Plant-based functional material

Abstract

The invention relates to a plant-based functional material. The present invention relates to a plant-based material, a method of making the same and a biological material comprising the plant-based material.

Description

[0001] This application is a divisional application of an application with an application date of March 6, 2020, application number 202080018725.7, and invention name “Plant-based functional materials”. Technical Field

[0002] The present invention relates to a plant based material, a method for producing the same and a biomaterial comprising the plant based material according to the present invention. Background Art

[0003] Synthetic polymers such as plastics exhibit excellent mechanical and chemical properties and have been widely used over the past 60 years. However, these polymers are non-biodegradable and can accumulate in the environment, causing economic damage and potentially affecting human health through the food chain and air.

[0004] The development of materials that exhibit high functionality as well as high biodegradability and biocompatibility is a key goal in meeting societal demands for improved material performance in areas ranging from packaging to pharmaceuticals.

[0005] Self-assembly emerges as an attractive route towards making such materials, but most building blocks developed to date are synthetic in origin.

[0006] Among the different types of biopolymers that can be used as building blocks for the production of new functional materials, proteins are interesting candidates due to their ability to self-assemble into functional structures.

[0007] Currently, the use of these materials in commercial applications is limited to highly soluble animal-derived proteins. Animal-based proteins commonly used in food, such as whey proteins, exhibit good biocompatibility, biodegradability, amphiphilicity, and functional properties such as water solubility, emulsification, and foaming abilities. However, there is an increasing demand to replace animal-derived proteins with plant-based proteins, not only because of their lower environmental impact, but also because of their lower allergenicity and reduced cost.

[0008] Self-assembled materials formed from plant-based proteins have been reported, with hydrogels being obtained from soy and pea proteins under a range of experimental conditions. However, the mechanical properties obtained from structured plant-based materials are generally lower than those obtained from animal-derived materials; and plant proteins are more difficult to process, at least in part due to their inherently low solubility in water.

[0009] Therefore, plant-based proteins have not been successfully used as biomaterials to date, and the production of structured protein materials from renewable and cost-effective sources and using environmentally sustainable methods remains a challenge. Summary of the invention

[0010] According to one aspect of the present invention, there is provided a method for producing a plant-based material, comprising:

[0011] a) forming a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases the solubility of the one or more plant-based proteins and a second co-solvent decreases the solubility of the one or more plant-based proteins; and

[0012] b) inducing a sol-gel transition of the protein in the solution to form a plant-based protein hydrogel.

[0013] In another embodiment, the method comprises the steps of:

[0014] c) forming the plant-based protein hydrogel into a structured material.

[0015] Forming the plant-based protein hydrogel into a structured material allows the formation of structured materials such as gels, films, microgels, microcapsules, etc. In a preferred embodiment, the protein hydrogel can be formed into a specified shape by molding. In another preferred embodiment, a microfluidic device can be used to form the protein hydrogel into a specified shape.

[0016] The present invention has identified a new method for making functional materials from plant-based proteins. By utilizing a cosolvent mixture, control can be exerted on the sol-gel transition, thereby allowing structurally strong materials to be formed from renewable plant protein sources. The method allows the generation of a number of structured materials, including hydrogels, films, microcapsules, microgels, microsponges, etc. Structured materials can be reliably formed without the need for crosslinking agents or any other hazardous materials, making them suitable for contact with the human body. These materials are also derived from renewable sources, thereby reducing environmental impact relative to synthetic analogs.

[0017] In another aspect, there is provided the use of a co-solvent mixture for adjusting the properties of a plant-based protein in solution to control the sol-gel conditions and thereby form a plant-based material.

[0018] Sol-gel conditions can be controlled by selecting a solvent system comprising miscible co-solvents, wherein a first co-solvent increases the solubility of the one or more plant-based proteins and a second co-solvent decreases the solubility of the one or more plant-based proteins.

[0019] The ratio of the first cosolvent to the second cosolvent can vary from about 20-80% v / v, 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. Such ratios result in functionally useful materials.

[0020] The solvent system may contain one or more first co-solvents and / or one or more second co-solvents.

[0021] For the first time, plant-based materials can be reliably and reproducibly formed using a scalable method. By being able to control the sol-gel conditions, the properties of the resulting material can be tuned and / or the manufacturing process can be adjusted to allow the production of useful biomaterials.

[0022] The method according to the present invention allows the formation of a plant-based structured material formed via a thermoreversible cold-set gelation process. The plant-based structured material may be a plant-based protein supramolecular structure; or may be a 3-dimensional network of aggregated and entangled plant-based protein supramolecular structures.

[0023] Thermoreversible cold-setting gelation processes can be considered to be processes in which plant-based protein molecules can be heated above a certain temperature to form a liquid solution that can be processed into a desired configuration and then cooled to enable a sol-gel transition to form a network of self-assembled protein aggregates held together by non-covalent intermolecular interactions. Thus, the gelation process according to the present invention does not require covalent chemical cross-linking and is therefore reversible. The present invention includes a thermoreversible cold gelation process.

[0024] Therefore, in another aspect, a plant-based structured material formed via a thermoreversible cold-setting gelation process is provided; wherein the structured material can optionally be a film, a thin film, a micropatterned film (or thin film), a micro- or nanostructured film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a micro-sponge, a hard capsule or a functional coating.

[0025] In another aspect, a plant-based thermoreversible gel is provided.

[0026] In another aspect, a plant-based thermoreversible hydrogel is provided.

[0027] In another aspect, a composite material is provided, which comprises the plant-based material according to the present invention, and one or more other biopolymers, such as proteins, polysaccharides, etc.

[0028] In another aspect, there is provided a material prepared according to the method of the present invention.

[0029] The plant-based materials of the present invention and methods for making them allow precise control of the sol-gel transition, thereby opening up the use of plant-based proteins for forming biomaterials that have heretofore only been successfully made using proteins of animal origin. Suitable biomaterials include membranes, microbeads, microcapsules, scaffolds, gels, sponges, and the like.

[0030] In another aspect, there is provided a microbead comprising a plant-based material according to the present invention.

[0031] In another aspect, a microcapsule comprising the plant-based material according to the present invention is provided.

[0032] In another aspect, there is provided a hard capsule comprising the plant-based material according to the present invention.

[0033] In another aspect, there is provided a sponge or micro-sponge comprising the plant-based material according to the present invention.

[0034] In another aspect, there is provided a film, preferably a thin film, comprising a plant-based material according to the present invention.

[0035] In another aspect, there is provided a nano-patterned or micro-patterned film comprising the plant-based material according to the present invention.

[0036] In another aspect, a bioscaffold comprising the plant-based hydrogel according to the present invention is provided.

[0037] In another aspect, a functional coating comprising the plant-based hydrogel according to the present invention is provided.

[0038] In another aspect, there is provided a food, cosmetic, medicine or medical device comprising the plant-based material according to the present invention.

[0039] In another aspect, there is provided a thermoreversible method for obtaining a plant-based material, comprising:

[0040] a) forming a solution comprising one or more plant-based proteins in a solvent system comprising miscible co-solvents; wherein a first co-solvent increases the solubility of the one or more plant-based proteins and a second co-solvent decreases the solubility of the one or more plant-based proteins; and

[0041] b) inducing a sol-gel transition of the protein in the solution to form a plant-based protein hydrogel.

[0042] After the material is obtained, the thermoreversible property of the material can be removed. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Shown are soy protein isolate (SPI) hydrogels formed under varying co-solvent ratios of acetic acid / DI water;

[0044] Figure 2 shows a SEM (scanning electron microscope) image of a hydrogel produced using a 30% v / v co-solvent ratio;

[0045] Figure 3 The size distribution of SPI particles estimated by dynamic light scattering (DLS) is shown for SPI dispersions in water (a), NaOH at pH 10 (b), HCl at pH 2 (c), and in 30% acetic acid (d). For each solvent, the samples were prepared by heating at 95C for 30 minutes (denoted as heated) or sonicating for 30 minutes (denoted as sonicated). All measurements were performed at 0.1% protein concentration and repeated twice. The inset shows the original correlation plot for each condition;

[0046] Figure 4a Rheological properties of SPI hydrogels as a function of H2O:acetic acid co-solvent ratio are shown; Figure 4b The shear-thinning behavior of SPI hydrogels prepared at different concentrations is shown; Figure 4c The thermoreversible rheological behavior of SPI hydrogel at different temperatures is shown;

[0047] Figure 5a and 5b The structural changes of the secondary structure of SPI hydrogels at different H2O:acetic acid cosolvent ratios are shown, which are calculated from the amide I band in the FTIR spectra; Figure 5c The structural changes of the secondary structure of SPI hydrogel at different temperatures are shown, which are calculated from the amide I band in FTIR;

[0048] Figure 6 shows an SDS-PAGE electropherogram showing that upon increasing amounts of acetic acid, the extent of protein hydrolysis increases;

[0049] Figure 7 Microbeads formed using the hydrogel of the present invention are shown. Figure 7 a shows a schematic diagram of microbead formation; Figure 7 b shows stable microbeads suspended in aqueous solution (pH = 2); Figure 7 c shows a SEM image of microbeads prepared by supercritical point drying; and Figure 7 d shows a SEM image of the gel network on the surface of the microbeads;

[0050] Figure 8aSchematic diagram showing a multilayer 3D microfluidic droplet generator for generating core-shell microcapsules with lipophilic and hydrophilic cores, Figure 8b Core-shell microcapsules containing a lipophilic core suspended in an aqueous solution are shown;

[0051] Fig. 9 Core-shell microcapsules containing a hydrophilic core containing a suspension of a soluble active ingredient (riboflavin) are shown;

[0052] Figure 10a-Figure 10c The results of the simulated degradation are shown, where Fig.10a shows microcapsules in aqueous solution; Fig.10b shows the microcapsules after 60 minutes in SGF (simulated gastric fluid); and Fig.10c shows the microcapsules after 120 minutes in SIF (simulated intestinal fluid);

[0053] Fig.11a Core-shell microcapsules containing a core consisting of a riboflavin solution in 1% (w / w) HMP pectin are shown; Fig.11b Results of a two-stage in vitro digestibility study followed by HPLC analysis are shown, showing the cumulative release of riboflavin under simulated conditions;

[0054] Fig.12a The distribution of fragrance oil loaded plant-based protein microsponges is shown. Figure 12b A single fragrance loaded plant-based protein microsponge at higher magnification (20x) is shown, where the protein microgel shell can be easily observed;

[0055] Fig.13 A schematic diagram showing an example of generating a stable protein film;

[0056] Fig.14a shows the stress-strain curve of the film prepared according to the present invention; Fig.14b shows the Young's modulus and Fig.14c The elongation at break % is shown; and

[0057] Fig.15 FTIR spectra of SPI films prepared without the use of glycerol, and spectra of commercial SPI dried samples are shown;

[0058] Fig.16 a shows a TEM image of a dilute SPI solution dried on a TEM grid, confirming the presence of a large number of β-sheet crystals (scale bar: 5 nm). Fig.16 b shows a close-up image of the β-pleated crystal of the SPI film (scale bar: 2 nm);

[0059] Fig.17aMicropatterning of uniform micro-pillar arrays on the soy protein film surface was shown to significantly increase the contact angle of a water droplet (99°) compared to the non-micropatterned control sample. Fig.17b Nanopatterning of uniform nanochannel arrays obtained by casting soy protein films on DVD discs is shown. The nanostructured patterns on the soy protein films exhibit photonic properties (Mie scattering);

[0060] Fig.18 A schematic process for producing a soy protein film coating on a paperboard substrate is shown. The soy protein film-coated substrate showed a 50% reduction in water absorption compared to the uncoated control sample;

[0061] Fig.19a The formation of a thin 3-dimensional hydrogel layer around the base of a 2-ml Eppendorf tube is shown. Fig.19b shows soy protein hard capsules obtained after drying the molded 3-dimensional hydrogel and removing it from the substrate;

[0062] Fig. 20 a shows a pea protein hydrogel produced using a 30% v / v co-solvent ratio; Fig. 20 b shows a SEM (scanning electron microscope) image of pea protein hydrogel produced using a 30% v / v co-solvent ratio; Fig. 20 c shows a pea protein isolate membrane. Fig. 20 d shows stable pea protein microgel suspended in aqueous solution (pH = 2);

[0063] Fig.21 A potato protein hydrogel prepared from a 100 mg / ml solution of potato protein isolate in 30% (v / v) acetic acid in water is shown;

[0064] Figure 22a-22b shows stress-strain curves of other films prepared according to the present invention;

[0065] Fig.23 Optical images of gelation using acetic acid and either HCl or NaOH according to the present invention are shown. DETAILED DESCRIPTION

[0066] The following features apply to all aspects of the invention.

[0067] Any suitable plant-based protein may be used in the present invention. Suitable plant sources include soybean, pea, rice, potato, wheat, zein, sorghum, etc. Particular plant proteins include soy protein and pea protein.

[0068] Suitable plant-based proteins also include:

[0069] - Brassicas: including Brassica balearica: Mallorca cabbage, Brassica carinata: Abyssinian mustard or Abyssinian cabbage, Brassica elongate: elongated mustard, Brassica fruticulosa: Mediterranean cabbage, Brassica hilarionis: St Hilarion cabbage, Brassica juncea: Indian mustard, brown and leaf mustards, Sarepta mustard, Brassica napus: napus: rapeseed, canola, rutabaga, Brassica narinosa: broadbeaked mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, kai-lan, Brussels sprouts, kohlrabi, Brassica perviridis: tender green, mustard spinach, Brassica rapa (synonym, B. campestris): Chinese cabbage cabbage, turnip, rapini, komatsuna, Brassica rupestris: brown mustard, Brassica tournefortii: Asian mustard;

[0070] -Solanaceae: includes tomatoes, potatoes, eggplants, bell peppers and hot peppers;

[0071] - Cereals: including corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, fonio;

[0072] - Pseudocereals: including amaranth (love-lies-bleeding, red amaranth, prince-of-Wales-feather), breadnut, 苦 Buckwheat, chia, cockscomb (also called quail grass or soko), pitseed goosefoot, quinoa and wattleseed (also called acacia seed);

[0073] -Fabaceae: including Acacia alata (Winged Wattle), Acacia decipiens, Acacia saligna (commonly known by various names including coojong, golden wreath wattle, orange wattle, blue-leafed wattle), Arachis hypogaea (peanut), Astragalus galegiformis, Cytisus labournum (commonly known as laburnum, golden chain or golden rain), Cytisus supinus, Dolichios lablab (common names include hyacinth bean, lablab-bean bonaviist bean / pea, dolichos bean, seim bean, lablab bean, Egyptian kidney bean, Indian lentil bean, bean), bataw and Australian pea, Ervum lens (Lentil), Genista tinctorial (common names include Dyder's whin, waxen woad and waxen wood), Glycine max (soybean), Lathyrus clymenum (peas or vetchlings), Lathyrus odoratus (peavines or vetchlings), Lathyrus staivus (peavines or vetchlings), Lathyrus Silvetris (peavines or vetchlings), Lotustetragonolobus (asparagus-pea or winged pea), Lupinus albus (Lupin), Lupinus angustifolius (lupin), Lupinus luteus (Lupin), Lupinus polyphyllus (Lupin), Medicago sativa (Alfalfa), Phaseolus aureus (Mung bean), bean)), Phaseoluscoccineus(Runnerbean), Phaseolus nanus (Mung bean / French bean), Phaseolus vulgaris (Green bean / French bean), Pisum sativum (Pea), Trifolium hybridum (Clover), Trifolium pretense (Redclover), Vicia faba (Broad bean), Vicia sativa (Vetch), Vignaunguiculate (cowpea);

[0074] - Non-leguminous plants: including: Acanshosicyos horrida (Acanshosicyos horrida), Aesculus hyppocastanum (Conker tree / Horsechestnut), Anacardium occidentale (Cashew tree), Balanites aegyptica, Bertholletia excels (Brazil nut), Beta vulgaris (Sugar beet), Brassica napus (Rapeseed), Brassica juncea, Brassica juncea (Brown mustard), Brassica nigra (Black mustard), Brassica hirta (Eurasian mustard), Cannabis sativa (marijuana), Citrullus vulgaris (watermelon seed), Citrus aurantiaca (Citrus), Cucurbita maxima (squash), Fagopyrum esculentum (knotweed), Gossypium barbadense (Extra long staple cotton), Heianthus annuus (sunflower), Nicotiana sp. (Tobacco plant), Prunus avium (cherry), Prunus cerasus (Sour cherry), Prunus domestica (plum), Prunus amygdalus (almond), Rricinus community (Castor / Castor Oil Plant), Sasamumindicum (Sesame), Sinapis alba (White mustard), Terlfalrea pedata (Oyster nut).

[0075] For the avoidance of doubt, the plant-based structuring material according to the present invention does not encompass plants in their natural state. For example, naturally occurring plant cells, organelles or vesicles are not the plant-based structuring material of the present invention.

[0076] One characteristic of proteins of plant origin is their inherently poor solubility in water. This has so far limited their use in the production of biomaterials. However, the present invention overcomes previous limitations associated with such proteins.

[0077] In the method according to the invention, the material is formed by adding the plant-based protein to a solvent system, wherein the solvent system comprises two or more miscible co-solvents as defined herein.

[0078] The first co-solvent increases the solubility of the one or more plant-based proteins. The first co-solvent can be considered a solubilizing co-solvent. One or more solubilizing co-solvents can be present and the one or more solubilizing co-solvents can fully or partially solubilize the one or more plant-based proteins.

[0079] Examples of solubilizing cosolvents are organic acids. Organic acids are organic compounds with acidic properties. Suitable organic acids include acetic acid or alpha-hydroxy acids. Suitable alpha-hydroxy acids include glycolic acid, lactic acid, malic acid, citric acid and tartaric acid. Preferred organic acids are acetic acid and lactic acid. The use of organic acids enables dissolution of plant proteins and also allows for gentle hydrolysis of proteins. For example, without wishing to be bound by theory, the solubility of plant-based proteins in organic acids is possible due to i) protonation of the protein and ii) the presence of an anionic solvation layer that helps reduce hydrophobic interactions. Once initially dissolved in an organic acid, the protonation of the plant-based proteins can help stabilize them in a non-solvent (e.g., water).

[0080] In a preferred embodiment, the first co-solvent is an organic acid.

[0081] The second co-solvent has reduced solubility for the one or more plant-based proteins compared to the first co-solvent. The second co-solvent may be considered a desolubilising co-solvent. One or more desolubilising co-solvents may be present.

[0082] The example of one or more desolvable second co-solvents is an aqueous buffer solution. In another embodiment, the second co-solvent can be ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, tert-butyl alcohol, ethyl acetate or hexafluoroisopropanol. In a particularly preferred embodiment, the second co-solvent is water and ethanol. In another particularly preferred embodiment, the second co-solvent is water.

[0083] In a preferred embodiment, the concentration of the one or more plant-based proteins in the solvent system is 25-200 mg / ml, preferably 50-150 mg / ml. The ratio of organic acid can vary depending on the protein concentration, for example, a higher organic acid ratio is used as the protein concentration increases.

[0084] In a preferred embodiment, the extent of protein hydrolysis is controlled to change the properties of the resulting hydrogel. For example, increasing the acid concentration present during formation will increase the extent of protein hydrolysis. A higher degree of protein hydrolysis results in the formation of a less rigid hydrogel.

[0085] In order to form a solution comprising one or more plant-based proteins, it may be necessary to apply a physical stimulus to the protein / solvent system mixture to enable the protein to dissolve. Suitable physical stimuli include ultrasonic treatment, stirring, high shear mixing or other physical techniques. The preferred technique is ultrasonic treatment.

[0086] In one embodiment, the solution is subjected to ultrasonic treatment for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or more than 30 minutes. The preferred ultrasonic treatment time is about 30 minutes.

[0087] The protein solution is heated so that the liquid solution remains above the sol-gel transition of the protein or proteins. The sol-gel transition temperature of the protein or proteins can be altered by changing the solvent system (e.g., by selection of organic acids, selection of ratios of organic acids to other solvents, or by other means). By appropriate selection of conditions, the sol-gel transition of the protein can be carefully controlled, thereby controlling the formation of the hydrogel.

[0088] In one embodiment, the protein solution is heated to about or above 70° C. In another embodiment, the protein is heated to about or above 75° C., about or above 80° C., about or above 85° C., or about 90° C. In a preferred embodiment, the protein is heated to 85° C.

[0089] The protein solution can be maintained at an elevated temperature for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes or 1 hour. A preferred period is at least 30 minutes to allow the protein to dissolve completely. The protein solution can be maintained at elevated temperatures for a longer period of time. This may be useful for commercial batch processes or for use in fluid processing steps where the protein solution needs to be retained in liquid form for a longer period of time.

[0090] After the protein solution has been heated to above the sol-gel transition temperature of the protein, the temperature of the protein solution can be reduced to a second temperature lower than the sol-gel transition temperature to promote the formation of the hydrogel. The second temperature can be room temperature. The protein solution can be kept at a reduced temperature for a time period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or about 30 minutes. The special reduced time period is about 5 minutes. However, the method of the present invention allows the protein to be kept in the solution for a long time. Therefore, depending on demand, the protein solution can be kept above the sol-gel transition temperature for a long time required to keep the protein in liquid form. This may be several hours, several days or longer. In addition, since the reaction is reversible, the solution can be, for example, kept at a lower temperature (e.g., room temperature) where a hydrogel will be formed, but then heated to above the sol-gel transition temperature so that the solution is restored to a liquid state for further processing. Since the hydrogel remains stable for a long time, the protein hydrogel can be stored for hours, days, weeks, months or years in this way.

[0091] The particular temperature will depend on the properties of the protein source, the solvent conditions used and therefore the sol-gel transition temperature. Alternatively, the elevated and lowered temperatures may be relatively fixed (e.g., about 85°C and then about room temperature), and the co-solvent mixture conditions adjusted to ensure the appropriate sol-gel transition temperature for the selected plant-based protein.

[0092] Thus, in one embodiment, there is provided a method for forming a plant-based material, comprising:

[0093] a) forming a protein solution comprising one or more plant-based proteins and a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases the solubility of the one or more plant-based proteins and a second co-solvent decreases the solubility of the one or more plant-based proteins;

[0094] b) mechanically stirring the protein solution for a period of time, such as ultrasonic treatment;

[0095] c) raising the temperature of the protein solution to a first elevated temperature above the sol-gel transition temperature for a period of time. The temperature increase may be caused by the mechanical stirring in b) or may be from an external heat source;

[0096] d) reducing the temperature of the protein solution to below the sol-gel transition temperature so that the plant-based protein self-aggregates into a hydrogel; and, optionally

[0097] e) forming the hydrogel into a defined shape, such as a microgel, microcapsule, microsponge, membrane, etc. The forming step may be a molding step, i.e. molding the hydrogel into a defined shape. The forming step may use a microfluidic device.

[0098] The protein solution may be maintained at an elevated temperature in step c) while the solution is conformed to the desired final shape. For example, within a microfluidic device, the protein solution may be maintained at an elevated temperature c) within the device reservoir, but as the solution is discharged from the device, its temperature decreases, thereby forming a microgel or microcapsule shell. Alternatively, the protein solution may be maintained at an elevated temperature while being shaped in a suitable mold, wherein after the temperature is reduced, the protein is allowed to form into a hydrogel.

[0099] Without wishing to be bound by theory, it is believed that when vegetable proteins are added to a solvent system, the vegetable proteins form a highly viscous dispersion of insoluble colloidal protein aggregates.

[0100] In addition, it is believed that the application of mechanical agitation (e.g., supersound process) destroys large colloidal protein aggregates into smaller aggregates, and destroys the interaction between protein molecules. Utilize this mode, the size of protein aggregates can be significantly reduced to a granularity lower than 100nm. In one embodiment, the present invention includes a mean size less than 200nm, preferably less than 150nm, less than 125nm, less than 100nm, less than 90nm, less than 80nm, less than 70nm, less than 60nm, less than 50nm, less than 40nm or less than 30nm protein aggregates.

[0101] In addition, it is believed that when the protein solution is heated to a temperature higher than the sol-gel temperature in the presence of a cosolvent system, the plant protein partially unfolds, resulting in the exposure of the hydrophobic amino acids initially buried in the native structure of the protein. Once partially unfolded, the cosolvent is able to interact with the unfolded protein molecules. For example, organic acids are more accessible to protonate the amino acid residues and can form anionic salt bridges that stabilize the hydrophobic interactions. In addition, after heating at elevated temperatures, protein-protein non-covalent intermolecular contacts are destroyed. In addition, it is believed that when the protein solution is cooled to a temperature lower than the sol-gel temperature, protein-protein non-covalent intermolecular contacts can be achieved, thereby promoting the self-assembly of plant protein molecules into a network of supramolecular aggregates.

[0102] It is believed that the method of the present invention allows plant proteins to aggregate into supramolecular structures held by intermolecular hydrogen bonding interactions, and in particular between the β-strands.

[0103] The method of the present invention enables the formation of materials in which high levels of beta-sheet intermolecular interactions exist. This leads to new materials that have not been made before.

[0104] In one embodiment, the plant-based material according to the present invention has a protein secondary structure having at least 40% intermolecular β-sheets, at least 50% intermolecular β-sheets, at least 60% intermolecular β-sheets, at least 70% intermolecular β-sheets, at least 80% intermolecular β-sheets, or at least 90% intermolecular β-sheets. In one embodiment, the plant-based material is a film. In one embodiment, the plant-based material is a dried material, such as a dried hydrogel. In one embodiment, the plant-based material is a hydrogel or other material described herein. It is believed that previous gels prepared from plant-based protein sources may have a lower amount of intermolecular β-sheets in the secondary structure, resulting in unfavorable properties of the prior art.

[0105] The plant-based material according to the present invention comprises β-pleated crystals. The plant-based material according to the present invention may exhibit a high degree of β-pleated crystal structure. The plant-based material may comprise at least 40% β-pleated crystals, at least 50% β-pleated crystals, at least 60% β-pleated crystals, at least 70% β-pleated crystals, at least 80% β-pleated crystals, or at least 90% β-pleated crystals. In one embodiment, the plant-based material is a film. In one embodiment, the plant-based material is a dried material, such as a dried hydrogel. In one embodiment, the plant-based material is a hydrogel or other material described herein.

[0106] The plant-based material having the said intermolecular β-pleated secondary structure and the said plant-based material containing β-pleated crystals can be any of the materials described in the present invention, including structured materials such as microcapsules, microbeads, bioscaffolds, biosupports, sponges, micro sponges, hard capsules or functional coatings, etc. They can also be membranes, films, micro-patterned films (or films), micro- or nano-structured films or microgels, etc.

[0107] Thus, the present invention encompasses, for example, plant-based films having a protein secondary structure with at least 40% intermolecular β-sheets, at least 50% intermolecular β-sheets, at least 60% intermolecular β-sheets, at least 70% intermolecular β-sheets, at least 80% intermolecular β-sheets or at least 90% intermolecular β-sheets. The same applies to other materials according to the present invention.

[0108] Furthermore, the present invention encompasses, for example, plant-based films comprising plant-based β-pleated crystals, comprising at least 40% β-pleated crystals, at least 50% β-pleated crystals, at least 60% β-pleated crystals, at least 70% β-pleated crystals, at least 80% β-pleated crystals or at least 90% β-pleated crystals. The same applies to other materials according to the present invention.

[0109] The present invention provides a plant-based structured material, which includes a film, a thin film, a micro-patterned film (or a thin film), a micro- or nano-structured film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a micro-sponge, a hard capsule or a functional coating.

[0110] The materials according to the invention have advantageous mechanical properties. For example, the ability to reversibly change from a gel to a liquid upon a change in temperature enables advantageous manufacturing capabilities.

[0111] In one embodiment, the hydrogel produced according to the present invention has a storage modulus (G') at 10 rad / s of greater than 500 Pa, greater than 1000 Pa, greater than 2500 Pa, greater than 3000 Pa, greater than 4000 Pa.

[0112] In one embodiment, the hydrogel exhibits shear thinning behavior, wherein viscosity decreases with increasing shear rate.

[0113] In one embodiment, the hydrogels produced according to the present invention exhibit unique thermally reversible gelation behavior. Upon heating at elevated temperatures and / or by applying mechanical agitation, the protein gel reverts to liquid form. This is a unique property not observed in the case of existing hydrogels, which do not revert to a fully liquid state upon heating. In contrast, gels prepared according to the present invention can. In one embodiment, upon heating at elevated temperatures and / or by applying mechanical agitation, the protein solution can have a storage modulus of less than 250Pa, less than 100Pa, less than 50Pa, less than 10Pa. This allows both the materials and methods of the present invention to have unique manufacturing capabilities.

[0114] Likewise, if desired, the thermoreversibility can be removed by removing the solvent system of the invention. For example, plant-based microcapsules can be prepared using the method of the invention. Once formed, the solvent system can be washed off, which will terminate the thermoreversible properties of the microcapsules. Thus, if the microcapsule is then heated, it will remain stable and will not remelt. This enables, for example, microcapsules according to the invention to be subjected to high temperature processes and still remain intact and stable.

[0115] Thus, the hydrogels formed according to the present invention possess unique properties not observed in prior plant-based hydrogels. These include the ability to form hydrogels from high concentrations (i.e., 5%-15% w / w) of plant-based proteins from commercial sources, and the ability to maintain such highly concentrated protein solutions in a liquid state after heat denaturation, which enables them to be molded into well-defined objects.

[0116] A feature of the material of the present invention is that there is no need to provide a crosslinking agent, as the plant protein will form a hydrogel on its own. Therefore, in one embodiment of the present invention, a plant protein material (eg, a hydrogel) is provided that does not contain or substantially does not contain a crosslinking agent.

[0117] However, in an alternative embodiment, the hydrogel according to the invention may comprise a cross-linking agent. Suitable cross-linking agents include microbial transglutaminase, glutaraldehyde, formaldehyde, glyoxal, phenolic compounds, epoxy compounds, genipin or dialdehyde starch.

[0118] Due to the porous network of the hydrogel, the solvent mixture within the hydrogel can be exchanged for another solvent mixture without compromising the mechanical stability of the hydrogel. The solvent exchange process can be performed to remove the organic acid from the hydrogel porous network.

[0119] In one embodiment of the invention, the method comprises the step of exchanging the solvent system in which the plant-based protein hydrogel is formed for an alternative solvent system. In another embodiment, this is performed using a solvent exchange process. This step can be performed after the hydrogel is formed, but can also be performed after the hydrogel has been formed into a structured material (e.g. after step b) or c) of the method of the invention). In a preferred embodiment, an aqueous buffer is used to replace the organic acid co-solvent mixture within the porous network of the hydrogel.

[0120] In one embodiment, the solvent mixture within the hydrogel is evaporated to produce a dried material, such as a film, a microstructured / nanostructured film, or microbeads. In another embodiment, one or more cosolvents in the solvent system are volatile solvents. In a preferred embodiment, the first cosolvent is a volatile organic acid, such as acetic acid. In a preferred embodiment, the second or additional cosolvent is a volatile alcohol, such as ethanol. In another preferred embodiment, the first cosolvent and the second or additional cosolvent are both volatile solvents.

[0121] In one embodiment, the material of the present invention may include one or more plasticizers. Possible plasticizers include 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.

[0122] The amount of plasticizer to be included will depend on the use of the material, such as a film. In one embodiment, the composition may include about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or more of a plasticizer. In another embodiment, the hydrogel may include about 5-50% plasticizer, about 10-50%, about 20-40%, about 15-35%, or about 20% plasticizer.

[0123] The addition of plasticizers may affect the mechanical properties of the material. Typically, the addition of plasticizers will increase the elasticity of the material, but this typically in turn reduces the strength of the resulting material.

[0124] The hydrogels of the present invention allow for the formation of a range of useful plant-based biomaterials. The use of plant-based materials has many advantages over previously used animal or petrochemical sources. First, plant sources are renewable and can be efficiently obtained in an environmentally efficient manner. Secondly, plant sources are biodegradable and are therefore environmentally friendly alternatives to other plastics. Thirdly, plant-based proteins have a significant advantage over animal-derived proteins in that they do not introduce animal-derived proteins into the human body. This has a positive impact from both a pharmacological and pharmaceutical point of view, where materials of animal origin must undergo rigorous inspection and processing to ensure that there are no adverse elements (e.g., removal of prions, etc.); and because these products are suitable for vegetarians / vegans.

[0125] Since plant-based proteins are naturally present in the human (or other animal) diet, the biomaterials prepared according to the invention exhibit a higher degree of digestibility than other biopolymers such as polysaccharides (e.g., alginate or chitosan). This makes them particularly suitable for pharmaceutical, food and / or cosmetic applications.

[0126] In one embodiment, the hydrogels of the invention can be used to form films, such as thin films. Plant protein derived films have many applications, including forming biodegradable flexible films for food packaging applications or for use with medical devices including implantable devices.

[0127] One advantage of the plant-based materials of the present invention over animal-based materials (or starch-based / cellulose materials) is their inherent insolubility in water. Most biopolymer films readily dissolve in water, thus rendering them inherently unusable for food packaging applications, requiring additional coatings utilizing synthetic polymers. The present invention overcomes these problems.

[0128] Typical thicknesses of the films may be 1-1000 μm, 1-100 μm, 10-100 μm, 20-60 μm, 30-50 μm, and the like.

[0129] The Young's modulus of the film may be over 20 MPa, over 50 MPa, over 80 MPa, over 100 MPa, over 200 MPa, over 300 MPa, over 400 MPa, over 500 MPa, over 600 MPa or more.

[0130] The elongation at break of the film may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 100%, or more.

[0131] Young's modulus / elongation at break These numbers describe films made using plant-based functional materials. Said materials can be used to replicate said properties in all structured materials as described herein.

[0132] The films can be micropatterned with features ranging from 100 nm to 1000 μm, enabling new functional properties such as superhydrophobicity (lotus effect) or structural color (attributed to Mie scattering).

[0133] Functional composite membranes can be produced by embedding inorganic nanoparticles, such as gold nanoparticles or silver nanoparticles, to generate flexible electronic devices or membranes with antimicrobial properties, respectively.

[0134] In another embodiment, the hydrogels of the present invention can be used to form microbeads. Forming microbeads from plant-based sources overcomes the environmental issues of utilizing current plastic-based microbeads. Microbeads are typically solid particles having a diameter of less than one millimeter in their largest dimension.

[0135] In one embodiment, the microbeads according to the invention have a maximum dimension of less than 1 mm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm or less than 100 μm.

[0136] In another embodiment, the hydrogels of the present invention can be used to form microcapsules. Microcapsules can be used to encapsulate a variety of substances and have a variety of industrial applications, including cosmetics, food, household products, agrochemicals, and pharmaceuticals.

[0137] Microcapsules prepared according to the present invention provide a fully biodegradable alternative to standard synthetic polymer microencapsulation shell materials.

[0138] Microcapsules prepared according to the present invention uniquely enable microfluidic assembly of plant-based materials.

[0139] The microcapsules prepared according to the present invention provide excellent stability, for example, against adverse storage conditions, and become a safer food-grade or pharmaceutical-grade solution to preserve active ingredients in food applications, such as vitamins, essential fatty acids or antioxidants, or pharmaceutical active ingredients (including small molecules and macromolecules).

[0140] Microcapsules prepared according to the present invention may also have the advantage that they can be produced using microfluidics technology, thereby ensuring high reproducibility, the ability to produce complex structures (core-shell) and / or can be prepared using mild processing conditions, thereby protecting the active agents they encapsulate.

[0141] Robust microcapsules can also be generated from plant-based proteins by controlling the self-assembly of protein aggregates in the absence of cross-linking agents or any other harmful substances.

[0142] In one embodiment, the microcapsules according to the present invention can encapsulate a hydrophobic internal composition. In another embodiment, the microcapsules according to the present invention can encapsulate a hydrophilic internal composition. In another embodiment, the microcapsules according to the present invention can encapsulate a living organism composition. In another embodiment, the microcapsules according to the present invention can encapsulate a powdered composition. In yet another embodiment, the microcapsules according to the present invention can encapsulate an oil-in-water, water-in-oil, oil-in-water-in-oil, or water-in-oil-in-water emulsion, etc.

[0143] The plant-based hydrogel according to the present invention can form a microcapsule shell. In one embodiment, the shell may have a thickness of about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 30 μm, 35 μm, 40 μm or 50 μm, etc. Other thicknesses include thicknesses between 10 nm-50000 μm, 10 μm-100 μm, 10 μm-50 μm, 1 μm-10 μm, etc.

[0144] The plant-based microcapsules according to the present invention can encapsulate any nutritional, cosmetic, pharmaceutical or agrochemical suitable active agents, including vitamins, essential fatty acids, antioxidants, small molecules, hydrophilic small molecules, hydrophobic small molecules, proteins, antibodies, antibody-drug conjugates, spices and other macromolecules.

[0145] Suitable encapsulating agents include one or more selected from the following:

[0146] Crosslinkers, hardeners, organic catalysts and metal-based catalysts (for example organic and inorganic complexes of platinum, palladium, titanium, molybdenum, copper or zinc) for the polymerization of elastomer formulations, rubber formulations, paint formulations, coating formulations, adhesive formulations or sealant formulations;

[0147] dyes, colorants, pigments for inks, personal care products, elastomer formulations, rubber formulations, paint formulations, coating formulations, adhesive formulations, sealant formulations, or paper formulations;

[0148] Fragrances for detergents, household cleaning products, personal care products, textiles (so-called smart textiles), coating formulations. Fragrances useful for the present invention are any compounds belonging to the standard list published and updated by the International Fragrance Association (IFRA);

[0149] Spices, flavorings, vitamins, amino acids, proteins, essential lipids, probiotics, antioxidants, preservatives (for feed and food);

[0150] fabric softeners and conditioners for detergents and personal care products;

[0151] Bioactive compounds such as enzymes, vitamins, proteins, plant extracts, moisturizers, disinfectants, antimicrobial agents, sunscreens, pharmaceuticals for personal care products, textiles (so called smart textiles). These compounds include but are not limited to vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, para-aminobenzoic acid, alpha-hydroxy acids, camphor, ceramides, ellagic acid, glycerol, glycine, glycolic acid, hyaluronic acid, hydroquinone, isopropyl isostearate, isopropyl palmitate, oxybenzone, panthenol, proline, retinol, retinyl palmitate, salicylic acid, sorbic acid, sorbitol, triclosan, tyrosine; and

[0152] Fertilizers, herbicides, insecticides, pesticides, fungicides, repellents and disinfectants (for agrochemicals).

[0153] The plant-based microcapsules according to the present invention can also be used for diagnosis and high-throughput screening.

[0154] The plant-based hydrogels according to the invention can also be used to make microgels or microsponges. Microgels are micro hydrogels. Microsponges can be considered as microgels loaded with substances, such as active ingredients or ingredients.

[0155] The plant-based hydrogels according to the present invention can also be used to make bio-scaffolds and bio-supports. Such materials can be used to grow cells and tissues in vitro and in vivo. The hydrogels can also be used to coat medical devices and implants.

[0156] The plant-based proteins according to the present invention may be functionalized and / or derivatized to alter the properties of the protein.

[0157] The invention will now be described with reference to the following non-limiting examples.

[0158] Material

[0159] -Soy protein isolate (SPI) (92% protein) was purchased from MP Biomedicals.

[0160] - Pea protein isolate (PPI) (80% protein) was purchased from Cambridge Commodities Ltd.

[0161] -Acetic acid (glacial) and lactic acid (natural, ≥85%), soybean oil (soybean oil from Glycine max), (-)-riboflavin, glycerol and PFO (1H,1H,2H,2H-perfluoro-1-octanol) were purchased from Sigma Aldrich. Pectin was kindly provided by Cargill.

[0162] - Fluorinert (FC-40) was purchased from Fluorochem.

[0163] -008- Fluorosurfactant was purchased from RAN Biotechnologies.

[0164] Example 1

[0165] Free-standing hydrogels were prepared according to the following procedure.

[0166] Glacial acetic acid was mixed with deionized water at different ratios (10% v / v, 30% v / v, 50% v / v, 70% v / v and 90% v / v). Soy protein isolate was added to the DI water / acetic acid solution at a final protein concentration of 100 mg / ml. A dispersion of insoluble protein was obtained. In order to dissolve the protein, the mixture was exposed to ultrasonic treatment for 30 minutes (high frequency power output = 70 W, frequency = 20 KHz, amplitude = 90%). During this process, the sample temperature was maintained at 85°C-90°C. After 30 minutes, a completely translucent liquid solution was obtained. The sample was cooled at room temperature for 5 min. During this process, the liquid sample became a transparent and self-supporting hydrogel observable after the vial was inverted.

[0167] For different acetic acid / DI water ratios, the hydrogels prepared according to Example 1 were as follows: Figure 1 shown.

[0168] Self-supporting hydrogels were observed at a ratio of 10%-70% v / v acetic acid / DI water. Note that for 30% v / v or higher solutions, completely translucent solutions were produced that rapidly formed self-supporting hydrogels. The fact that the gel was completely translucent indicates that it is composed of small soluble aggregate structures, rather than being composed of large insoluble aggregates (as those commonly found in protein cold solidified gels). The hydrogels prepared according to the present invention are stable and maintain their structure after multiple washing steps in both water and ethanol.

[0169] Scanning electron microscopy

[0170] SEM (Scanning Electron Microscopy) of hydrogels prepared using 30% v / v co-solvent ratio and Figure 2 The hydrogel microstructure confirms the presence of a densely packed network of thin protein aggregates. The figure also shows that the gel network remains intact after the removal of acetic acid.

[0171] Thus, plant-based thermoreversible gels were prepared for the first time.

[0172] In the last step, 100% anhydrous ethanol is used to dehydrate the soy protein hydrogel and microgel in ethanol. The sample is transferred to a microporous sample capsule (78 μm pore size, Agar Scientific), which is soaked and partially filled with 100% anhydrous ethanol to prevent the sample from drying during transfer. Then, the sample is critically dried using a Quorum E3100 critical point dryer, where liquid CO2 is rinsed 4-5 times and incubated for at least 15 minutes between each rinse. The sample is mounted on an aluminum SEM stump using a conductive carbon sticky pad (Agar Scientific) and 15nm iridium is coated using a Quorum K575X sputter coater. The sample is observed using a FEI Verios 460 scanning electron microscope operating at 2keV and 25-50pA probe current. Secondary electron images are acquired using an Everhard-Thornley detector in fieldless mode (low resolution) or a through-lens detector in full immersion mode (high resolution).

[0173] Particle size analysis

[0174] To characterize the size of SPI aggregates, SPI dispersions were prepared in various solvent systems: a) deionized water, b) deionized water adjusted to pH = 10 with NaOH, c) deionized water adjusted to pH = 2 with HCl, and d) 30% (v / v) acetic acid in water) and treated with three different methods: (1) untreated, (2) heated at 95°C in a water bath for 30 minutes, or (3) sonicated at 95°C for 30 minutes. The particle size (hydrodynamic diameter) was measured using a Zetasizer Nano (Malvern). All samples were diluted to 0.1% (v / v) immediately after sonication and subsequently measured.

[0175] Dynamic light scattering (DLS) analysis ( Figure 3 ) showed that protein aggregates prepared in 30% (v / v) acetic acid solution sonicated at 95°C for 30 min had significantly smaller particle size (29±9.1 nm) compared to the unsolubilized ones prepared under different conditions.

[0176] Rheological characterization

[0177] To characterize the rheological properties of the SPI hydrogels, the samples produced via Example 1 were cooled at room temperature for 1 h and then kept at 4° C. for 12 h.

[0178] Rheological measurements were performed using an ARES controlled strain rheometer. Tests were performed using 25 mm smooth parallel plates at a temperature of 20°C. Strain and frequency sweeps were performed on SPI hydrogel samples containing increasing amounts of acetic acid. Strain sweeps were performed from 0.01% to 100% strain using a frequency of 10 rad / s. Frequency sweeps were performed from 0.1 to 100 rad / s using a strain of 1% (within the linear viscoelastic region). All rheological measurements were performed under temperature control (20 ± 0.25°C).

[0179] Figure 4a The rheological properties of SPI hydrogels as a function of the H2O: acetic acid co-solvent ratio are shown. It can be seen that the addition of a small amount of acetic acid (10% v / v) leads to the formation of a weak hydrogel, which may be attributed to the incomplete dissolution of the protein and the presence of larger insoluble aggregates. On the other hand, a large amount of acetic acid also leads to the formation of a weak hydrogel (90% v / v). The hydrogels formed with a co-solvent ratio of 30-70% v / v lead to stronger hydrogels, with the hydrogels prepared with 30% and 50% acetic acid (v / v) showing a G' greater than 2500 Pa.

[0180] To determine the thermoreversible gelation properties of SPI hydrogels (75 mg / ml SPI dispersed in 30% v / v acetic acid aqueous solution), rheological measurements were performed using a Discovery HR-2 (TA Instruments) rheometer. Tests were performed using 20 mm smooth parallel plates. The SPI hydrogel samples were tested at different temperatures using an angular frequency of 10 rad / s. Initially, the ultrasonicated SPI solution was loaded into the preheated rheometer probe at 90 °C. After 500 seconds, the temperature was reduced to 10 °C at a rate of 5 °C / min. After the sample temperature reached 10 °C, it was kept at 10 °C for 500 seconds. Finally, the sample was heated to 90 °C at a rate of 5 °C / min. The results of the thermoreversible rheological properties of SPI hydrogels are presented in Figure 4c Shown in.

[0181] This demonstrates the unique manufacturing capabilities of the material of the present invention, as the gel moves completely thermoreversibly between the gel and liquid states. Such thermoreversibility has never been seen before.

[0182] FTIR

[0183] The changes in the secondary structure of hydrogels under different solvent ratios were studied. The SPI hydrogels were structurally analyzed using an FTIR-Equinox55 spectrometer (Bruker). The hydrogel samples were used without further pretreatment and loaded into an FTIR holder and analyzed by subtracting a 50% v / v (DI water / acetic acid) reference. The atmosphere compensation spectrum was subtracted from the original FTIR spectrum, and the second-order derivative was applied for further analysis. Each FTIR measurement was replicated 3 times for each sample (9 replicates per sample on average). The sensitivity of the detection instrument was 5%. In order to resolve the transformation of the native structure of soy protein isolate into supramolecular aggregates, the vibration changes of amide I, which is strictly related to the secondary structure of the protein, were tracked.

[0184] The results of FTIR measurements are shown in Figure 5a As shown in the figure, the SPI hydrogel has a high content of α-helix (1656 cm -1 ) and intermolecular parallel β-sheet (1625cm -1 ) secondary structure.

[0185] Figure 5b The FTIR measurements of the SPI hydrogel samples prepared using different solvent ratios are shown in Figure 2. The results show that the intermolecular antiparallel and parallel β-sheets (1620 cm -1 ) content, using increasing amounts of acetic acid resulted in an α-helical structure (1654 cm -1 )Increase.

[0186] The changes in the secondary structure of the hydrogels at different temperatures were also investigated. FTIR measurements of SPI hydrogel samples (SPI 100 mg / ml dispersed in 30% v / v acetic acid) were performed by incubating the samples at different temperatures (90°C, 55°C and 20°C) in a FTIR sample holder. Figure 5c It can be clearly observed that after heating the sample at 90 °C, the amount of the intermolecular parallel β-sheet structure decreases, while after cooling the sample to 20 °C, the content of the parallel β-sheet structure increases greatly.

[0187] This is demonstrated by the thermoreversibility of the present invention, thereby correlating this advantageous property to the protein secondary structure.

[0188] Example 2

[0189] The hydrogels were prepared using lactic acid as a co-solvent according to the following procedure.

[0190] Lactic acid is mixed with deionized water in different ratios (10% v / v, 30% v / v, 50% v / v, 70% v / v and 90% v / v). Soy protein isolate is added to DI water / lactic acid solution with a final protein concentration of 100 mg / ml. A dispersion of insoluble protein is obtained. In order to dissolve the protein, the mixture is exposed to ultrasonic treatment for 30 min (high frequency power output = 70 W, frequency = 20 KHz, amplitude = 90%). During this process, the sample temperature is maintained at 85 ° C-90 ° C. After 30 min, a completely clear liquid solution is obtained. The sample is cooled at room temperature for 5 min.

[0191] Gel electrophoresis

[0192] Analysis of the different hydrolyzed protein fragments at different water:lactic acid ratios (from 0 to 90% v / v) was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4 -12% gels with MES buffer.

[0193] The results are shown in Figure 6 , and it can be seen that the increase in the amount of lactic acid leads to a higher degree of protein hydrolysis.

[0194] Having established that stable hydrogels can be formed using the methods of the present invention, the use of such hydrogels can be employed in microfluidic methods to form discrete and uniform microscopic objects, such as microgels and microcapsules.

[0195] Example 3 - Microgel Fabrication

[0196] The microfluidic device (droplet generator) was fabricated using standard soft lithography using a negative master photoresist (SU8 3050). The continuous oil phase (2% 008-fluorosurfactant in Fluorinert FC-40) was loaded into a 2 ml tube, while the dispersed liquid SPI phase (85 mg / ml SPI in 40% v / v acetic acid, maintained at 85°C) was loaded into a 1.5 ml tube and quickly placed on a heating block at 85°C. To prevent gelation of the SPI solution during transfer to the microfluidic device, a custom silicone heater (Holroyd Components) (including 1 / 32" ID stainless steel tubing) was used to maintain the temperature of the PTFE tubing connecting the SPI reservoir and the inlet in the microfluidic device. The silicon heater temperature was controlled by a custom temperature controller. Both solutions were pumped into a standard flow focusing droplet generator via a pressure-driven system (Elveflow OB1) to generate droplets of ~100 μm diameter. Different pressure levels were tested until uniform and continuous generation of a monodisperse population of microdroplets was achieved. The final pressure level for the dispersed aqueous phase was 175 mbar, and the final pressure level for the continuous oil phase was 200 mbar. The generated droplets were collected in 1 ml pipette tips and kept at room temperature for 12 hours to ensure completion of the gelation process. The formed microgels were then washed by a standard de-emulsification procedure: first, the continuous oil phase containing the fluorosurfactant was removed from the vial. For 500 ul of microgel, an equal volume of 10% in Fluorinert FC-40 was added. PFO solution and mix thoroughly for 30 seconds. The 10% PFO solution in FluorinertFC-40 was then removed, and two subsequent oil washes were performed by adding an equal volume of pure Fluorinert FC-40. Finally, 500 μl of deionized water was added to the vial, causing the microgels to transfer from the oil phase to the water phase. The supernatant containing the microgel suspension was transferred to a separate vial. A subsequent series of washing steps were performed to remove acetic acid from the microgel suspension by adding 500 μl of deionized water that had been adjusted to pH = 2 with HCl to the microgel suspension, followed by centrifugation at 1000 rpm for 1 min. The supernatant was removed and the microgels were resuspended by adding 500 μl of deionized water adjusted to pH = 2 with HCl. Total A total of 3 washing steps were performed to remove acetic acid from the microgel suspension. Mechanically stable microgels suspended in deionized water (pH = 2) were obtained after the last washing step. To prepare microgel samples for SEM analysis, the microgels were washed with 100% ethanol in a 3-step procedure: the microgels were first suspended in a 25% v / v ethanol-water solution and placed under constant stirring (100 rpm) for 3 h. After centrifugation and removal of the supernatant, the microgels were then resuspended in a 50% v / v ethanol-water solution and placed under constant stirring (100 rpm) for 3 h.Finally, after centrifugation and removal of the supernatant, the microgels were then resuspended in a 100% v / v ethanol-water solution and placed under constant stirring (100 rpm) for 12 h.

[0197] The results of this experiment are Figure 7 shown. Figure 7 a shows a schematic diagram of microbead formation. Figure 7 b shows stable microgels suspended in aqueous solution (pH = 2). Figure 7 c shows a SEM image of microbeads prepared by supercritical point drying. Figure 7 d shows a SEM image of the gel network on the surface of the microbeads.

[0198] The resulting microgels are stable in aqueous solutions under acidic conditions and in the presence of 100% ethanol. Once self-assembled, the protein aggregates are completely insoluble in solution, thus contributing to the stability of the microgels.

[0199] These results demonstrate that mechanically strong microgels can be generated from plant-based proteins simply by controlling the self-assembly of protein aggregates at the nanoscale and in the complete absence of cross-linkers or any harmful substances.

[0200] Example 4 - Core-shell microcapsule preparation

[0201] Having established that mechanically strong microgels could be produced, the use of hydrogels for microencapsulation was explored.

[0202] To fabricate the coaxial flow focusing microfluidic device, a multi-step photolithography process was followed. Although the photolithography process is explained in Tran, TM, Cater, S. & Abate, AR Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices, Biomicrofluidics 8, 1–7 (2014), it is briefly described below.

[0203] Two SU8 photoresist masters are required. In the first master, a 25μm photoresist layer is made for the inner channel, followed by a 50μm layer for the middle channel, and a 75μm layer for the outer channel. The second master contains a 25μm layer for the medium channel and a 50μm layer for the outer channel. To make the final coaxial flow focusing device, two different PDMS sheets are peeled off from these masters and then aligned and bonded by plasma oxidation. After plasma oxidation, a small amount of sprayed water droplets are placed between the two PDMS sheets to allow the microfluidic channels to be aligned. The final aligned PDMS device is baked in an oven at 65°C overnight to complete the bonding between the two layers.

[0204] The microfluidic process of generating core-shell microcapsules is equivalent to the process for generating microbeads in Example 3. However, an additional inlet for the inner phase (core) is added to the system. In order to make the core-shell microcapsules containing lipophilic active ingredients, 450 μl of soybean oil is pre-emulsified in 550 μl SPI protein solution (20 mg / ml, 30% v / v acetic acid). By pressure-driven system (Elveflow OB1), water-in-water soybean oil emulsion (inner phase), SPI solution (85 mg / ml, 40% v / v acetic acid, middle phase) and FC-40 (2% v / v 008-fluorinated surfactant, outer phase) are pumped to the coaxial flow focusing microfluidic device. Test different pressure levels until the uniform and continuous generation of a monodisperse core-shell microcapsule group with a diameter of ~120 μm (pressure inner phase (100 mbar), pressure middle phase (150 mbar) and pressure outer phase (100 mbar)) is achieved. The core-shell ratio can be adjusted by simply controlling the relative pressure between the inner and middle phases.The microcapsules were collected and subsequently washed as described in the previous section.

[0205] As can be seen, in this embodiment, core material and shell material are kept apart, which is different from standard microencapsulation matrix method (wherein active ingredient is mixed with matrix material in advance). The reason for this method is that the pH of the starting protein solution is very low due to the presence of a large amount of acetic acid at first, and high temperature is also needed to keep the protein solution in liquid state. On the contrary, 3-D coaxial microfluidic device is used, which allows to easily produce core-shell structure without damaging the stability of the encapsulated component.

[0206] Figure 8a A schematic diagram of a microfluidic droplet generator is shown, which shows a 3D flow focusing microfluidic device. The core-shell microcapsules produced according to the present invention contain a lipophilic core suspended in an aqueous solution. Figure 8b The microcapsule shell contains only self-assembled SPI protein and the core is an oil-in-water microemulsion (lipophilic).

[0207] Example 5 - Preparation of core-shell microcapsules encapsulating active agents

[0208] The microfluidic process described in Example 4 was repeated to produce microcapsules comprising a hydrophilic core. The core comprised a 1% (w / w) HMP pectin solution containing a suspension of a soluble active ingredient (riboflavin).

[0209] The core-shell microcapsules prepared according to the present invention contain a hydrophilic core suspended in an aqueous solution. Fig. 9 The microcapsule shell contains only the self-assembled SPI protein and the core is a hydrophilic suspension of the active agent (riboflavin in this case).

[0210] Example 6 - Controlled Release Microcapsules

[0211] Since the protein shell consists only of protein aggregates, an experiment was performed to demonstrate, via a two-stage in vitro digestibility test, that the release of the payload can be triggered by degradation of the protein shell in the presence of digestive enzymes.

[0212] A 1 L stock solution of SGF (simulated gastric fluid) electrolyte solution was prepared by dissolving 0.257 g KCl, 0.061 g KH2PO4, 1.05 g NaHCO3, 1.38 g NaCl, 0.122 g MgCl2(H2O)6 and 0.024 g (NH4)2CO3 in 1 1 of deionized water. A 1 L stock solution of SIF (simulated intestinal fluid) electrolyte solution was prepared by dissolving 0.253 g KCl, 0.054 g KH2PO4, 3.57 g NaHCO3, 1.12 g NaCl, 0.335 g MgCl2(H2O)6, 0.44 g CaCl2·2H2O and 0.23 g bile extract in 1 1 of deionized water. 1 ml of SGF was prepared by dissolving 8 mg of pepsin in SGF electrolyte solution and the pH was adjusted to pH=2 by adding a small amount of 1 M HCl.

[0213] 50 μl of the core-shell microcapsules prepared according to Example 4 were washed from the oil phase using the method described in Example 4 (addition of 150 μl of deionized water at pH = 2). Then, 200 μl of SGF was mixed with the microcapsule suspension and then incubated in a Thermo Shaker at 37°C and 300 rpm for 60 min. For the simulated intestinal stage, 3 mg of pancreatin was dissolved in 1 ml of SIF electrolyte solution. 400 μl of SIF was added to the previous microcapsules in the SGF solution. The pH was adjusted to 7 by adding a small amount of 1 M NaOH. The microcapsules were then incubated at 37°C for 120 min.

[0214] The results of the experiment are as follows Figure 10a-Figure 10c As shown, Fig.10a Microcapsules in aqueous solution are shown; Fig.10b shows that the microcapsules are still intact after 60 minutes in SGF; and Fig.10c Release from microcapsules after 120 min in SIF is shown.

[0215] This experiment confirmed that incubation under SGF conditions (8 mg / ml pepsin, pH=2) triggered a slow degradation of the protein shell without compromising the stability of the lipophilic core retained within the microcapsules. Subsequent incubation in SIF (1.5 mg / ml pancreatin, pH=7) resulted in significant degradation of the protein shell and complete release of the lipophilic core. Control experiments without enzymes but under the same pH conditions showed that protein shell degradation was mainly triggered by enzymatic digestion.

[0216] It can thus be seen that the microcapsules remain intact under simulated gastric conditions but rapidly disintegrate under simulated intestinal conditions, which confirms that the microcapsules according to the present invention can be used for the controlled delivery of nutritional or pharmaceutical active ingredients.

[0217] Example 7 - Controlled Release Microcapsules Containing Active Agents

[0218] The experiment of Example 6 was repeated, but using microcapsules prepared according to Example 5 comprising a hydrophilic core containing the active agent riboflavin.

[0219] Fig.11a Riboflavin microcapsules (ie, microcapsules with a hydrophilic core (1% HMP pectin + riboflavin)) are shown. Fig.11b Results of a two-stage in vitro digestibility study are shown, followed by HPLC analysis showing the cumulative release of riboflavin under simulated conditions. It can be seen that controlled release of riboflavin under simulated gastric and intestinal conditions can be achieved by encapsulating the hydrophilic active ingredient within a plant-based protein microcapsule shell. Encapsulation was shown to prevent rapid diffusion of riboflavin and promote controlled release.

[0220] Example 8 - Preparation of micro sponges

[0221] Soy protein micro sponge is prepared according to the present invention. The microfluidic process described in Example 2 is repeated to produce soy protein microgel (diameter is 100 μm). The microgel is washed with 100% ethanol in a 3-step procedure: first, the microgel is suspended in a 25% v / v ethanol-water solution and placed under constant stirring (100 rpm) for 1 h. After centrifugation and removal of the supernatant, the microgel is resuspended in a 50% v / v ethanol-water solution and placed under constant stirring (100 rpm) for 1 h. Finally, after centrifugation and removal of the supernatant, the microgel is resuspended in a 100% v / v ethanol-water solution and placed under constant stirring (100 rpm) for 1 h. At this point, ethanol should have completely replaced the water in the microgel porous network. The microgel suspension is centrifuged (1000 rpm, 1 min) and the ethanol supernatant is removed. Then, 250 μl of geranium oil (Natural) is added to the microgel. The microgel is suspended in the oil solution by gently shaking the Eppendorf bottle. At this stage, the ethanol-miscible oil phase infiltrates the microgel porous network. After adding 500 μl of deionized water to the oil-microgel suspension, the oil-loaded SPI microsponges were then transferred to the water phase. The remaining oil phase was removed from the Eppendorf tube.

[0222] Fig.12a Shown is the distribution of plant-based protein microsponges loaded with fragrance oil. Figure 12b A single fragrance loaded plant-based protein micro-sponge is shown at higher magnification (20x), where the protein microgel shell can be easily observed.

[0223] By using this method, water-immiscible solvents such as fragrance oils can be loaded into plant-based protein microsponges. This method enables high loading efficiency without the use of complex microcapsule generation methods such as core-shell structures.

[0224] Example 9 - Preparation of membranes

[0225] Soy protein film is prepared according to the present invention. 500mg soy protein isolate) is dissolved in 5mL of 30% (v / v) acetic acid (100mg / ml protein concentration). The solution is then exposed to ultrasonic treatment (high frequency power output = 70W, frequency = 20KHz, amplitude = 90%) for 30min. A few minutes before the ultrasonic treatment is completed, 125mg of glycerol is added to the SPI solution and mixed for the remaining time of the ultrasonic treatment step. After the ultrasonic treatment step, the solution is immediately cast into a glass culture dish to form a hydrogel (2mm thickness). The glass culture dish is heated to about 100°C to avoid any gelation during the casting process. The glass dish is then removed from the heating plate and air dried overnight. After drying, the film is removed from the glass culture dish and stored in a 50% humidity chamber until further use. The bottom surface of the glass culture dish is coated with a Teflon sheet so that the film can be easily peeled off.

[0226] A schematic diagram of the method according to Example 9 together with the resulting membrane is shown in Fig.13 As can be seen, a mechanically strong and transparent film was produced.

[0227] Tensile properties

[0228] The tensile properties of the films produced in Example 9 were tested as a function of the amount of plasticizer added. 10%, 20%, 30%, 40% and 50% (w / w) glycerol were used to make the films.

[0229] The tensile properties of the film were tested using a Tinius Olson 5kN with a 10N load cell. The film was cut into 5mm wide pieces and the two ends of the film were bonded with a paper holder with a gap length of 1mm. The holder and the film were mounted on a mechanical testing machine and then the paper fold was cut before measurement to ensure that the load was only applied to the film. The measurement was performed at a speed of 2mm / min. A digital caliper was used to measure the film thickness of a single sample. Typical film thickness is 30-50um.

[0230] The stress-strain curve of the film of the present invention is shown in Fig.14a The Young's modulus of the membrane is Fig.14b It is shown that increasing the amount of plasticizer reduces the Young's modulus of the film. Fig.14c , where the maximum elongation is shown at 30% plasticizer.

[0231] The mechanical properties of the membranes obtained by this method are superior to those of previously reported membranes made from commercial soy proteins. This is due to the high degree of intermolecular interaction and subsequent self-assembly of the protein molecules, in contrast to the weak intermolecular interactions that are typically present in soy protein membranes produced by casting monomeric soy proteins in high concentrations of chaotropes (i.e., 8 M urea).

[0232] FTIR

[0233] To investigate the secondary structure and intermolecular interactions in the films, FTIR analysis was performed on SPI films prepared without the addition of glycerol.

[0234] Data were collected using 128 scans at 4 cm-1 resolution with background subtraction. For structural analysis of proteins, spectra were smoothed and normalized using second-order and seven-point window Savitzky–Golay filters. Second-order derivatives in the amide I band (1600–1700 cm-1) were calculated from the smoothed data to deconvolute and quantify secondary and quaternary structure contributions.

[0235] FT-IR analysis of the resulting films showed the presence of a higher amount of intermolecular β-sheet structures (65%) in the films compared to the starting soy protein isolate ( Fig.15 ).

[0236] Transmission electron microscopy (TEM) analysis

[0237] TEM analysis was performed to investigate the morphology of protein self-assembled structures present in the films. For transmission electron microscopy (TEM) imaging, SPI samples used for film formation as detailed in Example 9 were diluted to 0.02% concentration and deposited on TEM grids (C400Cu, EM resolution) and stained with uranyl acetate. Fig.16 The presence of abundant β-sheet crystals was shown, which correlates with the high amount of intermolecular β-sheet structure determined by FTIR.

[0238] β-pleated crystals have never been observed before in the case of plant protein materials. Such crystals have similarities to silk materials known to have enhanced mechanical properties (including strength). Without wishing to be bound by theory, it is believed that the β-pleated crystals are due to formation by the method described in the present invention. In contrast to plant-based gels described in the prior art, such β-pleated crystals can contribute to the enhanced mechanical properties observed in the case of the plant-based materials of the present invention. The β-pleated crystal data together with FTIR clearly show that the materials prepared according to the present invention have a high degree of intermolecular interaction, which enables them to have properties that have not been reported before for plant materials (such as tensile strength of the film).

[0239] Thus, the resulting plant-based material of the present invention differs from known plant-based gels, and these differences result in significantly improved properties.

[0240] Example 10 - Microstructure and Nanostructure Patterning

[0241] The film prepared according to Example 9 was micropatterned according to the following procedure.

[0242] In order to pattern microstructures such as microcolumns on protein films, SU-8 3025 was used as a photoresist to make a negative pattern of a 20μm×20μm microcolumn array on a silicon wafer by standard photolithography. A mixture of polydimethylsiloxane elastomer and curing agent (Sylgard 184, Dow Corning) in a 10:1 ratio was poured on the wafer and cured at 65°C for 1 hour. The cured PDMS was peeled off from the wafer and used as a negative pattern of the soybean protein film. The soybean protein solution prepared as in Example 9 was cast onto the PDMS and dried overnight. The SPI film was peeled off from the PDMS master and the resulting micropatterned structure was observed using a scanning electron microscope (SEM) (MIRA 3FEG-SEM, TESCAN) with a 10nm platinum coating. The contact angle of the film was measured using FTA1000B, First Ten Angstroms.

[0243] By simply casting the sonicated soy protein solution onto the micropatterned substrate, a uniform array of micropillars can be fabricated on the film surface. The contact angle of the micropatterned film was 99°, which was significantly higher than that of the control film sample with a non-micropatterned surface ( Fig.17a ). This clearly demonstrates that the hydrophobic surface properties can be greatly enhanced by simply patterning micropillars (such as those naturally present on lotus leaves) on the surface of plant-based protein films.

[0244] In order to generate nanostructured protein films with photonic properties, a DVD disc was used as a mold substrate. First, the outer plastic layer was carefully removed from the DVD disc and the remaining intermediate layer was used. PDMS was poured onto the DVD substrate and cured at 65°C for 1 hour. The patterned PDMS was peeled off from the DVD substrate, and the film-forming soy protein solution prepared as in Example 9 was cast on the PDMS substrate and dried overnight at room temperature. The SPI film was peeled off from the PDMS master, and the resulting nano-patterned structure was observed using a scanning electron microscope (SEM) (MIRA 3FEG-SEM, TESCAN) with a 10nm platinum coating. The photonic properties (Mie scattering) of the film can be easily observed by the naked eye ( Fig.17b ).

[0245] Example 11 - Preparation of coating

[0246] To test the coating properties of the plant-based protein film, a small piece of cardboard (2×2 cm) was immersed in the film-forming SPI solution prepared as in Example 9 in the absence of a plasticizer. After immersing the cardboard in the film-forming solution for about 5 seconds, the cardboard was removed from the solution and air-dried overnight at room temperature. After complete drying, the protein-coated cardboard was immersed in 10 mL of deionized water to measure the water absorption of the cardboard. The water absorption was calculated by measuring the weight increase of the cardboard when it was immersed in deionized water at different time intervals.

[0247] As control samples, uncoated paperboard pieces and soy protein monomer coated paperboard (prepared by immersing the paperboard in a soy protein solution prepared under alkaline conditions (pH = 9)) were tested. The water absorption was normalized using the initial weight measured before soaking the paperboard samples in water.

[0248] As in Fig.18 As observed in the previous study, the uncoated paperboard control sample exhibited very high water absorption after immersion in water for 10 seconds. The water absorption of the SPI-coated paperboard was reduced by almost 50% compared to the uncoated control sample or the paperboard samples coated with soy protein solutions prepared under alkaline conditions, which also exhibited very low water resistance. These results highlight that coatings produced from plant-based protein films assembled from proteins with a high degree of intermolecular interactions exhibit enhanced water barrier properties.

[0249] Example 12 - Hard Capsule Manufacturing

[0250] Soy protein hard capsules are manufactured according to the present invention. First, a 100 mg / ml SPI solution is prepared by dissolving 5 g of SPI into 50 ml of an aqueous solution containing acetic acid (40% v / v). The protein dispersion is heated at 95° C. for 30 min, then exposed to ultrasonic treatment for another 5 min (high frequency power output = 70 W, frequency = 20 KHz, amplitude = 90%). The solution is maintained at 85° C. to prevent gelation. A 2 ml Eppendorf tube is immersed in the heated solution, then maintained in the liquid solution for 5 seconds, and then removed. A thin layer of SPI hydrogel is formed around the outer surface of the 2 ml Eppendorf tube within a few seconds after it is removed from the liquid SPI solution ( Fig.19a The vial was then placed in an oven at 45°C for one hour to ensure evaporation of the water / acetic acid solvent fraction within the hydrogel layer, thereby generating a 3-dimensional film. After the solvent evaporated, the hard capsule was removed from the Eppendorf tube ( Fig.19b ).

[0251] This is the first example showing the formation of hard capsules produced from plant-based proteins. This process is only possible due to the thermoreversible properties of these plant-based materials, which enable the formation of thin 3-dimensional hydrogels on substrates.

[0252] Example 13 - Pea and potato protein materials

[0253] Protein hydrogels, microgels and membranes were produced following the procedures described in Example 1, Example 3 and Example 9 by using pea protein isolate (80%) and potato protein isolate as starting materials.

[0254] Fig. 20 a shows a translucent and free-standing pea protein hydrogel observable after the vial was inverted.

[0255] like Fig.21 Similar properties as described herein were also observed with potato protein as shown in Figure 1, which shows a potato protein hydrogel prepared from 100 mg / ml potato protein isolate in 30% (v / v) acetic acid in water.

[0256] Example 13 demonstrates that the method of the present invention is applicable to a variety of plant-based protein sources.

[0257] Example 14 - Stress-Strain Film Analysis

[0258] Figure 22a-22b The stress-strain curve of the film of the present invention is shown in FIG. Fig.22a The stress-strain curves of the films prepared according to the present invention are shown. The strain-stress curve of the self-assembled sample (blue) corresponds to the SPI film prepared from a 30% (v / v) aqueous acetic acid solution in the presence of 30% glycerol (w / w, relative to the dry protein weight). The strain-stress curve of the unstructured sample (red) corresponds to the SPI film prepared in an alkaline aqueous solution adjusted to pH = 10 using NaOH in the presence of 30% glycerol (w / w, relative to the dry protein weight).

[0259] Figure 22b Shown are strain-stress curves of SPI films prepared from 30% (v / v) acetic acid in water in the presence of different amounts of glycerol as plasticizer (20% to 40% w / w, relative to dry protein weight).

[0260] The self-assembled SPI film prepared under 30% (w / w) glycerol exhibited high tensile strength (15.6±2.07 MPa) and Young's modulus (209±39.1 MPa).

[0261] It can be seen that changing the amount of glycerol can achieve the adjustment of the mechanical properties of the hydrogel. For example, when the concentration of glycerol changes from 20-40% (w / w), the Young's modulus of the mechanical properties changes from 483±58.4MPa to 92.7±25.3MPa, and the tensile strength changes from 25.0±3.49MPa to 6.18±0.98MPa. It was found that the tensile strength and Young's modulus of the self-assembled film using 30% (w / w) glycerol were higher than the values ​​of the unstructured film using 30% (w / w) glycerol (Young's modulus and tensile strength were 131±22.6MPa and 9.30±1.53MPa, respectively).

[0262] The sample preparation in Example 14 is as follows:

[0263] 500 mg of SPI was dispersed in 5 mL of 30% (v / v) acetic acid aqueous solution and shaken thoroughly until a turbid and highly viscous dispersion was obtained. It was then ultrasonically treated for 30 minutes using an ultrasonic homogenizer (Bandelin, HD4200) at 25% power (pulse duration was 0.7 seconds on time and 0.3 seconds off time). After ultrasonic treatment, different concentrations (20, 30 or 40 w / w%) of glycerol (≥99.5%, Sigma-Aldrich) were added as a plasticizer, and the solution was ultrasonically treated for another 1 minute. The hot liquid solution was immediately cast onto a 7 cm glass petri dish preheated at 90 ° C. The casting solution was then dried for 3 days at room temperature (19-22 ° C) and ambient humidity (typically 50-70%). The dried film was then taken out from the mold and stored in a humidity controlled room (50%) until further use.

[0264] As a control experiment, films were also prepared in alkaline aqueous solution, where a 100 mg / ml SPI dispersion was prepared in aqueous solution at pH = 10 (adjusted with NaOH) and heated at 95°C for 30 minutes without ultrasonic treatment (referred to as unstructured SPI). 30% (w / w) glycerol was then added, and the hot liquid solution was cast onto a glass petri dish and dried for 3 days.

[0265] Example 15 - Comparison of Acetic Acid with HCl and NaOH

[0266] Comparative experiments were conducted to evaluate the differences between the hydrogels prepared using acetic acid according to the present invention and the methods using HCl or NaOH (which do not undergo a sol-gel transition). Fig.23 Shown.

[0267] Fig.23Optical images of 100 mg / ml SPI dispersions prepared by sonication at elevated temperature in different aqueous solutions are shown. (a) SPI dispersions in 30% (v / v) acetic acid aqueous solution (left), acidic aqueous solution adjusted to pH=2 using HCl (center), and alkaline aqueous solution adjusted to pH=10 using NaOH after 30 minutes of sonication (right). (b) Inverting the glass vials shows that gelation is only observed for the SPI solution prepared in 30% (v / v) acetic acid. The HCl and NaOH vials show that no sol-gel transition occurred.

[0268] In summary, the present invention provides a solvent system as described herein that enables a method of disrupting intermolecular interactions at high temperature and high shear. This allows selective promotion of the formation of intermolecular interactions when the temperature changes. Such a method allows materials to be molded into different shapes. This has not been possible so far for plant-derived materials.

[0269] Thus, the present invention may relate to a method of moulding a plant-derived material comprising the method as described herein.

[0270] In addition, the present invention creates a protein secondary structure of the self-assembling material, which has a higher amount of intermolecular β-folding structure (compared to the protein starting material). Such a new type of material has not been seen in existing plant-derived materials. This new type of secondary structure gives the material unique properties, such as higher tensile film strength.

[0271] No doubt many other effective alternatives will occur to the skilled person.It should be understood that the invention is not limited to the embodiments described and encompasses modifications obvious to those skilled in the art and within the spirit and scope of the invention.

[0272] References

[0273] 1. Ahnert, SE, Marsh, JA, Hernández, H., Robinson, CV & Teichmann, SA Principles of assembly reveal a periodic table of protein complexes. Science (80-.). 350, (2015).

[0274] 2. Mezzenga, R. & Fischer, P. The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions. Reports Prog. Phys. 76, (2013).

[0275] 3. Knowles, TPJ & Mezzenga, R. Amyloid Fibrils as Building Blocks for Natural and Artificial Functional Materials. Adv. Mater. 6546–6561 (2016). doi: 10.1002 / adma.201505961

[0276] 4. Shimanovich, U. et al. Protein Microgels from Amyloid Fibril Networks. ACS Nano 9, 43–51 (2014).

[0277] 5. Zhou, XM et al. Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry. ACS Nano 9, 5772–5781 (2015).

[0278] 6. Shen, Y. et al. Amyloid fibril systems reduce, stabilize and deliver bioavailable nanosized iron. Nat. Nanotechnol. 12, 642–647 (2017).

[0279] 7. G., Fong, W.K. & Mezzenga, R. Ice-Templated and Cross-Linked Amyloid Fibril Aerogel Scaffolds for Cell Growth. Biomacromolecules 18, 2858–2865 (2017).

[0280] 8. Amagliani, L. & Schmitt, C. Globular plant protein aggregates for stabilization of food foams and emulsions. Trends Food Sci. Technol. 67, 248–259 (2017).

[0281] 9. Munialo, CD, Martin, AH, Van Der Linden, E. & De Jongh, HHJ Fibrilformation from pea protein and subsequent gel formation. J. Agric. Food Chem. 62, 2418–2427 (2014).

[0282] 10. Munialo, CD, van der Linden, E. & de Jongh, HHJ The ability to store energy in pea protein gels is set by network dimensions smaller than 50 nm. Food Res. Int. 64, 482–491 (2014).

[0283] 11. Lee, KH, Ryu, HS & Rhee, KC Protein solubility characteristics of commercial soy protein products. JAOCS, J. Am. Oil Chem. Soc. 80, 85–90 (2003).

[0284] 12. Lee, H. et al. Soy protein nano-aggregates with improved functional properties prepared by sequential pH treatment and ultrasonication. Food Hydrocoll. 55, 200–209 (2016).

[0285] 13. Jiang, S. et al. Modifying the physicochemical properties of pea protein by pH-shifting and ultrasound combined treatments. Ultrason. Sonochem. 38, 835–842 (2017).

[0286] 14. Mason, TO et al. Expanding the solvent chemical space for self-assembly of dipeptide nanostructures. ACS Nano 8, 1243–1253 (2014).

[0287] 15. Li, Y. et al. Understanding the dissolution of α-zein in aqueous ethanol and acetic acid solutions. J. Phys. Chem. B 116, 12057–12064 (2012).

[0288] 16. Akkermans, C. et al. Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate. J. Agric. Food Chem. 55, 9877–9882 (2007).

Claims

1. A plant-based structured material, wherein the structured material is: a film, a thin film, a micropatterned film (or film), a micro- or nanostructured film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a microsponge, a hard capsule or a functional coating. The plant-based structured material according to claim 1 , wherein the structured material is a microcapsule.

3. The plant-based structured material according to claim 1, wherein the structured material is microbeads. The plant-based structural material according to claim 1 , wherein the structural material is a bioscaffold or a biosupport. The plant-based structured material according to claim 1 , wherein the structured material is a sponge or a micro sponge. The plant-based structured material according to claim 1 , wherein the structured material is a hard capsule. The plant-based structured material according to claim 1 , wherein the structured material is a film or a thin film.

8. The plant-based structured material according to claim 1, wherein the structured material is a micro-patterned film, a micro-patterned film, or a micro- or nano-structured film.

9. The plant-based structured material according to claim 1, wherein the structured material is a microgel.

10. The plant-based structured material according to claim 1, wherein the structured material is a functional coating.

11. A plant-based structured material according to any preceding claim, wherein the structured material comprises plant protein β-pleated crystals.

12. The plant-based structured material of claim 11, wherein the structured material comprises at least 40% β-pleated crystals, at least 50% β-pleated crystals, at least 60% β-pleated crystals, at least 70% β-pleated crystals, at least 80% β-pleated crystals, or at least 90% β-pleated crystals.

13. A plant-based structured material according to any preceding claim, wherein the structured material comprises a plant-based protein having a secondary structure having at least 40% intermolecular β-sheets, at least 50% intermolecular β-sheets, at least 60% intermolecular β-sheets, at least 70% intermolecular β-sheets, at least 80% intermolecular β-sheets or at least 90% intermolecular β-sheets.

14. The plant-based structured material according to any preceding claim, wherein the storage modulus (G') of the structured material at 10 rad / s is greater than 500 Pa, greater than 1000 Pa, greater than 2500 Pa, greater than 3000 Pa, greater than 4000 Pa.

15. A plant-based structured material according to any preceding claim, wherein the Young's modulus of the structured material exceeds 20 MPa; preferably exceeds 50 MPa, exceeds 80 MPa, exceeds 100 MPa, exceeds 200 MPa, exceeds 300 MPa, exceeds 400 MPa, exceeds 500 MPa or exceeds 600 MPa.

16. A plant-based structured material according to any preceding claim, wherein the structured material comprises protein aggregates having 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.

17. A plant-based structured material according to any preceding claim, wherein the plant-based structured material is capable of thermoreversibly gelling.

18. The plant-based structured material according to claim 17, wherein upon heating at elevated temperature and / or by applying mechanical agitation, the plant-based structured material will form a protein solution having a storage modulus below 250 Pa, preferably below 100 Pa, below 50 Pa or below 10 Pa.

19. A plant-based structured material according to any preceding claim, formed via a thermo-reversible cold-set gelling process.

20. A plant-based thermoreversible hydrogel.

21. The plant-based thermoreversible hydrogel of claim 20, wherein the hydrogel is molded into a structured shape, formed into a structured shape by a microfluidic device, or otherwise formed into a structured shape; thereafter the structured hydrogel is optionally dried.

22. A composite material comprising a plant-based material according to any preceding claim, and one or more other biopolymers, such as proteins and / or polysaccharides.

23. A food, cosmetic, medicine, medical device or biomaterial comprising the plant-based structured material according to claims 1 to 19, the plant-based thermoreversible hydrogel according to claim 20 or 21, or the composite material according to claim 22.

24. A method for producing a plant-based material, the method comprising: a) forming a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises a miscible co-solvent; wherein the first co-solvent increases the solubility of the one or more plant-based proteins and the second co-solvent decreases the solubility of the one or more plant-based proteins; and b) inducing a sol-gel transition of the protein in the solution to form a plant-based protein hydrogel.

25. The method according to claim 24, comprising: c) forming the plant-based protein hydrogel into a structured material.

26. The method of claim 25, wherein the plant-based protein hydrogel is formed into a structured material before, during or after the sol-gel transition.

27. The method of any one of claims 24 to 26, wherein the plant-based protein hydrogel is molded into a structured material or wherein the plant-based protein hydrogel is formed into a structured material using a microfluidic device.

28. A method according to any one of claims 24 to 27, wherein the method is used to produce a structured material; for example, a membrane, a film, a micropatterned film (or film), a micro- or nanostructured film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a microsponge, a hard capsule or a functional coating.

29. The method according to any one of claims 24 to 28, wherein the one or more plant proteins are obtained from soy, pea, rice, potato, wheat, zein or sorghum; preferably the one or more plant proteins are selected from soy protein, pea protein, potato protein and / or rice protein.

30. The method according to any one of claims 24 to 29, wherein the first co-solvent is an organic acid; preferably acetic 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; wherein particularly preferred organic acids are acetic acid and / or lactic acid.

31. The method according to any one of claims 24 to 30, wherein the second co-solvent or the additional co-solvent(s) is an aqueous buffer; preferably selected from water, ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, tert-butanol, ethyl acetate or hexafluoroisopropanol, particularly preferably water and / or ethanol; further particularly preferably water.

32. The method of any one of claims 24 to 31, wherein the solvent system comprises a co-solvent ratio 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.

33. The method according to any one of claims 24 to 32, further comprising subjecting the protein solution comprising one or more plant-based proteins and a solvent system to mechanical shearing, preferably sonication.

34. The method of any one of claims 24 to 33, wherein the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant-based proteins and then reduced to a second temperature below the sol-gel temperature of the one or more plant-based proteins to form a hydrogel.

35. A plant-based hydrogel formed using the method of any one of claims 24 to 33.

36. A thermoreversible method for obtaining a plant-based material, the thermoreversible method comprising: a) forming a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases the solubility of the one or more plant-based proteins and a second co-solvent decreases the solubility of the one or more plant-based proteins; and b) inducing a sol-gel transition of the protein in the solution to form a plant-based protein hydrogel.

37. The thermoreversible method of claim 36, wherein the solvent system is subsequently removed such that the plant-based protein hydrogel no longer has thermoreversible properties.

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