Improved processing of plant-derived material

Abstract

The invention relates to a process for the production of a biodegradable modified plant protein-containing powder, to the biodegradable modified plant protein-containing powder thus obtained, to a process for the production of a biodegradable body from the biodegradable modified plant protein-containing powder, to the biodegradable body thus obtained, to a process for the production of a biodegradable secondary body from the biodegradable modified plant protein-containing powder, and to the biodegradable secondary body thus obtained.

Description

[0001] IMPROVED PROCESSING OF PLANT-DERIVED MATERIAL

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a process for the production of a biodegradable modified plant protein-containing powder, to the biodegradable modified plant protein-containing powder thus obtained, to a process for the production of a biodegradable body from the biodegradable modified plant protein-containing powder, to the biodegradable body thus obtained, to a process for the production of a biodegradable secondary body from the biodegradable modified plant protein-containing powder, and to the biodegradable secondary body thus obtained.

[0004] BACKGROUND

[0005] There is a benefit to replacing conventional, non-biodegradable petrochemically derived plastics with materials having an improved environmental profile. Plastic pollution is an increasingly recognized problem and a wide range of approaches are being used to address this problem, ranging from increased recycling to the use of biodegradable plastics, such as PLA. Such materials need to have sufficiently similar properties to conventional petrochemical plastics to act as replacements and need to have high levels of biodegradability. Materials such as PLA will degrade over time but are often slow to do so, especially in sea water. In addition, the raw materials used in any such compositions ideally need to be sustainable and provide a real environmental benefit. There is little benefit in using a plant-based biomaterial that has taken so much processing and pre-treatments before use in a thermoplastic process that it would have been better just to use a conventional, petrochemical material. Ideally, the plant-based biomaterials of the present invention need to comprise waste materials or other low value materials.

[0006] Virtually all plastic processing uses some form of thermal treatment. These processes can include melt-extrusion, blown extrusion, injection-moulding, calendaring, vacuum forming and so on. In all these processes, heat is applied to melt the plastic or otherwise make it more deformable and mouldable into the desired shapes. Such materials are termed thermoplastics. In order for any plant-based biomaterial to be industrially viable as a thermoplastic, it needs to be able to use as much of the existing manufacturing base as possible. Preferably, the biomaterial needs to be adapted to current equipment rather than the other way round. Much of the art concerning the processing of plant materials, including plant proteins, into forms and products such as films, relate to the processing and handling of plant proteins and other plant materials in solutions and using solution chemistry. Learnings and techniques from such solution chemistry and processing is simply not relevant to thermoplastic processing. The same applies to the processing of plant proteins and other plant-derived materials in the food industry.

[0007] Polysaccharides, such as starches, have been used for many years as thermoplastics. Starch is widely used to make packaging, such as films and foam beads, by melt and blown extrusion. Very typically the starch needs some level of chemical modification to be processable as a thermoplastic. This can limit its biodegradability. For example, much starch packaging is only biodegradable in industrial composting facilities. Starch is also often limited in its applications as it is sensitive and susceptible to moisture.

[0008] Cellulose is also widely used in packaging films, but the process typically depends on dissolving the cellulose material in an organic solvent and evaporating the solvent. It is not a thermoplastic process and could not be processed as a thermoplastic using thermoplastic equipment.

[0009] Plastics based on caseins have been used for many years. Caseins are a family of phosphoproteins found in milk. An early plastic based on casein was widely used to make buttons for clothing. Casein can be thermoplastically processed and is commercially available as pellets for use as a replacement for some petrochemical plastics in extrusion processes. All proteins typically undergo permanent changes on heating. An example is the change in egg albumen upon boiling. The albumen changes from a clear gel to a white solid as the protein cooks and denatures and cross-links. A boiled egg does not change back to an uncooked state upon cooling.

[0010] Animal-derived proteins, such as casein and hydrolysed collagen, are smaller, more soluble and more hydrophilic than plant-based proteins. Typically, they are easier to process and process differently to plant-based proteins. However, often the availability of the raw materials is limited - for example the amount of casein that is available is limited since it is a dairy product - and increasing numbers of people have general ethical issues with the use of animal products.

[0011] Hence there are advantages to the use of plant-based biomaterials in thermoplastic compositions, especially if the biomaterial can comprise a waste or low value material. However, most plant-based biomaterials, especially proteins, present a variety of major challenges for thermal processing compared to conventional petrochemical plastics. They are typically more thermally sensitive and typically undergo permanent changes upon heating.

[0012] Another issue with virtually all naturally-derived materials is that they are more susceptible to moisture than many synthetic materials. Starch films, for example, become very weak with increasing moisture level. Another issue for starch is that it is susceptible to retrogradation at low temperatures. This can weaken starch-based films and packaging and limits the range of applications. The incorporation of plant proteins has been shown to make compositions containing them more robust and resistant to moisture and yet still be highly biodegradable.

[0013] There has been extensive work looking at soy proteins as plastics. Henry Ford made components for the Model T out of pressed soy protein and there is extensive art in the field. Much of this looks at the use of chemical cross-linkers to bond protein together when the protein is compressed under heat and pressure. This type of cross-linking is effective for compression moulding but does inherently have the effect of reducing the biodegradability of the resulting bioplastic. One paper reported that, after treatment with formaldehyde, soy protein formed a strong bioplastic but only had limited biodegradability.

[0014] There therefore continues to be extensive research on the use and incorporation of plant proteins into bioplastic compositions. Typically, a plant protein source, such as pea protein isolate or PPI, is mixed with a plasticiser and subjected to shear and heat. Typically, this will cause the protein to unfold and simultaneously cross-link to form a permanent bond and a solid. Such a material could potentially be used in processes such as injection moulding and should be regarded as a thermoset material. Processes where pea protein is mixed with a plasticiser, such as glycerol, and then injected into a mould with heating are described in the literature. However, the applicability of the processes and products described in the art is not broad enough to be used as a general replacement for petrochemical materials and conventional plastics.

[0015] The major problem for thermoplastic processing most biomaterials is that they can undergo many other reactions when heated, as well as any unfolding. In practice, the inventors have seen that many plant protein-containing mixes simply cannot be melt-processed “as is”. When the mix is hot enough to unfold the protein, the protein is rapidly “cooking” to form some sort of cross-linked solid that is unsuitable for further processing. Plant protein mixes are often heated and sheared to produce a fibrous material for use in plant-based meat products. Such processing is diametrically opposed to what is needed for a thermoplastic material. There has been extensive work done on processing protein blends with different plasticisers at different levels and with different types of equipment. For example, pea and soy proteins have been extruded with high levels of glycerol and glycerol / water at high temperatures in a twin-screw extruder. This has formed some form of molten “paste” but many of the mixes were reported as being unsuitable for forming a film. Many of the tested compositions had very high levels of glycerol or other plasticisers. Some mixes had more than 60% glycerol.

[0016] The use of different plasticisers (in addition to glycerol, the most common plasticiser) has also been tested but there is relatively little benefit from the use of different plasticisers with unmodified plant protein material. They do not fundamentally change the nature of the effect of heat on the protein. In addition, many plasticisers also are relatively volatile and evaporate over time, giving materials that become progressively more brittle. Glycerol is also known to act as a protein stabilizer and can actually inhibit the unfolding of proteins at high levels.

[0017] Prior art examples are either limited in scope and / or material or in the performance of the resulting material. Kluver et al (Polymer Engineering and Science, 2015, 1913-1918) investigated the properties of extruded mixes of various mixes containing plant proteins. These included soy and pea proteins. Unmodified plant protein mixes could be extruded but only at high temperatures and very high levels of glycerol. The extruded mixes were weak or unsuitable to form into films. This is consistent with observations by the inventors. Brauer et al (Macromolecular Materials and Engineering, 2007, 292, 176-183) extruded various plant protein mixes but only by acylating the proteins - which introduces complexity and would be expected to reduce biodegradability - and the physical properties of the resulting films were described as poor. Other processes can only use specific materials such as zein and hence are limited in their applicability. WO2011080623 (to Kimberley Clark) describes thermoplastically processable blends of materials that include plant polymers but also include high levels of synthetic polymers. WO200183597 describes a biodegradable zein thermoplastic resin made of zein protein and fatty acids which can be thermoplastically formed into shaped articles. CN104144984 and corresponding W02013029018 describe the use of macrophyte biomass, for example duckweed, as a source of plant protein. Most of the protein in this biomass is Rubisco protein, which is part of the photosynthetic process and does not have the structural properties of storage proteins, and globulin proteins in particular. Moreover the bioplastic composition uses the raw milled plant biomass which contains many other plant polymer materials that interfere with the plastic properties of the bioplastic composition making it difficult to produce a material with consistent properties. In addition the biomass must also be blended with synthetic polymers meaning it is not possible to have a highly biodegradable bioplastic.

[0018] Zein and other prolamine proteins are known to be thermoplastically processable and this is believed to be due to their naturally low level of inter-molecular beta sheets. However, the extraction and purification of zein is complex and this makes it too expensive to be used as a primary material for many large-scale applications. There is an advantage in developing biopolymer materials for thermoplastic processes that can use more readily available or easily processable raw materials, though it is still possible to include some prolamine protein in these mixes as a processing aid.

[0019] There is therefore a need for developing plant protein compositions that can be thermoplastically processed.

[0020] SUMMARY OF THE INVENTION

[0021] According to the present invention, this need is met in a first aspect by a process for the production of a biodegradable modified plant protein-containing powder comprising the steps of: a) providing a dry plant protein-containing powder; b) subjecting the plant protein-containing powder to mechanical action to form a modified plant protein-containing powder having a particle size dso of less than 30 microns, wherein the mechanical action in step b) is performed by an impact milling process, wherein the mechanical action in step b) changes the physical properties of the plant proteincontaining powder, and wherein the physical properties of the plant protein-containing powder that are changed include the reduction in the level of intermolecular beta sheets of the plant protein.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Preferably, the mechanical action in step b) also changes the chemical properties of the plant protein-containing powder. The term “dry” relates to powders that have a powdery form and nature, formed of discrete particles. “Dry” powders can contain liquids such as water, plasticisers and liquid reagents provided that the level of liquid does not change the powdery nature of the mix, for example by forming a paste or dough. The term “dry” does not mean that the powder cannot comprise any liquid. There is the question of how to best enable the interaction of any chemical additives with plant proteins and other materials. One possible option is to make an aqueous slurry of the plant derived materials, including the plant protein plus any additives, and process the mix as a slurry. The aqueous environment makes chemical blending and reactions simpler, and the slurry processing can be combined with other mechanical processes, such as size reduction via high shear stirring, ultrasonication and / or high-pressure homogenization. Due to the viscosity of such slurries, these slurries typically need to be quite dilute when being processed and this can affect the interaction of the plant protein with the additive - for example by reducing the localised concentration of the additive. There is also an issue with the removal of the large amounts of water coming from the use of dilute systems.

[0024] Thermoplastic processes depend on the heat treatment of solid materials directly, rather than incorporating additional processing steps such as solvent removal.

[0025] Processes such as spray-drying are widely used to remove solvent but the inventors have observed that the spray-drying process itself then has a major effect on the materials that are being spray-dried. Many of the advantages from the previous chemical and physical modification can be completely negated by the spray-drying step. There is little benefit in reducing the particle size of materials such as proteins or reacting a protein with a reagent in a slurry if the spray-drying step then re-aggregates the proteins into a larger particle that does not re-disperse on further heating. The inventors have seen that spray-drying a plant protein slurry that had been highly sheared to reduce particle size, produced a powder that processed much more poorly than the original plant protein powder when hot pressed into a film.

[0026] However, in accordance with the present invention, size reduction can also be done on the plant protein, and / or other biomaterial, when in solid form. Milling of plant product solids is widely practiced in the food industry, typically to alter texture or rate and level of dissolution or to extract a material. Often this is done as an aqueous slurry.

[0027] However, the potential benefits of modifying a protein material as a solid by subjecting it to the mechanical action of milling to make it more suitable for thermoplastic processing have not been recognized. Impact milling is a process for size reduction that uses repeated mechanical impacts on particles to cause them to fracture and then fragment. Pin mills and ball mills are examples of impact mills. In a pin mill, the material being ground is hit by the pins of the rotor. In a media mill, such as a ball or rod mill, the material being ground is impacted and fragmented by the impacts of the media balls or rods. A ball mill or rod mill maybe a tumbling mill, or a vibratory mill, or a planetary mill depending on the direction and nature of the motion. A particular type of impact mill is a jet mill. In a jet mill, high velocity jets of fluid (typically air) cause powder particles to impact each other, thus causing their fragmentation.

[0028] Many grinders and mills are suitable for size reduction but media mills, such as ball mills and rod mills, and variants thereof, are preferred due to reasons of cost and availability. Ballmilling is very widely used in ceramics processing and many other industries. Equipment is low cost and available at large scale. Ball mills include variants such as rod mills. Ball mills can either be batch mills or continuous mills. Ball milling is often carried out in sequential steps, such as in sequential separate mills, with conditions changing at each step, such as changing the media balls, to reduce the particle sizes. Very small, milled sizes can be obtained.

[0029] Impact mills are different to attrition mills and mills that rely on compression, such as roller mills.

[0030] The inventors have seen that there is a general benefit from subjecting a protein material to a size reduction step before any thermoplastic processing step. Reducing the particle size of a protein material, such as a protein isolate, can make it easier for it to unfold or interact with other protein particles or otherwise respond to heat. The particle size of a typical processed protein material (for example a foodstuff) will be a result of the process used to produce that material, such as precipitation, and are much larger than the size of the actual protein itself. Many biomaterials are best understood as particles that become softened and partially open and unfold on heat treatment but which retain some particulate structure. It is how these softened particles then interact with their neighbors that determines how strong the resulting macro and nanoscale structure will be. The process is somewhat analogous to sintering in ceramics. Reducing the particle size of particles used in a sintering process will increase the strength of the resulting solid due to the increased number of particle interactions even if there is no change in the nature of the chemical interactions. It is believed that a similar effect can occur with plant proteins. The inventors have seen, for example, that milling pea protein makes the material easier to thermally process.

[0031] Therefore, in a preferred aspect of the present invention, the mechanical action in step b) is performed by ball milling including tumbling, vibrating and planetary ball milling. The mechanical action of milling can modify a protein or bio-material by reducing and / or changing the nature of crystallinity in a material. The inventors have seen that intense milling can modify the protein secondary structure of plant proteins, as well as reduce the particle size.

[0032] Milling may also increase or change the ability and nature of how a protein interacts with a plasticiser. It is widely reported that plasticisers will typically form a solvation shell around the protein core. For example, glycerol is reported to interact with proteins in two ways - one by the hydrophilic outer shell of the protein globule and one - to a much lesser extent - fitting into the more hydrophobic core. Fracturing a protein particle open by intense milling may make it easier for plasticisers to access these more hydrophobic “internal” regions. It may mean that more hydrophobic plasticisers should be used, for example propylene glycol or dipropylene glycol rather than glycerol with highly milled protein materials.

[0033] The structures and conformations that proteins can adopt can be described by various primary, secondary and tertiary structures. The primary structure of a protein is the peptide sequence forming the primary strand. The secondary structure describes the various ways that peptide strands can interact with each other. The tertiary structure describes how the protein is folded overall. On top of the tertiary structure will be any macro-structure - for example larger particles formed by the processing of the protein, such as precipitated or spray-dried particles. The most important secondary structures in proteins are beta sheets, alpha-helices, beta turns and other amorphous forms.

[0034] Beta-sheets are formed by peptide strands aligning together and hydrogen bonding to form planar structures. These are robust structures - for example beta-sheets give silk its strength. There are two beta-sheet forms that are important. “Intermolecular” beta sheets are beta-sheets formed between two different protein molecules. “Intramolecular1’ beta-sheets are formed by peptide strands within the same folded protein molecule. Intermolecular beta sheets provide strength to protein-containing materials as they provide the bonding mechanism for long-range bonds. It is believed that modifying a plant protein to reduce the level of intermolecular beta sheets before thermoplastic processing would be beneficial as this may make it easier for the protein to unfold on heating. The ideal process is one that modifies a plant protein to have a lower level of intermolecular beta sheets before thermoplastic processing and the thermoplastic processing then reforms some of these intermolecular beta sheets so as to provide strength to the formed body. Some plant proteins, most notably prolamins such as zein, naturally have a low level of beta sheets and a high level of alpha helices which makes it easier to thermoplastically process them. However, these typically need a high level of extraction and purification. Most other plant proteins, such as globulins, will naturally have higher levels of beta sheets, and are widely available from a variety of biomass sources and are more easily extracted and purified.

[0035] The inventors have seen that the secondary structure of plant proteins can be modified by intense and extended milling which can change the secondary structure, such as reducing the level of intermolecular beta sheets. This makes subsequent thermal processing easier as the strong intermolecular beta sheet structures will typically hold the protein globule together and inhibit the protein from unfolding. The inventors have seen that intense ball-milling of a plant protein can reduce the level of intermolecular beta sheets and result in a higher level of amorphous material, for example alpha helixes. This will make it more susceptible to melting or thermal processing. This is expected to typically increase the general processability of the material. Milling will also reduce the particle size of any macro-scale protein particles which will help processing for the reasons described earlier.

[0036] Therefore, in a preferred aspect of the present invention, the level of intermolecular beta sheets of the plant protein is reduced by greater than 10%, more preferably greater than 20%, even more preferably greater than 30%, still more preferably greater than 40%, even more preferably greater than 50%, and most preferably greater than 60%.

[0037] As the size of a milled protein-containing particle gets progressively smaller, this effect may be expected to increase, and the change in the secondary structure to increase. It is therefore reasonable to expect that milled protein will be more sensitive to temperature, and denature at progressively lower temperatures, as the material is progressively milled.

[0038] The relative levels of beta-sheets and other structures in a protein sample can be determined by Fourier Transform Infrared (FTIR) spectroscopy according to the protocols described by Yang et al in Nature Protocols, Vol 10, No. 3 (2015). FTIR spectroscopy is an established technique in the field to determine the secondary structure of proteins. In this process, the amide I band, a major band of protein IR absorption found between 1600-1700cm'1is analysed. This amide I originates mainly from C=O stretching vibration of the amide group, and is highly sensitive to small changes in hydrogen bonding and geometric orientation of amide bonds, therefore each type of secondary structures gives rise to a different C=O stretching frequency. In particular, the absorption of intermolecular beta-sheets is assigned for 2 frequency regions: 1610-1625 cm-1and 1685-1695 cm-1for parallel and anti-parallel intermolecular beta sheets respectively. Other secondary structures are also assigned to other specific frequency regions in the spectra. Table 1 of the above reference provides typical frequencies for different structures. Further examples can be found of such analysis, for example in Yang et al in Foods, 12 (22), 409 (2023) where the secondary structures of plant proteins have been analysed by this method and peaks have been assigned as intermolecular p-sheets (1610-1625 cm-1and 1685-1695 cm-1).

[0039] Since the absorption of amide I is a single broad band, it is usually analysed through curvefitting process to resolve the overlapping bands of different secondary structures. The area of peaks in the 2 regions mentioned above are compared to the area of all the peaks in the amide I band, in order to calculate the relative contribution of intermolecular beta-sheets to the overall protein secondary structure.

[0040] Many biomaterials will not truly melt - unless subject to a very high level of modification. For example, a highly hydrolysed protein will consist of short peptide chains. Such a material could be much more deformable, especially if additionally treated with a reducing agent. One potential issue is that some modifications that might be expected to make a material much more deformable and ductile may impact its ability to subsequently form strong links with neighboring particles and hence give robust structures for the finished products. Process choices are typically a balance of competing requirements.

[0041] The present invention relates to a biodegradable modified plant protein-containing powder comprising at least 40 wt% plant protein. Plant protein naturally exists in combination with other plant-materials the most abundant of these being cellulose and starches and during the protein extraction process the level of protein in increased. Typically protein concentrates have greater than 50 wt% protein and protein isolates have greater than 70 wt% protein.

[0042] One or more additives may be co-milled with the plant protein. The additives may be added simultaneously to the plant protein or they may be added sequentially. For example, the plant protein may be milled with sodium sulphite and then subsequently milled with a chaotropic agent or a non-plant protein plant-derived material, such as a starch or a cellulose.

[0043] The plant protein may be contacted with an additive prior to the application of the milling step. For example, the plant protein could be contacted with a solution of the additive and then allowed to dry before being milled. This could enhance the effect of the additive. The inventors have seen good results from pre-treating pea protein isolate powder with a solution of SDS surfactant, drying the mix and then processing further.

[0044] The non-plant protein plant derived material may include starch or other polysaccharides. It may include milled celluloses or fibrous materials. The inventors had observed that simply adding (tapioca) starch to un-milled pea protein isolate powder resulted in opaque films when pressed at 150 °C. Co-milling the mixture resulted in transparent / translucent films even at considerably lower temperatures. Combinations of starches and plant proteins are interesting as thermoplastic materials as the inclusion of the protein can potentially overcome many of the limitations of low-cost materials such as starch. The modification of the plant protein can be expected to allow a wider range of plant protein sources to be used, including low-value protein sources such a rapeseed protein, as the combination of additive and processing will allow high levels of selective modification to the protein.

[0045] The inventors have realized that milling a composition comprising a plant protein is also an opportunity to combine and modify the plant protein with other additives. The ability to modify the properties and behaviour of a plant protein by subjecting it to heat and mechanical action by co-milling the protein with an additive is not known in the art in the context of thermoplastic processing. In this context, modification of the behaviour of a plant protein can include both (i) a very intimate mixing of the protein with another material from the co-milling without a chemical or structural change prior to thermo-processing and (ii) a chemical or structural change to the protein caused by the co-milling.

[0046] Forming a very intimate mixture can result in different outcomes when processing materials even if the additive does not chemically modify the molecular structure of the protein. The very intimately mixed materials are less able to separate during any subsequent heating and processing step and can form mixes and blends that would otherwise not be possible due to the tendency to phase separate.

[0047] This behaviour can typically be seen in changes to the Differential Scanning Calorimetry (DSC) profile of a material compared to the raw materials before co-milling. Co-milling materials can result in the mixture having a mono-modal endotherm as compared to a multimodal endotherm. The benefits of very intimate mixing can typically be seen in an increased normalised heat flow at the principal (largest) endotherm, showing an increased tendency for proteins to unfold as temperature increases. Preferably, the normalised heat flow of a comilled mixture, such as a mixture of plant protein (for example pea protein) and SDS surfactant, is more than 50%, or more than 70%, or more than 100% greater than the weight- averaged normalised heat flow of the materials forming the mixture.

[0048] The inventors have seen that milling with a stirred media mill such as a ball mill is preferred for any co-milling process. This is believed to be due to the amount of time needed for the milling to happen. Certain types of mill, for example a hammer mill, provide intense but short- lived impacts and energy inputs and are effective for size reduction but may not allow the time for chemical reactions to happen.

[0049] The invention provides a process for the thermoplastic processing of mixes containing plant proteins. It modifies protein-containing powders and then combines plasticiser with the modified protein-containing powder in a thermoplastic process. The basic modification of the plant protein to make it more suitable for thermoplastic processing is a reduction in the dso particle size to less than 30 microns, with a highly preferred further modification being a change in the secondary protein structure and especially a reduction in the intermolecular beta sheet level. The inventive process comprises the following.

[0050] In a preferred aspect of the present invention, the plant protein-containing powder further comprises one or more additives.

[0051] The term “additive” encompasses additives that are deliberately added to the plant proteincontaining powder as well as naturally occurring additives that are present in the plant protein-containing powder when purchased already, particularly for protein-containing powders of low grade and low purity such as protein concentrates and flours.

[0052] In an even more preferred aspect of the present invention, the one or more additives are selected from the group consisting of polysaccharides, chemical modification agents and combinations thereof.

[0053] The cross-linking of proteins is primarily due to the formation of disulphide bonds between neighboring protein chains. It is known that this can be counteracted by the pretreatment of the protein with a reducing agent such as sodium sulphite or tris(2-carboxyethyl)phosphine (TCEP). One example in the literature adds a solution of sodium sulphite to soy protein, allowing the protein to react with the sulphite before blending the treated soy protein with other materials, including starch, in a high torque mixer and processing further. This pretreatment reportedly gave a major improvement in the high temperature rheology of the protein-containing mix and allowed it to be used in an injection-moulding device. Such treatments are attractive because they do not reduce the biodegradability of the plant protein.

[0054] This shows one option for improving the thermoplastic processability of plant protein containing blends, which is to chemically modify the plant protein (or other biomaterial) before subjecting it to thermal treatment, such as injection moulding or calendering. There are multiple chemical modifications that can be done to a protein material. Various materials, such as chaotropic agents, are known to interact with plant proteins and other plant-derived materials. Many plant-based materials, including plant proteins, will also hydrolyze in conditions of strong alkalinity, forming shorter-chain peptides and protein fragments. Hydrolysis of plant proteins is known to increase the solubility of the protein. Hydrolysis of plant proteins is used to improve the aqueous solubility of plant proteins but its uses and benefits for thermal processing have not been reported and is believed to be due to the reduction in the molecular weight and size of the protein.

[0055] Chaotropic agents disrupt hydrogen bonding. As such, a chaotropic agent can help proteins denature when heat is applied or to remain denatured once unfolded. A chaotropic agent will not stop cross-linking reactions but it is plausible that a chaotropic agent could reduce the temperature needed for a protein to unfold and hence reduce the level of cross-linking reactions by reducing the required temperatures. A plant protein could be modified by the intimate mixing of a chaotropic agent.

[0056] In a preferred aspect of the present invention, the polysaccharides are selected from the group consisting of starches, alginates, gums including carrageenan, locust bean gum, guar gum and xanthan gum, celluloses and combinations thereof.

[0057] In the present invention polysaccharides include plant derived polysaccharides, algae derived polysaccharides, fungi derived polysaccharides and microbial derived polysaccharides.

[0058] Plant derived polysaccharides are the most abundant polysaccharides and include starch and cellulose. Starches maybe be native or modified chemically, enzymatically or physically.

[0059] In a preferred aspect of the present invention, the polysaccharide is from the same plant source as the plant protein. For example, the plant protein and plant polysaccharide(s) may be supplied as one raw material, such as a partially purified plant protein concentrate containing protein, starch and cellulose. In a preferred aspect of the present invention, the cellulose may be in the form of fibrous particles or fragments. Commercially available plant protein containing powders are typically available in a range of concentrations depending on the level of purification and can range from 15% up to > 80%.

[0060] In preferred aspects of the present invention, the starch is selected from wheat starch, potato starch, pea starch, waxy potato starch, maize starch, waxy maize starch, high amylose maize starch, tapioca starch, cassava starch, rye starch, sorghum starch, chickpea starch, soy starch, or a mixture thereof, preferably potato starch. In alternative preferred aspects of the present invention, the starch is a modified starch selected from acid-treated starch, dextrin, alkaline-modified starch, bleached starch, oxidized starch, enzyme-treated starch, maltodextrin, cyclodextrin monostarch phosphate, distarch phosphate, acetylated starch, hydroxypropylated starch, hydroxyethyl starch, starch sodium octenyl succinate, starch aluminium octenyl succinate or cationic starch, or a mixture thereof.

[0061] Some starches are classified as waxy starches. A waxy starch consists essentially of amylopectin and lacks an appreciable amount of amylose. Typical waxy starches include waxy maize starch, waxy rice starch, waxy potato starch, and waxy wheat starch. Alternatively, some starches are classified as high amylose starches.

[0062] Modified starches are prepared by physically, enzymatically, or chemically treating native starch to change its properties. Starches may be modified, for example, by enzymes, by heat treatment, oxidation, or reaction with various chemicals.

[0063] In preferred aspects of the present invention, the starch is selected from wheat starch, potato starch, pea starch, waxy potato starch, maize starch, waxy maize starch, high amylose maize starch, tapioca starch, cassava starch, rye starch, sorghum starch, chickpea starch, soy starch, rapeseed starch or a mixture thereof.

[0064] In alternative preferred aspects of the present invention, the starch is a modified starch selected from acid-treated starch, dextrin, alkaline-modified starch, bleached starch, oxidized starch, enzyme-treated starch, maltodextrin, cyclodextrin monostarch phosphate, distarch phosphate, acetylated starch, hydroxypropylated starch, hydroxyethyl starch, starch sodium octenyl succinate, starch aluminium octenyl succinate or cationic starch, or a mixture thereof, preferably acid-treated starch.

[0065] Cellulose is a complex polysaccharide, consisting of 3,000 or more glucose units. It is the basic structural component of plant cell walls, and is the most abundant of all naturally occurring organic compounds. Cellulose can be extracted from plant or algae sources. Preferably the cellulose comes from the same plant source as the plant protein or can come from another source. The cellulose can be in the form of microfibri Hated cellulose (MFC), nanofibrillated cellulose (NFC), microbial nanocellulose or cellulose nanocrystals (CNC). Nanofibrillated cellulose (NFC), also referred to as cellulose nanofibrils (CNF) or cellulose nanofibers (CNF), is a material composed of nanosized cellulose fibrils typically having a high aspect ratio (length to width ratio). NFC is typically obtained from wood pulp or another natural source of cellulose fibres, typically by a process that includes subjecting the pulp / fibres to mechanical shear forces.

[0066] The polysaccharides can also include algae derived polysaccharides include red, brown and green algae derived polysaccharide, preferably salts of alginic acid, whether cross-linked or not, carrageenan, furcellaran, agar, ulvans and gums. In preferred aspects of the present invention, the red algae- or brown algae-derived polysaccharides are extracted from macroalgae. In preferred aspects of the present invention, the red algae- or brown algae- derived polysaccharides are extracted from microalgae. Microalgae are microscopic algae not visible to the naked eye. They are unicellular organisms but can be found in aggregates.

[0067] Other plant derived polysaccharides include polyglucans such as those commonly referred to as "polydextroses", fructose polymers or polyfructans such as, for example, inulin and levan, or polyxylans, pectin, dextrans, natural gums such asxanthan gum arabic, guar gum, karaya gum, gum tragacanth, ghatti gum, carob gum, locust bean gum etc.).

[0068] In an alternatively preferred aspect of the present invention, the chemical modification agents are selected from the group consisting of chaotropic agents, hydrolysing agents, reducing agents and combinations thereof.

[0069] Chaotropic agents include urea, thiourea, guanidinium chloride, magnesium chloride, surfactants such as sodium dodecyl sulphate and lithium acetate. Preferably, the chaotropic agents are selected from the group consisting of urea and surfactants.

[0070] Preferably, the hydrolysing agents are selected from the group consisting of sodium hydroxide, ammonium hydroxide, sodium carbonate, calcium oxide and calcium hydroxide.

[0071] Preferably, the reducing agent is sodium sulphite.

[0072] Co-milling the plant protein powder with a powder additive will ensure very intimate mixing and can ensure the interaction of the protein plus additive. The inventors have observed that co-milling pea protein isolate with solid sodium hydroxide in a ball mill rapidly results in the generation of an unpleasant amine odor. This must indicate the pea protein is being highly hydrolysed by contact with the solid sodium hydroxide. A similar odour was noted when pea protein was treated with aqueous sodium hydroxide. Co-milling pea protein with sodium sulphite caused a mild odour of rotten eggs typical of sulphur, again indicating a reaction between the pea protein and the additive.

[0073] In a preferred aspect of the present invention, the one or more additives are added to the plant protein-containing powder either in step a) or during step b).

[0074] In a further aspect, the present invention relates to a biodegradable modified plant proteincontaining powder obtainable by a process as described before.

[0075] The present invention also relates to a biodegradable modified plant protein-containing powder comprising between 20 and 99 wt% plant protein and between 1 and 80 wt% of one or more additives and having a primary particle size dso of less than 30 microns.

[0076] The biodegradable modified plant protein-containing powder can be mixed with other thermoplastic materials prior to further processing. An example of such a material is polyvinyl alcohol (PVA), preferably biodegradable grades of PVA.

[0077] In another aspect, the present invention relates to a process for the production of a biodegradable body comprising the steps of: a) providing the biodegradable modified plant protein-containing powder as described before; b) mixing the biodegradable modified plant protein-containing powder with at least one plasticiser to form a plasticised modified plant protein mix; c) subjecting the plasticised modified plant protein mix to a heating step to form a biodegradable body.

[0078] The inventive process takes the modified plant protein-containing powder as described before and blends it with a plasticiser, such as glycerol or dipropylene glycol, followed by subjecting the mix to a thermoplastic processing step with the application of heat and pressure to form a processed material. Suitable processes include extrusion, calendering, injection-moulding and combination thereof. The processed material can be a finished article such as a film or can be an intermediate used in subsequent process steps. For example, the modified material powder comprising plant protein could be formed into a processed material, such as an extrudate, and then processed further. This would be very similar to the pre-compounding of different petrochemical plastics to form pellets which are then extruded or processed further. In a preferred aspect of the present invention, the plasticizer is selected from glycerol, ethylene glycol, propylene glycol, dipropylene glycol, triethylene glycol, polypropylene glycols, polyethylene glycols such as PEG600, sorbitol, mannitol, xylitol, triethyl citrate, low MW (< 600 dalton) fatty acids, sugars including glucose, mannanose, fructose, sucrose, lecithin, waxes, organic acids including acetic acid and lactic acid, urea, amino acids and short chain peptides, water and mixtures thereof.

[0079] Plant proteins, for example soy and pea protein, are typically globular proteins. These are large, quite hydrophobic proteins, with the protein chains typically folded into the form of a ball or “globule”. They adopt this form as it shields the more hydrophobic core from the aqueous external environments. In order for these proteins to be useful as structural materials, they need to be at least partially unfolded so the protein chains can interact with nearby protein or polysaccharide chains. A tightly wrapped protein globule will only interact weakly with neighboring particles, so would form a weak material such as a film. A thermoplastic plastic consists of entangled polymer chains. When heated, the polymer chains become more flexible and can slide and move relative to each other. This is the material melting. When the material is cooled, the polymer chains lose flexibility and remain entangled, thus forming a solid. The nature of the interactions between the polymer chains determines the properties of the plastic.

[0080] Plant-based proteins are mainly comprised of globular proteins which are storage proteins and can be classified as albumins (soluble in water), globulins (soluble in dilute salt solutions), prolamins (soluble in aqueous ethanol solutions), and glutelins (soluble in dilute acid / alkaline solutions or insoluble in water).

[0081] Albumins and globulins are predominately present in all pulses (at greater than 50%) and some pseudo cereals (such as quinoa and amaranth). Globulins represent between about 70 and 78 wt% of the protein found in legume seeds, whereas albumins constitute between about 10 and 20 wt% of the protein. Globulins are the storage proteins of most legume seeds. Globulins have higher molecular weights than albumins and are insoluble in pure water but dissolve in dilute salt solutions. Globulins are typically more water soluble than prolamins.

[0082] In typical commercial protein isolates, there are generally only residual amounts of albumins present as they are generally removed during the protein extraction process. Globulins are typically obtained from soybean, pea, rice, potato, rapeseed, sunflower, cottonseed, lentil, chickpea, bean, fava bean, mung bean, sunflower seed, pumpkin seed, flax, chia, canola, lupine, alfalfa, moringa, borage, hemp seed, and cotton seed; preferably obtained from pea protein, potato protein, rapeseed protein, cottonseed protein and / or sunflower protein.

[0083] Prolamins and glutelins make up 85% of protein in the cereal and pseudo cereal families. Prolamins are typically found in wheat, corn, barley and rye whilst glutelins are typically only found in wheat and rice.

[0084] Prolamins are high in proline and glutamine amino acid content. They have a relatively high fraction of non-polar functionalities. They are less abundant than globulins and are found across fewer plant species. They include gliadin from wheat, hordein from barley, secalin from rye, zein (alpha, beta, gamma) from corn, kafirin from sorghum, avenin from oats. Prolamins are typically much less water soluble than Globulins.

[0085] Rubisco protein, Ribulose bisphosphate carboxylase / oxygenase, is the most abundant plant protein found in every green leaf. It is the key enzyme in photosynthesis responsible for the fixing carbon dioxide from the atmosphere. Rubisco is a globular protein with very specific functionality and is significantly different from plant storage proteins, in particular significantly different to globulin proteins. There is growing interest in commercialising it and extracting it from agricultural waste streams for its nutritional and functional properties. It has been shown to be extracted from leaves of sugar beet, mulberry, alfalfa, aubergine, radish, duckweed and other vegetable byproducts.

[0086] In a preferred aspect of the present invention, the plant protein-containing powder comprises one or more plant proteins selected from albumin and globulin proteins, most preferably globulin proteins.

[0087] In a preferred aspect of the present invention, the plant protein-containing powder comprises one or more plant proteins which have been extracted from the natural plant material, such as seeds, leaves, roots or other plant organs, and purified to increase the protein content.

[0088] Preferably the level of plant protein in the plant protein-containing powder is 40% or greater by dry weight, more preferably 50% or greater by dry weight, even more preferably 55% or greater by dry weight, still more preferably 60% or greater by dry weight, even more preferably 65% or greater by dry weight, still more preferably 70% or greater by dry weight, even more preferably 75% or greater by dry weight, still more preferably 80% or greater by dry weight, even more preferably 85% or greater by dry weight, and most preferably by 90% or greater by dry weight. By “dry weight” it is meant that the material is dried in an oven at a temperature of about 70 °C until there is no further change in weight.

[0089] In another preferred aspect of the present invention, the plant protein-containing powder comprises one or more plant proteins which remain in the natural plant material without having been extracted from the seeds, leaves, roots or other plant organs.

[0090] In a preferred aspect of the present invention, the plant protein-containing powder comprises one or more plant-based proteins selected from the group consisting of soybean protein, pea protein, rice protein, potato protein, rapeseed protein, cottonseed protein, lentil protein, chickpea protein, bean protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupine protein, alfalfa protein, moringa protein, borage protein, hemp seed protein and cotton seed protein, preferably selected from pea protein, potato protein, rapeseed protein, and / or sunflower protein. Most preferably, the plant protein-containing powder comprises one or more plantbased proteins selected from pea protein, rapeseed protein, bean protein, soy protein and sunflower protein.

[0091] A material that forms cross-links between neighboring polymer chains when molten is called a thermoset. Such materials are liquid or deformable when initially heated but then cross-link due to the temperature and solidify. A thermoset is typically regarded as being different to a thermoplastic. A thermoset material can be melted once but then solidifies due to the formation of permanent cross-links. A true thermoplastic is a material that can be repeatedly melted and solidified. This is due to the lack of formation of permanent cross-linking bonds when the material is heated and molten. A material such as polypropylene will behave as a thermoplastic due to the lack of bonds formed between neighboring chains when molten.

[0092] Materials can have properties of both a thermoplastic and a thermoset. If the rate at which cross-linking bonds is formed is low, it may be able to go through multiple heating / cooling cycles before becoming too cross-linked and difficult to process. What is important for processability is that the period of time when the material is molten and can be pumped or deformed into shape before hardening is sufficiently long to allow the required process steps to happen. The term “thermoplastic” used herein describes both true thermoplastics and materials having thermoplastic and / or thermoset properties. The application of heat to a globular (or other) protein typically causes the protein to unfold. This is beneficial for the formation of bonds and links to neighboring particles and forming larger structures.

[0093] However, plant proteins (and many plant-derived materials) are difficult materials to process thermally. This is due to the number of rapid and permanent cross-linking reactions that occur to proteins upon heating. Essentially, they “cook” due to the formation of irreversible cross-links between proteins that happen near simultaneously with the protein unfolding due to temperature. This can happen simultaneously with applied shear. Meat does not melt when it is cooked. The experience of the inventors, when exposing samples of pea protein isolate and glycerol to elevated temperatures to test the rheology and Melt Flow Index, was that heating the mixture to sufficiently high temperatures to cause the protein / glycerol mix to soften, very rapidly caused it to “over-cook” and become a blackened solid.

[0094] The Melt Flow Index (MFI) is a measure of the ease of flow of the melt of a thermoplastic material and is defined as the mass of material, in grams, flowing in ten minutes through a capillary of a specific diameter and length by a pressure applied via a 10kg weight (typically) at a material-specific temperatures. The method is described byASTM D1238.

[0095] During the Melt Flow Index test at 190 deg C, material started to extrude from the test rig but then rapidly blackened and solidified.

[0096] There is therefore a benefit to enabling plant proteins to unfold or react at lower temperatures - thereby reducing the intensity of temperature-dependent reactions and increasing the processing window. It can also be beneficial to treat the protein to prevent the cross-linking reactions from happening or to combine both approaches. This means that the plant protein, plus other materials, preferably needs to be modified before being thermally processed in order to significantly increase thermoplastic processability. This modification can include both physical changes and chemical changes and combinations of the two. Physical and chemical changes or modifications can be achieved through only the mechanical action of the process without the need for any additional materials other than the energy imparted by the process. Additional chemical changes can be achieved by the inclusion of additives, as herein described. The invention describes a process for thermoplastically processing plant protein containing compositions wherein the plant protein is modified in a chemical or physical manner by a mechanical action so as to make it easier to thermally process. The modified mixes can be used in a range of thermoplastic processes. Typical processes include extrusion, either in a single flight or in a twin-screw extruder, injection moulding and calendaring.

[0097] In a preferred aspect of the present invention, step c) is performed by an extrusion step, an injection moulding step, a calendaring step or combinations thereof.

[0098] In an alternatively preferred aspect of the present invention, step c) is carried out with the simultaneous application of pressure and / or shear.

[0099] In accordance with the present invention, the biodegradable body may be selected from one of films, coating, extrudates, granules and mouldings. A film is a relatively thin sheet of material, often used for packaging goods, which has sufficient structural integrity to be a stand-alone material. A coating is a very thin film that is formed on a substrate, wherein the substrate provides the structural integrity. An extrudate is a material that has been forced out of an extruder, typically through a die, to create a specific shape. It is the product of an extrusion process, often a continuous length of material with a consistent cross-section. A granule is a powder material with a larger particle size than a fine powder, the particles having a controlled specific shape and specific bulk density. A moulding is a 3-D body formed by a process of one of injection moulding, block moulding, spin moulding, compression moulding or cast moulding.

[0100] Accordingly, the process as herein described can also be modified in such a way that the plasticised modified plant protein mix of step b) is applied to a substrate before carrying out step c).

[0101] The substrate required for a coating preferably is a fibre-based material. More preferably, the fibre-based material is a cellulosic material selected from the group consisting of wood, wood pulp, cotton fibres, hemp fibres, jute fibres, sisal fibres, flax fibres, cellulose-based fibres, and silica-based fibres. Most preferably, the fibre-based material is selected from the group consisting of paper, cardboard, and corrugated board

[0102] The biodegradable body can be a consumer good, or a packaging material or product, or some other commercial object or product, such as pre-compounded extrudates suitable for further processing. A consumer good is typically a product directly used by a consumer. A commercial product is typically used by a business to form a consumer product. In a preferred aspect of the present invention, the biodegradable body is subjected to a further step d) selected from one or more of extrusion, injection moulding, compression moulding, block moulding, spin moulding, calendaring and hot pressing to form a biodegradable secondary body.

[0103] In accordance with the present invention, the biodegradable secondary body may be selected from one of films, sheets and mouldings. Typically, the biodegradable secondary body is a consumer goods item or part of a consumer goods item, such as a disposable cup or piece of cutlery or part of the structural elements of an item or is used as a packaging material such as a film or sheet.

[0104] The present invention also relates to a biodegradable body obtainable by a process as described before.

[0105] The present invention further relates to a biodegradable secondary body obtainable by a process as described before.

[0106] Figure 1 : Pea protein isolate particle size dso and % intermolecular beta sheets versus milling time

[0107] Figure 2: 2ndderivative normalized for pea protein isolate samples as measured by FTIR Figure 3: % of protein structures in pea protein isolate samples as measured by FTIR Figure 4: normalized FTIR spectrum for soy protein isolate samples

[0108] Figure 5: 2ndderivative normalized for soy protein isolate samples as measured by FTIR Figure 6: % of protein structures in soy protein isolate samples as measured by FTIR Figure 7: Pressed tablets of soy protein isolate samples: no milling on the left, 1 hour milled on the right-hand side

[0109] The benefits of milling plant material compositions are shown by the following, non-limiting examples.

[0110] EXAMPLES

[0111] Protein Structural Determination

[0112] Data on beta-sheet and other structural forms can be determined by Fourier Transform Infrared (FTIR) techniques. Suitable spectrometers include the Thermo-Fisher Nicolet iS10, the Bruker Equinox 55 / S FTIR spectrometer, the JASCO FT / IR 6200, and the Cary 630 (Agilent) FTIR spectrometer. The Bruker spectrometer is preferred due to its greater sensitivity sensitivity as a function of spectral resolution and signal-to-noise ratio. Secondary structural components including random coils, a-helices, intermolecular (parallel and antiparallel) beta-sheets, intramolecular beta-sheets and beta-turns were evaluated using Fourier self-deconvolution of the infrared absorbance spectra. Background measurements were taken on an empty cell and the background was subtracted from the reading of samples.

[0113] Data was collected using 128 or 256 scans (depending on the equipment) at 4cnr1resolution with background subtractions. For the structural analysis of proteins, the original data was smoothed applying a Savitzky-Golay filter (2nd order, 9 points with the Bruker spectrometer) and the second derivative was calculated applying a Savitzky-Golay filter (2nd order, 15 points with the Bruker spectrometer) and normalized.

[0114] The second derivative in the Amide I band (1600 - 1700 cm-1) was calculated from the smoothed data to deconvolve and quantify the secondary and quaternary structural contributions using the peaks at different wavelengths. The peaks were allocated to the various structures according to the following wavelengths:

[0115] The percentage of each structure relative to all the structures was calculated from the peak areas. Intermolecular beta sheet levels are the combination of parallel and anti-parallel intermolecular beta-sheet levels.

[0116] Typically all measurements were done in triplicate, and the averages are quoted herein.

[0117] Measurement of normalised heat flow and onset melting temperature by Differential Scanning Calorimetry

[0118] Suitable Differential Scanning Calorimetry_equipment includes the DSC882e from Mettler- Toledo. A small sample (10-30mg) of the material being tested is placed in an aluminium pan such as a 40 pL aluminium pans (#51119870, purchased from Mettler Toledo), and heated from 25°C to 180°C at a heating rate of 10°C / min in a nitrogen atmosphere. The pan lid should be pierced prior to the sealing and an empty pan used as a reference. The normalised heat flow is recorded and plotted as a function of temperature.

[0119] The onset melting point of a sample is defined as the first inflection point in the DSC curve showing a rate increase in the heat flow to the sample with increasing temperature. As the sample starts to melt, the heat flow to the sample increases, thus creating a change of gradient and an inflection point in the graph.

[0120] Normalised heat flow plots can be visually assessed by an operator to determine the inflection point in the graph. However, this analysis is now typically done using software analysis tools. Such analysis tools are typically included as part of the equipment operating system. Suitable software includes the STARe evaluation software supplied by Mettler- Toledo.

[0121] Measurement of Particle Size

[0122] The particle size distribution is typically measured by laser diffraction. A suitable standard for size analysis by laser diffraction is given in ISO 13320:2009. The measurement is typically done as per the manufacturer’s instruction manual and test procedures. Suitable analysers include the Anton Paar Particle Size Analyser PSA 1190 and the Mastersizer 3000 by Malvern Instruments.

[0123] The sample to be tested is dispersed in isopropanol. A concentration of 0.1g solid in 100 mL of isopropanol is suitable and the sample should be dispersed by application of ultrasound for 30 - 60 seconds. The mixture was diluted to the required concentration in order to have the desired optical density (normally 5-15% obscuration) for the measurement.

[0124] Typically all measurements were done in triplicate, and the averages are quoted herein. Particle size dso quoted is for the volume distribution, d and ds values for the volume distribution can also be obtained in this way using laser diffraction.

[0125] Raw Materials

[0126] ProEarth Pea Protein Isolate (80% protein) was sourced from Cambridge Commodities. All PPI work was done with the same batch.

[0127] Soy Protein Isolate (90.8% protein) was sourced from Biomedicals LLC Sodium hydroxide pellets (98.5% lab grade) were sourced from ThermoScientific. Glycerol (99.5%) and Propylene Glycol (99.8%) were sourced from APC Pure. Urea (Technical Grade) was sourced from VWR.

[0128] Sodium Sulphite (lab grade reagent) was sourced from Sigma Aldrich. Sodium Dodecyl Surfactant (SDS) 99% was sourced from VWR.

[0129] Tapioca starch was sourced from the Bakerite Company, UK. Isopropanol was sourced from Fisher Scientific, UK Water used was lab-grade Reverse Osmosis water.

[0130] Comparative Example 1

[0131] Commercially available Pea protein isolate (PPI) was measured by laser diffraction (using an Anton Par PSA 1190) to have a dso particle size of 68.2 microns. FTIR analysis was carried out according to the method herein using a Bruker spectrometer and the spectrum, 2ndderivative (Figure 2) and % of secondary structures was obtained (Figure 3). FTIR analysis showed it to have an intermolecular beta-sheet level of 33.8%, an intramolecular beta-sheet level of 15.8% and an alpha-helix and random coil level of 26.4% (Sample A).

[0132] 3.0g of the PPI was then hand mixed with a mixture of 2.01g of Propylene Glycol and 0.44g of water for between 1-2 minutes until a uniform powdery paste was formed. This was further stirred for 1 minute to form a damp powdery mass. 0.4g of this mass was then placed in a rectangular stainless steel metal rectangular mould of 2cm by 1cm and pressed by a matching metal die with a weight of 10 kg (hence applying a pressure of 0.49 MPa) for 1 minute.

[0133] The mould and die had been pre-heated to 150 °C in an oven and taken out immediately prior to use. After 1 minute, the pressed PPI mix was removed from the mould. It had formed a uniform, light brown partially opaque disc (a biodegradable body) with limited flexibility and did not have the properties of a thermoplastic.

[0134] These tests were repeated to form biodegradable bodies but now with the mould at 100 °C. The un-milled material made a disc that actually snapped in two when being flexed. It was completely opaque. This demonstrated that the material was not thermoplastic.

[0135] Inventive Example 1 Pea protein isolate, PPI, (Sample A) underwent modification by mechanical action on the plant protein by milling in a Retsch MM 400 lab tumbling ball mill at 20 Hz and 30 Hz using a custom made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel balls. FTIR analysis was carried out according to the method herein using a Bruker spectrometer and the spectrum, 2ndderivative (Figure 2) and % of secondary structures (Figure 3) was obtained.

[0136] Figure 1 shows the change in both the material particle size dso and % intermolecular betasheets with increasing milling time.

[0137] At 20Hz there was only a small decreased in particle size dso but this remained above 30 microns as not sufficient energy was imparted. There was also only a small 10.9% reduction in intermolecular beta sheet from 33.8 to 30.1%

[0138] At 30Hz there was a much greater decreased in particle size dso to below 30 microns within 2 minutes of milling as sufficient energy was imparted. There was also a much greater reduction in intermolecular beta sheet after 10 minutes of milling. FTIR analysis showed the material (Sample B) to have an inter-molecular beta-sheet level of 19.2%, an intramolecular beta-sheet level of 22.0% and an alpha-helix and random coil level of 35.9%. This was a 43.2% reduction in intermolecular beta-sheets. The particle size was measured using an Anton Par PSA 1190 in triplicate to be 16.6 microns.

[0139] Comparative Example 2 and Inventive Example 2

[0140] Sample B of PPI tumbling ball milled at 30 Hz for 10 mins was hand-mixed with an 85:15 mixture of glycerol and water to form a 60:40 solids: plasticiser mix. One gram test samples of this mix were put through a hot roller test rig consisting of two parallel, counter rotating, 15 cm diameter rollers at 100°C with a controllable gap between the rollers set to 0.15 mm. The milled mix samples formed smooth, homogenous, semi-transparent thin films with good flexibility.

[0141] Testing a similar mix made with un-milled PPI (Sample A) under the same conditions gave a much thicker, more opaque film sample that was hard to remove from the roller.

[0142] Inventive Example 3

[0143] Extended milling times can be used to further change the secondary structure as measured with a Bruker spectrometer. Milling PPI in the Retsch MM400 tumbling ball mill using a custom made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel ball at 20 Hz for 2 hours further reduced the inter-molecular beta-sheet level to 16.5%, a 51.2% reduction. The intramolecular beta-sheet level was 26.1 % and an alpha-helix and random coil level was 33.5% (Sample C). This compares to the values seen for Sample B milled at 30 Hz for 10 mins, which were an inter-molecular beta-sheet level of 19.2%, an intramolecular beta-sheet level of 22.0% and an alpha-helix and random coil level of 35.9%

[0144] Milling PPI in the Retsch MM400 tumbling ball mill using a custom made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel ball at 30 Hz for 30 mins (as compared to 10 mins for Sample B) further reduced the intermolecular beta-sheet level to 13.1 %, a 61.2% reduction, with the intramolecular beta-sheet level of 28.0% and the alpha-helix and random coil level of 33.4% and the particle size (measured using an Anton Par PSA 1190 in triplicate) to 25.0 microns. (Sample D).

[0145] Different types of mills can be used. The different levels of intensity in different mills mean that materials will need to be milled for different times in different mills. It took 30 minutes of milling in a Retsch PM 100 lab planetary ball mill to reduce the intermolecular beta sheets in the PPI to 24.3%, a reduction of 28.1 %. The intramolecular beta-sheet level was 22.1 % and an alpha-helix and random coil level was 29.6% (Sample E). It took a further 90 mins, to a total of 2 hours, to reduce the intermolecular beta sheet level to 18.4%, a reduction of 45.6%, and the particle size dso to 23.3 microns. The intramolecular beta-sheet level was 25.9% and an alpha-helix and random coil level was 31.5% (Sample F).

[0146] Inventive Example 4

[0147] The same commercial PPI powder as in the other examples was mixed with SDS, urea and sodium sulphite additive powders to make the following three different compositions on a weight basis:

[0148] 95% PPI - 5% SDS 95% PPI - 5% urea 95% PPI - 5% sodium sulphite

[0149] These compositions were individually tumbling ball milled in the Retsch MM 400 mill using a custom made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel ball for 10 mins at 20 Hz (3g samples per pot). 3g of each milled sample was then mixed with a glycerol: propylene glycol: water mix by hand to make the following compositions on a weight basis:

[0150] 56.5% (PPI + additive)

[0151] 15.5% glycerol

[0152] 18.9% propylene glycol 9.1% water

[0153] 0.4g samples of each freshly prepared composition were tested in the hot (150 °C) mould (2cm x 1cm) as per previous examples. All the milled samples formed transparent flexible discs with evidence of material extruded up the side gaps during pressing. The sample with urea was the most flexible and plastic and the sodium sulphite sample the least flexible. The same compositions were prepared with un-milled PPI and additive and tested under the same conditions. The sample discs were all opaque and with much more limited flexibility.

[0154] A sample of the same PPI was ball milled with 6.7% solid NaOH in a Retsch MM400 tumbling mill using a custom made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel ball for 10 minutes at 20 Hz. The milled powder material was then mixed with an 85:15 glycerol : water mix to make a 65% solids (PPI + NaOH) and 35% plasticiser. When tested in a similar manner to the samples above, the mix was so plastic that most of the mix was extruded into the gaps in the mould and did not form a reasonable disc.

[0155] Inventive Example 5

[0156] A sample of the same PPI as the other examples was ball milled with 7% solid NaOH in a Retsch MM400 tumbling ball mill using a custom made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel ball at 30 Hz. After 30mins the intermolecular beta-sheet level measured with a Bruker spectrometer was reduced to 11.7%, a reduction of 65.4%, the intramolecular beta-sheet level was 29.6% and an alpha-helix and random coil level to 33.2% (Sample G). After 2 hours of milling, the intermolecular beta-sheet level was further reduced to 6.4%, a reduction of 81.1%, the intramolecular beta-sheet level was 30.4% and the alpha-helix and random coil level was 40.4% (Sample H).

[0157] This shows the benefits of milling a plant material plus the opportunity to modify the properties of a mix by co-milling PPI plus additives. Comparative Example 6 and Inventive Example 6

[0158] 3720g of the same PPI batch as used in earlier examples was mixed with 280g of crushed NaOH pellets and loaded into a rotating drum of length 0.8m and diameter 0.75m. The drum was loaded with 10 kg of ceramic grinding balls having a diameter of 3.5cm and then rotated at 37 rpm for 7 hours. The post-ball milled material had a dso particle size of 25.6 microns.

[0159] Samples of this material were hand mixed with a glycerokwater mix to make a composition of 55% PPI / NaOH, 37% glycerol and 8% water. When 0.4g was hand-pressed in the heated mould (at 130 °C), the material did not form a disc but again was squeezed into the gaps, indicating high plasticity, leaving fragments of a transparent brown film. PPI “as is” when tested under exactly the same conditions gave a brown, opaque and rigid disc indicating a much less plastic material.

[0160] A similar mix to above was freshly prepared and then 3g of the mix was hot-pressed between 2 mylar sheets using platens at a temperature of 90 °C for 30 s at a nominal force of 50 tons. This formed a transparent and flexible film. Repeating the experiment with PPI “as is” gave a brittle film with large amounts of powder. A platen temperature of 150 °C was required to form the un-milled PPI composition into a similar film. The NaOH co-milled films could be folded over and pre-pressed under similar conditions multiple times, thus demonstrating a high level of thermoplasticity. This was not possible with films formed from un-milled PPI.

[0161] This NaOH co-milled PPI was then extruded using a Thermofisher 16 Twin-Screw Extruder having a diameter of 25mm and a Length to Diameter ratio of 40. The extruder was set up with two sets of kneading blocks at 45° at barrels 4 and 7 and the four barrels at the outlet were heated to 100 °C. The mix was extruded through a die plate having 3 orifices of 3mm diameter. A composition of 65% PPI / NaOH solids and 35% of an 85:15 glycerol: water plasticiser mix was easily extruded at a rate of 2.6 kg / hr with the plasticiser being injected into barrel 2. The treated PPI formed elastic and rubbery translucent brown extrudate strands. The extrudates were then easily further processed, for example by being pressing into films using the hot platens at 90 °C.

[0162] Other 4 kg batches were co-milled using the larger drum as above, under similar conditions. The following compositions were prepared on a weight basis:

[0163] Batch 1 96.5% PPI, 3.5% NaOH Batch 2 92.5% PPI, 3.5% NaOH, 4% SDS

[0164] Batch 3 92% PPI, 4% urea, 4% SDS

[0165] Batch 4 68.5% PPI, 20% tapioca starch, 3.5% NaOH, 4% SDS, 4% urea

[0166] All samples had dso particle sizes between 23 microns and 27 microns when measured using a Mastersizer 3000 according to the described method.

[0167] Comparative Example 7 and Inventive Example 7

[0168] The Batch 3 material was tested in a hot roller to test film formation via calendering.

[0169] The test rig consisted of two parallel, counter rotating 15 cm diameter rollers with a controllable gap between the rollers. The rollers were heated to a target temperature of 100 °C and set to a speed to 12 rpm. Batch 3 material was mixed in a kitchen mixer with the 85:15 glycerol: water plasticiser mix to form a 60% solids, 40% plasticiser mix. The mix was a soft granular powder and was sieved through a 2mm sieve to ensure uniformity. This mix was then continuously added by scoop onto the rollers, which were set to a target gap of 0.15mm though this was controlled by a maximum force and powder mixes could push the rollers apart, increasing film thickness. The rollers pressed the mix into an opaque or semi-opaque film that could be collected and rolled up. The film was then be further processed - e.g., by passing the film through the rollers again at higher temperatures (here 120 °C) - as would happen in a typical calendaring process for film making - or by hot pressing etc. Repeated roller pressing resulted in thinner, more uniform, more flexible and less opaque films. This shows the suitability of the mix for use in a calendering process.

[0170] The film made with the milled Batch 3 mix was much more flexible, robust and easier to handle than calendared films made with the same composition but using un-milled materials. The un-milled films were thicker, very opaque and weaker - even after repeated passes through the rollers.

[0171] A 10cm x10cm sample of the “first pass” calendared film with milled Batch 3 material above was then hot pressed between platens at 150 °C for 1 minute at a nominal force of 25 tons. This gave a clear transparent film having good physical properties showing the benefits of the intimate mixing of the additives and reduction in size of the protein and how the further processing of a biodegradable body can further change and improve the properties of the resulting secondary biodegradable body. As mentioned earlier, a sample of PPI milled at 30 Hz for 10 mins was made into a 60 solids: 40 plasticiser mix using a plasticiser mix of 85% glycerol and 15% water. 1g of a freshly prepared sample was dropped in the hot rollers at 100 °C and 12 rpm and formed into a film on the first pass. This formed a smooth piece of film of about 10cm length which could be easily peeled off the rollers to give a flexible, semi-opaque material. The same composition made with un-milled PPI stuck to the rollers and gave a small and much less uniform piece of film. This shows the benefit of a small plant protein particle size as well as modifying the secondary structure of the plant protein.

[0172] Comparative Example 8 and Inventive Example 8

[0173] Batch 4 material (68.5% PPI, 20% tapioca starch, 3.5% NaOH, 4% SDS, 4% urea) was processed in a Thermofisher 16 Twin-Screw Extruder having a diameter of 25mm and a Length to Diameter ratio of 40.

[0174] The extruder was set up with two sets of kneading blocks at 45° at barrels 4 and 7 and the two barrels at the outlet were heated to 100 °C followed by the next two barrels at 90 °C and the fifth barrel (from the outlet) at 60 °C. A die-plate having 3 orifices of 3mm diameter was initially used. Compositions ranging from 65% to 75% solids using an 85:15 glycerol: water plasticiser mix were easily extruded at a rate of 2.6 - 2.8 kg / hr with the plasticiser being injected into barrel 2. Extrudates having a composition of 70% solids were collected for further processing. The extrudates could be easily hot-pressed into transparent films using hot platens at 100 °C for 30 seconds.

[0175] The die-plate was then changed to a slit die of 1mm diameter and 10 mm width. Using similar conditions to previously, material was very easily extruded through the slit to form a film strip. The film strip was robust and could be easily handled.

[0176] The thermoplastic nature of the mix and ability of the mix to be processed by extrusion was demonstrated by re-extruding the extrudates collected previously, rather than the powder blend and plasticiser. The extrudates could be processed with very similar process conditions (such as die-plate pressures) to the initial materials and could be formed into film strips of very similar appearance to the earlier samples. The re-extruded film strips could also be hot- pressed into transparent films.

[0177] Most previous pea protein containing mixes produced unsatisfactory extrudates when processed with the TSE. Comparative Example 9

[0178] Commercially available Soy protein isolate (SPI) with no milling (Sample J), had its particle size measured by laser diffraction (using an Anton Par PSA 1190) and found to have a dso of 40.9 microns. FTIR analysis was carried out according to the method herein using a Cary 630. Due to the reduced sensitivity of this equipment smoothing was done with 22 points for the original spectrum and 34 points for the 2ndderivative. The spectrum, 2ndderivative and % of secondary structures are shown in Figures 4, 5 and 6 respectively. Sample J with no milling was found to have an intermolecular beta-sheet level of 21.5%, an intramolecular beta-sheet level of 31.6% and an alpha-helix and random coil level of 34.4%.

[0179] 82 wt% Propylene Glycol was mixed into reverse osmosis water. 9 parts by weight of the Propylene Glycol solution was mixed well with 11 parts by weight of the SPI powder by hand with a spatula.

[0180] 0.4g of this mass was then placed in a rectangular stainless steel metal rectangular mould of 2cm by 1cm. The mould and die had been pre-heated to 150 °C in an oven and taken out immediately prior to use. The mould was returned to the oven at 150°C and was pressed by the matching metal die with a weight of 15 kg for 1 minute. The pressed plasticised SPI mix was removed from the mould. It had formed a uniform, light brown opaque tablet (a biodegradable body) with very limited flexibility and did not have the properties of a thermoplastic. The sample can be seen on left-hand side of Figure 7.

[0181] Inventive Example 9

[0182] The Soy protein isolate (SPI) of Comparative Example 9 underwent modification by mechanical action on the plant protein material by ball milling in a Retsch MM 400 lab tumbling ball mill for times ranging between 1 minute and 1 hour at 30 Hz using a custom- made stainless steel jar (approximately 40 mis) filled with three 12mm diameter stainless steel balls. The particle size was measured using an Anton Par PSA 1190 (in triplicate). FTIR analysis was carried out according to the method herein using a Cary 630 as described in Comparative Example 9. The spectrum, 2ndderivative and % of secondary structures are shown in Figures 4, 5 and 6 respectively. All of the milled samples had a reduced level of intermolecular beta sheets in comparison with the un-milled sample, of at least 10%, as shown in Figure 6. The sample milled for 10 minutes, Sample K, had a dso of 18.5 microns. FTIR analysis showed the material to have a reduced intermolecular beta-sheet level of 10.5%, a reduction due to processing of 51.2%, and an intramolecular beta-sheet level of 33.5% and an alphahelix and random coil level of 37.3%.

[0183] The sample milled for 1 hour, Sample L, had a dso of 25.5 microns. FTIR analysis showed the material to have a reduced intermolecular beta-sheet level of 8.9%, a reduction due to processing of 58.6%, and an intramolecular beta-sheet level of 34.8% and an alpha-helix and random turn level of 35.6%.

[0184] The 1-hour milled material of Sample L was mixed with Propylene glycol and moulded according to the formulation and method of Comparative Example 9. The pressed plasticised milled SPI mix was removed from the mould. It had formed a transparent pale yellow tablet (a biodegradable body) that was flexible and had the properties of a thermoplastic. The sample can be seen on the right-hand side of Figure 7.

Claims

CLAIMS1 . A process for the production of a biodegradable modified plant protein-containing powder comprising the steps of: a) providing a dry plant protein-containing powder; b) subjecting the plant protein-containing powder to mechanical action to form a modified plant protein-containing powder having a particle size dso of less than 30 microns, wherein the mechanical action in step b) is performed by an impact milling process, wherein the mechanical action in step b) changes the physical properties of the plant protein-containing powder, and wherein the physical properties of the plant proteincontaining powder that are changed include the reduction in the level of intermolecular beta sheets of the plant protein.

2. The process of claim 1 , wherein the mechanical action in step b) changes the chemical properties of the plant protein-containing powder.

3. The process of claim 2, wherein the mechanical action in step b) is performed by ball milling.

4. The process of any of claims 1 to 3, wherein the level of intermolecular beta sheets of the plant protein is reduced by greater than 10%, preferably greater than 20%, more preferably greater than 30%, still more preferably greater than 40%, even more preferably greater than 50%, and most preferably greater than 60%.

5. The process of any of claims 1 to 4, wherein the plant protein-containing powder comprises one or more plant proteins selected from the group consisting of soybean protein, pea protein, rice protein, potato protein, rapeseed protein, sunflower protein, lentil protein, chickpea protein, bean protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupine protein, alfalfa protein, moringa protein, borage protein, hemp seed protein and cotton seed protein, preferably wherein the plant protein-containing powder comprises one or more plant proteins selected from the group consisting of pea protein, potato protein, rapeseed protein, sunflower protein, and soybean protein.

6. The process of any of claims 1 to 5, wherein the plant protein-containing powder further comprises one or more additives, preferably wherein the one or more additives are added to the plant protein-containing powder either in step a) or during step b).

7. The process of claim 6, wherein the one or more additives are selected from the group consisting of polysaccharides, chemical modification agents, prolamin proteins and combinations thereof, wherein the polysaccharides are preferably selected from the group consisting of starches, alginates, gums including carrageenan, locust bean gum, guar gum, xanthan gum, celluloses and combinations thereof, wherein the chemical modification agents are preferably selected from the group consisting of chaotropic agents, hydrolysing agents, reducing agents and combinations thereof, wherein the chaotropic agents are preferably selected from the group consisting of urea and surfactants, wherein the hydrolysing agents are preferably selected from the group consisting of sodium hydroxide, ammonium hydroxide, sodium carbonate, calcium oxide and calcium hydroxide, and wherein the reducing agent preferably is sodium sulphite.

8. A process for the production of a biodegradable body comprising the steps of: a) providing the biodegradable modified plant protein-containing powder of any of claims 1 to 7; b) mixing the biodegradable modified plant protein-containing powder with at least one plasticiser to form a plasticised modified plant protein mix; c) subjecting the plasticised modified plant protein mix to a heating step to form a biodegradable body.

9. The process of claim 8, wherein the plasticised modified plant protein mix of step b) is applied to a substrate before carrying out step c).

10. The process of claim 9, wherein the substrate is a fibre-based material, preferably wherein the fibre-based material is a cellulosic material selected from the group consisting of wood, wood pulp, cotton fibres, hemp fibres, jute fibres, sisal fibres, flax fibres, cellulose-based fibres, and silica-based fibres, most preferably wherein the fibre-based material is selected from the group consisting of paper, cardboard, and corrugated board.

11. The process of any of claims 8 to 10, wherein the biodegradable body is selected from one of films, coating, extrudates, granules and mouldings.

12. The process of any of claims 8 to 11 , wherein step c) is performed by an extrusion step, an injection moulding step, a calendaring step or combinations thereof, preferably wherein step c) is carried out with the simultaneous application of pressure and / or shear.

13. The process of any of claims 8 to 12, wherein the biodegradable body is subjected to a further step d) selected from one or more of extrusion, injection moulding, calendaring and hot pressing to form a biodegradable secondary body, preferably wherein the biodegradable secondary body is selected from one of films, sheets and mouldings.

14. A biodegradable modified plant protein-containing powder obtainable by a process according to any of claims 1 to 7.

15. A biodegradable body obtainable by a process according to any of claims 8 to 12.

16. A biodegradable secondary body obtainable by a process according to claim 13.

17. A biodegradable modified plant protein-containing powder comprising between 20 and 99% plant protein and between 1 and 80% of one or more additives, and having a primary particle size dso of less than 30 microns.

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