A BIO-BASED LEATHER SUBSTITUTE MATERIAL

FIELD OF THE INVENTION

The present invention relates to a bio-based leather substitute material that provides a sustainable non-synthetic alternative to animal-derived leather.

BACKGROUND

Animal-derived leather is resource intensive and cruel to animals. However, alternative products such as synthetic leather are typically composed of plastic. Not only are these alternative products difficult to work with, but they are also non- biodegradable and contribute to microplastic pollution.

Recently, other bio-based alternatives have been considered. However, such materials typically 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, non-synthetic leather substitute material that mimics the behaviour of animal-derived leather and can be produced via an easily scalable production process.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provided a bio-based leather substitute material comprising the reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker.

According to a second aspect of the present invention, there is provided a biobased leather substitute material comprising Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein, a crosslinker, and an isolated algal polysaccharide or salt thereof.

According to a third aspect of the present invention, there is provided a construct comprising: two or more layers of the bio-based leather substitute material according to the first aspect of the present invention; or the bio-based leather substitute material according to the first aspect of the present invention, and a scaffold support.

According to a fourth aspect of the present invention, there is provided a construct comprising: two or more layers of the bio-based leather substitute material according to the second aspect of the present invention; or the bio-based leather substitute material according to the second aspect of the present invention and a scaffold support.

According to a fifth aspect of the present invention, there is provided an article formed of: a bio-based leather substitute material according to the first aspect of the present invention; or a construct according to the third aspect of the present invention.

According to a sixth aspect of the present invention, there is provided an article formed of: a bio-based leather substitute material according to the second aspect of the present invention; or a construct according to the fourth aspect of the present invention.

According to a seventh aspect of the present invention, there is provided a biobased leather substitute material according to the first aspect of the present invention, wherein the bio-based leather substitute material is produced by a method comprising: providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a crosslinker, and forming the bio-based leather substitute material.

According to an eighth aspect of the present invention, there is provided a method of producing a bio-based leather substitute material according to the first aspect of the present invention, the method comprising providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a crosslinker, and forming the bio-based leather substitute material.

According to a ninth aspect of the present invention, there is provided a bio-based leather substitute material according to the second aspect of the present invention, wherein the bio-based leather substitute material is produced by a method comprising: subjecting the isolated algal polysaccharide or salt thereof to a heat treatment; combining with Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein, and a crosslinker; and forming the bio-based leather substitute material.

According to a tenth aspect of the present invention, there is provided a method of producing a bio-based leather substitute material according to the second aspect of the present invention, the method comprising: subjecting the isolated algal polysaccharide or salt thereof to a heat treatment; combining with Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein, and a crosslinker; and forming the bio-based leather substitute material.

According to an eleventh aspect of the present invention, there is provided a use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a bio-based leather substitute material according to the first aspect of the present invention.

According to a twelfth aspect of the present invention, there is provided a use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a bio-based leather substitute material according to the second aspect of the present invention.

According to a thirteenth aspect of the present invention, there is provided a biobased leather substitute material comprising a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a plasticiser.

According to a fourteenth aspect of the present invention, there is provided a construct comprising: two or more layers of the bio-based leather substitute material according to the thirteenth aspect of the present invention; or the biobased leather substitute material according to the thirteenth aspect of the present invention and a scaffold support.

According to a fifteenth aspect of the present invention, there is provided an article formed of: a bio-based leather substitute material according to the thirteenth aspect of the present invention; or a construct according to the fourteenth aspect of the present invention. According to a sixteenth aspect of the present invention, there is provided a biobased leather substitute material according to the thirteenth aspect of the present invention, wherein the bio-based leather substitute material is produced by a method comprising: providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a plasticiser, and forming the bio-based leather substitute material.

According to a seventeenth of the present invention, there is provided a method of producing a bio-based leather substitute material according to the thirteenth aspect of the present invention, the method comprising providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a plasticiser, and forming the bio-based leather substitute material.

According to an eighteenth aspect of the present invention, there is provided a use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a biobased leather substitute material according to the thirteenth aspect of the present invention.

DETAILED DESCRIPTION

It has been surprisingly and advantageously found that the bio-based leather substitute material according to the present invention provides a sustainable, nonsynthetic alternative to animal-derived leather. This novel material is formed utilising an abundant by-product of the brewing industry, Brewer’s Spent Grain, or an abundant by-product of ethanol production, Distiller’s Spent Grain. Accordingly, production of the bio-based leather substitute material taps into an already existing supply chain (e.g. breweries or ethanol distilleries) and makes use of waste-feedstock that is widely available and low-cost.

The bio-based leather substitute material according to the present invention is plastic-free, fully biodegradable, and mimics properties of animal-derived leather such as flexibility and hand feel. It can therefore be used advantageously as a non-synthetic alternative to animal-derived leather in, for example, the fashion, textile, packaging and furniture industries. It is particularly surprising and advantageous that Brewer’s Spent Grain or Distiller’s Spent Grain, and protein extract derived therefrom, can be utilised to produce bio-based leather substitute material according to the present invention.

Brewer’s Spent Grain or Distiller’ Spent Grain is not a pure grain, but a waste feedstock, an unwanted 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. This intensive processing results not only in the Brewer’s Spent Grain or Distiller’s Spent Grain comprising a variable, and potentially unpredictable, mixture of components, but there is also no guarantee that there is no irreparable damage to the protein remaining in the Brewer’s Spent Grain or Distiller’s Spent Grain. During the intensive processing, protein degradation, typically hydrolysis, can occur resulting in shorter protein chains that lack uniformity and may be unsuitable for further processing and use. This will vary from batch to batch. Furthermore, Brewer’s Spent Grain or Distiller’s Spent Grain is not easy to handle, particularly in the form in which it is obtained from the breweries, distilleries, or ethanol biofuel plants. It is a wet biomass that is highly prone to decomposition and the formation of unfavourable bacterial and/or fungal material therein.

It is therefore very surprising and advantageous that a bio-based leather substitute material may be produced from Brewer’s Spent Grain or Distiller’ Spent Grain given the highly unpredictable nature of the starting material. Brewer’s Spent Grain or Distiller’s Spent Grain typically comprises multiple different types of proteins, all having different properties. There are many considerations that need to be made.

By ‘leather substitute material’ is meant an alternative material that can be used to replace animal-derived leather. By ‘bio-based’ is meant derived from plants and other renewable agricultural materials, as opposed to non-renewable materials such as petroleum. The bio-based leather substitute material according to the present invention is a bio-based material that is suitable for use as a leather substitute material. As used herein, the term ‘Brewer’s Spent Grain or Distiller’s Spent Grain’ refers to the protein-containing grain by-product of beer (Brewer’s) or spirit (Distiller’s) production, such as whiskey, in which a grain is utilised in a fermentation process. Distiller’s Spent Grain may also refer to the protein-containing grain by-product of ethanol biofuel production. Brewer’s Spent Grain typically comprises barley grain by-products, typically comprising protein, lignin, lipid, and cellulose, and optionally by-products of other grain components introduced during the brewing process. The Brewer’s Spent Grain may further comprise additional by-products 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 rice, wheat, rye and/or com grain by-products, typically comprising protein, lignin, lipid and cellulose, and optionally by-products of other grain components introduced during the fermentation process. The Distiller’s Spent Grain may further comprise additional by-products 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 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 or Distiller’s Spent Grain. Typically, Brewer’s Spent Grain 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 or Distiller’s Spent Grain. Typically, Brewer’s Spent Grain or Distiller’s Spent Grain may comprise 1 to 15 wt.% lipid, based on the dry weight of Brewer’s Spent Grain or Distiller’s Spent Grain. Typically, Brewer’s Spent Grain 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 or Distiller’s Spent Grain. As referred to herein, the ‘cellulose’ of the Brewer’s Spent Grain or Distiller’s Spent Grain also encompasses any hemi-cellulose and starch present in the Brewer’s Spent Grain or Distiller’s Spent Grain. 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 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).

By the term ‘protein extract’ is meant a protein composition that has been extracted from Brewer’s Spent Grain or Distiller’s Spent Grain by solubilisation of the protein therein. Without being bound by theory, the present inventors consider that the protein extract has a different structure following extraction compared to when it is present in Brewer’s Spent Grain or Distiller’s Spent Grain. The present inventors consider that the protein of the protein composition unravel and are assumed to denature upon extraction. During production of the bio-based leather substitute material the proteins are able to both bind with each other to form a protein network and also able to bind other components of the bio-based leather substitute material, as required. The protein extract may comprise a complex mixture of proteins. The term ‘protein extract’ as used herein, encompasses a ‘protein isolate’, a protein isolate being a protein composition that has been extracted from the Brewer’s Spent Grain or Distiller’s Spent Grain by solubilisation of the protein therein, and then separated from one or more of the other components of Brewer’s Spent Grain or Distiller’s Spent Grain, typically the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain, and optionally further purified. An insoluble component may be the insoluble husk of the grain. During the production of the bio-based leather substitute material, the protein extract or isolate may be present in solution or in solid form.

Preferably, the protein extract in the bio-based leather substitute material according to the first aspect of the present invention is derived from Brewer’s Spent Grain.

The presence of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain in the bio-based leather substitute material according to the first aspect of the present invention enables the formation of a strong and supple material having increased abrasion resistance, elasticity, and hand feel.

The bio-based leather substitute material according to the first aspect of the present invention comprises the reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker. The reaction product is a crosslinked protein extract. It will be appreciated that the bio-based leather substitute material according to the first aspect of the present invention may also comprise protein extract and/or crosslinker that are not crosslinked, e.g. excess protein extract and/or crosslinker.

Without being bound by theory, the present inventors consider that Brewer’s Spent Grain or Distiller’s Spent Grain contains prolamins. Prolamins include hordeins, albumins, gliadins and glutelins.

Accordingly, the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain may comprise prolamins selected from hordeins, albumins, gliadins and/or glutelins, preferably hordeins.

The protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain may be present in the bio-based leather substitute material according to the first aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise from 6 to 90 wt.% protein extract, such as from 12 to 80 wt.% protein extract. When the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is a protein isolate, the bio-based leather substitute material may comprise from 55 to 90 wt.% protein isolate, preferably from 55 to 80 wt.% protein isolate. It will be appreciated that this amount refers to all protein extracts in the bio-based leather substitute material, whether crosslinked or otherwise. When the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is a protein extract but not a protein isolate, i.e. has been extracted from Brewer’s Spent Grain or Distiller’s Spent Grain but not separated nor optionally purified, the bio-based leather substitute material may comprise from 6 to 60 wt.% protein extract, preferably 12 to 50 wt.% protein extract, or 12 to 30 wt.% protein extract. The bio-based leather substitute material according to the first aspect of the present invention may further comprise one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain (other than the protein extract). For example, the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain. The components may be retained throughout the production of the biobased leather substitute material following extraction of the protein from the Brewer’s Spent Grain or Distiller’s Spent Grain such that the components are present in the resulting bio-based leather substitute material. Alternatively, during the production of the bio-based leather substitute material according to the first aspect of the present invention, the protein extract may be separated from one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain, typically the insoluble component(s) of the Brewer’s Spent Grain or Distiller’s Spent Grain, and optionally purified. However, these one or more other components may be reintroduced during the production process, either to be part of the bio-based leather substitute material, or in another form such as in the scaffold support, as described below. By ‘insoluble component(s)’ of the Brewer’s Spent Grain or Distiller’s Grain as discussed herein, is meant the component(s) of the Brewer’s Spent Grain or Distiller’s Spent Grain other than protein extract that are insoluble in water and/or the solution, such as an alkali solution, used for protein extraction.

The bio-based leather substitute material according to the first aspect of the present invention may further comprise lignin, lipid, and/or cellulose. The biobased leather substitute material according to the first aspect of the present invention may therefore comprise 2 to 32 wt.% lignin, preferably 2 to 26 wt.% lignin. The bio-based leather substitute material according to the first aspect of the present invention may therefore comprise 1 to 15 wt.% lipid, 1 to 10 wt.% lipid. The bio-based leather substitute material according to the first aspect of the present invention may therefore comprise 15 to 60 wt.% cellulose, preferably 20 to 50 wt.% cellulose.

The bio-based leather substitute material according to the first aspect of the present invention may comprise a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, as well as a protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain. In such instances, the bio-based leather substitute material according to the first aspect of the present invention may comprise from 6 to 60 wt.% protein extract and protein isolate, preferably 12 to 50 wt.% protein extract and protein isolate.

The bio-based leather substitute material according to the first aspect of the present invention may further comprise an isolated algal polysaccharide or salt thereof, preferably an isolated algal polysaccharide. More than one isolated algal polysaccharide or salt thereof may be utilised.

By ‘isolated algal polysaccharide’ as used herein is meant a polysaccharide extracted and separated from algae such as brown, red or green algae, and optionally purified.

Suitable isolated algal polysaccharides include, but are not limited to: isolated algin; isolated fucoidan; isolated laminarin; isolated alginic acid; isolated agar agar; isolated carrageenan such as K-carrageenan, l-carrageenan or L- carrageenan; and isolated ulvan; or combinations thereof. Preferably, the isolated algal polysaccharide is an anionic polysaccharide. Preferably, the isolated algal polysaccharide is an isolated carrageenan, such as K-carrageenan, or an isolated agar agar, or a combination of both.

Salts of the isolated algal polysaccharides include ionic analogues of the isolated algal polysaccharides, for example, sodium or calcium salts such as sodium or calcium algin.

Presence of an isolated algal polysaccharide in the bio-based leather substitute material according to the first aspect of the present invention enables the formation of a strong material having further increased tensile strength and increased tear strength. Without being bound by theory, the present inventors consider that, when present in the bio-based leather substitute material, the isolated algal polysaccharide provides advantageous strength as it forms tightly packed chains of polysaccharides with significant intermolecular bonding. The isolated algal polysaccharide may be present in the bio-based leather substitute material according to the first aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise 4 to 50 wt.% isolated algal polysaccharide, such as from 4 to 40 wt.% isolated algal polysaccharide.

According to a second aspect of the present invention, there is provided a biobased leather substitute material comprising: Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein; a crosslinker; and an isolated algal polysaccharide or salt thereof.

All features of the second aspect of the present invention, and as discussed below as preferred or optional, are applicable to all other aspects described herein. Similarly, all features of all other aspects described herein, whether preferred or optional, are applicable to the second aspect of the present invention.

The isolated algal polysaccharide or salt thereof of the bio-based leather substitute material according to the second aspect of the present invention are as described above in accordance with the first aspect of the present invention.

Suitable isolated algal polysaccharides include, but are not limited to: isolated algin; isolated fucoidan; isolated laminarin; isolated alginic acid; isolated agar agar; isolated carrageenan such as K-carrageenan, l-carrageenan or L- carrageenan; and isolated ulvan; or combinations thereof. Preferably, the isolated algal polysaccharide is an anionic polysaccharide. Preferably, the isolated algal polysaccharide is an isolated carrageenan, such as K-carrageenan, or an isolated agar agar, or a combination of both.

Salts of the isolated algal polysaccharides include ionic analogues of the isolated algal polysaccharides, for example, sodium or calcium salts such as sodium or calcium algin.

Presence of an isolated algal polysaccharide in the bio-based leather substitute material according to the second aspect of the present invention enables the formation of a strong material having further increased tensile strength and increased tear strength. Without being bound by theory, the present inventors consider that, when present in the bio-based leather substitute material, the isolated algal polysaccharide provides advantageous strength as it forms tightly packed chains of polysaccharides with significant intermolecular bonding.

The isolated algal polysaccharide may be present in the bio-based leather substitute material according to the second aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise 4 to 50 wt.% isolated algal polysaccharide, such as from 4 to 40 wt.% isolated algal polysaccharide.

The bio-based leather substitute material according to the second aspect of the present invention comprises Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein.

Preferably, the bio-based leather substitute material according to the second aspect of the present invention comprises Brewer’s Spent Grain containing a protein.

The Brewer’s Spent Grain or Distiller’s Spent Grain may be present in the biobased leather substitute material according to the second aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise 40 to 98 wt.% Brewer’s Spent Grain or Distiller’s Spent Grain, such as from 50 to 95 wt.% Brewer’s Spent Grain or Distiller’s Spent Grain. Preferably, the bio-based leather substitute material comprises 55 to 95 wt.% Brewer’s Spent Grain or Distiller’s Spent Grain.

The Brewer’s Spent Grain or Distiller’s Spent Grain contains a protein. The protein may be present in the bio-based leather substitute material according to the second aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise 5 to 28 wt.% protein, such as from 6 to 28 wt.% protein.

The Brewer’s Spent Grain or Distiller’s Spent Grain typically comprises lignin, lipid, and/or cellulose as discussed above. Accordingly, the bio-based leather substitute material according to the second aspect of the present invention may comprise 2 to 30 wt.% lignin. The bio-based leather substitute material according to the second aspect of the present invention may comprise 1 to 13 wt.% lipid. The bio-based leather substitute material according to the second aspect of the present invention may comprise 10 to 58 wt.% cellulose.

For the bio-based leather substitute material according to the first or second aspects of the present invention, the Brewer’s Spent Grain or Distiller’s Spent Grain may be substituted with rapeseed meal, a protein-containing grain byproduct of rapeseed oil production. Accordingly, when the bio-based leather substitute material according to the first and second aspects of the present invention are referenced herein for other aspects of the present invention, the Brewer’s Spent Grain or Distiller’s Spent Grain may be substituted with rapeseed meal in those aspects. The rapeseed meal may comprise protein, lignin, lipids and cellulose. The rapeseed meal may comprise 15 to 45 wt.% protein, such as 30 to 36 wt.% protein, based on the dry weight of the rapeseed meal. The rapeseed meal may comprise 15 to 50 wt.% cellulose, based on the dry weight of the rapeseed meal. The rapeseed meal may comprise 5 to 15 wt.% lignin, based on the dry weight of the rapeseed meal. The rapeseed meal may comprise 2 to 22 wt.% lipids, based on the dry weight of the rapeseed meal. As referred to herein, the ‘cellulose’ of the rapeseed meal also encompasses any hemi-cellulose and starch present in the rapeseed meal.

The bio-based leather substitute material according to the first aspect of the present invention comprises a reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker. More than one crosslinker may be used, preferably two crosslinkers are utilised, such as two different crosslinkers.

The crosslinker may be selected from a bridging reagent and/or a crosslinking catalyst. One or more bridging reagents and/or crosslinking catalysts may be utilised. Preferably, a bridging reagent and a crosslinking catalyst are utilised. The bridging reagent and crosslinking catalyst are different. Where a bridging reagent and crosslinking catalyst are utilised, the bridging catalyst and crosslinking catalyst may be used together, or sequentially. Preferably, the bio-based leather substitute material comprises a reaction product of a sequential reaction of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging reagent and a crosslinking catalyst. For example, the bio-based leather substitute material may comprise a reaction product of a sequential reaction of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging reagent and then a crosslinking catalyst, or alternatively a crosslinking catalyst and then a bridging reagent. Preferably, the bio-based leather substitute material comprises a reaction product of a sequential reaction of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging reagent and then a crosslinking catalyst. In the context of the present invention, the term ‘sequential reaction’ includes the reaction of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging agent followed by the reaction of the resulting product with a crosslinking catalyst. The term also includes the reaction of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging agent, followed by other reaction steps therebetween, for example a heat treatment and/or ultrasonication treatment step, before the reaction of the resultant product thereof with a crosslinking catalyst. Preferably, the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is reacted with a bridging agent and the resulting product is then reacted with a crosslinking catalyst.

Suitable bridging reagents include, but are not limited to: citric acid, sebacic acid, formaldehyde, glutaraldehyde, benzaldehyde, oxalic acid, phosphoric acid, glucuronic acid, fumaric acid, benzoic 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.

Preferably, the bridging reagent is a naturally derived crosslinker. By ‘naturally derived’ as used herein, is meant a component that is obtained or derived from natural sources. Such components are not derived from petroleum. Such components are not synthetic.

Preferably, the bridging reagent is selected from citric acid, formaldehyde, urea, genipin, azetidinium, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, ferulic acid and caffeic acid, or combinations thereof.

More preferably, the bridging reagent is selected from citric acid, urea, genipin, isosorbide, tannic acid, gallic acid, malic acid, ellagic acid, ferulic acid and caffeic acid, or combinations thereof. These bridging reagents are not only naturally derived, but have good biodegradability and low toxicity. More preferably, the bridging reagent is selected from citric acid, malic acid and tannic acid, or is a combination thereof. Most preferably, the bridging reagent is selected from citric acid and malic acid, or is a combination thereof.

Presence of the bridging reagent in the bio-based leather substitute material according to the first or second aspect of the present invention enables the formation of a bio-based leather substitute material having increased tensile strength, tear strength and/or abrasion resistance.

The bridging reagent may be present in the bio-based leather substitute material according to the first or second aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise 1 to 25 wt.% bridging reagent. Preferably, the bio-based leather substitute material comprises 5 to 20 wt.% bridging reagent. It will be appreciated that this includes all bridging reagent, whether present as bridging reagent or bridging moiety as discussed below, or otherwise, e.g. excess bridging reagent.

It will be appreciated that a bridging reagent reacts with the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain to form a crosslinked protein extract, in which the protein extract is linked via the bridging reagent or a bridging moiety of the bridging reagent, dependent on the type of crosslinking occurring. The bridging reagent or bridging moiety is part of the crosslinked protein extract structure. The bridging reagent has reactive groups which interact with the protein extract. The bridging moiety of the bridging reagent is the moiety that forms part of the crosslinked protein extract structure. It is a moiety retained in the crosslinked structure after the reaction between the protein extract and bridging reagent, once the reactive groups of the bridging reagent have interacted with the protein extract. For example, the chemical formula of citric acid (bridging reagent) is CO2HCH2C(CO2H)(OH)CH ₂ CO2H. 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. The reactive groups of citric acid may be the two CO2H groups, and the bridging moiety in the reaction product (crosslinked protein extract) structure may be X)2CCH2C(CO2H)(OH)CH ₂ CO2'. The protein extract may be linked via the O atoms (COO) of the CO2H groups.

Suitable crosslinking catalysts include, but are not limited to: enzymes. When an enzyme is utilised as the crosslinking catalyst (‘enzymatic crosslinking'), the enzyme facilitates the crosslinking of the protein extract itself. Typically, the protein extract is linked together through the formation of bonds between reactive moieties of the protein extract. A crosslinked protein extract 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 extract structure. The enzyme remains unchanged. Exposure of the protein extract to the enzyme induces protein extract crosslinking, enhancing the chain length of the protein extract itself.

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 protein extract itself to form a crosslinked protein extract 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 protein extract.

Accordingly, the bio-based leather substitute material according to the first aspect of the present invention may comprise a reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker, the reaction product comprising a protein extract 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 bio-based leather substitute material according to the first aspect of the present invention. Alternatively, it may be removed during the production of the bio-based leather substitute material once it has completed its role of catalysing the formation of the bonds between the protein extract, for example by washing or centrifugation, or other appropriate mechanisms.

When retained in the bio-based leather substitute material according to the first aspect of the present invention, the crosslinking catalyst may be present in the bio-based leather substitute material in any suitable amount. The bio-based leather substitute material according to the first aspect of the present invention 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 extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, i.e. with respect to the amount of protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain. Use of a crosslinking catalyst in the formation of the bio-based leather substitute material according to the first aspect of the present invention enables the production of a material having increased tear strength, tensile strength, abrasion resistance and/or flexibility. This leads to a bio-based leather substitute material of improved texture and/or hand feel.

The term ‘crosslinked protein extract’ as used herein refers to protein extract linked by bridging reagent as discussed above, as well as protein extract linked following catalysation by a crosslinking catalyst, for example, protein extract linked by isopeptide bonds, or a combination thereof. The crosslinked protein extract may comprise both protein extract linked by bridging reagent and protein extracts linked following catalysation by a crosslinking catalyst. This is discussed further below.

The bio-based leather substitute material according to the first aspect of the present invention comprises a reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker, preferably two crosslinkers, more preferably a bridging reagent and a crosslinking catalyst. The bridging reagent and crosslinking catalyst are different. Preferably the bio-based leather substitute material comprises a reaction product of a sequential reaction of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging reagent and a crosslinking catalyst. Preferably, the bio-based leather substitute material comprises a reaction product of a sequential reaction of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a bridging reagent and then a crosslinking catalyst. For example, in such a scenario, it will be appreciated that the first reaction forms crosslinked protein extract linked by bridging moieties. The crosslinked protein extract, and any other protein extract, are then induced to itself crosslink in a second reaction by the crosslinking catalyst. As discussed above, the bridging moiety of the bridging group will be present in the crosslinked structure, but not the crosslinking catalyst.

Use of a both a crosslinking catalyst and a bridging reagent in the formation of the bio-based leather substitute material according to the first aspect of the present invention enables the production of a material having increased tensile strength, tear strength, abrasion resistance, and/or flexibility. This leads to a bio-based leather substitute material of improved texture and/or hand feel.

The bio-based leather substitute material according to the first or second aspect of the present invention may further comprise a plasticiser. More than one plasticiser may be present. Preferably, the bio-based leather substitute material according to the first or second aspect of the present invention further comprises a plasticiser.

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

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

Presence of a plasticiser in the bio-based leather substitute material according to the first or second aspect of the present invention enables the production of a material having further increased flexibility, softness, and elasticity.

The plasticiser may be present in the bio-based leather substitute material according to the first or second aspect of the present invention in any suitable amount. The bio-based leather substitute material may comprise 1 to 35 wt.% plasticiser, such as from 5 to 30 wt.% plasticiser. Preferably, the bio-based leather substitute material comprises 5 to 25 wt.% plasticiser. It will be appreciated that if the bio-based leather substitute material according to the first or second aspect of the present invention is to be formed by extrusion, as discussed below, the bio-based leather substitute material preferably comprises a plasticiser.

The bio-based leather substitute material according to the first or second aspect of the present invention may further comprise one or more additional components. Suitable one or more additional components may include, but are not limited to: 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 used as one or more additional components. Preferably, carboxymethylcellulose is used as a one or more additional component. The one or more additional components may be present in any suitable amount. The one of more additional components may each be present in the bio-based leather substitute material in an amount of from 1 to 50 wt.%.

The bio-based leather substitute material according to the first or second aspect of the present invention does not comprise a component derived from petroleum. The bio-based leather substitute material does not comprise a petroleum-based component such as a petroleum-based resin. The bio-based leather substitute material according to the first or second aspect of the present invention is typically provided as a flat piece of material, typically a broad flat piece of material. The bio-based leather substitute material may be described as a sheet of flexible material.

The bio-based leather substitute material according to the first or second aspect of the present invention may have any suitable thickness. Preferably, the biobased leather substitute material may have a thickness of 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.

The bio-based leather substitute material according to the first or second aspect of the present invention may have any suitable surface area.

When the bio-based leather substitute material according to the first aspect of the present invention comprises a protein isolate, the bio-based leather substitute material may comprise a core comprising one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain. These may be the one or more components of Brewer’s Spent Grain or Distiller’s Spent Grain from which the protein extract has been separated during production of the bio-based leather substitute material according to the first aspect of the present invention, typically the insoluble component(s) of Brewers’s Spent Grain or Distiller’s Spent Grain. Preferably the core is in the form of a foam, through which the protein isolate is dispersed.

It has been surprisingly and advantageously found that the bio-based leather substitute material according to the first or second aspect of the present invention is able to mimic the properties of animal-derived leather. The bio-based leather substitute material demonstrates advantageous high tensile strength, tear strength, flexibility, abrasion resistance, and elasticity. The bio-based leather substitute material also has advantageous texture and ‘hand feel’.

The bio-based leather substitute material according to the first or second 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 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 bio-based leather substitute material according to the first or second aspect of the present invention may be assessed by measurement of tear load. The bio-based leather substitute material according to the first or second aspect of the present invention 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 flexibility of the bio-based leather substitute material according to the first or second aspect of the present invention may be assessed by measurement of flex resistance. The bio-based leather substitute material according to the first or second aspect of the present invention 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 bio-based leather substitute material according to the first or second 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 bio-based leather substitute material according to the first or second aspect of the present invention may be assessed by measurement of elongation at load. The bio-based leather substitute material according to the first or second aspect of the present invention may have an elongation at load of 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 bio-based leather substitute material according to the first or second 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, advantage hand feel is represented by a softness of material and the ability of the material to feel like traditional tanned leather and not plastic-based leather alternatives.

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

The bio-based leather substitute material according to the first or second aspect of the present invention may be utilised to form a construct as discussed below.

An additional material may be attached to or integrated into the bio-based leather substitute material according to the first or second aspect of the present invention. The additional material may be a scaffold support as described below.

Alternatively, or in addition to the use of an additional material, one or more topcoat layers may be present on the bio-based leather substitute material. A topcoat layer may impart further advantageous properties, such as water repellence. This topcoat layer may comprise a coating, which may be applied to the bio-based leather substitute material in any suitable manner, for example, spray applied to the bio-based leather substitute material. A topcoat layer may comprise components such as bio-derived polymers, dyes or colourants, waxes, oils, or combinations thereof. For example, a topcoat layer comprising a wax typically improves hydrophobicity of the bio-based leather substitute material, and a dye or colorant will affect the aesthetic appearance thereof. Suitable waxes include, but are not limited to carnauba wax, sunflower wax, candelilla wax, and beeswax. Suitable bio-derived polymers include but are not limited to bacterial derived polylactic acid (PLA), polyethylene terephthalate (PET) and polyurethane (PU). One or more topcoat layers may be present on the bio-based leather substitute material. These layers may be of the same or different types of topcoats. Preferably one or two topcoat layers may be present on the bio-based leather substitute material. Preferably, two topcoat layers are present on the biobased leather substitute material, for example, a topcoat containing a dye or colourant, and a topcoat applied thereover containing a wax.

A construct according to the third or fourth aspect of the present invention may also comprise two or more layers of the bio-based leather substitute material according to the first or second aspect of the present invention. It will be appreciated that such layers may be bound together, typically by an adhesive, crosslinker (for example, a bridging reagent such as dilute citric acid solution) or stitching. This may be of the same bio-based leather substitute material, or different bio-based leather substitute materials according to the present invention. For example, the construct may comprise two or more layers of the bio-based leather substitute material according to the first aspect of the present invention, or a layer of the bio-based leather substitute material according to the first aspect of the present invention, as well as a layer of the bio-based leather substitute material according to the second aspect of the present invention.

The bio-based leather substitute material according to the first or second aspect of the present invention may also be treated. For example, the bio-based leather substitute material may be bleached or dyed. Bleaching may be achieved through application of a peroxide source to the bio-based leather substitute material. Suitable peroxide sources include hydrogen peroxide and benzyl peroxide. The bio-based leather substitute material is then allowed to dry. This is typically done to enhance aesthetic properties of the material. Dyeing may be achieved through application of a dye to the bio-based leather substitute material, typically via coating or submerging the material in an appropriate dye solution.

The bio-based leather substitute material according to the first or second aspect of the present invention may be embossed with a mark, pattern, or image. This may be achieved by pressing the bio-based leather substitute material under pressure, or when the bio-based leather substitute material is formed by solution casting, by solution casting on top of a pattern or template or in a marked, patterned or imaged mold. ‘Mark, pattern, or image’ incorporates, but is not limited to: logos, codes, graphics, figures, words, pictures, symbols, and text.

According to a third aspect of the present invention, there is provided a construct comprising two or more layers of the bio-based leather substitute material according to the first aspect of the present invention, or the bio-based leather substitute material according to the first aspect of the present invention, and a scaffold support.

According to a fourth aspect of the present invention, there is provided a construct comprising two or more layers of the bio-based leather substitute material according to the second aspect of the present invention, or the bio-based leather substitute material according to the second aspect of the present invention, and a scaffold support.

All features of the third and fourth aspects of the present invention, and as discussed below as preferred or optional, are applicable to all other aspects described herein. Similarly, all features of all other aspects described herein whether preferred or optional, are applicable to the third and fourth aspects of the present invention.

The scaffold support may be attached to or integrated into the bio-based leather substitute material. The scaffold support may be attached to the bio-based leather substitute material by adhesion, mechanical pressing, or may be solvent cast in layers so as to produce a multi-layer construct. When adhered together, the adhesive may be a naturally-derived adhesive or crosslinker (e.g. bridging reagent). The construct may be considered to have a layer of a scaffold support attached to a layer of the bio-based leather substitute material. It will be appreciated that more than one layer of the scaffold support can be provided. Alternatively, the scaffold support may be integrated into the bio-based leather substitute material to form the construct. The scaffold support may be embedded in the bio-based leather substitute material, or the bio-based leather substitute material allowed to form around the scaffold material, for example, during solvent evaporation, so as to cause adhesion/crosslinking between the bio-based leather substitute material and the scaffold support. It will be appreciated that the construct may comprise more than one scaffold support. Each of the scaffold supports may be attached to or integrated into the bio-based leather substitute material. For example, the construct may comprise a bio-based leather substitute material having a scaffold support integrated therein, and a scaffold support attached thereto.

Suitable examples of scaffold supports include, but are not limited to cotton, viscose, natural cellulosic fibres, perforated mesh, mesh, cheesecloth, linen or muslin mesh.

The scaffold support may be formed from one or more other components of the Brewer’s Spent Grain or Distiller’s Spent Grain from which the protein extract has have been separated during production of the bio-based leather substitute material according to the first aspect of the present invention, typically the insoluble component(s) of the Brewer’s Spent Grain or Distiller’s Spent Grain, in addition to other components such as, for example, a plasticiser and crosslinker.

The scaffold support enhances the strength of the construct, particularly with respect to its tensile strength and/or tear strength. It will be appreciated that the scaffold support may be provided between layers of the bio-based leather substitute material where two or more layers are utilised, or may be provided on one, or both, sides of a layer of the bio-based leather substitute material.

It will be appreciated that, as discussed above, the construct according to the third and fourth aspects of the present invention may comprise two or more layers of the bio-based leather substitute material. This may be of the same bio-based leather substitute material, or different bio-based leather substitute materials according to the present invention. For example, the construct according to the third aspect of the present invention may comprising a layer of the bio-based leather substitute material according to the first aspect of the present invention, as well as a layer of the bio-based leather substitute material according to the second aspect of the present invention. The construct according to the third and fourth aspects of the present invention may further comprise one or more topcoat layers as discussed above with regard to topcoat layers of the bio-based leather substitute material. A topcoat layer may impart further advantageous properties, such as water repellence.

According to a fifth aspect of the present invention, there is provided an article formed of the bio-based leather substitute material according to the first aspect of the present invention, or the construct according to the third aspect of the present invention.

According to a sixth aspect of the present invention, there is provided an article formed of the bio-based leather substitute material according to the second aspect of the present invention, or the construct according to the fourth aspect of the present invention.

All features of the fifth and sixth aspects of the present invention, and as discussed below as preferred or optional, are applicable to all other aspects described herein. Similarly, all features of all other aspects described herein, whether preferred or optional, are applicable to the fifth and sixth aspects of the present invention.

Examples of articles according to the fifth and sixth aspects of the present invention 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 bio-based leather substitute material can be made to form anything of which animal-derived leather is currently made.

The bio-based leather substitute material according to the first aspect of the present invention may be produced by any suitable method.

According to a seventh aspect of the present invention, there is provided a biobased leather substitute material according to the first aspect of the present invention, wherein the bio-based leather substitute material is produced by a method comprising: providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a crosslinker, and forming the bio-based leather substitute material.

According to an eighth aspect of the present invention, there is provided a method of producing a bio-based leather substitute material according to the first aspect of the present invention, the method comprising providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a crosslinker, and forming the bio-based leather substitute material.

All features of the seventh and eighth aspects of the present invention, and as discussed below as preferred or optional, are applicable to all other aspects described herein. Similarly, all features of all other aspects described herein, whether preferred or optional, are applicable to the seventh and eighth aspects of the present invention.

In the seventh and eighth aspects of the present invention, the crosslinker is as described herein in relation to the first aspect of the present invention. Where a crosslinking catalyst is utilised, the combination of the protein extract and crosslinking catalyst preferably takes place at a temperature of less than 60 °C, such as less than 50 °C. This is to prevent denaturisation of the crosslinking catalyst.

Preferably, in the production of the bio-based leather substitute material according to the method of the seventh or eighth aspects of the present invention, the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is combined with both a bridging reagent and crosslinking catalyst. Preferably, as discussed above, this is a sequential reaction and takes place in two stages. Preferably, the protein extract is combined with a bridging reagent, and then the crosslinking catalyst. More preferably, the protein extract is combined with a bridging reagent, and then the resulting product is then combined with the crosslinking catalyst. The present inventors consider such two-stage crosslinking to be advantageous as it creates enhanced tensile strength, tear strength, abrasion resistance and/or flexibility, as well as improved hand feel. The protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain may remain in solution throughout the method for producing the bio-based leather substitute material according to the seventh or eighth aspects of the present invention.

For the seventh and eighth aspects of the present invention, following combination of the protein extract and crosslinker, the bio-based leather substitute material according to the first aspect of the present invention is formed. The bio-based leather substitute material may be formed by any suitable technique. Suitable techniques include, but are not limited to casting and optionally rolling, solution casting, thermal extrusion, thermal compression molding or baking, die casting, pultrusion, electrospraying, wet spinning, dry spinning, melt spinning and gel spinning, preferably casting, solution casting, thermal extrusion, thermal compression molding or baking. The bio-based leather substitute material may also be subject to further drying. Suitable drying conditions include microwave drying, radiowave drying, vacuum drying, and freeze drying. The bio-based leather substitute material may also be subjected to further pressing to achieve the required thickness.

Preferably, the bio-based leather substitute material according to the seventh or eighth aspect of the present invention is formed by solution casting.

The solution casting is preferably carried out in a mold, such as a silicone mold. The mold may be marked, patterned, imaged or shaped in such a way so as to create a mark, pattern or image on the bio-based leather substitute material formed. For solution casting, any suitable temperature may be utilised. The temperature may be an ambient temperature or above, such as up to 60 °C. Preferably, the mold is the same temperature as the solution to be introduced therein.

Following formation, such as by solution casting, the bio-based leather substitute material may be dried by any suitable method. Suitable drying conditions include microwave drying, radiowave drying, vacuum drying, and freeze drying. Prior to formation of the bio-based leather substitute material, the method according to the seventh and eighth aspects of the present invention may comprise a heat treatment and/or ultrasonication treatment step. Preferably, the method according to the seventh and eighth aspects of the present invention comprises a heat treatment step and an ultrasonication treatment step.

Preferably, the method according to the seventh and eighth aspects of the present invention comprises a heat treatment step and/or ultrasonication treatment step prior to the formation of the bio-based leather substitute material. Preferably, the heat treatment and/or ultrasonication treatment takes place after extraction of the protein from Brewer’s Spent Grain or Distiller’s Spent Grain (as discussed below) and prior to the formation of the bio-based leather substitute material. Preferably, the heat treatment and/or ultrasonication step takes place following combination of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with a crosslinker. When more than one crosslinker is utilised, such as more than one bridging reagent and/or crosslinking catalyst, preferably a bridging reagent and crosslinking catalyst as discussed above, the heat treatment and/or ultrasonication step may take place between reaction steps using the different crosslinkers, such as between the addition of the bridging agent and the addition of the crosslinking catalyst, or prior to or following the addition of both, preferably between reaction steps using the different crosslinkers or following the addition of both.

This heat treatment step may be a heating, pasteurisation, curing and/or thermal annealing step. Without being bound by theory, the present inventors consider that this heat treatment step facilitates the formation of protein nanofibrils (alteration of the protein structure), dissolution of components, dispersion of components, and/or enhances crosslinking of the bio-based leather substitute material where taking place subsequent to the addition of a crosslinker. The present inventors consider the heat treatment step to lead to enhanced tensile strength of the bio-based leather substitute material formed. The heat treatment step may comprise heating to a temperature of from 50 to 100 °C, such as from 60 to 90 °C, or from 70 to 90 °C. The heat treatment step may take place for any suitable length of time, such as from 15 minutes to 4 hours, or from 30 minutes to 3 hours. Stirring may also take place during the heat treatment step.

When the method according to the seventh and eighth aspects of the present invention comprises a heat treatment step and an ultrasonication treatment step, the ultrasonication treatment may take place at the same time as the heat treatment, or prior or subsequent thereto. Preferably, the ultrasonication treatment takes place following, i.e. subsequent to, the heat treatment step. This is typically directly after the heat treatment step.

The ultrasonication treatment involves sonication and the application of ultrasonic frequencies. This may be using a ultrasonication device. Suitable ultrasonication devices include sonic dismembrators provided by Fischerbrand®. For the ultrasonication treatment, the power applied may be from 15 to 700 watts, such as 50 watts, 120 watts, 500 watts or 700 watts, preferably from 15 to 500 watts, such as from 15 to 120 watts. The frequency applied may be of 20 KHz. The ultrasonication treatment may be carried out in pulses, such as 5 to 30 second pulses. The ultrasonication may be carried out for 1 to 20 minutes, such as for 1 to 15 minutes, such as from 1 to 10 minutes, or 1 to 5 minutes. Without being bound by theory, the present inventors consider that this ultrasonication treatment step facilitates the formation of protein nanofibrils (alteration of the protein structure), dissolution of components, dispersion of components and/or enhances crosslinking of the bio-based leather substitute material where taking place subsequent to the addition of a crosslinker. The present inventors consider the ultrasonication step to lead to enhanced tensile strength of the bio-based leather substitute material formed.

In the seventh and eighth aspects of the present invention, the method may further comprise the step of obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain. Preferably, in the seventh and eighth aspects of the present invention, the method further comprises the step of obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain. The protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is obtained by solubilisation of the protein in Brewer’s Spent Grain or Distiller’s Spent Grain. The protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain may be obtained 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 protein extract is 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 an aqueous alkaline solution. The temperature at which the alkali extraction takes place may be from 50 to 75 °C, preferably from 50 to 70 °C, or 55 to 70 °C. 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.

Preferably, the protein extract remains in solution following extraction. After obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, the solubilised protein extract may be retained in the same solution as one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain from which the protein has been extracted, and the solution utilised in the production of the bio-based leather substitute material. It will thus be appreciated that in such instances, the bio-based leather substitute material will comprise other components of Brewer’s Spent Grain or Distiller’s Spent Grain. These one or more other components include the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain, for example the husks of the grain.

When the protein extract is a protein isolate, after obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, the solubilised protein extract is separated from one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain, typically the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain, and optionally purified. The separated components of the Brewer’s Spent Grain or Distiller’s Spent Grain, typically the insoluble component(s) of Brewer’ Spent Grain or Distiller’s Spent Grain, may be re-combined later during production of the bio-based leather substitute material. In some instances, the components may be utilised to form a core through which the protein isolate penetrates and disperses. Alternatively, after separation, the one or more other components, typically the insoluble component(s), may not be re-combined. In this case, after separation, the one or more other components may be utilised to form a scaffold support which, as discussed above, may be attached or integrated into the bio-based leather substitute material to form a construct. On the other hand, after separation, the one or more other components, typically the insoluble component(s), may be discarded and not further utilised in the production of the bio-based leather substitute material, or a construct or article comprising such.

Accordingly, when the protein extract is a protein isolate, in the seventh and eighth aspects of the present invention, the method may further comprise the step of obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, separating the protein extract from one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain, typically the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain, and optionally purifying, to form a protein isolate. The protein extract may be obtained by solubilisation of the protein in Brewer’s Spent Grain or Distiller’s Spent Grain as outlined above. The protein extract may then be separated to provide the protein isolate by filtration, for example of the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain from the solution, and/or by centrifuge separation, for example to separate the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain. Preferably, separation is by filtration and/or centrifuge separation, more preferably filtration. Suitable purification techniques for the protein isolate include precipitation and/or centrifugation or filtration. The one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain from which the protein isolate is separated are typically insoluble in the solution used for protein extraction. Purification of the protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain may comprise precipitation, for example, cold precipitation or acid precipitation, preferably acid precipitation.

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 protein is 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. 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 where the protein has been extracted using an alkali solution.

Purification of the protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain may alternatively or further comprise centrifugation 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.

Preferably, the protein extract or protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain remains in solution. The protein extract may then remain in solution during combination with the crosslinker in the process of production of the bio-based leather substitute material. If the protein isolate is purified as discussed above, it is 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 is preferably water, or an aqueous solution of water and an acid, for example HCI, or an aqueous solution of water and an alkali salt, such as NaOH.

Preferably, in the seventh and eight aspects of the present invention, the method further comprises the step of obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, separating the protein extract from one or more other components of the Brewer’s Spent Grain, typically the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent Grain, preferably by filtration and/or centrifuge separation and more preferably filtration, to obtain a protein isolate and optionally purifying the protein isolate. Preferable methods of purification are as discussed above.

The Brewer’s Spent Grain or Distiller’s Spent Grain from which the protein extract is to be derived may be in wet or dry form. Preferably, the Brewer’s Spent Grain or Distiller’s Spent Grain is dry Brewer’s Spent Grain or Distiller’s Spent Grain. In the seventh and eighth aspects of the present invention, the method may therefore comprise an additional step of drying the Brewer’s Spent Grain or Distiller’s Spent Grain prior to obtaining a protein extract derived therefrom. By ‘dry form’ is meant Brewer’s Spent Grain 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 or Distiller’s Spent grain is typically obtained from breweries or other sources, in wet form.

When the Brewer’s Spent Grain or Distiller’s Spent Grain is utilised in dry form, the Brewer’s Spent Grain or Distiller’s Spent Grain may be shredded or milled before the protein is extracted therefrom. In the seventh and eighth aspects of the present invention, the method may therefore comprise a step of shredding or milling the Brewer’s Spent Grain or Distiller’s Spent Grain prior to obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain. Preferably, the Brewer’s Spent Grain or Distiller’s Spent Grain is milled before the protein is extracted therefrom. When the Brewer’s Spent Grain 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 or Distiller’s Spent Grain is milled, the texture of the bio-based leather substitute material formed is advantageously smoother.

The Brewer’s Spent Grain or Distiller’s Spent Grain may be washed in water, preferably distilled water, prior to use. This washing may be of the Brewer’s Spent Grain or Distiller’s Spent Grain in wet or dry form, preferably in dry form. In the seventh and eighth aspects of the present invention, the method may therefore comprise a step of washing the Brewer’s Spent Grain or Distiller’s Spent Grain. This step will be prior to the extraction of protein therefrom.

When the bio-based leather substitute material according to the first aspect of the present invention further comprises an isolated algal polysaccharide or salt thereof, for the seventh and eighth aspects of the present invention, the method may further comprise heat treatment of the isolated algal polysaccharide or salt thereof. Preferably, the heat treatment is carried out in water or distilled water. Heat treatment of the isolated algal polysaccharide or salt thereof may comprise heating the isolated algal polysaccharide or salt thereof in solution, preferably an aqueous solution and more preferably water or distilled water, to a temperature of 80 °C or more, such as from 80 °C to 100 °C, preferably from 85 to 100 °C, and more preferably from 85 °C to 95 °C. Without being bound by theory, the present inventors consider that the isolated algal polysaccharides undergo dissolution when heated, such that their networks break up and they are separated. Upon cooling, the isolated algal polysaccharides are then able arrange into tightly packed chains with significant intramolecular bonding such that they form a binding network for other components of the bio-based leather substitute material. The heat treatment may take place over a period of time of from 5 to 120 minutes.

When the bio-based leather substitute material according to the first aspect of the present invention further comprises an isolated algal polysaccharide or salt thereof, the step of obtaining the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain may be carried out separately to the heat treatment of the isolated algal polysaccharide. The resulting solutions can then be combined in the method for producing the bio-based leather substitute material, and any other required components introduced. Alternatively, the heat treatment of the isolated algal polysaccharide and the step of obtaining the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain can be carried out together, in the same solution. It will be appreciated that when this is the case, the temperature at which the heat treatment and extraction are carried out may be from 80 °C to 95 °C, or from 85 to 95 °C. In addition, the solution may be an aqueous solution of the alkaline reagent as discussed above. In the seventh and eighth aspects of the present invention, after the provision (extraction) of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, the method for producing the bio-based leather substitute material may further comprise a neutralisation step. Preferably, if present, the neutralisation step takes after the provision (extraction) of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and prior to the formation of the bio-based leather substitute material. Preferably, if present, the neutralisation step takes place prior to the combination of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with the crosslinker. The present inventors consider such neutralisation to prevent unwarranted oxidation/hydrolyzation and damage to the protein extract, especially if the protein extraction has been achieved by alkali extraction. The neutralisation reduces the pH of a solution comprising the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain to a neutral pH, such as a pH of from 6 to 8, preferably 7. Neutralisation may be achieved through the addition of a neutralising agent. When the 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. The amount of acid added is that required to reach the desired pH.

Where a bridging reagent is utilised in the method of production of the bio-based leather substitute material, 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 protein extract and a bridging reagent. Suitable bridging reagents that can also act as a neutralising agent include, but are not limited to: 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 the method according to the seventh and eighth aspects of the present invention may comprise a neutralisation step utilising an acid other than one suitable to act as a bridging reagent, for example hydrochloric acid, and then the use of a bridging reagent as a crosslinker in the production of a bio-based leather substitute material according to the present invention. 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 is also combined with the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain in the same reaction step, i.e. introduced together, the neutralisation may take place before the reaction with the bridging reagent and crosslinking catalyst.

Following neutralisation, the resulting solution comprising the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain may undergo centrifugation. The protein collected may be 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 is preferably water, or an aqueous solution of water and an acid, for example HCI, or an aqueous solution of water and an alkali salt, such as NaOH.

For the seventh and eighth aspects of the present invention, where the crosslinker is a bridging reagent, and no crosslinking catalyst is utilised, instead of neutralisation, the method according to the seventh or eighth aspect of the present invention may further comprise acidification following extraction and optional isolation of the protein from the Brewer’s Spent Grain or Distiller’s Spent Grain. The acidification reduces the pH of a solution comprising the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain to an acidic pH, such as a pH of less than 6, preferably from 2 to 4. 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 extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a bridging reagent. In the seventh and eighth aspects of the present invention, once the protein extract has been combined with the crosslinker, and optionally the plasticiser and/or one or more additional components, any excess crosslinker (either bridging reagent and/or crosslinking catalyst), plasticiser, one or more additional components, and/or another non-protein components may be removed. This may be achieved through filtration and/or centrifugation, preferably centrifugation. The resulting product may be re-dissolved in aqueous solution, typically an aqueous solution of a pH of from 4 to 10, such as from 6 to 8 , or used to form the bio-based leather substitute material in its resulting form. 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 salt, such as NaOH.

In the seventh and eighth aspects of the present invention, the plasticiser and/or any of the one or more additional components, may be introduced at any time following obtaining the protein extract from the Brewer’s Spent Grain or Distiller’s Spent Grain but before formation of the bio-based leather substitute material. In particular, the plasticiser and/or any of the one of more additional components may be introduced before, during and/or after combination of the protein extract with the crosslinker. The plasticiser and/or any of the one or more additional components, may be introduced before, during and/or after heat treatment and/or ultrasonication treatment. Preferably prior to the heat treatment and/or ultrasonication treatment stage, and more preferably after the combination with the crosslinker. If more than one crosslinker is used, such as more than one bridging reagent and/or crosslinking catalyst, preferably a bridging reagent and crosslinking catalyst as discussed above, the plasticiser and/or any of the one or more additional components may be introduced between reaction steps using the different crosslinkers, such as between addition of the bridging agent and the addition of the crosslinking catalyst, or prior to or following the addition of both. Different components may be introduced at different stages. For example, the introduction of a plasticiser may take place after combination of the protein extract with crosslinker, but introduction of the one or more additional components, such as filler, may take place during the combination of the protein extract and crosslinker. Preferably, the plasticiser is introduced after combination of the protein extract and crosslinker, i.e. to crosslinked protein extract.

When more than one crosslinker is used, in addition to the above, the plasticiser, and/or any of the one or more additional components, may be introduced during the combination of the combination of the protein extract 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 protein extract 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 seventh and eighth aspects of the present invention, a preferred method of production of the bio-based leather substitute material involves steps (1) to (3) as detailed below. All preferred or optional features detailed above for the seventh and eighth aspects of the present invention are applicable to the relevant steps.

(1) Obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, preferably obtaining a protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain;

(2) Combining the protein extract with a crosslinker to form a reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker; and

(3) Forming the bio-based leather substitute material, preferably by solution casting.

For step (1) of the preferred method, preferably the extraction takes place in solution. Preferably, the protein extract is extracted by alkali extraction, such as using an aqueous alkaline solution. Preferably, following extraction, the protein extract is separated from one or more other components of Brewer’s Spent Grain or Distiller’s Spent Grain, preferably the insoluble component(s) of Brewer’s Spent Grain or Distiller’s Spent by filtration and/or centrifuge separation to form protein isolate, and optionally purified.

For step (2) of the preferred method, preferably the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is combined with a bridging reagent and/or a crosslinking catalyst. Preferably, the protein extract is combined with a bridging reagent and a crosslinking catalyst. Preferably, the protein extract is combined with a bridging reagent and crosslinking catalyst in sequential reactions. Preferably, the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is reacted with a bridging reagent and then a crosslinking catalyst in sequential reactions. Preferably, the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain is reacted with a bridging reagent and the resulting product is then reacted with a crosslinking catalyst.

Optionally, after step (1) and prior to step (2), the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain undergoes a neutralisation step. Preferably, the bridging reagent acts as a neutralising agent.

Preferably, between step (2) and (3), product resulting from the combination of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a crosslinker is subjected to heat treatment and/or ultrasonication treatment, preferably both a heat treatment and ultrasonication treatment step. Where a bridging reagent and crosslinking catalyst are used as crosslinkers as described herein, the heat treatment and/or ultrasonication treatment step may take place between the addition of the bridging reagent and the addition of the crosslinking catalyst, or prior to or after the addition of both, preferably between the addition of the different crosslinkers or after the addition of both. Preferably the heat treatment comprises heating to a temperature of 50 to 100 °C, such as from 70 to 90 °C.

For step (3), preferably formation of the bio-based leather substitute material is achieved by solution casting. For the preferred method, preferably plasticiser and/or one or more additional components may be added before step (2) following step (1), during step (2), and/or after step (2) and before step (3).

Preferably Brewer’s Spent Grain is utilised in the preferred method.

Preferably the Brewer’s Spent Grain or Distiller’s Spent Grain from which protein extracts are obtained is in dried form in the preferred method.

According to a ninth aspect of the present invention, there is provided a bio-based leather substitute material according to the second aspect of the present invention; wherein the bio-based leather substitute material is produced by a method comprising subjecting the isolated algal polysaccharide or salt thereof to a heat treatment; combining with Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein, and a crosslinker; and forming the bio-based leather substitute material.

According to a tenth aspect of the present invention, there is provided a method of producing a bio-based leather substitute material according to the second aspect of the present invention, the method comprising: subjecting the isolated algal polysaccharide or salt thereof to a heat treatment; combining with Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein, and a crosslinker; and forming the bio-based leather substitute material.

All features of all other aspects described herein, whether preferred or optional, are applicable to the ninth and tenth aspects of the present invention. Similarly, all features of all other aspects described herein, whether preferred or optional, are applicable to the ninth and tenth aspects of the present invention.

For the ninth and tenth aspects of the present invention, following combination of the protein extract and crosslinker, the bio-based leather substitute material according to the first aspect of the present invention may be formed by any suitable technique. Suitable techniques include, but are not limited to casting, solution casting, thermal extrusion, thermal compression molding or baking, die casting, pultrusion, electrospraying, wet spinning, dry spinning, melt spinning and gel spinning, preferably casting, solution casting, thermal extrusion, thermal compression molding or baking. The bio-based leather substitute material may also be subject to further drying. The bio-based leather substitute material may also be subjected to further pressing to achieve the required thickness.

The Brewer’s Spent Grain or Distiller’s Spent Grain for use in the ninth and tenth aspects of the present invention may be used in dry or wet form. Preferably, the Brewer’s Spent Grain or Distiller’s Spent Grain is dry Brewer’s Spent Grain or Distiller’s Spent Grain. In the ninth and tenth aspects of the present invention, the method may therefore comprise an additional step of drying the Brewer’s Spent Grain or Distiller’s Spent Grain prior to use.

When the Brewer’s Spent Grain or Distiller’s Spent Grain is utilised in dry form, the Brewer’s Spent Grain or Distiller’s Spent Grain may be shredded or milled prior to use. In the ninth and tenth aspects of the present invention, the method may therefore comprise a step of shredding or milling the Brewer’s Spent Grain or Distiller’s Spent Grain. Preferably, the Brewer’s Spent Grain or Distiller’s Spent Grain is milled. When the Brewer’s Spent Grain 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 or Distiller’s Spent Grain is milled, the texture of the bio-based leather substitute material formed is advantageously smoother.

For the ninth and tenth aspects of the present invention, the method comprises heat treatment of the isolated algal polysaccharide or salt thereof. Preferably, the heat treatment is carried out in water or distilled water. Heat treatment of the isolated algal polysaccharide or salt thereof may comprise heating the isolated algal polysaccharide or salt thereof in solution, preferably aqueous solution and more preferably water or distilled water, to a temperature of 80 °C or more, such as from 80 °C to 100 °C, preferably from 85 to 100 °C, such as from 85 °C to 95 °C. Without being bound by theory, the present inventors consider that the isolated algal polysaccharides undergo dissolution when heated, such that their networks break up and they are separated. Upon cooling, the isolated algal polysaccharides are then able arrange into tightly packed chains with significant intramolecular bonding such that they form a binding network for other components of the bio-based leather substitute material. The heat treatment may take place over a period of time of from 5 to 120 minutes.

It will be appreciated that for the ninth and tenth aspects of the present invention, any other components being utilised to make the bio-based leather substitute material according to the second aspect of the present invention, such as plasticiser, may be introduced at the same time as the crosslinker and Brewer’s Spent Grain or Distiller’s Spent Grain, or may be part of the isolated algal polysaccharide solution that is subjected to heat treatment.

According to an eleventh aspect of the present invention, there is provided a use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a bio-based leather substitute material according to the first aspect of the present invention.

According to a twelfth aspect of the present invention, there is provided a use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a bio-based leather substitute material according to the second aspect of the present invention.

All features of all other aspects described herein, whether preferred or optional, are applicable to the eleventh and twelfth aspects of the present invention. Similarly, all features of all other aspects described herein, whether preferred or optional, are applicable to the eleventh and twelfth aspects of the present invention.

According to a thirteenth aspect of the present invention, there is provided a biobased leather substitute material comprising a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a plasticiser.

According to a fourteenth aspect of the present invention, there is provided a construct comprising two or more layers of the bio-based leather substitute material according to the thirteenth aspect of the present invention, or the biobased leather substitute material according to the thirteenth aspect of the present invention and a scaffold support. According to a fifteenth aspect of the present invention, there is provided an article formed of: a bio-based leather substitute material according to the thirteenth aspect of the present invention; or a construct according to the fourteenth aspect of the present invention.

According to a sixteenth aspect of the present invention, there is provided a biobased leather substitute material according to the thirteenth aspect of the present invention; wherein the bio-based leather substitute material is produced by a method comprising: providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a plasticiser, and forming the bio-based leather substitute material.

According to a seventeenth of the present invention, there is provided a method of producing a bio-based leather substitute material according to the thirteenth aspect of the present invention; the method comprising providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, combining with a plasticiser, and forming the bio-based leather substitute material.

According to an eighteenth aspect of the present invention, there is provided a use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a biobased leather substitute material according to the thirteenth aspect of the present invention.

All features of all other aspects described herein, whether preferred or optional, are applicable to the thirteenth to eighteenth aspects of the present invention. Similarly, all features of all other aspects described herein, whether preferred or optional, are applicable to the thirteenth to eighteenth aspects of the present invention.

For the bio-based leather substitute material according to the thirteenth aspect of the present invention, the protein extract derived from Brewer’s Spent Grain or Distiller’s Grain and plasticiser may be as described above for the bio- based leather substitute material according to the first aspect of the present invention. Preferably, for the bio-based leather substitute material according to the thirteenth aspect of the present invention, the plasticiser is selected from glycerol, ethylene glycol, polyethylene glycol, propylene glycol, lecithin, sunflower lecithin, mannitol, xylitol, diethylene glycol, tetraethylene glycol, ethanolamine, triethanolamine, acetic acid, glycol, fatliquor, sugar alcohols such as sorbitol, sorbitan, didecyldimethylammonium chloride (DDAC), polysorbate 20, polysorbate 80, erythritol, triethyl citrate, epoxidized oil and acetylated monoglyceride.

Preferably, the plasticiser is a polyol-based plasticiser. Suitable polyol-based plasticisers include glycerol, ethylene glycol, polyethylene glycol, propylene glycol, diethylene glycol, tetraethylene glycol, sugar alcohols such as sorbitol, sorbitan, and erythirol.

More preferably, the plasticiser is selected from glycerol, acetic acid, fatliquor, sorbitol, sorbitan, polysorbate 20, polysorbate 80, erythritol, triethyl citrate, epoxidized oil and acetylated monoglyceride. Most preferably, the plasticiser is glycerol.

For the method according to the fifteenth or sixteenth aspect of the present invention, the method of providing the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, preferably a protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain, is as described above in relation to the seventh and eighth aspects of the present invention. Additionally, for the method according to the fifteenth or sixteenth aspect of the present invention, the method of forming the bio-based leather substitute material is as described above in relation to the seventh and eighth aspects of the present invention.

Preferably, for the methods according to the fifteenth or sixteenth aspect of the present invention, the method comprises a neutralisation following provision (extraction) of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain. Preferably, for the methods according to the fifteenth or sixteenth aspects of the present invention, the neutralisation step takes place following provision (extraction) of the protein extract from Brewer’s Spent Grain or Distiller’s Spent Grain and prior to the formation of the bio-based leather substitute material. Preferably, for the methods according to the fifteenth or sixteenth aspects of the present invention, the neutralisation step takes place before combination of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with the plasticiser. The neutralisation step is as described above in relation to the method according to the seventh and eighth aspects of the present invention. The present inventors consider such neutralisation to prevent unwarranted oxidation/hydrolyzation and damage to the protein extract, especially if the protein extraction has been achieved by alkali extraction. The neutralisation reduces the pH of a solution comprising the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain to a neutral pH, such as a pH of from 6 to 8, preferably 7. Neutralisation may be achieved through the addition of a neutralising agent. When the 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. The amount of acid added is that required to reach the desired pH.

Preferably, for the methods according to the fifteenth or sixteenth aspect of the present invention, the method further comprises a heat treatment and/or ultrasonication treatment step prior to the formation of the bio-based leather substitute material. More preferably, the method according to the fifteenth or sixteenth aspect of the present invention further comprises a heat treatment and ultrasonication treatment step. The heat treatment and/or ultrasonication treatment may take place after extraction of the protein from Brewer’s Spent Grain or Distiller’ Spent Grain, and prior to the formation of the bio-based leather substitute material. The heat treatment and/or ultrasonication treatment may take place before or after combination of the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain with the plasticiser, preferably afterwards. The heat treatment and/or ultrasonication treatment step are as described above in relation to the method according to the seventh and eighth aspects of the present invention.

The heat treatment step may comprise heating to a temperature of from 50 to 100 °C, such as from 60 to 90 °C, or from 70 to 90 °C. When the method according to the fifteenth or sixteenth aspect of the present invention comprises a heat treatment and ultrasonication treatment step, the ultrasonication treatment may take place at the same time as the heat treatment, or prior or subsequent thereto. Preferably, the ultrasonication treatment takes place following, i.e. subsequent to, the heat treatment step. This is typically directly after the heat treatment step.

The ultrasonication treatment involves sonication and the application of ultrasonic frequencies. This may be using a ultrasonication device. Suitable ultrasonication devices include sonic dismembrators provided by Fischerbrand®. For the ultrasonication treatment, the power applied may be from 15 to 700 watts, such as 50 watts, 120 watts, 500 watts or 700 watts, preferably from 15 to 500 watts, such as from 15 to 120 watts. The frequency applied may be of 20 KHz. The ultrasonication treatment may be carried out in pulses, such as 5 to 30 second pulses. The ultrasonication may be carried out for 1 to 20 minutes, such as for 1 to 15 minutes, such as from 1 to 10 minutes, or 1 to 5 minutes.

Without being bound by theory, the present inventors consider that the inclusion of a heat treatment and/or ultrasonication step bypasses the need for a crosslinker as the heat treatment and/or ultrasonication treatment step for the method according to the fifteenth or sixteenth aspect alters the structure of the protein and facilitates the formation of protein nanofibrils which bind together to form the biobased leather substitute material according to the thirteenth aspect of the present invention.

Accordingly, preferably, the bio-based leather substitute material according to the eleventh aspect of the present invention does not further comprise a crosslinker.

Preferably, the method according to the fifteenth and sixteenth aspects of the present invention comprises the steps of:

(1) Obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, preferably obtaining a protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain; (2) Combining the protein extract with a plasticiser to form a reaction product of a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and a plasticiser;

(3) Preferably, subjecting the resulting product to a heat treatment and/or ultrasonication treatment step; and

(4) Forming the bio-based leather substitute material, preferably by solution casting.

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 bio-based leather substitute or intermediates in the production thereof are exposed, or procedures for the production of the bio-based leather substitute material are carried out, i.e. the range of temperatures, pressures and atmospheric conditions to which the bio-based leather 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, a pressure of from 20 to 100 kPa, and the environment is typically an oxygencontaining atmosphere. EXAMPLES

Example 1

A bio-based leather substitute material according to the first aspect of the present invention.

Milled dry Brewer’s Spent Grain (<50 pm, 4.2 g) was added to 0.1 M NaOH solution, heated to 90 °C, and stirred for 20 minutes. Glycerol (1.7 g) was added aliquots of citric acid were added until the pH of the solution was neutral (pH=7). The solution was stirred for an additional 5 minutes, cast in a 10x10cm silicone tray, and dried at ambient temperature over 16 hours. The material was removed from the tray and dried at 35 °C in a dehydrator for an additional 3 hours, yielding a brown bio-based leather substitute material (surface area 10 cm ² , thickness 0.6 to 0.73 mm).

Example 2

A bio-based leather substitute material according to the first aspect of the present invention.

Milled dry Brewer’s Spent Grain (<50 pm, 4.2 g) and K-carrageenan (0.4 g) were added to 40 mL of 0.1 M NaOH solution, heated to 90 °C, and stirred for 25 minutes. Glycerol (1.79 g) was added and aliquots of citric acid were added until the pH of the solution was neutral (pH = 7). The solution was stirred for an additional 5 minutes, cast in a 10x10 cm silicone tray, and dried at ambient temperature over 16 hours. The material was removed from the tray and dried at 35 °C in a dehydrator for an additional 3 hours, yielding a brown tan bio-based leather substitute material (surface area 10 cm ² , thickness 0.81 to 0.87 mm).

Example 3

A bio-based leather substitute material according to the second aspect of the present invention. K-carrageenan (0.4 g), agar agar (0.4 g), sorbitol (0.085 g), CaCOs (0.06 g), KCI (0.02 g) and glycerol (0.3 g) were dissolved in 40 mL of distilled water. The mixture was heated to 95 °C and stirred for 30 minutes, before cooling to 60 °C upon which milled dry Brewer’s Spent Grain (<50 pm, 3.5 g), tannic acid (0.3 g) and citric acid (0.05 g) were added. The solution was stirred for 5 minutes at 60 °C, cast in a 10x10 cm silicone tray, and dried at ambient temperature over 16 hours. The material was removed from the tray and dried at 35 °C in a dehydrator for an additional 3 hours, yielding a brown tan material (surface area 10 cm ² , thickness 0.93 to 0.95 mm, tensile strength 49.9 MPa).

Example 4

Dried Brewer’s Spent Grain was sieved through a fine mesh to remove unwanted fragments other than the grain and washed with distilled water (400 ml x 3). The washed Brewer’s Spent Grain was added to 0.1 M NaOH solution (1200 ml) and stirred for 1 hour at a temperature of 72 °C. The solution was filtered to separate the insoluble components. The solution was then slowly brought down to a pH of 7 with the slow addition of 0.2M citric acid solution (30 ml). After cooling to 45 °C under ambient temperatures, transglutaminase (Stabizym® TGL, 0.6 g) and nanocellulose (0.315 g) were added, the solution was sonicated for 7 minutes to ensure even mixing, and then left to stir for 16 hours. Glycerol (8.34 g) was added and the solution heated to 72 °C for 2.5 hours, before casting into two silicone trays (20 cm x 25 cm). The cast solution was left to dry under ambient conditions, resulting in the formation of two sheets of brown bio-based leather substitute material (0.4 mm thick) according to the first aspect of the present invention. One of the sheets was sprayed with dilute citric acid (adhesive/crosslinker) and the other placed on top and the sheets adhered together to from a construct according to the third aspect of the present invention (thickness 0.8 mm, tensile strength 7.1 MPa). One of the sheets was then embossed. The construct was formed into an article, a cardholder, according to the fifth aspect of the present invention.

Example 5 Dried Brewer’s Spent Grain (150.4 g, provided by Kernel Brewery) was sieved over a mesh, to remove unwanted fragments other than the grain, and washed with distilled water (1000 ml x 3). The washed Brewer’s Spent Grain was added to a 0.1 M NaOH solution (1200 ml) and stirred for 1 hour at a temperature of 70 °C. The solution was filtered to separate the insoluble components and 500 ml kept for this experiment. The solution was then slowly brought down to a pH of 7 with the slow addition of 0.1 M HCI solution, before heating to 72 °C for an additional 2.5 hours.

The solution was split into 3x 80 mL solution aliquots A, B and C.

(A)

1 g of glycerol was added to solution A and mixed for 5 minutes before casting into 2 square silicone molds (10cm x 10cm). The solutions were left to dry under ambient conditions (20 °C) resulting in the formation of two sheets of brown biobased leather substitute material according to the first aspect of the present invention (0.28 mm thick, elasticity (elongation at load) 18.3%, tensile strength 3.74 MPa).

(B)

1 g of glycerol and 0.127 g of citric acid were added to solution B and mixed for 5 minutes before casting into 2 square silicone molds (10cm x 10cm). The solutions were left to dry under ambient conditions (20 °C) resulting in the formation of two sheets of brown bio-based leather substitute material according to the first aspect of the present invention (0.28 mm thick, elasticity (elongation at load) 20.2%, tensile strength 4.44 MPa).

(C)

1 g of glycerol and 0.124 g of citric acid were added to solution B and mixed for 5 minutes. The solution was left to cool to below 55 °C before transglutaminase (0.133 g) was added. After stirring for 5 minutes the solution was cast into 2 square silicone molds (10cm x 10cm). The solutions were left to dry under ambient conditions (20 °C) resulting in the formation of two sheets of brown bio-based leather substitute material according to the first aspect of the present invention (0.28 mm thick, elasticity (elongation at load) 23.7%, tensile strength 5.47 MPa).

Example 6

Dried Brewer’s Spent Grain (150.6 g, provided by Kernel Brewery) was sieved over a mesh to remove unwanted fragments other than the grain, and washed with distilled water (750 ml x 3). The washed Brewer’s Spent Grain was added to a 0.1 M NaOH solution (1200 ml) and stirred for 1 hour at a temperature of 72 °C. The solution was filtered to separate the insoluble components. The solution was then slowly brought down to a pH of 7.4 with the slow addition of 0.2M citric acid solution. After cooling to 35 °C under ambient temperatures, transglutaminase (0.67 g) and Ca(OH) ₂ (0.62 g) were added. The pH was slowly brought back down to a pH of 7.4 with the slow addition of 0.2M citric acid solution before leaving the solution to stir for 16 h. The solution was heated to 72 °C for 2 h. 200 mL of the solution was used for this experiment. Nanocellulose (15.4 g) and glycerol (3.1 g) were added and left to stir for 5 minutes. The solution was cast into a square silicone mold (20cm x 20cm) and left to dry under ambient conditions (20 °C) resulting in the formation of a sheet of brown bio-based leather substitute material according to the first aspect of the present invention (0.45 mm thick, elasticity (elongation at load) 12.1%, tensile strength 9.76 MPa).

EMBODIMENTS

1. A bio-based leather substitute material comprising: a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain; and a crosslinker.

2. The bio-based leather substitute material according to embodiment 1 , wherein the bio-based leather substitute material further comprises an isolated algal polysaccharide or salt thereof, preferably an isolated algal polysaccharide. The bio-based leather substitute material according to embodiment 2, wherein the isolated algal polysaccharide is selected from isolated algin, isolated alginic acid, isolated fucoidan, isolated laminarin, isolated agar agar, isolated carrageenan, and isolated ulvan, or combinations thereof, preferably wherein the isolated algal polysaccharide is an anionic polysaccharide, and more preferably wherein the isolated algal polysaccharide is an isolated carrageenan or isolated agar agar, or combination thereof. The bio-based leather substitute material according to any of embodiment 1 to 3, wherein the protein extract is a protein isolate. A bio-based leather substitute material comprising: Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein; a crosslinker; and an isolated algal polysaccharide or salt thereof. The bio-based leather substitute material according to embodiment 5, wherein the bio-based leather substitute material further comprises an isolated algal polysaccharide or salt thereof, preferably an isolated algal polysaccharide. The bio-based leather substitute material according to embodiment 5 or 6, wherein the isolated algal polysaccharide is selected from isolated algin, isolated alginic acid, isolated fucoidan, isolated laminarin, isolated agar agar, isolated carrageenan, and isolated ulvan, or combinations thereof, preferably wherein the isolated algal polysaccharide is an anionic polysaccharide, and more preferably wherein the isolated algal polysaccharide is an isolated carrageenan or isolated agar agar, or combination thereof. The bio-based leather substitute material according to any of embodiments 1 to 7, wherein the crosslinker is a naturally derived crosslinker, preferably wherein the crosslinker is selected from citric acid, formaldehyde, acetone, urea, gum arabic, alginate, genipin, transglutaminase, azetidinium, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid, caffeic acid, and vanillin, or combinations thereof, more preferably from citric acid, formaldehyde, acetone, urea, gum arabic, alginate, genipin, transglutaminase, azetidinium, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid and caffeic acid, or combinations thereof, more preferably from citric acid, urea, gum arabic, alginate, genipin, transglutaminase, rosin, isosorbide, tannic acid, gallic acid, ellagic acid, ferulic acid and caffeic acid, or combinations thereof, and more preferably from citric acid and tannic acid, or the combination thereof. The bio-based leather substitute material according to any of embodiments 1 to 8, wherein the bio-based leather substitute material further comprises a plasticiser, preferably a naturally derived plasticiser, more preferably the plasticiser is selected from glycerol, ethylene glycol, acetic acid, water, ethanol, glycol, fatliquor, sorbitol, sorbitan, didecyldimethylammonium chloride (DDAC), polysorbate 20, polysorbate 80, erythritol, triethyl citrate, epoxidized oil and acetylated monoglyceride, more preferably from glycerol, acetic acid, water, ethanol, fatliquor, sorbitol, sorbitan, polysorbate 20, polysorbate 80, erythritol, triethyl citrate, epoxidized oil and acetylated monoglyceride, and more preferably from glycerol and water, or a combination thereof. The bio-based leather substitute material according to any of embodiments 1 to 9, wherein the protein is derived from Brewer’s Spent Grain. The bio-based leather substitute material according to any of embodiments 1 to 10, wherein the Brewer’s Spent Grain or Distiller’s Spent Grain is a protein-containing grain by-product of beer or spirit production, and preferably, wherein the Brewer’s Spent Grain or Distiller’s Spent Grain comprises 5 to 40 wt.% protein based on the dry weight of the Brewer’s Spent Grain or Distiller’s Spent Grain, optionally 1 to 15 wt.% lipid based on the dry weight of the Brewer’s Spent Grain or Distiller’s Spent Grain, optionally 5 to 35 wt.% lignin based on the dry weight of the Brewer’s Spent Grain or Distiller’s Spent Grain, and optionally 20 to 60 wt.% of cellulose based on the dry weight of the Brewer’s Spent Grain or Distiller’s Spent Grain. A multi-layer construct comprising the bio-based leather substitute material according to any of embodiments 1 to 11 , and a scaffold support, preferably provided as a layer of the multi-layer construct, and preferably wherein the scaffold support is selected from cotton, viscose, natural cellulosic fibres, perforated mesh, mesh, cheesecloth, linen or muslin mesh. An article formed of: the bio-based leather substitute material according to any of embodiments 1 to 11 ; or the multi-layer construct according to embodiment 12. A method of forming the bio-based leather substitute material according to any of embodiments 1 to 4, and embodiment 8, 9, 10 or 11 where dependent thereon, the method comprising providing a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and combining with a crosslinker to form the bio-based leather substitute material. The method according to embodiment 14, wherein the method comprises the steps of: obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain; providing the protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain and combining with a crosslinker to form the bio-based leather substitute material. The method according to embodiment 14, wherein the method comprises the steps of: obtaining a protein extract derived from Brewer’s Spent Grain or Distiller’s Spent Grain, separating the protein extract, and optionally purifying the protein extract, to provide a protein isolate; providing a protein isolate derived from Brewer’s Spent Grain or Distiller’s Spent Grain and combining with a crosslinker to form the bio-based leather substitute material. The method according to embodiment 15 or 16, wherein the protein extract is obtained 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, more preferably alkali extraction in solution, and more preferably using an alkaline reagents selected from 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, and optionally at a temperature of from 50 to 95 °C, preferably from 55 to 95 °C. The method according to embodiment 16 or 17, wherein the protein is separated by filtration and/or centrifuge separation. The method according to any of embodiment 14 to 18, wherein the biobased leather substitute material further comprises an isolated algal polysaccharide or salt thereof, and the method further comprises heat treatment of the isolated algal polysaccharide or salt thereof. The method according to embodiment 19, wherein the heat treatment is carried out in solution at a temperature of 80 °C or more, such as from 80 to 100 °C, preferably from 80 to 98 °C, and more preferably from 80 to 95 °C. and wherein the solution is preferably an aqueous solution and more preferably water or distilled water. A method of forming the bio-based leather substitute material according to any of embodiments 5 to 11 , the method comprising: subjecting the isolated algal polysaccharide or salt thereof to a heat treatment; and combining with Brewer’s Spent Grain or Distiller’s Spent Grain containing a protein, and a crosslinker, to form the bio-based leather substitute material. The method according to embodiment 21 , wherein the heat treatment is carried out in solution at a temperature of 80 °C or more, such as from 80 to 100 °C, preferably from 80 to 98 °C, and more preferably from 80 to 95 °C. and wherein the solution is preferably an aqueous solution and more preferably water or distilled water. The method according to any of embodiments 14 to 22, wherein the Brewer’s Spent Grain or Distiller’s Spent Grain is dry and preferably, is shredded or milled, preferably milled, prior to use. Use of Brewer’s Spent Grain or Distiller’s Spent Grain in the production of a bio-based leather substitute material according to any of embodiments 1 to 11.