4alkyl, and -CH2-E, where E is

For R5, Ci-C4alkly is preferably Ci-C2alkyl, such as methyl. Optionally, n is 1 , for example, as in compounds of formula (VI) or (VII) below.

Optionally, n may be from 2 to 12, such as from 2 to 10, or from 2 to 8. For example, compound (V) below.

Preferably, the one or more poly-epoxy functionalised compounds are of formula (la). The one or more poly-aldehyde functionalised compounds may have the following formula (Ila), (lib), (lie), or (lid): wherein n is from 1 to 12; each occurrence of L is a direct bond or -C(=O)-; each occurrence of R4 is independently selected from a divalent linking moiety; and each occurrence of R5 is independently selected from hydrogen and C1- C4alkyl.

Optionally, each occurrence of R4 may be independently selected from Ce-C aryl and C2-C7alkylene, preferably C2-C7alkylene. C2-C7alkylene may be selected from C2-C7alkylene, or C2-C5alkylene, or C^alkylene.

Each occurrence of R4 may optionally be independently substituted with one or more of -C(=O)H or -ORe, where Re is independently selected from hydrogen and -C(=O)H.

Optionally, each occurrence of R4 may be independently based on a sugar alcohol or reduced dimer-acid di-alcohols, more preferably independently based on glycerol, sorbitol, isosorbide, propanediol, xylitol, tannic acid, or combinations thereof, and more preferably glycerol, sorbitol or xylitol.

Optionally, each occurrence of R4 is the same.

Optionally R4 may be a divalent oligomeric or polymeric moiety, such as an oligosaccharide, polysaccharide, oligopeptide or polypeptide.

Optionally R4 may be a monosaccharide or peptide.

For R5, Ci-C4alkly is preferably Ci-C2alkyl, such as methyl.

Optionally, n is 1. Optionally, n may be from 2 to 12, such as from 2 to 10, or from 2 to 8.

Preferably, the one or more poly-aldehyde functionalised compounds are of formula (Ila).

Preferably, the one or more poly-epoxy functionalised compounds are selected from formula (III) to (VIII): wherein n is 1 to 3, preferably 2 or 3, and more preferably 3;

combinations thereof. Preferably, the one or more poly-epoxy functionalised compound is selected from formula (III), (IV), (V), (VII), or combinations thereof. For formula (V), n is preferably 2 or 3, such as 3. The one or more poly-epoxy functionalised compound may be selected from formula (III), (IV), and (VII), or combinations thereof. Preferably, the one or more poly-epoxy functionalised compound is selected from formula (V), preferably where 2 or 3, such as 3.

Preferably, the one or more poly-epoxy functionalised compounds are selected from: glycerol diglycidyl ether, glycerol polyglycidyl ether, polyglycerol polyglycidyl ether, polyethylene glycol)diglyciyl ether, polypropylene glycol)diglycidol ether, sorbitol polyglycidyl ether, diglycidyl ether of isosorbide, diepoxy xylitol, triepoxy xylitol, tetraepoxy xylitol, pentaepoxy xylitol, or combinations thereof.

Suitable commercially available poly-epoxy functionalised compounds include GEX-313, GEX-512, GEX-521 , GEX-622 and GEX-614b sold under the tradename DENACOL™ by DENACOL and BRIOZEN® RD 124 G, RD 131 G, RD 133 G, RD 135 G and RD 143 G obtained from Aditya Birla Chemicals.

The one or more poly-epoxy functionalised compounds may be epoxyfunctionalised monosaccharides, disaccharides or polysaccharides. The one or more poly-aldehyde functionalised compounds may be aldehyde-functionalised monosaccharides, disaccharides or polysaccharides.

The one or more poly-epoxy functionalised compounds may be protein-derived poly-epoxy functionalised compounds. The one or more poly-aldehyde functionalised compounds may be protein-derived poly-aldehyde functionalised compounds.

In particular, the use of one or more poly-epoxy functionalised compounds and/or one or more poly-aldehyde functionalised compounds, or combinations thereof, as a crosslinker advantageously increases water stability and resistance to shrinkage and/or swelling in water of the protein-based plastic substitute material, once formed. This is advantageous as it means materials or articles formed thereof will not be damaged or altered upon exposure to moisture, such as in rainy conditions. The materials formed are able to retain shape, structure and mechanical properties such as flexibility and strength, after submersion in, or contact with, water. Resistance to shrinkage and/or swelling in water may be measured by the % difference in dimensions of the material in water, over time. A value as close to 0% as possible is desirable. A value of ±30% or less, such as ±20% or less, or ±10% or less may be satisfactory. The ± accounts for shrinking or swelling depending on which property is being assessed. The use of one or more poly-epoxy functionalised compounds as a crosslinker, in particular those of formula (III), (IV), (V) and (VII), or combinations thereof, also advantageously provides both a strengthening and plasticising effect to the material. This may be demonstrated by tensile strength and elongation. Without being bound by theory, the present inventors consider this to be due to pendant moieties increasing free volume in the protein matrix of the material.

Preferably, the bridging reagent is selected from citric acid, formaldehyde, urea, genipin, azetidinium, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, ferulic acid, caffeic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof. Such bridging reagents may be obtained or derived from natural sources, i.e. ‘naturally- derived’. Such components are preferably not derived from petroleum. Such components are preferably not synthetic.

More preferably, the bridging reagent is selected from citric acid, urea, genipin, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, ferulic acid, caffeic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof. These bridging reagents are naturally derived and have good biodegradability and low toxicity. More preferably, the bridging reagent is selected from citric acid, malic acid, tannic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or is a combination thereof. Most preferably, the bridging reagent is selected from citric acid, malic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds or a combination thereof. Presence of the bridging reagent in the solid fused protein-containing structure or the protein-based plastic substitute material enables the formation of a proteinbased plastic substitute material having increased tensile strength, tear strength and/or abrasion resistance.

The bridging reagent(s) may be present in the protein composition in any suitable amount, for example from 1 to 40 wt.% based on the total solids content of the protein composition, such as from 5 to 35 wt.%, or from 5 to 30 wt.%, or from 10 to 25 wt%, or even from 1 to 30 wt.%, or from 1 to 20 wt.%, or 5 to 20 wt.%, such as 5 to 15 wt.%, or even 7.5 wt.% to 12.5 wt.%, such as 10 wt.%.

The bridging reagent(s) may be present in the solid fused protein-containing structure or the protein-based plastic substitute material in any suitable amount, for example from 1 to 40 wt.%, such as from 5 to 35 wt.%, or from 5 to 30 wt.%, or from 10 to 25 wt%, or even from 1 to 30 wt.%, or from 1 to 20 wt.%, or 5 to 20 wt.%, such as 5 to 15 wt.%, or even 7.5 wt.% to 12.5 wt.%, such as 10 wt.%.

The bridging reagent has reactive groups which interact with the proteins. The bridging moiety of the bridging reagent is the moiety that forms part of the crosslinked protein composition structure. It is a moiety retained in the crosslinked structure after the reaction between the proteins and bridging reagent, once the reactive groups of the bridging reagent have interacted with the proteins. For example, citric acid may crosslink via hydrogen bond donation or acceptance, the formation of ion bridges from the conjugate base, or by covalent bonding through the reactive groups of the crosslinker as an electrophile or as a nucleophile.

Suitable crosslinking catalysts include enzymes. When an enzyme is utilised as the crosslinking catalyst (‘enzymatic crosslinking'), the enzyme facilitates the crosslinking of the proteins of the protein composition themselves. Typically, the proteins of the protein composition are linked together through the formation of bonds between reactive moieties of the proteins of the protein composition. A crosslinked protein composition is formed. The crosslinking catalyst functions differently to the bridging reagent detailed above in that it does not itself become part of the crosslinked protein structure. The enzyme remains unchanged. Exposure of the proteins of the protein composition to the enzyme induces protein crosslinking, enhancing the chain length of the proteins.

When the crosslinking catalyst is an enzyme, the enzyme may be selected from a transglutaminase, a lysyl oxidase and a laccase, or combinations thereof.

Preferably, the enzyme is a transglutaminase, preferably a non-animal derived transglutaminase. Preferably, the transglutaminase is a microbial transglutaminase, such as a bacterial transglutaminase. A suitable transglutaminase is available under the tradename Stabizym®, such as Stabizym® TGL.

The crosslinking catalyst acts to catalyse the formation of bonds between the proteins of the protein composition themselves to form a crosslinked protein structure. For example, where a transglutaminase is utilised, the crosslinking catalyst catalyses the formation of isopeptide bonds between carboxyamide groups and amine groups of the proteins of the protein composition.

Accordingly, the solid fused protein-containing structure or the protein-based plastic substitute material may comprise a reaction product of the proteins of the protein composition and a crosslinker, the reaction product comprising the proteins of the protein composition crosslinked by isopeptide bonds.

The crosslinking catalyst may be accompanied by a metal salt, such as a calcium salt, for example Ca(OH)2, that enhances the effect of the crosslinking catalyst. For example, an enzyme such as transglutaminase may be accompanied by a metal salt, such as a calcium salt, for example Ca(OH)2.

The crosslinking catalyst may be present in the solid fused protein-containing structure or protein-based plastic substitute material. Alternatively, it may be removed once it has completed its role of catalysing the formation of the bonds between the proteins, for example by washing or centrifugation, or other appropriate mechanisms. When retained in the solid fused protein-containing structure or the protein-based plastic substitute material, the crosslinking catalyst may be present in any suitable amount. The solid fused protein-containing structure or protein-based plastic substitute material may comprise from 0.1 to 8 wt.% of the crosslinking catalyst, such as from 1 to 4 wt.% of the crosslinking catalyst. The crosslinking catalyst may be used in an amount of 0.1 to 4 wt.% with respect to the wt% of protein composition.

When a crosslinker is used, the proteins may be combined with the crosslinker at any suitable temperature. If a poly-epoxy functionalised compound, polyaldehyde functionalised compound, or crosslinking catalyst is used as a crosslinker, the combination of the proteins and bridging reagent or crosslinking catalyst preferably takes place at a temperature of less than 60 °C, such as less than 50 °C. This is to prevent deterioration or denaturing of the bridging agent or crosslinking catalyst.

Use of a crosslinking catalyst in the formation of the protein-based plastic substitute material enables the production of a material having increased tear strength, tensile strength, abrasion resistance and/or flexibility. This leads to a protein-based plastic substitute material of improved texture and/or hand feel.

The term ‘crosslinked protein composition’ or ‘crosslinked protein structure’, or like terms used herein, refer to proteins of the protein composition linked by bridging reagent as discussed above, and/or proteins of the protein composition themselves linked following reaction with a crosslinking catalyst, for example, proteins linked by isopeptide bonds.

When a crosslinker is used, the proteins may be combined with the crosslinker: in step (a); during formation of the protein composition into the solid fused proteincontaining structure in step (b); or following formation of the solid fused proteincontaining structure in step (b).

The protein composition may further comprise a plasticiser, optionally more than one plasticiser. The proteins of the protein composition may be combined with a plasticiser in step (a), preferably the proteins of the protein composition are combined with a plasticiser in a liquid carrier.

The protein composition in step (a) may comprise from 5 to 60 wt.%, or from 10 to 60 wt.%, or from 10 to 50 wt.%, such as from 20 to 50 wt.%, or from 15 to 45 wt.%, such as 15 to 40 wt.%, or from 20 to 40 wt.%, or from 30 to 40 wt.% of the plasticiser, based on the total solids content of the protein composition.

Preferably, the plasticiser is a naturally derived plasticiser. Suitable plasticisers include, but are not limited to sugar alcohols such as mannitol, xylitol, glycerol, erythritol, and sorbitol; water; ethylene glycol; polyethylene glycol; propylene glycol; lecithin; sunflower lecithin; diethylene glycol; tetraethylene glycol; ethanolamine; triethanolamine; acetic acid; glycol; fatliquor; fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid; sorbitan; didecyldimethylammonium chloride (DDAC); polysorbate 20; polysorbate 80; erythritol; triethyl citrate; and acetylated monoglyceride.

Preferably, the plasticiser is selected from sugar alcohols such as glycerol, sorbitol, and erythritol; water; ethanol; acetic acid; fatliquor; fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid; sorbitan; polysorbate 20; polysorbate 80; triethyl citrate; and acetylated monoglyceride. These plasticisers are not only naturally derived but have good biodegradability and low toxicity. Preferably, the plasticiser is selected from glycerol and water. More preferably, the plasticiser is selected from glycerol and water, or a combination thereof.

The presence of a plasticiser in the protein-based plastic substitute material enables the production of a protein-based plastic substitute material having further increased flexibility, softness, and elasticity.

The plasticiser may be present in the solid fused protein-containing structure or the protein-based plastic substitute material in any suitable amount. The solid fused protein-containing structure or the protein-based plastic substitute material may comprise 1 to 35 wt.% plasticiser, such as from 5 to 30 wt.% plasticiser, or 5 to 25 wt.% plasticiser. The solid fused protein-containing structure or the proteinbased plastic substitute material may comprise from 5 to 60 wt.%, or from 10 to 60 wt.%, or from 10 to 50 wt.%, such as from 20 to 50 wt.%, or from 15 to 45 wt.%, such as 15 to 40 wt.%, or from 20 to 40 wt.%, or from 30 to 40 wt.% of the plasticiser.

The protein composition in step (a) preferably further comprises a plasticiser. The protein composition preferably further comprises one or more crosslinker and a plasticiser. The solid fused protein-containing structure and the protein-based plastic substitute material preferably further comprises a plasticiser. The solid fused protein-containing structure and the protein-based plastic substitute material preferably further comprises one or more crosslinker and a plasticiser.

The protein composition in step (a) may further comprise one or more additional components. Suitable additional components may include antimicrobial agents; antifungal agents; inorganic components such as inorganic metal salts, including calcium carbonate (CaCOs), calcium hydroxide (Ca(OH)2, potassium chloride (KCI); binding agents such as wheat gluten, resins, bio-based polymers such as alginates, chitosan and starch; dyes or colorants; fillers including titanium dioxide, calcium carbonate, sodium carbonate, carbon fibres, birch bark powder, nano silica, olive stone powder, coconut shell powder, cork powder, rice hull char, carboxymethylcellulose, methylcellulose, fibrillated nanocellulose, nanocellulose, gum arabic, agar, chitosan, montmorillonite, talc, calcium silicate, alumina, graphene, kaolin, nanoclay, mica, sodium citrate, wollastonite, rosin, and alignate; pigment or lightening agents such as titanium dioxide, waxes such as candelilla wax and carnauba wax, and aqueous wax emulsion; oils such as linseed oil, vegetable oil, tea tree oil, coconut oil, almond oil, and soybean oil, sunflower oil, bergamot oil, cinnamon oil, olive oil, and rice bran oil; lanolin; benzyl alcohol; salicylic acid; sorbic acid; and turmeric; natural rubber; or combinations thereof. Preferably, carboxymethylcellulose, methylcellulose, fibrillated nanocellulose, nanocellulose, or combinations thereof may be included in the protein composition in step (a). Preferably, carboxymethylcellulose is included in the protein composition in step (a). The one of more additional components may each be present in the protein composition in step (a) in any suitable amount, e.g. from 1 to 50 wt.%, based on the total solids content of the protein composition. The solid fused protein-containing structure and the protein-based plastic substitute material may therefore further comprise one or more additional component, as described above.

The protein composition may further comprise a liquid carrier. The liquid carrier is preferably an aqueous liquid carrier. The liquid carrier may be water or a mixture of water with one or more other water-miscible solvents. Preferably the liquid carrier is water. The liquid carrier may optionally comprise or consist of water and/or the solvent used to extract the seed storage proteins of the protein composition from the plant source.

The protein composition may comprise at least 50 wt.% liquid carrier, based on the total weight of the protein composition (total solids and liquid carrier). For example, the protein composition may comprise at least 60 wt.% liquid carrier, or at least 70 wt.% liquid carrier, or at least 80 wt.% liquid carrier, or at least 90 wt.% liquid carrier. The protein composition preferably comprises 70 to 98 wt.% liquid carrier.

Preferably, the seed storage proteins of the protein composition are solubilised and/or dispersed in the liquid carrier. Any crosslinker and/or plasticiser, and optionally one or more additional components may be solubilised, dispersed or precipitated in the liquid carrier.

If the protein composition comprises a liquid carrier, the total solids content of the protein composition may be 30 wt.% or less, such as 20 wt.% or less, or even 10 wt.% or less, optionally 1 to 30 wt.%, such as 2 to 30 wt.%, or from 3 to 25 wt.%.

The protein composition may comprise 15 wt.% or less, such as 10 wt.% or less, or 5 wt.% or less, such as 0 wt.%, of synthetic polymers, in particular, 15 wt.% or less, such as 10 wt.% or less of non-proteinaceous polymers, such as polymethyl acrylic acid, polyurethane, polyurethane-polyamide copolymers, polyurethanepolyester copolymers, polyacrylic acid-polyester copolymers, polyacrylic acid- polyamide copolymers, natural rubber latex, natural rubber, technically specified or block natural rubber (TSR), ribbed smoked sheet natural rubber (RSS), high ammonia natural rubber latex concentrate, low ammonia natural rubber latex concentrate, deproteinized natural rubber latex concentrate with a dry rubber content in a range of about 50% to about 62%, polyurethane-polycarbonate copolymers, polyurethane-polyether copolymers, or any combination thereof.

In step (b), the protein composition (of step (a)) is formed into a solid fused proteincontaining structure by any suitable method. The method will depend upon the desired form of the protein-based plastic substitute material.

The protein composition may be formed by rolling, tape casting, casting and optionally rolling, electrospinning, solution casting, thermal extrusion, or thermal compression molding or baking. Preferably, in step (b), the protein composition is formed by electrospinning, tape casting, casting or solution casting. More preferably, in step (b), the protein composition is formed by tape casting, casting or solution casting, such as by solution casting.

Step (b) may comprise applying the protein composition to a forming surface and forming the solid fused protein-containing structure on the forming surface. The protein composition may be applied to the forming surface by any suitable method. The protein composition may be applied to the forming surface and then rolled. The protein composition may be cast onto the forming surface.

Step (b) may comprise collecting the solid fused protein-containing structure on a forming surface. For example, for electrospinning, the protein composition is not applied to a forming surface but electrospun fibres formed by electrospinning are collected on a forming surface such as a charged collector surface. The technique of electrospinning is well known in the art. Electrospun fibres are formed by exposure of a solution (protein composition comprising a liquid carrier) to a high voltage. During electrospinning, the liquid carrier is evaporated and electrospun fibres are collected on a forming surface such as a charged collector. The forming surface may be any suitable surface. Preferably, the forming surface is a flat surface. Optionally, the forming surface may be patterned, or embossed (raised or recessed) with a mark, pattern or image that will then be present on the surface of the protein-based plastic substitute material. The forming surface may be a mould, container, or any such space having a surface contained within boundaries orwalls. The forming surface may be a conveyor. The forming surface may not require boundaries orwalls.

When the protein composition further comprises a liquid carrier, the protein composition may be coated onto the forming surface, cast onto the forming surface, or poured on to the forming surface. Formation of the solid fused proteincontaining structure in step (b) by solution casting involves applying a protein composition further comprising a liquid carrier to a forming surface and removing some or all of the liquid carrier. The liquid carrier may be removed by evaporation of the liquid carrier.

Preferably, step (b) is not carried out under any applied pressure. Preferably, the protein composition is not subjected to any applied pressure in the formation of the solid fused protein-containing structure of step (b). Preferably, step (b) is carried out at atmospheric pressure (around 0.1 MPa). Preferably, step (b) is carried out at a pressure of 0.2 MPa or less.

The solid fused protein-containing structure may have any suitable shape. The solid fused protein-containing structure may have any suitable surface area.

The solid fused protein-containing structure may be in the form of a layer or sheet. The solid fused protein-containing structure may be a film. The solid fused protein-containing structure may be three dimensional. The solid fused proteincontaining structure may be formed of fibres. Preferably, the solid fused proteincontaining structure may be in the form of a layer or sheet.

The ‘solid fused protein-containing structure’ refers to a structure in which the protein composition, in particular the protein thereof, agglomerates or otherwise comes together and interacts, and forms a cohesive structure. The solid fused protein-containing structure is solid. It is preferably firm and stable. The proteins of the protein composition interact to form the solid fused structure, this may be through the presence of an optional crosslinker, or the interactions between the proteins themselves.

In the case that the protein composition comprises a liquid carrier, step (b) generally comprises the removal of some or all of the liquid carrier such that the protein composition attains a solid form. However, it is not excluded that some of the liquid carrier may be retained within the solid fused protein-containing structure.

Step (b) may comprise removing some or all of the liquid carrier to form the solid fused protein-containing structure. This may be done on the forming surface, such as in solution casting. Alternatively, the liquid carrier may be removed before collection of the solid fused protein-containing structure on the forming surface, such as in electrospinning. The liquid carrier may be removed by allowing the liquid carrier to evaporate, as outlined below.

The solid fused protein-containing structure may be formed in step (b) by allowing the liquid carrier to evaporate. Evaporation of the liquid carrier may be carried out under ambient conditions, including ambient temperature (10 to 35 °C, for example 18 to 25 °C) and 30 to 60% RH (relative humidity), for example 40 to 55% RH. Suitable methods of evaporation also include microwave drying, vacuum drying and radiowave drying. Evaporation of the liquid carrier may also be carried out at low relative humidity such as less than 30% RH, and/or under vacuum conditions. Evaporation of the liquid carrier may be carried out in a desiccator. Air flow may be increased across the forming surface to facilitate drying. Evaporation of the liquid carrier may be carried out in drying chambers, as a forming surface is placed or passes therethrough. Evaporation of the liquid carrier may be carried out using any combination of these methods. The length of time of drying may vary. The drying may take 72 hours or less, such as 24 hours or less, or 12 hours or less. The drying may take at least 30 minutes, such as at least 1 hour, or at least 12 hours, or 24 hours, or at least 72 hours. Drying in a desiccator, microwave drying, vacuum drying, radiowave drying, drying at low relative humidity such as 30% RH or less and/or under vacuum conditions, or combinations thereof may be used if increased drying is required, for example if the thickness of the solid fused protein-containing structure being formed is greater than 0.7 mm in thickness.

The solid fused protein-containing structure preferably has a moisture content of from 25 wt.% or less, such as 20 wt.% or less, or 15 wt.% or less, such as 10 wt.% or less. The solid fused protein-containing structure preferably has a moisture content of from 5 to 25 wt.%, such as from 5 to 20 wt.%, or from 10 to 20 wt.%. A moisture content within these ranges avoids deformation of the solid fused proteincontaining structure upon heating to more than 60 °C, for example due to the formation of bubbles of water vapour during step (c).

‘Moisture content’ as used herein with reference to any entity (e.g. solid fused protein-containing structure, protein-based plastic substitute material etc.) refers to the content of water and/or other solvent in the entity. The moisture content may be measured by weighing the entity, heating the entity till dry, and reweighing to determine the moisture content lost. This may be known in the art as a ‘loss on drying’ method. Suitable apparatus for determining the moisture content include a thermogravimetric moisture analyser or moisture analyser.

Where step (b) of the method according to the first aspect of the present invention includes application of the protein composition to a forming surface, the method may further comprise removing the solid fused protein-containing structure from the forming surface prior to step (c). Alternatively, where step (b) of the method according to the first aspect of the present invention includes application of the protein composition to a forming surface, the protein-based plastic substitute material may be removed from the forming surface after step (c).

In step (c), the solid fused protein-containing structure is heated to a temperature of more than 60 °C. Such heating advantageously enables the protein-based plastic substitute material formed to be water stable. Without being bound by theory, the present inventors consider that the heating promotes structural changes to the protein composition that increase water stability. These structural changes are thought to include rearrangement of the folding/packing of proteins to a more thermodynamically stable arrangement, including the rearrangement of crosslinks between the amino acid residues of the protein, as well as further reaction of the crosslinker, for example bridging reagents, if present.

For the method according to the second aspect of the present invention, a solid fused protein-containing structure is heated to a temperature of more than 60 °C. The solid fused protein-containing structure is as described herein for the first aspect of the present invention.

Preferably, for both the first and second aspects of the present invention, the solid fused protein-containing structure is heated to a temperature of 65 °C or more, or 70 °C or more, such as 80 °C or more, or 100 °C or more, or 110 °C or more, or 120 °C or more. The temperature refers to the internal temperature of the solid fused protein-containing structure that is attained during step (c). For thin planar or sheet-like structures, the internal temperature may be assumed to be equivalent to the surface temperature. For three-dimensional structures, the internal temperature of the solid fused protein-containing structure may be calibrated using a temperature probe in a test sample.

Preferably, the solid fused protein-containing structure is heated to a temperature of 160 °C or less, such as 140 °C or less.

Preferably, the solid fused protein-containing structure is heated to a temperature of from 65 to 160 °C, preferably from 70 to 150 °C, more preferably from 80 to 140 °C, such as from 90 to 130 °C, and more preferably from 90 to 120 °C.

Preferably, for both the first and second aspects of the present invention, the solid fused protein-containing structure is heated for 5 minutes or more, such as for 15 minutes or more, or for 30 minutes or more, such as for 45 minutes or more.

Preferably, the solid fused protein-containing structure is heated for 3 days or less, such as for 2 days or less, or for 1 day or less, such as for 18 hours or less, or for 15 hours or less, such as for 14 hours or less, or for 12 hours or less, or for 10 hours or less, or even for 8 hours or less, such as for 6 hours or less, or 4 hours or less, or 120 minutes or less, such as for 90 minutes or less, or for 60 minutes or less.

Preferably, the solid fused protein-containing structure is heated for from 5 minutes to 3 days, such as from 10 minutes to 3 days, or from 15 minutes to 1 day, or from 20 minutes to 18 hours, or from 25 minutes to 16 hours, or from 30 minutes to 15 hours, or from 35 minutes to 14 hours, or from 40 minutes to 12 hours, or from 45 minutes to 10 hours, or from 45 minutes to 8 hours, or 45 minutes to 6 hours, or 50 minutes to 4 hours.

Preferably, the solid fused protein-containing structure is heated for from 5 to 120 minutes, such as from 10 to 90 minutes, or from 15 to 90 minutes, or 15 to 60 minutes.

Preferably, for both the first and second aspects of the present invention, the solid fused protein-containing material is heated to a temperature of more than 60 °C, such as 65 °C or more, or 70 °C or more, such as 80 °C or more, or 100 °C or more, such as 110 °C or more, or 120 °C or more, for 5 minutes or more, such as for 15 minutes or more, or for 30 minutes or more, such as 45 minutes or more. This may be for 120 minutes or less, such as for 90 minutes or less, or for 60 minutes or less, or for 5 to 120 minutes, such as from 10 to 90 minutes, or from 15 to 90 minutes, or 15 to 60 minutes.

Preferably, for both the first and second aspects of the present invention, the solid fused protein-containing structure is heated to a temperature of 160 °C or less, such as 140 °C or less, for 5 minutes or more, such as for 15 minutes or more, or for 30 minutes or more, such as 45 minutes or more. This may be for 120 minutes or less, such as for 90 minutes or less, or for 60 minutes or less, or for 5 to 120 minutes, such as from 10 to 90 minutes, or from 15 to 90 minutes, or 15 to 60 minutes.

Preferably, for both the first and second aspects of the present invention, the solid fused protein-containing structure is heated to a temperature of from 65 to 160 °C, preferably from 70 to 150 °C, more preferably from 80 to 140 °C, such as from 90 to 130 °C, and more preferably from 100 to 120 °C, for 5 minutes or more, such as for 15 minutes or more, or for 30 minutes or more, such as for 45 minutes or more. This may be for 120 minutes or less, such as for 90 minutes or less, or for 60 minutes or less, or for 5 to 120 minutes, such as from 10 to 90 minutes, or from 15 to 90 minutes, or 15 to 60 minutes.

Heating of the solid fused protein-containing structure may be achieved through conventional heating methods. For example, heating of the solid fused proteincontaining structure may be achieved through heating in an oven, on a hot plate, or other suitable heating apparatus.

Preferably, step (c) of the first aspect of the present invention, or the heating of the solid fused protein-containing structure in the second aspect of the present invention, is not carried out under any applied pressure. Preferably, the solid fused protein-containing structure is not subjected to any applied pressure in the formation of the protein-based plastic substitute material, e.g. in step (c). Preferably, step (c) of the first aspect of the present invention, or the heating of the solid fused protein-containing structure in the second aspect of the present invention, is carried out at atmospheric pressure (around 0.1 MPa). Preferably, step (c) of the first aspect of the present invention, or the heating of the solid fused protein-containing structure in the second aspect of the present invention, is carried out at a pressure of 0.2 MPa or less.

After the heating of step (c) of the first aspect of the present invention, or the heating of the solid fused protein-containing structure in the second aspect of the present invention, the protein-based plastic substitute material may have a moisture content of 10 wt.% or less, such as 5 wt.%, or less or 4 wt.% or less, such as 3 wt.% or less, or even 1 wt.% or less, such as 0%. After the heating of step (c), the protein-based plastic substitute material may have a moisture content of 0 to 10 wt.%, or 0 to 5 wt%. Moisture, such as water and/or solvent, has been driven from the material during heating. The protein structure may be fragile or brittle with such a low water content and therefore a rehydration step may be used prior to any further processing of the material, such that the protein-based plastic substitute material achieves sufficient plasticity for further processing.

Accordingly, the method of the invention (of either the first or second aspect of the present invention) preferably further comprises the step of:

(d) equilibrating the protein-based plastic substitute material in an atmosphere of 10-90% relative humidity (RH) and at a temperature in the range from 10 to 50 °C.

In step (d), the protein-based plastic substitute material will absorb a certain amount of atmospheric moisture until it reaches equilibrium with the surroundings. This is referred to herein as ‘rehydration’ of the protein-based plastic substitute material.

Preferably, the protein-based plastic substitute material is preferably equilibrated in step (d) at a temperature in the range from 15 to 40 °C, preferably from 18 to 35 °C, more preferably from 20 to 30 °C.

Preferably, the protein-based plastic substitute material is preferably equilibrated in step (d) at a relative humidity in the range from 30 to 80% RH or from 40 to 75% RH or from 55 to 70% RH.

The duration of step (d) depends on the form of the protein-based plastic substitute material. However, it is suitably in the range from 1 to 100 hours.

The resulting protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may have a moisture content of 5 to 25 wt.%, such as from 5 to 20 wt.%, or from 10 to 20 wt.%.

The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may comprise less than 1 wt.%, such as less than 0.5 wt.%, such as 0 wt.% of a component derived from petroleum. The protein-based plastic substitute material may not comprise a petroleum-based component such as a petroleum-based resin. The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may be provided as a layer or sheet of material. The protein-based plastic substitute material may be a film. The protein-based plastic substitute material may be three dimensional. The protein-based plastic substitute material may be formed of fibres. Preferably, the protein-based plastic substitute material is in the form of a layer or sheet of material. The protein-based plastic substitute material may be planar or flat.

The protein-based plastic substitute material (the protein-based substitute material according to the third aspect of the present invention) may have any suitable shape. The protein-based plastic substrate material may have any suitable surface area.

The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may have any suitable thickness. Preferably, the protein-based plastic substitute material has a thickness of from 0.2 to 10 mm, or from 0.2 to 5 mm, such as from 0.2 to 2 mm, or 0.2 to 1 .5 mm, such as from 0.4 to 1 .5 mm, or 0.5 to 1 .5 mm, or from 0.8 to 1 .4 mm.

If the protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) is electrospun fibres, the fibres may have a micrometre-scale average diameter or width.

It has been surprisingly and advantageously found that the protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) can mimic the properties of animal-derived or synthetic leather. The protein-based leather substitute material demonstrates advantageous high tensile strength, tear strength, flexibility, abrasion resistance, and elasticity. The protein-based plastic substitute material also has advantageous texture and ‘hand feel’.

The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may have a tensile strength of from 0.5 to 130 MPa, such as from 0.5 to 60 MPa, or even from 2 to 60 MPa, or from 3 to 60 MPa, such as from 4 to 60 MPa, such as from 4 to 45 MPa, or from 4 to 40 MPa, such as from 4 to 30 MPa, or 4 to 25 MPa, such as 7 to 25 MPa. Tensile strength may be measured on a Z3 X500 Universal Testing Machine with an ISO 37 type 2 dumbell cutter. Tensile strength is measured according to ISO 3376:2020.

The tear strength of the protein-based plastic substitute material may be assessed by measurement of tear load. The protein-based plastic substitute material may have a tear load of from 4 to 25 N, such as from 5 to 20 N. Tear load is measured according to ISO 3377-1 :2011.

The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may be a flexible sheet of material. The flexibility of the protein-based plastic substitute material may be assessed by measurement of flex resistance. The protein-based plastic substitute material may have a flex resistance of from 15,000 to 300,000 cycles, such as from 20,000 to 200,000 cycles. Flex resistance is measured according to ISO 5402-1 :2022.

The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may have an abrasion resistance of from 80 to 250 cycles, such as from 100 to 200 cycles. Abrasion resistance is measured according to ISO 17076-1 :2020.

The elasticity of the protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) may be assessed by measurement of elongation at load. The protein-based plastic substitute material may have an elongation at load of from 5 to 120%, or from 10 to 100%, or from 10 to 80%, or from 10 to 65%, such as from 15 to 65%, or 15 to 60%, or from 20 to 50%, or even from 5 to 40%, such as from 5 to 30%, or from 5 to 25%, such as 10 to 25%, or from 10 to 20%. Elongation at load is measured according to ISO 3376:2020. The elongation at load of a material is a measure of deformation that occurs when the material is subjected to the highest tensile load it can withstand. The protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) has an advantageous ‘hand feel’. By hand feel is meant how a material feels against the skin or in the hand. In the context of the present invention, hand feel is represented by a softness of material and the ability of the material to feel, for example, like traditional tanned leather and plastic-based leather alternatives.

The protein-based plastic substitute material has advantageous water stability. Preferably, the protein-based plastic substitute material does not lose its shape or structure (e.g. does not disintegrate or fall apart) when completely submerged in water, partially submerged in water, or when directly contacted with water, preferably for at least 1 day (24 hours), or for at least 2 days, or 4 days, or for at least 7 days, such as for at least 14 days, or even at least 21 days. The proteinbased plastic substitute material preferably does not lose its shape or structure upon removal from, or upon removal of, the water. The protein-based plastic substitute material preferably does not lose its shape or structure after removal from, or after removal of, the water. The water in which the protein-based plastic substitute material is completely or partially submerged, or with which the proteinbased plastic substitute material is contacted, is preferably at ambient temperature. The protein-based plastic substitute material is preferably retained in the water, or contacted therewith, under ambient conditions. The water is preferably still water (e.g. not running water). The water may be deionised and/or distilled water.

In light of its advantageous properties, the protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention) can be advantageously used as a non-synthetic alternative in industries such as fashion for items such as bags, shoes, and garments, and furniture in upholstery. The protein-based plastic substitute material may be used to replace animal-derived leather or synthetic leather in any product formed therefrom.

An additional material may be attached to or integrated into the protein-based plastic substitute material (the protein-based plastic substitute material according to the third aspect of the present invention). The additional material may be a scaffold support. Accordingly, the method according to the first or second aspect of the present invention may comprise an additional step of attaching or integrating the protein-based plastic substitute material to, or into, an additional material. The additional material may enhance the strength of the protein-based plastic substitute material, particularly with respect to tensile strength and/or tear strength. The additional material will typically reflect the shape of the proteinbased plastic substitute material, for example where the protein-based plastic substitute material is provided as a layer, the additional material is also provided as a layer attached thereto or integrated therein. Suitable examples of scaffold supports include, but are not limited to cotton, viscose, natural cellulosic fibres, perforated mesh, mesh, cheesecloth, linen or muslin mesh.

An article formed of the protein-based plastic substitute material (the proteinbased plastic substitute material according to the third aspect of the present invention) may be provided. Examples of articles include, but are not limited to bags, shoes, garments, upholstery, packaging, apparel, saddles, book binders, book covers, luggage tags, jackets. It will be appreciated that the protein-based plastic substitute material can be made to form anything currently made of plastic material, for example, of synthetic leather.

Step (a) may further comprise the step of obtaining the protein of the protein composition from a plant source containing the one or more seed storage proteins.

Step (a) may further comprise the step of obtaining the one or more seed storage proteins of the protein composition from a plant source containing the one or more seed storage proteins.

Step (a) may further comprise the step of separating the one or more seed storage proteins of the protein composition from non-protein components of the plant source.

The seed storage proteins may be separated from the plant source containing the one or more seed storage proteins by solubilisation of the seed storage proteins. The seed storage proteins may be separated from the non-protein components of the plant source containing the one or more seed storage proteins by solubilisation of the seed storage proteins.

The seed storage proteins may be separated by alkali extraction, ethanol extraction, organic solvent extraction, acid extraction, salt solution extraction, hydrothermal extraction, enzymatic extraction or sonication (ultrasonic-assisted extraction). Preferably, the extraction takes place in solution. Preferably, the seed storage proteins are obtained by alkali extraction. For alkali extraction, an aqueous alkaline solution may be utilised. Suitable alkaline reagents for use in the alkali extraction include, but are not limited to NaOH, KOH or Ca(OH)2 in aqueous solution, preferably at a concentration of from 0.05 M to 1 M, such as 0.1 M. The aqueous solution of the alkaline reagents utilised in the alkali extraction is preferably an aqueous alkaline solution. The temperature at which extraction, preferably the alkali extraction, takes place may be from 50 to 75 °C, preferably from 50 to 70 °C, or 55 to 70 °C. The extraction, preferably the alkali extraction, may take place over a period of time of from 20 to 300 minutes, preferably from 20 to 100 minutes, such as from 20 to 80 minutes.

After extraction, the seed storage proteins preferably remain in the solvent used for extraction. After extraction, the seed storage proteins preferably remain in the solution used for extraction. The seed storage proteins are preferably solubilised and/or dispersed in the solvent. The seed storage proteins are preferably solubilised and/or dispersed in the solution. The solvent may be the liquid carrier as described above. The solvent may be water and/or other solvents. The seed storage proteins are preferably solubilised and/or dispersed in the liquid carrier.

After extraction, the seed storage proteins may be separated from non-protein components of the plant source. This may be by filtration and/or centrifuge separation. The seed storage proteins may be separated from insoluble components of the plant source, such as insoluble components of a grain or legume, e.g. hulls or husks. The seed storage proteins may be optionally purified, for example by precipitation and/or centrifugation. Suitable purification techniques include precipitation and/or centrifugation and/or filtration. When purified, the seed storage proteins are preferably purified after separation from the non-protein components of the plant source.

Purification of the seed storage proteins may comprise precipitation, for example, cold precipitation or acid precipitation, preferably acid precipitation, or may comprise solvent evaporation. For cold precipitation, the solution may be maintained at a temperature of from 0 to 4 °C. The solution may be maintained at this temperature from a period of time of from 12 to 48 hours. The seed storage proteins are thus precipitated from the solution. For acid precipitation, an acid reagent may be added to the solution. The acid reagent may be selected from any suitable acid, for example, hydrochloric acid, citric acid, or acetic acid, preferably hydrochloric acid. For acid precipitation, the acid reagent is added in an amount suitable to provide a pH of from 1 to 6, preferably 3, 4 or 5. It will be appreciated that the amount of acid reagent introduced will thus vary. Acid precipitation is preferably utilised. Acid precipitation is preferably utilised where the protein has been extracted using an alkali solution. Following cold precipitation or acid precipitation, following precipitation of the protein from solution, a protein suspension may be obtained. For solvent evaporation, the solution may be left at a temperature of from 15 to 75 °C, such as from 30 to 65 °C, or 50 °C, preferably for 1 to 5 hours, such as from 1 to 3 hours, or 2 hours, optionally under continuous stirring, until all solvent has evaporated.

Purification of the seed storage proteins may alternatively, or further, comprise centrifugation and/or filtration. Preferably, this may be at ambient temperature, or at a temperature of from 0 to 20 °C, such as from 0 to 10°C. For centrifugation, this may take from 10 minutes to 1 hour, such as 30 minutes. The centrifugation may take place at 2000 to 9000 x g, preferably at 3000 x g. Typically, the liquid fraction contains the seed storage proteins.

The seed storage proteins are preferably in a liquid carrier as defined herein. The seed storage proteins are preferably solubilised and/or dispersed in a liquid carrier as defined herein. The protein composition preferably further comprises a liquid carrier. If the seed storage proteins are purified, they may be re-dissolved in a liquid carrier, for example an aqueous solution, typically an aqueous solution of a pH of from 4 to 10, such as from 6 to 8. This aqueous solution may be an aqueous alkaline solution, an aqueous organic solution, an aqueous ammonia solution, aqueous acetic acid solution, or an aqueous acid solution, preferably an aqueous alkaline solution. The aqueous solution may be water, or an aqueous solution of water and an acid, for example HCI, or an aqueous solution of water and an alkali reagent, such as NaOH.

Preferably, step (a) of the method according to the first aspect of the present invention further comprises separating the one or more seed storage proteins of the protein composition from non-protein components of the plant source.

Prior to obtaining the seed storage proteins therefrom, the plant source containing the one or more seed proteins may be dried. The method according to the first aspect of the present invention may therefore comprise an additional step of drying the plant source prior to obtaining the seed storage proteins therefrom.

Prior to obtaining the seed storage proteins, the plant source containing the one or more seed storage proteins may be shredded or milled. The method according to the first aspect of the present invention may therefore comprise an additional step of shredding or milling the plant source prior to obtaining the seed storage proteins therefrom. The plant source may be milled to have a particle size of from 10 to 600 pm, such as from 30 to 600 pm. This may be achieved using a high speed multi form grinder and sieving to the desired particle size.

Prior to obtaining the seed storage proteins, the plant source containing the one or more seed proteins may be washed, preferably in water, such as distilled water. The method according to the first aspect of the present invention may therefore comprise a step of washing the plant source.

For example, if Brewer’s Spent Grain and/or Distiller’s Spent Grain is used to obtain the seed storage proteins, the Brewer’s Spent Grain and/or Distiller’s Spent Grain may be in wet or dry form. Preferably, the Brewer’s Spent Grain and/or Distiller’s Spent Grain is dry Brewer’s Spent Grain and/or Distiller’s Spent Grain. By ‘dry form’ is meant Brewer’s Spent Grain and/or Distiller’s Spent Grain comprising less than 20 wt.% moisture content, preferably less than 15 wt.% moisture content. It will be appreciated that Brewer’s Spent Grain and/or Distiller’s Spent grain is typically obtained from breweries or other sources, in wet form.

For example, if Brewer’s Spent Grain and/or Distiller’s Spent Grain is used to obtain the seed storage proteins, and the Brewer’s Spent Grain and/or Distiller’s Spent Grain is utilised in dry form, the Brewer’s Spent Grain and/or Distiller’s Spent Grain may be shredded or milled before the protein composition is extracted therefrom. Preferably, the Brewer’s Spent Grain and/or Distiller’s Spent Grain is milled before the protein is extracted therefrom. When the Brewer’s Spent Grain and/or Distiller’s Spent Grain is milled, it is preferably milled to have a particle size of from 10 to 600 pm, such as from 30 to 600 pm. This may be achieved using a high speed multi form grinder and sieving to the desired particle size. When the Brewer’s Spent Grain and/or Distiller’s Spent Grain is milled, the texture of the protein-based plastic substitute material formed is advantageously smoother.

For example, if Brewer’s Spent Grain and/or Distiller’s Spent Grain is used to obtain the seed storage proteins, the Brewer’s Spent Grain and/or Distiller’s Spent Grain may be washed in wet or dry form, preferably in dry form.

If the method according to the first aspect of the present invention includes separating the seed storage proteins by extraction as discussed herein, step (a) of the method according to the first aspect of the present invention may further comprise a neutralisation step. The neutralisation step preferably takes place after the separation of the seed storage proteins from non-protein components of the plant source. The neutralisation step preferably takes place after purification of the seed storage proteins. Without being bound by theory, the present inventors consider the neutralisation step can prevent unwarranted oxidation/hydrolyzation and damage to the seed storage proteins, especially if the extraction has been achieved by alkali extraction. Preferably, the neutralisation step takes place prior to the combination of the seed storage proteins with a crosslinker and/or plasticiser. The neutralisation reduces or increases the pH of a solution comprising the seed storage proteins to a neutral pH, such as a pH of from 6 to 8, preferably 7. Preferably, the solution is of the seed storage proteins in a liquid carrier as defined herein. Neutralisation may be achieved through the addition of a neutralising agent. Suitable neutralising agents include acidic and alkaline reagents. It will be appreciated that the neutralising agent required is depending upon the pH of the solution. For example, when extraction has been achieved through alkali extraction, suitable neutralising agents include acids. Any acid may be utilised. For example, suitable acids include citric acid, malic acid, hydrochloric acid, nitric acid, and sulfuric acid. For example, where purification such as precipitation has been achieved through acid precipitation, suitable neutralising agents include alkaline reagents such as sodium hydroxide, NaOH. The amount of neutralising agent added is that required to reach the desired pH.

Where a bridging reagent is utilised as a crosslinker, in addition to its function as a bridging reagent as described herein, the bridging reagent may also act as a neutralising agent. Accordingly, the neutralisation may be achieved upon combination of the seed storage proteins and a bridging reagent. Suitable bridging reagents that can also act as a neutralising agent include citric acid, malic acid, tannic acid, gallic acid, ellagic acid, ferulic acid, caffeic acid. However, this is not always the case. It will be appreciated that step (a) of the method according to the first aspect of the present invention may comprise a neutralisation step utilising an acidic reagent or alkaline reagent other than one suitable to act as a bridging reagent, for example hydrochloric acid or sodium hydroxide, and then the use of a bridging reagent as a crosslinker, if present, in the production of a proteinbased plastic substitute material. Where a bridging reagent is used as a crosslinker, neutralisation may take place prior to the reaction with the bridging reagent.

Preferably, where a crosslinking catalyst is used as a crosslinker, neutralisation takes place prior to the reaction with the crosslinking catalyst. If a bridging reagent is also utilised in an earlier reaction step, the neutralisation may either take place before reaction with the bridging reagent, or the neutralisation step may be carried out by the bridging reagent acting as a neutralising agent as discussed above. If a bridging reagent and a crosslinking catalyst are combined with the seed storage proteins in the same reaction step, i.e. introduced together, the neutralisation may preferably take place before the reaction with the bridging reagent and crosslinking catalyst.

Following neutralisation, the resulting solution comprising the seed storage proteins may undergo centrifugation. The seed storage proteins collected may be present in, or re-dissolved in an aqueous solution, typically an aqueous solution of a pH of from 4 to 10, such as from 6 to 8. This aqueous solution may be an aqueous alkaline solution, an aqueous organic solution, an aqueous ammonia solution, aqueous acetic acid solution, or an aqueous acid solution, preferably an aqueous alkaline solution. This aqueous solution may be water, or an aqueous solution of water and an acid, for example HCI, or an aqueous solution of water and an alkali reagent, such as NaOH.

Where a crosslinker is used and the crosslinker is a bridging reagent, and no crosslinking catalyst is used, instead of neutralisation, step (a) of the method according to the first aspect of the present invention may further comprise an acidification step. The acidification reduces the pH of a solution comprising the protein composition to an acidic pH, such as a pH of less than 6, preferably from 2 to 4. Preferably, the solution is of the seed storage proteins in a liquid camer as defined herein. The acidification may occur through the addition of an acid, for example an acid selected from nitric acid, hydrochloric acid, citric acid and sulfuric acid. Preferably, the acid is introduced in an amount so as to reduce the pH of the solution to below 6, such as from 2 to 4.

For the acidification mentioned above, the bridging reagent may also act as the acid detailed above for the acidification. Accordingly, the acidification may be achieved upon combination of the protein composition and a bridging reagent.

If the seed storage proteins are combined with a crosslinker and/or a plasticiser, and optionally one or more additional components, any excess crosslinker (either bridging reagent and/or crosslinking catalyst), plasticiser, one or more additional components, may be removed in step (a) of the method according to the first aspect of the present invention. This may be achieved through filtration and/or centrifugation, preferably centrifugation. The resulting product may be present in, or re-dissolved in aqueous solution, typically an aqueous solution of a pH of from 4 to 10, such as from 6 to 8. This aqueous solution may be an aqueous alkaline solution, an aqueous organic solution, an aqueous ammonia solution, aqueous acetic acid solution, or an aqueous acid solution, preferably an aqueous alkaline solution. The aqueous solution may be water, or an aqueous solution of water and an acid, for example HCI, or an aqueous solution of water and an alkali reagent, such as NaOH.

If required, in step (a) of the method according to the first aspect of the present invention, one or more additional components may be introduced at any time. In particular, the one of more additional components may be introduced before, during and/or after combination of the seed storage proteins with a crosslinker and/or plasticiser. If more than one crosslinker is used, such as more than one bridging reagent and/or crosslinking catalyst, the one or more additional components may be introduced between reaction steps using the different crosslinkers, such as between addition of the bridging reagent and the addition of the crosslinking catalyst, or prior to or following the addition of both. If required, the one or more additional components are preferably introduced after the separation of the seed storage proteins from non-protein components of the plant source. If required, the one or more additional components are preferably introduced after the optional purification of the seed storage proteins.

Different components may be introduced at different stages in step (a) of the method according to the first aspect of the present invention. For example, the introduction of a plasticiser may take place after combination of the seed storage proteins with a crosslinker, but introduction of the one or more additional components, such as filler, may take place during the combination of the seed storage proteins and crosslinker. Preferably, where crosslinker and plasticiser are present, the plasticiser is introduced after combination of the seed storage proteins and crosslinker, i.e. to crosslinked protein composition. Preferably, where used, a crosslinker, plasticiser, and one or more additional components are each introduced after the separation of the seed storage proteins from non-protein components of the plant source. Preferably, where used, a crosslinker, plasticiser, and one or more additional components are each preferably introduced after the optional purification of the seed storage proteins.

When more than one crosslinker is used, in addition to the above, if also used, the plasticiser, and/or any of the one or more additional components, may be introduced during the combination of the combination of the seed storage proteins with one or each of the more than one crosslinkers, or in between reaction steps using the different crosslinkers. For example, when both a bridging reagent and a crosslinking catalyst are used, in addition to the above, the plasticiser and/or any of the one or more additional components may be introduced during the reaction of the proteins with the bridging reagent and/or the crosslinking catalyst, or in between the two, as well as before or after combination with both the bridging reagent and crosslinking catalyst (crosslinkers) as discussed above.

For the method according to the second aspect of the present invention, and the protein-based plastic substitute material according to the third aspect of the present invention, where they are the same, all of the features described herein with respect to the method according to the first aspect of the present invention are applicable to features of the method according to the second aspect of the present invention and the protein-based plastic substitute material according to the third aspect of the present invention. All features described herein for step (c) of the method according to the first aspect of the present invention are applicable to the method according to the second aspect of the present invention. All features described herein for the solid fused protein-containing structure and protein-based plastic substitute material in relation to the first aspect of the present invention are applicable to the method according to the second aspect of the present invention.

All of the features contained herein may be combined with any of the above aspects and in any combination.

All references to chemical compounds herein are to be interpreted as covering the compounds per se, and also, where appropriate, derivates, hydrates, solvates, complexes, isomers and tautomers thereof. For a better understanding of the present invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the following examples.

By the term “ambient temperature” as used herein is meant a temperature of from 10 to 35 °C, typically 18 to 28 °C. Ambient temperature is encompassed by the broader definition of “ambient conditions”, which refers to the normal range of conditions of the surrounding environment to which the protein-containing plastic substitute material or intermediates in the production thereof are exposed, or procedures for the production of the protein-based plastic substitute material are carried out, i.e. the range of temperatures, pressures and atmospheric conditions to which the protein-based plastic substitute material or intermediates in the production thereof are exposed during use, storage and otherwise. This includes solar radiation including electromagnetic radiation of X-rays, ultraviolet (UV) and infrared (IR) radiation. Typically, ambient conditions include a temperature of from 10 to 35 °C (for example 18 to 25 °C), 80 to 120 kPa, 30 to 60% RH (relative humidity) (for example 40 to 55% RH), a pressure of around 0.1 MPa (around 1 bar), and an environment that is typically an oxygen-containing and water vapourcontaining (from 0.01 % to 0.8%, such as 0.2 to 0.8% water) atmosphere.

EXAMPLES

Example 1

Methodology

Protein-based plastic substitute materials A to D (10cm x 10cm) were provided.

Sample A: protein-based plastic substitute material comprising a protein composition, a plasticiser (glycerol) and a crosslinker (citric acid).

Sample B: protein-based plastic substitute material comprising a protein composition and a plasticiser (glycerol).

Sample C: protein-based plastic substitute material comprising a protein composition. Sample D: Same as Sample A (Control).

Samples A to C were each heated in a fan oven to 120 °C for 1 hour. The samples were then retained for 72 hours under ambient conditions (around 22 °C and 50% RH) to re-absorb moisture which was lost during heating (around 15 wt.% of the sample). Sample D was the control and was not heated.

A 2 x 10 cm piece of each of Samples A to D was then submerged in 50 ml of still distilled water at ambient temperature. The Samples A to D were kept in the water at ambient conditions.

Results

Sample A: The sample was found to maintain its shape and structure after being submerged in water for 24 hours.

Sample B: The sample was found to maintain its shape and structure after being submerged in water for 24 hours.

Sample C: The sample was found to maintain its shape and structure after being submerged in water for 24 hours.

Sample D: The sample fell apart (disintegrated) in the water after 3 hours.

Formation

Samples A to D were formed as outlined below.

Samples A and D:

200g of dried Brewer’s Spent Grain was washed with distilled water (3 x 1 L). A further 1.2 L of distilled water was then added, and the sample was made alkali with the addition of 8 g of NaOH resulting in a solution with roughly 0.1 M NaOH. Next, the solution was stirred for 1 hour at 65 °C, separated by gravity filtration and then the filtrate was acidified to pH 7 with citric acid (2.1 g). Then, carboxymethyl cellulose (CMC, 5.49 g) was added with stirring and the solution was heated to 72 °C for 2h to facilitate full dissolution of the CMC. Finally, 20 g of glycerol was added with further stirring. To prepare a 10 x 10 cm sample, 80 ml of this solution was poured into a silicone mould. After drying under ambient conditions (around 22 °C and 50% RH) for 72 hours, a brown-coloured leatherlike material was yielded.

Sample B:

150g of dried Brewer’s Spent Grain was washed with distilled water (3 x 1 L). A further 1.2 L of distilled water was then added, and the sample was made alkali with the addition of 4.85 g of NaOH resulting in a solution with roughly 0.1 M NaOH. Next, the solution was stirred for 1 hour at 72 °C, separated by gravity filtration and then the filtrate was acidified with hydrochloric acid to pH 7 (1 .6 ml of 12 M HCI to acidify 500 ml of filtrate). To prepare a 10 x 10 cm sample, glycerol (1g) was added to 80 ml of the acidified filtrate, and this was poured into a silicone mould. After drying under ambient conditions (around 22 °C and 50% RH) for 72 hours, a brown-coloured leather-like material was yielded.

Sample C:

200g of dried Brewer’s Spent Grain was washed with distilled water (3 x 1 L). A further 1.2 L of distilled water was then added, and the sample was made alkali with the addition of 5.4 g of NaOH resulting in a solution with roughly 0.1 M NaOH. Next, the solution was stirred for 1 hour at 70 °C and separated by gravity filtration. A 300 ml portion of this filtrate was then placed into centrifuge tubes and centrifuged at 4 °C for 30 minutes at 10,000 rpm. The liquid fraction was then separated (leaving behind a puck of solid impurities) and then acidified to pH 7 with HCI (1.0 ml of 12 M HCI). 80 ml of this solution was poured into a silicone mould. After drying under ambient conditions (around 22 °C and 50% RH) for 72 hours, a brown-coloured leather-like material was yielded.

Example 2

Methodology

A 20 x 20 cm protein-based plastic substitute material comprising a protein composition, a plasticiser (glycerol) and a crosslinker (citric acid) was hand cut into four evenly sized pieces (Samples E, F G, and H). Samples E, F and G were each heated for 60 minutes in a fan oven at the temperature shown in Table 1 . The samples were then left for 72 hours under ambient conditions (around 22 °C and 50% RH) to re-absorb moisture which was lost during heating (around 15 wt.% of the sample). Sample H was the control and was not heated.

Table 1

A 2 x 10 cm piece of each of Samples E to H was then submerged in 50 ml of still distilled water at ambient temperature. The Samples E to H were kept in the water at ambient conditions.

Results

Samples E, F and G were water stable. The samples maintained their shape and structure after being submerged in water for 24 hours. Sample H fell apart (disintegrated) when removed from the water. The water caused Sample H to disintegrate, and this was evidenced upon removal from the water.

Formation

200g of dried Brewer’s Spent Grain was washed with distilled water (3 x 1 L). A further 1.2 L of distilled water was then added, and the sample was made alkali with the addition of 5.7 g of NaOH resulting in a solution with roughly 0.1 M NaOH. Next, the solution was stirred for 1 h at 65 °C, separated by gravity filtration and then the filtrate was acidified to pH 7 with citric acid (2.1 g). Then, to a 300 ml portion of the acidified filtrate, carboxy-methyl cellulose (CMC, 1.34 g) was added with stirring and the solution was heated to 72 °C for 2 hours to facilitate full dissolution of the CMC. Finally, 4.25 g of glycerol was added with further stirring. To prepare a 20 x 20 cm sample, 300 ml of this solution was poured into a silicone mould. After drying under ambient conditions (around 22 °C and 50% RH) for 72 hours a brown-coloured leather-like material was yielded.

Example 3

Methodology

A 20 x 20 cm protein-based plastic substitute material comprising a protein composition, a plasticiser (glycerol) and a crosslinker (citric acid) was hand cut into five evenly sized pieces (Samples J, K, L, M and N).

Samples J, K, L and M were each heated at 120 °C in a fan oven for the time shown in Table 2. The samples were then left for 72 hours under ambient conditions (around 22 °C and 50% RH) to re-absorb moisture which was lost during heating (around 15 wt.% of the sample). Sample N was the control and was not heated.

Table 2 A 2 x 10 cm piece of each of Samples J, K, L, M and N was then submerged in 50 ml of still distilled water at ambient temperature. The Samples J, K, L, M and N were kept in the water at ambient conditions.

Results

Samples J, K, L and M were water stable. The samples maintained their shape and structure after being submerged in water for 24 hours. Sample N fell apart (disintegrated) when removed from the water. The water caused Sample N to disintegrate, and this was evidenced upon removal from the water.

Formation

200g of dried Brewer’s Spent Grain was washed with distilled water (3 x 1 L). A further 1.2 L of distilled water was then added, and the sample was made alkali with the addition of 5.4 g of NaOH resulting in a solution with roughly 0.1 M NaOH. Next, the solution was stirred for 1 hour at 65 °C, separated by gravity filtration and then the filtrate was acidified to pH 7 with citric acid (2.1 g). Then, carboxymethyl cellulose (CMC, 3.52 g) was added with stirring and the solution was heated to 72 °C for 2h to facilitate full dissolution of the CMC. Finally, 20 g of glycerol was added with further stirring. To prepare a 20 x 20 cm sample, 300 ml of this solution was poured into a silicone mould. After drying under ambient conditions (around 22 °C and 50% RH) for 72 hours, a brown-coloured leatherlike material was yielded.

Claims

1. A method of forming a protein-based plastic substitute material, the method comprising:

(a) providing a protein composition comprising at least 30 wt.% of protein, based on the total solids content of the protein composition, wherein the protein includes one or more seed storage proteins selected from albumins, globulins, prolamins, and/or glutelins;

(b) forming the protein composition into a solid fused proteincontaining structure; and

(c) heating the solid fused protein-containing structure to a temperature of more than 60 °C for at least 5 minutes to provide the protein-based plastic substitute material.

2. The method according to claim 1 , wherein in step (c), the solid fused protein-containing structure is heated to a temperature in the range from 65 to 160 °C, or from 70 to 150 °C, or from 80 to 140 °C, or from 90 to 130 °C, or from 90 to 120 °C.

3. The method according to claim 1 or 2, wherein in step (c), the solid fused protein-containing structure is heated for from 5 minutes to 3 days, or from 10 minutes to 3 days, or from 15 minutes to 1 day, or from 20 minutes to 18 hours, or from 25 minutes to 16 hours, or from 30 minutes to 15 hours, or from 35 minutes to 14 hours, or from 40 minutes to 12 hours, or from 45 minutes to 10 hours, or from 45 minutes to 8 hours, or 45 minutes to 6 hours, or 50 minutes to 4 hours, preferably from 5 to 120 minutes, or from 10 to 90 minutes, or from 15 to 60 minutes.

4. The method according to any of claims 1 to 3, wherein the seed storage proteins of the protein composition of step (a) comprise or consist of prolamins, preferably hordeins.

5. The method according to any of claims 1 to 4, wherein the seed storage proteins of the protein composition of step (a) are obtained from Brewer’s Spent Grain and/or Distiller’s Spent Grain, more preferably from Brewer’s Spent Grain.

6. The method according to any of claims 1 to 5, wherein the protein composition of step (a) comprises at least 40 wt.% protein, or at least 50 wt.% protein, or at least 60 wt.% protein, based on the total solids content of the protein composition.

7. The method according to any of claims 1 to 6, wherein the protein composition of step (a) further comprises a crosslinker, preferably wherein the crosslinker is selected from a bridging reagent, a crosslinking catalyst or a combination thereof.

8. The method according to claim 7, wherein the crosslinker includes a bridging reagent selected from citric acid, sebacic acid, formaldehyde, glutaraldehyde, benzaldehyde, oxalic acid, phosphoric acid, glucuronic acid, fumaric acid, ascorbic acid, tartaric acid, maleic acid, tyrosine, riboflavin, bis(sulfosuccinimidyl)suberate, N-hydroxysulfo-succinimide, urea, genipin, azetidinium, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, feruic acid, caffeic acid, vanillin, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, preferably from citric acid, formaldehyde, urea, gum arabic, alginate, genipin, azetidinium, rosin, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, ferulic acid, caffeic acid, vanillin, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, more preferably from citric acid, malic acid, formaldehyde, urea, gum arabic, alginate, genipin, azetidinium, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid, caffeic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, more preferably from citric acid, malic acid, urea, gum arabic, alginate, genipin, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid, caffeic acid, one or more polyepoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, more preferably from citric acid, malic acid, tannic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or a combination thereof, more preferably citric acid, malic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or a combination thereof.

9. The method according to claim 7 or 8, wherein the crosslinker includes an enzyme crosslinking catalyst, preferably wherein the enzyme is selected from a transglutaminase, a lysyl oxidase and a laccase, or combinations thereof, more preferably wherein the crosslinking catalyst is a transglutaminase.

10. The method according to any of claims 1 to 9, wherein the protein composition of step (a) further comprises a plasticiser.

11 . The method according to claim 10, wherein the plasticiser is selected from sugar alcohols such as glycerol, mannitol, xylitol, sorbitol and erythritol; ethylene glycol; polyethylene glycol; propylene glycol; lecithin; sunflower lecithin; diethylene glycol; tetraethylene glycol; ethanolamine; triethanolamine; acetic acid; glycol; fatliquor; fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid; sorbitan; didecyldimethylammonium chloride (DDAC); polysorbate 20; polysorbate 80; triethyl citrate; and acetylated monoglyceride, preferably wherein the plasticiser is selected from sugar alcohols such as glycerol, sorbitol, and erythritol; ethylene glycol; acetic acid; ethanol; glycol; fatliquor; fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid; sorbitan; didecyldimethylammonium chloride (DDAC); polysorbate 20; polysorbate 80; triethyl citrate; and acetylated monoglyceride, or a combination thereof, more preferably wherein the plasticiser is selected from a polyol-based plasticiser, more preferably wherein the plasticiser is selected from a polyol-based plasticiser selected from sugar alcohols such as glycerol, sorbitol and erythritol; ethylene glycol; polyethylene glycol; propylene glycol; diethylene glycol; tetraethylene glycol; and sorbitan; or a combination thereof, and more preferably wherein the plasticiser is selected from glycerol, acetic acid, ethanol, fatliquor, fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid, sorbitol, sorbitan, polysorbate 20, polysorbate 80, erythritol, triethyl citrate, and acetylated monoglyceride, and more preferably wherein the plasticiser is selected from glycerol and water, or a combination thereof.

12. The method according to any of claims 1 to 11 , wherein the protein composition of step (a) further comprises a liquid carrier, preferably wherein the liquid carrier is water.

13. The method according to any of claims 1 to 12, wherein the protein-based plastic substitute material is in the form of a layer or sheet of material.

14. The method according to any of claims 1 to 13, wherein step (b) comprises thermal extrusion, or thermal compression molding or baking, of the protein composition.

15. The method according to any of claims 1 to 13, wherein step (b) comprises collecting the solid fused protein-containing structure on a forming surface, preferably wherein the solid fused protein-containing structure is formed by electrospinning.

16. The method according to any of claims 1 to 13, wherein step (b) comprises applying the protein composition to a forming surface and forming the solid fused protein-containing structure on the forming surface.

17. The method according to any of claims 1 to 13 or 16, wherein the solid fused protein-containing structure is formed by casting, solution casting, or tape casting, preferably by solution casting.

18. The method according to any of claims 15 to 17, wherein the protein composition further comprises a liquid carrier, and step (b) comprises removing the liquid carrier to form the solid fused protein-containing structure.

19. The method according to any of claims 15 to 18, wherein the solid fused protein-containing structure is removed from the forming surface prior to step (c), orwherein the protein-based plastic substitute material is removed from the forming surface after step (c).

20. The method according to any of claims 1 to 19, wherein the solid fused protein-containing structure formed in step (b) has a moisture content of 25% or less, preferably 20 wt.% or less, more preferably 15 wt.% or less, more preferably 10 wt.% or less, and optionally 5 wt%. or less.

21 . The method according to any of claims 1 to 20, further comprising the step of:

(d) equilibrating the protein-based plastic substitute material in an atmosphere of 10-90% relative humidity and at a temperature in the range from 10 to 50 °C.

22. The method according to any of claims 1 to 21 , wherein step (a) comprises obtaining the one or more seed storage proteins of the protein composition from a plant source containing said one or more seed storage proteins selected from albumins, globulins, prolamins, and/or glutelins, preferably prolamins, more preferably hordeins.

23. The method according to claim 22, wherein the plant source is Brewer’s Spent Grain and/or Distiller’s Spent Grain, preferably Brewer’s Spent Grain.

24. The method according to claim 22 or 23, wherein step (a) comprises separating the one or more seed storage proteins of the protein composition from non-protein components of the plant source.

25. The method according to any of claims 22 to 24, wherein the one or more seed storage proteins are separated by alkali extraction, ethanol extraction, organic solvent extraction, acid extraction, salt solution extraction, hydrothermal extraction, enzymatic extraction or sonication (ultrasonic-assisted extraction), preferably alkali extraction, preferably alkali extraction in aqueous alkaline solution, preferably using an alkaline reagent selected from NaOH, KOH or Ca(OH)2 in aqueous solution, and more preferably at a concentration of from 0.05 M to 1 M, such as 0.1 M, and optionally at a temperature of from 50 to 75 °C, preferably from 50 to 70 °C, or 55 to 70 °C.

26. The method according to claim 22 to 25, wherein, after extraction, the one or more seed storage proteins are separated from non-protein components of the plant source by filtration and/or centrifuge separation and optionally purified.

27. A method of forming a protein-based plastic substitute material, the method comprising heating a solid fused protein-containing structure to a temperature of more than 60 °C for at least 5 minutes to provide the protein-based plastic substitute material, wherein the solid fused proteincontaining structure comprises one or more seed storage proteins selected from albumins, globulins, prolamins and/or glutelins.

28. The method according to claim 27, wherein the solid fused proteincontaining structure is heated to a temperature in the range from 65 to 160 °C, or from 70 to 150 °C, or from 80 to 140 °C, or from 90 to 130 °C, or from 90 to 120 °C.

29. The method according to claim 27 or 28, wherein the solid fused proteincontaining structure is heated for from 5 minutes to 3 days, or from 10 minutes to 3 days, or from 15 minutes to 1 day, or from 20 minutes to 18 hours, or from 25 minutes to 16 hours, or from 30 minutes to 15 hours, or from 35 minutes to 14 hours, or from 40 minutes to 12 hours, or from 45 minutes to 10 hours, or from 45 minutes to 8 hours, or 45 minutes to 6 hours, or 50 minutes to 4 hours, preferably from 5 to 120 minutes, or from 10 to 90 minutes, or from 15 to 60 minutes.

30. The method according to any of claims 27 to 29, wherein the seed storage proteins comprise or consist of prolamins, preferably hordeins.

31 . The method according to any of claims 27 to 30, wherein the seed storage proteins are obtained from Brewer’s Spent Grain and/or Distiller’s Spent Grain, more preferably from Brewer’s Spent Grain.

32. The method according to any of claims 27 to 31 , wherein the solid fused protein-containing structure further comprises a crosslinker, preferably wherein the crosslinker is selected from a bridging reagent, a crosslinking catalyst or a combination thereof.

33. The method according to claim 32, wherein the crosslinker includes a bridging reagent selected from citric acid, sebacic acid, formaldehyde, glutaraldehyde, benzaldehyde, oxalic acid, phosphoric acid, glucuronic acid, fumaric acid, ascorbic acid, tartaric acid, maleic acid, tyrosine, riboflavin, bis(sulfosuccinimidyl)suberate, N-hydroxysulfo-succinimide, urea, genipin, azetidinium, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, feruic acid, caffeic acid, vanillin, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, preferably from citric acid, formaldehyde, urea, gum arabic, alginate, genipin, azetidinium, rosin, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, ferulic acid, caffeic acid, vanillin, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, more preferably from citric acid, malic acid, formaldehyde, urea, gum arabic, alginate, genipin, azetidinium, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid, caffeic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, more preferably from citric acid, malic acid, urea, gum arabic, alginate, genipin, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid, caffeic acid, one or more polyepoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or combinations thereof, more preferably from citric acid, malic acid, tannic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or a combination thereof, more preferably citric acid, malic acid, one or more poly-epoxy functionalised compounds, one or more poly-aldehyde functionalised compounds, or a combination thereof.

34. The method according to claim 32 or 33, wherein the crosslinker includes an enzyme crosslinking catalyst, preferably wherein the enzyme is selected from a transglutaminase, a lysyl oxidase and a laccase, or combinations thereof, more preferably wherein the crosslinking catalyst is a transglutaminase.

35. The method according to any of claims 27 to 34, wherein the solid fused protein-containing structure further comprises a plasticiser.

36. The method according to claim 35, wherein the plasticiser is selected from sugar alcohols such as glycerol, mannitol, xylitol, sorbitol and erythritol; ethylene glycol; polyethylene glycol; propylene glycol; lecithin; sunflower lecithin; diethylene glycol; tetraethylene glycol; ethanolamine; triethanolamine; acetic acid; glycol; fatliquor; fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid; sorbitan; didecyldimethylammonium chloride (DDAC); polysorbate 20; polysorbate 80; triethyl citrate; and acetylated monoglyceride, preferably wherein the plasticiser is selected from sugar alcohols such as glycerol, sorbitol, and erythritol; ethylene glycol; acetic acid; ethanol; glycol; fatliquor; fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid; sorbitan; didecyldimethylammonium chloride (DDAC); polysorbate 20; polysorbate 80; triethyl citrate; and acetylated monoglyceride, or a combination thereof, more preferably wherein the plasticiser is selected from a polyol-based plasticiser, more preferably wherein the plasticiser is selected from a polyol-based plasticiser selected from sugar alcohols such as glycerol, sorbitol and erythritol; ethylene glycol; polyethylene glycol; propylene glycol; diethylene glycol; tetraethylene glycol; and sorbitan; or a combination thereof, and more preferably wherein the plasticiser is selected from glycerol, acetic acid, ethanol, fatliquor, fatty acids including saturated and unsaturated fatty acids such as oleic acid and stearic acid, sorbitol, sorbitan, polysorbate 20, polysorbate 80, erythritol, triethyl citrate, and acetylated monoglyceride, and more preferably wherein the plasticiser is selected from glycerol and water, or a combination thereof.

37. The method according to any of claims 27 to 36, wherein the solid fused protein-containing structure is in the form of a layer or sheet of material.

38. The method according to any of claims 27 to 37, further comprising the step of: equilibrating the protein-based plastic substitute material in an atmosphere of 10-90% relative humidity and at a temperature in the range from 10 to 50 °C.

39. A protein-based plastic substitute material obtained by the method according to any of claims 1 to 38.