PROTEIN DISPERSIONS

Abstract

The present invention relates to a method for preparing a plant-based protein hydrogel slurry, and to a method for preparing a plant-based structured material (e.g. a film, a casting, a moulding etc.) from the plant-based protein hydrogel slurry.

Claims

1. A method for the preparation of a plant-based protein hydrogel slurry, the method comprising: (a) forming a solution comprising one or more plant-based protein(s) in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases solubility of the plant-based protein(s), and a second co-solvent decreases solubility of the plant-based protein(s); (b) inducing the protein in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel; and (c) subjecting the plant-based protein hydrogel to a shear treatment to form a plant-based protein hydrogel slurry.

2. The method according to claim 1, wherein the plant protein(s) is selected from soybean protein, pea protein, rice protein, potato protein, wheat protein, corn zein protein or sorghum protein.

3. The method according to any preceding claim, wherein the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant-based protein(s) solution, then reduced to a second temperature below the sol-gel temperature of the one or more plant-based protein(s) solution to form a hydrogel.

4. A method according to any preceding claim, wherein said shear treatment comprises a high-shear step; preferably wherein said high-shear step involves fragmenting the plant-based protein hydrogel into fragments; even more preferably wherein said fragments produced in said high-shear step have a d.sub.50 as determined by DLS of less than 500 nm, preferably less than 300 nm, more preferably less than 200 nm, even more preferably less than 50 nm.

5. A method according to any one of claims 1 to 3, wherein said shear treatment comprises a high-shear step; preferably wherein said high-shear step involves fragmenting the plant-based protein hydrogel into fragments; even more preferably wherein said fragments produced in said high-shear step have a d.sub.50 as determined by laser diffraction of 0.5 to 150 microns, preferably 0.6 to 100 microns, more preferably 0.7 to 70 microns, even more preferably 0.8 to 50 microns, more preferably 0.9 to 25 microns, more preferably 1 to 20 microns, more preferably 1 to 10 microns, even more preferably 1 to 5 microns.

6. A method according to any preceding claim, wherein said shear treatment comprises a low-shear step followed by a high-shear step.

7. A method according to claim 5, wherein at least 80% of said fragments produced in said low-shear step have a particle size in the range 1 mm to 50 mm, preferably 1 mm to 30 mm, more preferably 10 mm to 30 mm, more preferably 15 mm to 30 mm, even more preferably 20 mm to 30 mm, as determined by sieving.

8. A method according to claim 6 or claim 7, wherein step (c) further comprises subjecting the plant-based protein hydrogel slurry to a solvent reduction step, preferably a solubilising solvent reduction step, between said low-shear step and said high-shear step; wherein said solvent reduction step comprises the steps of: (i) contacting the fragments of the plant-based hydrogel slurry with a non-solubilising solvent; (ii) separating the fragments of the plant-based hydrogel slurry from the non-solubilising solvent to give a washed plant-based protein hydrogel; and (iii) optionally repeating steps (i) and (ii).

9. A method according to claim 8, wherein prior to washing said plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of between about 1000 to 20,000 Pa, between about 1000 to 15,000 Pa, between about 1000 to 10,000 Pa, between about 2000 to 20,000 Pa, between about 2000 to 15,000 Pa, between about 2000 to 10,000 Pa.

10. A method according to claim 8 or claim 9, wherein said washed plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of between about 500 to 20,000 Pa, between about 500 to 15,000 Pa, between about 500 to 10,000 Pa, between about 1000 to 20,000 Pa, between about 1000 to 15,000 Pa, or between about 1000 to 10,000 Pa.

11. A method according to any preceding claim, further comprising the step of: (d) altering the pH of the plant-based protein hydrogel slurry such that it is different to the isoelectric point of the protein hydrogel by more than 1 pH unit.

12. A method according to any preceding claim, further comprising adding an additional ingredient to the plant-based protein hydrogel slurry; wherein said additional ingredient is selected from plasticisers, opacifiers, preservatives, pigments and nanoparticles, or mixtures thereof.

13. A method according to any preceding claim, wherein the plant-based protein hydrogel slurry has a viscosity in the range 10 to 10000 cps at 50 s.sup.−1, preferably 10 to 8000 cps at 50 s.sup.−1, preferably 12 to 6000 cps at 50 s.sup.−1, preferably 15 to 5000 cps at 50 s.sup.−1.

14. A plant-based protein hydrogel slurry prepared according to the method of any one of claims 1 to 13.

15. A method for the preparation of a plant-based structured material, the method comprising: (a) preparing a plant-based protein hydrogel slurry according to the method of any preceding claim; and (b) subjecting the plant-based protein hydrogel slurry to one or more solvent level reduction step(s) to reduce the level of said first co-solvent and/or said second co-solvent to give said plant-based structured material.

16. A method according to claim 15, wherein the plant-based structured material is a film, a casting or a coating.

17. A method according to claim 16, wherein said plant-based structured material is a coating which is a food coating, a seed coating, a pharmaceutical coating, or a surface coating (e.g. a paper coating).

18. A method according to any one of claims 15 to 17, wherein the plant-based structured material comprises a plant-based protein(s) having secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet.

19. A plant-based structured material prepared according to the method of any one of claims 15 to 18.

20. Use of a plant-based protein hydrogel slurry according to claim 14 to produce a plant-based structured material.

21. Use according to claim 20, wherein said plant-based structured material is a film, a casting, a moulding, or a coating.

22. Use according to claim 21, wherein said plant-based structured material is a coating which is a food coating, a seed coating, a pharmaceutical coating, or a surface coating (e.g. a paper coating).

23. A plant-based protein hydrogel slurry having a protein solids content of 5 wt % to 25 wt % based upon the total weight of the plant-based protein hydrogel slurry and a viscosity in the range 10 to 10,000 cps at 50 s.sup.−1 and 20° C., wherein the plant-based protein hydrogel slurry comprises fragments having a d.sub.50 particle size as determined by laser diffraction of 0.5 to 150 microns.

24. A plant-based protein hydrogel slurry as claimed in claim 23, wherein the plant-based protein hydrogel slurry comprises fragments having a d.sub.50 particle size as determined by laser diffraction of 0.6 to 100 microns.

25. A plant-based protein hydrogel slurry having a protein solids content of 5 wt % to 25 wt % based upon the total weight of the plant-based protein hydrogel slurry and a viscosity in the range 10 to 10,000 cps at 50 s.sup.−1 and 20° C., wherein the plant-based protein hydrogel slurry comprises fragments having a d.sub.50 particle size as determined by Dynamic Light Scattering of less than 500 nm.

26. A plant-based protein hydrogel slurry as claimed in claim 25, wherein the plant-based protein hydrogel slurry comprises fragments having a d.sub.50 particle size as determined by Dynamic Light Scattering of less than 300 nm.

27. A plant-based protein hydrogel slurry as claimed in any one of claims 23 to 26, wherein the protein solids content is 6 wt % to 20 wt % based upon the total weight of the plant-based protein hydrogel slurry.

28. A plant-based protein hydrogel slurry as claimed in any one of claims 23 to 27, wherein the viscosity is in the range 10 to 8000 cps at 50 s.sup.−1 and 20° C.

29. A film comprising a plant-based protein hydrogel slurry as claimed in any one of claims 23 to 28.

30. A film according to claim 29, wherein the film: (a) comprises a plant-based protein(s) having secondary structure with at least 40% intermolecular β-sheet; and/or (b) has a tensile strength of 4 to 20 MPa; and/or (c) has an elongation break percentage of above 10%.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0174] FIG. 1 shows a transmission electron microscopy (TEM) image of the diluted pea protein-based slurry prepared in Example 1.

[0175] FIG. 2a is a photograph demonstrating the transparency of the film prepared in Example 2 and FIG. 2b is a photograph demonstrating the transparency of the film prepared in Example 3.

[0176] FIG. 3 shows a scanning electron microscopy (SEM) image of a coating layer on a paper substrate prepared from a pea protein-based slurry as described in Example 5. A section of uncoated paper is also shown.

[0177] FIG. 4a shows a scanning electron microscopy (SEM) image of a coating layer on a strawberry prepared from a pea protein-based slurry as described in Example 6 and FIG. 4b the uncoated strawberry.

[0178] FIG. 5a shows a scanning electron microscopy (SEM) image of a coating layer on a paracetamol tablet prepared from a pea protein-based slurry as described in Example 7 and FIG. 5b the uncoated tablet.

[0179] FIG. 6a shows a scanning electron microscopy (SEM) image of a coating layer on a wheat seed prepared from a pea protein-based slurry as described in Example 8 and FIG. 6b the uncoated seed.

EXAMPLES

Materials

[0180] Pea Protein Isolate (PPI) (80% protein) was purchased from Cambridge Commodities Ltd.

[0181] Soy Protein Isolate was purchased from Cambridge Commodities Ltd.

[0182] Lactic acid (food-grade, >80%) was purchased from Cambridge Commodities Ltd. Acetic acid (glacial) was purchased from Fisher Scientific.

Measurement Methods

[0183] Viscosity measurements were made using an Anton Paar MCR 92 Rheometer using a plate and cone measurement geometry with a 50 mm plate and 1 degree angle and a constant shear of 50 s.sup.−1 at 20° C.

[0184] Storage Modulus (G′) can be measured using an Anton Paar MCR 92 Rheometer using a plate and cone measurement geometry with a 50 mm plate and 1 degree angle with 1% strain at an oscillatory frequency of 1 Hz.

[0185] Particle size measurements were carried out using a Dynamic Light Scattering (DLS) technique or a laser diffraction technique. DLS measurements were taken using using a Zeta Sizer Nano S from Malvern Panalytical and operated according to the manufacturer's instructions. It is important that the slurry is sufficiently diluted so as to avoid misleading results due to particles coagulating during testing. The hydrogel slurries were diluted by a factor of 100 with deionised water. It is also important that the pH of a sample is away from the isoelectric point of that sample so as to avoid misleading results due to coagulation. The isoelectric point of the Pea Protein Isolate material tested here was 4.5 and the pH of the slurries was adjusted to 3 with acetic acid or lactic acid prior to measurement. Typically, several dilutions and/or buffer solutions should be tested to ensure proper dispersion. A 200 μL of the diluted slurry was placed in a cuvette and positioned in the equipment. Testing was then carried out according to the standard equipment procedures. The d.sub.50 measured is for the wt/volume distribution. DLS can generally be used to measure particle size up to 500 nm. The upper limit is primarily governed by the onset of sedimentation. For particles greater than this the particle size measurements were carried out using laser diffraction with an Anton Paar PSA 1190. Measurements were carried out by diluting the protein slurry in an aqueous solution with acetic acid or lactic acid adjusted to the same pH. The slurry was diluted to the required concentration in order to have the desired optical density (normally 5-15% obscuration) for the measurement. The d.sub.50 quoted is for the volume distribution. d.sub.10 and d.sub.5 values for the volume distribution can also be obtained in this way using laser diffraction.

[0186] Other equipment, such as the NANO-flex II® system from Colloid Metrix, can be used.

[0187] The particle sizes of the fragments of hydrogel from the low-shear step can be determined by sieving. A suitable technique is to take 200 g of the hydrogel mixture from the low-shear step and to rapidly disperse it in 1000 mL of DI water. The dispersed mixture is then quickly poured through a series of stacked sieves of decreasing mesh size between 50 mm and 1 mm. Sieves from Endecotts are suitable. The % of the slurry within a specific size rage can be calculated by combining the weights of the fragments on the different meshes and calculating this as a % of the total amount of slurry. Errors caused by any additional solvent exchange are minimal due to the short test period.

[0188] Transmission Electron Microscopy (TEM) measurements were taken using a FEI Talos F200X G2 TEM from Thermo Scientific. A suitable technique is to prepare a test sample by diluting the fine plant-based hydrogel slurry to a 1:100 dilution with a 3% acetic acid solution and deposit the sample on a TEM grid (C400Cu, EM resolutions), followed by staining with uranyl acetate. The maximum fragment size is then determined by optical examination of at least 30 fragments chosen at random from within the field of view of a test sample. The maximum length is the maximum length of a line drawn between any two opposing boundaries of a fragment that does not cross any external boundaries.

[0189] Scanning Electron Microscopy (SEM) images were taken using MIRA 3 FEG-SEM, TESCAN with a 10 nm coating of platinum.

[0190] Structural analysis was performed by using an FTIR-Equinox 55 spectrometer (Bruker). The samples were used without further pre-treatment and were loaded into the FTIR holder. The atmospheric compensation spectrum was subtracted from the original FTIR spectra and a secondary derivative was applied for further analysis. Each FTIR measurement was repeated 3 times. The sensitivity of the instrument was detected to be 5%. To resolve the transformation of the native structure of Pea Protein Isolate into supramolecular aggregates, vibrational changes in amide I, which is strictly correlated with protein secondary structure, were followed. Structural analysis was either performed in solution (e.g. using the plant-based protein hydrogel slurry directly) or on a resultant dried film. In the latter case, film samples were prepared for structural analysis by drying 200 μL of the relevant plant-based protein hydrogel slurry at 37° C. for 6 hours.

[0191] The Youngs Modulus and Tensile Strength of structured objects such as films can be measured using a 5ST electromechanical Universal tester from Tinius Olsen. A 10 cm by 1 cm strip is placed between grips and extended at 12.5 mm/min and the forces and extension recorded. The thickness of the film prior to testing can be measured by a micrometer.

[0192] Light transmittance of the structured objects such as films can be measured using a Cary 500 UV-vis spectrometer. The measurement was performed at wavelengths ranging from 300 to 800 nm with average time of 0.1 seconds at 600 nm/min scanning speed.

Example 1: Preparation of a Pea Protein Gel Structured Object

[0193] (a) Protein Hydrogel Formation

[0194] 500 ml of a mixture was prepared consisting of 12.5% (w/v) Pea Protein Isolate in 40% (v/v) lactic acid solution.

[0195] The mixture was then heated in a water bath at 80° C. for 30 minutes, followed by a short sonication step to disrupt large colloidal aggregates (Hielscher UIP1000hdT (1000 W, 20 kHz)), after which a transparent solution was obtained. The energy applied was 16 Wh over 7 minutes.

[0196] The solution was then poured into a 220 mm petri dish and left to cool down at 10° C. for 72 h to obtain a self-standing protein hydrogel.

[0197] (b) Application of Shear to Protein Hydrogel

[0198] Shear was then applied to the hydrogel formed in step (a) as follows. The protein hydrogel was cut into ˜1 cm cubes via a low-shear cutting step. The cubes were placed inside a 75 μm filter bag, which was then submerged inside a bucket containing 5 L of deionised water. This formed a coarse protein hydrogel slurry within the filter bag. The hydrogel cubes were left to soak for 1 h, with occasional gentle agitation. This step was performed to reduce to concentration of lactic acid in the hydrogel by diffusion to the continuous aqueous phase and was repeated 5 more times until the final pH of the aqueous solution was 3.28.

[0199] The strained gel cubes were transferred into a 500 ml bottle and were exposed to probe sonication in a high shear step for 10 minutes (˜0.2 kJ/ml) so as to form a homogeneous low-viscosity protein dispersion. The viscosity of the slurry was 12 cps. The d.sub.50 size of the fine slurry fragments was 90 nm as measured by DLS.

[0200] (c) Preparation of a Pea Protein Film

[0201] The fine hydrogel slurry prepared in step (b) was adjusted to pH 2.6 by adding a small amount of lactic acid, and was then poured onto a heated surface (which was held at 80° C.) and dried for 1 hour to form a structured object which was a film, with an average thickness of 18.1 μm. The resultant film was transparent and had a Youngs Modulus of 361 MPa and a tensile strength of 14 MPa.

Example 2: Preparation of a Pea Protein Gel Structured Object

[0202] (a) Protein Hydrogel Formation

[0203] 800 ml of a mixture was prepared consisting of 10% (w/v) Pea Protein Isolate in 40% (v/v) acetic acid solution.

[0204] The mixture was then heated in a water bath at 85° C. for 20 minutes, followed by a short sonication step to disrupt large colloidal aggregates (Hielscher UIP500hdT (500 W, 20 kHz)), after which a transparent solution was obtained. The energy applied was 200 kJ over 30 minutes.

[0205] The solution was then poured into two 220 mm petri dishes. The dishes were sealed and left to cool down by storage in a fridge at 4° C. for 20 h to obtain a self-standing protein hydrogel. The storage modulus of this hydrogel was 2640 Pa.

[0206] (b) Application of Shear to Protein Hydrogel

[0207] Shear was then applied to the hydrogel formed in step (a) as follows. The protein hydrogel was cut into ˜1 cm cubes via a low-shear cutting step. The cubes were placed inside a 75 μm filter bag, which was then submerged inside a bucket containing 7 L of deionised water. This formed a coarse protein hydrogel slurry within the filter bag. The hydrogel cubes were left to soak for 1.5 h, with occasional gentle agitation. This step was performed to reduce to concentration of acetic acid in the hydrogel by diffusion to the continuous aqueous phase and was repeated one more time until the final pH of the aqueous solution was 3.1.

[0208] The strained gel cubes were transferred into a 1 L bottle and were exposed to high shear with a rotor stator (15,000 rpm for 5 min) and probe sonication on ice for 30 minutes (˜0.5 kJ/ml) so as to form a homogeneous low-viscosity protein dispersion. The viscosity of the slurry was 23 cps. The d.sub.50 size of the fine slurry fragments was 103 nm as measured by DLS.

[0209] (c) Preparation of a Pea Protein Film

[0210] The fine hydrogel slurry prepared in step (b) was mixed with 20 w/w % glycerol and poured onto a plastic petri dish and dried for 24 hours at room temperature to form a structured object which was a film, with an average thickness of 78.5 μm. The PSD and optical properties of the pea protein film of Example 2 was investigated. The results are shown in Table 1. The resultant film was transparent, as demonstrated in FIG. 2a and by its transmittance value of 81.1% at 600 nm.

TABLE-US-00001 TABLE 1 DLS PSD Optical Example Protein Solvent (d.sub.50) properties 2 10 wt % PPI 40 vol % acetic 103 nm Transparent, acid (pH 1.8) transmittance 81.1% at 600 nm

[0211] The results show that functional films having high transparency can be formed using the plant-based protein hydrogel slurries of the present invention. Without wishing to be bound by theory, it is thought that achieving a controlled particle size distribution allows for the high levels of transparency observed.

Example 3: Preparation of a Soy Protein Gel Structured Object

[0212] (a) Protein Hydrogel Formation

[0213] 430 g of a mixture was prepared consisting of 7.0% (w/w) Soy Protein Isolate in 30% (v/v) acetic acid solution.

[0214] The mixture was then heated in a water bath at 85° C. for 30 minutes, followed by a short sonication step to disrupt large colloidal aggregates (Bandelin HD4200, TS 113 probe), after which a transparent solution was obtained. The energy applied was 200 kJ over 30 minutes.

[0215] The solution was then poured into two 220 mm petri dishes. The dishes were sealed and left to cool down by storage in a fridge at 4° C. for 20 h to obtain a self-standing protein hydrogel.

[0216] (b) Application of Shear to Soy Protein Hydrogel

[0217] Shear was then applied to the hydrogel formed in step (a) as follows. The protein hydrogel was cut into ˜1 cm cubes via a low-shear cutting step. The cubes were placed inside a 75 μm filter bag, which was then submerged inside a bucket containing 5 L of deionised water. This formed a coarse protein hydrogel slurry within the filter bag. The hydrogel cubes were left to soak for 1.5 h, with occasional gentle agitation. This step was performed to reduce to concentration of acetic acid in the hydrogel by diffusion to the continuous aqueous phase and was repeated one more time until the final pH of the aqueous solution was 3.07.

[0218] The strained gel cubes (469 g) were transferred into a 0.5 L bottle and were mixed with 40 g of deionised water. A colloidal suspension of large gel particles was obtained and divided into 50 g aliquots, which were then subjected to different levels of high shear to obtain samples with varying degrees of particle size distribution as detailed in Table 2:

TABLE-US-00002 TABLE 2 High-shear Speed/Energy d.sub.50 Sample type Instrument applied (μm) A Ultrasonication Hielscher 0.2 KJ/g 141.6 UIP500hdT (500 W, 20 kHz) B Ultrasonication Hielscher 0.6 KJ/g 73.7 UIP500hdT (500 W, 20 kHz) C Ultrasonication Hielscher 1.1 KJ/g 11.5 UIP500hdT (500 W, 20 kHz)

[0219] (c) Preparation of a Soy Protein Film

[0220] The fine hydrogel slurry of sample B prepared in step (b) was mixed with 20 w/w % glycerol and poured onto a plastic petri dish and dried at room temperature for 24 h to form a structured object which was a film, with an average thickness of 61 μm. The PSD and optical properties of the soy protein film of Example 3 was investigated. The results are shown in Table 3. The resultant film was transparent, as demonstrated in FIG. 2b and by its transmittance value of 89.8% at 600 nm.

TABLE-US-00003 TABLE 3 PSD Optical Sample Protein Solvent (d.sub.50) properties B 7 wt % Soy 30 vol % acetic 73.7 μm Transparent, Protein acid (pH 2.03) transmittance Isolate 89.8% at 600 nm
The results show that functional films having high transparency can be formed using the plant-based protein hydrogel slurries of the present invention. Without wishing to be bound by theory, it is thought that achieving a controlled particle size distribution allows for the high levels of transparency observed.

Example 4: Preparation of a Pea Protein Gel Structured Object

[0221] (a) Protein Hydrogel Formation

[0222] 450 g of a mixture was prepared consisting of 11.11% (w/w) Pea Protein Isolate in 30% (v/v) acetic acid solution.

[0223] The mixture was then heated in a water bath at 85° C. for 30 minutes, followed by a short sonication step to disrupt large colloidal aggregates (Bandelin HD4200, TS 113 probe), after which a transparent solution was obtained. The energy applied was 200 kJ over 30 minutes.

[0224] The solution was then poured into two 220 mm petri dishes. The dishes were sealed and left to cool down by storage in a fridge at 4° C. for 20 h to obtain a self-standing protein hydrogel.

[0225] (b) Application of Shear to Protein Hydrogel

[0226] Shear was then applied to the hydrogel formed in step (a) as follows. The protein hydrogel was cut into ˜1 cm cubes via a low-shear cutting step. The cubes were placed inside a 75 μm filter bag, which was then submerged inside a bucket containing 5 L of deionised water. This formed a coarse protein hydrogel slurry within the filter bag. The hydrogel cubes were left to soak for 1.5 h, with occasional gentle agitation. This step was performed to reduce to concentration of acetic acid in the hydrogel by diffusion to the continuous aqueous phase and was repeated one more time until the final pH of the aqueous solution was 3.02.

[0227] The strained gel cubes (400 g) were transferred into a 0.5 L bottle and were mixed with 100 g of deionised water. A colloidal suspension of large gel particles was obtained and divided into 50 g aliquots, which were then subjected to different levels of high shear to obtain samples with varying degrees of particle size distribution as detailed in Table 4:

TABLE-US-00004 TABLE 4 Speed/ % Inter- High-shear Energy d.sub.50 molecular Sample type Instrument applied (μm) β-sheet A Ultrasonication Hielscher 0.2 KJ/g 67.7 50.4 UIP500hdT (500 W, 20 kHz) B Ultrasonication Hielscher 0.6 KJ/g 1.24 51.3 UIP500hdT (500 W, 20 kHz) C Ultrasonication Hielscher 1.1 KJ/g 0.41 50.7 UIP500hdT (500 W, 20 kHz)

[0228] (c) Preparation of a Pea Protein Film

[0229] The fine hydrogel slurries prepared in step (b) were mixed with 20 w/w % glycerol and poured onto a PTFE evaporating dish and dried at room temperature for 24 h to form a structured object which was a film. The characterisation of the resultant films is described in the Table 5:

TABLE-US-00005 TABLE 5 Film thickness Tensile Strength Elongation at break Sample (μm) (MPa) (%) A 140.8 ± 6.5  4.9 ± 0.1 17.7 ± 6.6  B 111.0 ± 49.9 12.0 ± 0.5  35.9 ± 20.4 C 97.7 ± 4.0 8.0 ± 0.4 7.8 ± 5.5

[0230] The results show that the methods of the present invention allow for the preparation of plant-based protein hydrogel slurries having a controlled particle size distribution. The results also show that the plant-based protein hydrogel slurries of the present invention can be used to prepare structured objects, such as films, with superior tensile properties that can be controlled through control of the slurry particle size. The films produced have a secondary structure with a high level of intermolecular β-sheet (e.g. at least 50% intermolecular n-sheet). This high degree of intermolecular interactions is thought to contribute to the enhanced mechanical properties observed.

Example 5: Using Pea Protein Dispersion as a Coating Layer for Paper

[0231] A colloidal pea protein dispersion obtained from Example 2 was spray-coated on an uncoated cardboard substrate with an airbrush. One layer was first applied followed by drying in the oven for 1 min at 80° C. to evaporate the remaining solvent. This procedure was repeated 15 times until a uniform transparent coating was applied. The results are shown in FIG. 3.

Example 6: Using Pea Protein Dispersion as a Coating Layer for Food Products

[0232] A colloidal pea protein dispersion obtained from Example 2 was applied as a coating to a fresh strawberry via a dip-coating step. A strawberry was first submerged into 50 ml of pea protein dispersion for 5 seconds, followed by removal of the excess dispersion by briefly applying compressed air. The coated strawberry was left to dry at room temperature for 10 min. This procedure was repeated 5 times until a uniform transparent coating was applied. The results are shown in FIG. 4.

Example 7: Using Pea Protein Dispersion as a Coating Layer for Pharmaceutical Products

[0233] A colloidal pea protein dispersion obtained from Example 2 was spray-coated on a uncoated Paracetamol tablet with an airbrush. One layer was first applied followed by drying in the oven for 1 min at 80° C. to evaporate the remaining solvent. This procedure was repeated 15 times until a uniform transparent coating was applied. The results are shown in FIG. 5.

Example 8: Using Pea Protein Dispersion as a Coating Layer for a Seed

[0234] A colloidal pea protein dispersion obtained from Example 2 was applied as a coating to a wheat seed via a dip-coating step. A wheat seed was first submerged into 50 ml of pea protein dispersion for 5 seconds, followed by removal of the excess dispersion by briefly applying compressed air. The coated wheat seed was left to dry at room temperature for 10 min. This procedure was repeated 2 times until a uniform transparent coating was applied. The results are shown in FIG. 6.

CLAUSES

[0235] 1. A method for the preparation of a plant-based protein hydrogel slurry, the method comprising: [0236] (a) forming a solution comprising one or more plant-based protein(s) in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases solubility of the plant-based protein(s), and a second co-solvent decreases solubility of the plant-based protein(s); [0237] (b) inducing the protein in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel; and [0238] (c) subjecting the plant-based protein hydrogel to a shear treatment to form a plant-based protein hydrogel slurry. [0239] 2. The method according to clause 1, wherein the plant protein(s) is selected from soybean protein, pea protein, rice protein, potato protein, wheat protein, corn zein protein or sorghum protein. [0240] 3. The method according to clause 1 or clause 2, wherein the first co-solvent is an organic acid; preferably acetic acid, formic acid, propionic acid and/or an α-hydroxy acid; wherein the α-hydroxy acid may preferably be selected from glycolic acid, lactic acid, malic acid, citric acid and/or tartaric acid; with particularly preferred organic acids being acetic acid and/or lactic acid. [0241] 4. The method according to any one of clauses 1 to 3, wherein a second or further co-solvent(s) is an aqueous buffer solution; preferably selected from water, ethanol, methanol, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate or hexafluoroisopropanol; particularly preferably water and/or ethanol; further particularly preferably water. [0242] 5. The method according to any one of clauses 1 to 4, wherein the solvent system has a co-solvent ratio of first co-solvent to second co-solvent of about 20-80% v/v, preferably about 20-60% v/v, about 25-55% v/v, about 30-50% v/v, about 20%, about 30%, about 40% about 50% or about 60% v/v, most preferably about 30-50% v/v. [0243] 6. The method according to any one of clauses 1 to 5, wherein the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant-based protein(s) solution, then reduced to a second temperature below the sol-gel temperature of the one or more plant-based protein(s) solution to form a hydrogel. [0244] 7. A method according to any one of clauses 1 to 6, wherein said shear treatment comprises a high-shear step. [0245] 8. A method according to clause 7, wherein said high-shear step involves fragmenting the plant-based protein hydrogel into fragments. [0246] 9. A method according to clause 8, wherein said fragments produced in said high-shear step have a d.sub.50 as determined by DLS of less than 500 nm, preferably less than 300 nm, more preferably less than 200 nm, even more preferably less than 50 nm. [0247] 10. A method according to any one of clauses 7 to 9, wherein said high-shear step involves ultrasonication, high-shear mechanical stirring, or cavitation. [0248] 11. A method according to any one of clauses 1 to 6, wherein said shear treatment comprises two steps. [0249] 12. A method according to clause 11, wherein said shear treatment comprises a low-shear step followed by a high-shear step. [0250] 13. A method according to clause 12, wherein said low-shear step involves fragmenting the plant-based protein hydrogel into fragments. [0251] 14. A method according to clause 13, wherein at least 80% of said fragments produced in said low-shear step have a particle size in the range 1 mm to 50 mm, preferably 1 mm to 30 mm, more preferably 10 mm to 30 mm, more preferably 15 mm to 30 mm, even more preferably 20 mm to 30 mm, as determined by sieving. [0252] 15. A method according to any one of clauses 12 to 14, wherein said low-shear step involves mechanical cutting. [0253] 16. A method according to any one of clauses 13 to 15, wherein said high-shear step involves further fragmenting the plant-based protein hydrogel. [0254] 17. A method according to clause 16, wherein said fragments produced in said high shear-step have a D50 as determined by DLS of less than 500 nm, preferably less than 300 nm, more preferably less than 200 nm, even more preferably less than 50 nm. [0255] 18. A method according to any one of clauses 12 to 17, wherein said high-shear step involves ultrasonication, high-shear mechanical stirring, or cavitation. [0256] 19. A method according to any one of clauses 12 to 18, wherein step (c) further comprises subjecting the plant-based protein hydrogel slurry to a solvent reduction step, preferably an organic solvent reduction step, between said low-shear step and said high-shear step. [0257] 20. A method according to clause 19, wherein said solvent reduction step comprises the steps of: [0258] (i) contacting the fragments of the plant-based hydrogel slurry with a non-solubilising solvent; [0259] (ii) separating the fragments of the plant-based hydrogel slurry from the non-solubilising solvent to give a washed plant-based protein hydrogel; and [0260] (iii) optionally repeating steps (i) and (ii). [0261] 21. A method according to clause 20, wherein step (ii) involves mesh filtration. [0262] 22. A method according to clause 20 or 21, wherein prior to washing said plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of greater than 1000 Pa, preferably greater than 2000 Pa, more preferably greater than 5000 Pa, even more preferably greater than 6000 Pa, most preferably greater than 8000 Pa. [0263] 23. A method according to any one of clauses 20 or 22, wherein prior to washing said plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of less than 20,000 Pa, preferably less than 15,000 Pa, more preferably less than 10,000 Pa.; [0264] optionally wherein prior to washing said plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of between about 1000 to 20,000 Pa, preferably between about 2000 to 15,000 Pa, more preferably between about 2000 to 10,000 Pa. [0265] 24. A method according to any one of clauses 20 to 23, wherein said washed plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of greater than 500 Pa, preferably greater than 1000 Pa, more preferably greater than 2500 Pa, even more preferably greater than 3000 Pa, most preferably greater than 4000 Pa. [0266] 25. A method according to any one of clauses 20 to 24, wherein said washed plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of less than 20,000 Pa, preferably less than 15,000 Pa, more preferably less than 10,000 Pa; [0267] optionally wherein said washed plant-based protein hydrogel has a storage modulus (G′) at 10 rad/s of between about 500 to 20,000 Pa, between about 500 to 15,000 Pa, between about 500 to 10,000 Pa, between about 1000 to 20,000 Pa, between about 1000 to 15,000 Pa, or between about 1000 to 10,000 Pa. [0268] 26. A method according to any one of clauses 1 to 25, further comprising the step of: [0269] (d) altering the pH of the plant-based protein hydrogel slurry such that it is different to the isoelectric point of the protein hydrogel by more than 1 pH unit. [0270] 27. A method according to clause 26, wherein step (d) is carried out after step (c). [0271] 28. A method according to clause 26, wherein step (d) is carried out sequentially with step (c). [0272] 29. A method according to any one of clauses 26 to 28, wherein step (d) involves adding a pH-modification material to the plant-based protein hydrogel slurry. [0273] 30. A method according to clause 29, wherein said pH-modification material is a solution comprising monovalent metal ions, divalent metal ions or ammonium ions, preferably an aqueous alkaline solution comprising monovalent metal ions, divalent metal ions or ammonium ions. [0274] 31. A method according to clause 30, wherein said pH-modification material is an aqueous hydroxide solution, preferably sodium hydroxide, potassium hydroxide, or ammonium hydroxide. [0275] 32. A method according to any one of clauses 26 to 31, wherein the pH of the plant-based protein hydrogel slurry after step (d) is below the isoelectric point of the plant-based protein by at least 1 pH unit. [0276] 33. A method according to any one of clauses 26 to 31, wherein the pH of the plant-based protein hydrogel slurry after step (d) is above the isoelectric point of the plant-based protein by at least 1 pH unit. [0277] 34. A method according to any one of clauses 1 to 33, further comprising adding an additional ingredient to the plant-based protein hydrogel slurry. [0278] 35. A method according to clause 34, wherein said additional ingredient is selected from plasticisers, opacifiers, preservatives, pigments and nanoparticles, or mixtures thereof. [0279] 36. A method according to clause 35, wherein said additional ingredient is a plasticiser. [0280] 37. A method according to clause 36, wherein said plasticiser is selected from ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, ethanolamine, urea, triethanolamine, vegetable oils, lecithin, waxes and amino acids. [0281] 38. A method according to any one of clauses 1 to 37, wherein the plant-based protein hydrogel slurry has a viscosity in the range 10 to 10000 cps at 50 s.sup.−1, preferably in the range 15 to 5000 cps at 50 s.sup.−1. [0282] 39. A method according to any one of clauses 1 to 38, wherein the plant-based protein hydrogel slurry comprises protein aggregates with an average size of less than 200 nm, preferably less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, or less than 30 nm. [0283] 40. A method according to any one of clauses 1 to 39, wherein the plant-based protein hydrogel slurry has a protein solids content in the range 5 wt % to 25 wt % based upon the total weight of the plant-based protein hydrogel slurry. [0284] 41. A plant-based protein hydrogel slurry prepared according to the method of any one of clauses 1 to 40. [0285] 42. A method for the preparation of a plant-based structured material, the method comprising: [0286] (a) preparing a plant-based protein hydrogel slurry according to the method of any one of clauses 1 to 40; and [0287] (b) subjecting the plant-based protein hydrogel slurry to one or more solvent level reduction step(s) to reduce the level of said first co-solvent and/or said second co-solvent to give said plant-based structured material. [0288] 43. A method according to clause 42, wherein step (b) involves placing the plant-based protein hydrogel slurry on a surface before forming the one or more solvent level reduction step(s). [0289] 44. A method according to clause 42 or clause 43, wherein said solvent level reduction step involves heating. [0290] 45. A method according to clause 44, wherein said solvent level reduction step involves heating at a temperature in the range 50 to 100° C. [0291] 46. A method according to clause 42 or clause 43, wherein said solvent level reduction step involves forced convection of dry air. [0292] 47. A method according to any one of clauses 42 to 46, wherein the plant-based structured material is a film. [0293] 48. A method according to any one of clauses 42 to 46, wherein the plant-based structured material is a casting. [0294] 49. A method according to any one of clauses 42 to 46, wherein the plant-based structured material is a coating. [0295] 50. A method according to any one of clauses 42 to 49, wherein the plant-based structured material comprises a plant-based protein(s) having secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet. [0296] 51. A method according to any one of clauses 42 to 50, wherein the plant-based structured material has a Young's modulus over 20 MPa; preferably over 50 MPa, over 80 MPa, over 100 MPa, over 200 MPa, over 300 MPa, over 400 MPa, over 500 MPa, or over 600 MPa. [0297] 52. A method according to any one of clauses 42 to 51, wherein the plant-based structured material is a film having a thickness in the range 1 to 1000 μm, preferably 1 to 100 μm, more preferably 10 to 100 μm, even more preferably 20 to 60 μm, most preferably 30 to 50 μm. [0298] 53. A method according to any one of clauses 42 to 52, wherein the plant-based structured material is a film having a Tensile Strength greater than 1 MPa, preferably greater than 5 MPa, preferably greater than 10 MPa and most preferably greater than 25 MPa. [0299] 54. A method according to any one of clauses 42 to 53, wherein the plant-based structured material is a film having an elongation break percentage of above 10%, above 20%, above 30%, above 40%, above 50%, above 60%, above 70%, above 80%, above 90%, above 100% or more. [0300] 55. A plant-based structured material prepared according to the method of any one of clauses 42 to 54. [0301] 56. Use of a plant-based protein hydrogel slurry according to any one of clauses 1 to 40 to produce a plant-based structured material. [0302] 57. Use according to clause 56, wherein said plant-based structured material is a film, a casting, or a coating.