Plant-based functional materials

Abstract

To provide plant-based materials, methods for manufacturing the same, and biomaterials incorporating the plant-based materials.SOLUTION: A plant-based structured material is a film, a thin film, a micropatterned film (or thin film), a micro- or nanostructured thin film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a microscale sponge, a hard capsule, or a functional coating. The plant-based structured material comprises a plant-based protein having a secondary structure with at least 40% intermolecular β-sheet.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to plant based materials, methods for their production, and biological materials incorporating the plant material of the invention. [Background technology]

[0002] Synthetic polymers such as plastics exhibit excellent mechanical and chemical properties and have been widely used for the past 60 years. However, these polymers are not biodegradable and can accumulate in the environment, causing economic damage and potentially affecting human health through the food chain and the atmosphere.

[0003] The development of materials that exhibit high levels of functionality and a high degree of biodegradability and biocompatibility is a key objective in meeting societal needs for improved materials performance in areas ranging from packaging to pharmaceuticals.

[0004] Self-assembly has emerged as an attractive avenue for the fabrication of such materials, but the majority of the building blocks utilized to date are of synthetic origin.

[0005] Among the various types of biopolymers that can serve as building blocks for generating new functional materials, proteins are interesting candidates given their ability to self-assemble into functional structures.

[0006] Currently, the commercial use of these materials is limited to highly soluble animal-derived proteins.Animal proteins commonly used in food products, such as whey protein, exhibit good biocompatibility, biodegradability, amphiphilicity, and functional properties, such as water solubility, emulsifying ability, and foaming ability.However, there is an increasing demand to replace animal-derived proteins with plant-derived proteins, not only because of their smaller impact on the environment, but also because of their lower allergenicity and reduced cost.

[0007] The formation of self-assembled materials from plant proteins has been reported, with hydrogels being obtainable from soybean and pea proteins under a variety of experimental conditions. However, the mechanical properties obtained from structured plant-based materials are generally lower compared to those obtained from animal-derived materials, and plant proteins are more difficult to process, at least in part due to their inherent low solubility in water.

[0008] Thus, to date, plant proteins have not been successfully utilized as biomaterials, and it remains a challenge to produce structured protein materials from renewable and cost-effective feedstocks using environmentally sustainable methods. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Tran, TM, Cater, S. & Abate, AR Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices. Biomicrofluidics 8, pp. 1-7 (2014) Summary of the Invention [Means for solving the problem]

[0010] In one aspect of the invention there is provided a method for producing plant material comprising the steps of: a) forming a solution comprising one or more vegetable proteins in a solvent system, the solvent system comprising a plurality of miscible co-solvents, a first co-solvent increasing the solubility of the vegetable protein and a second co-solvent decreasing the solubility of the vegetable protein; and b) subjecting the protein in solution to a sol-gel transition to form a vegetable protein hydrogel. A method is provided, comprising:

[0011] In a further embodiment, the method comprises: c) forming a structured material from the vegetable protein hydrogel Includes.

[0012] Forming structured materials from vegetable protein hydrogels allows for the formation of structured materials such as gels, films, microgels, microcapsules, etc. In a preferred embodiment, the protein hydrogels can be formed into specific shapes by molding. In a further preferred embodiment, the protein hydrogels can be formed into specific shapes using a microfluidic device.

[0013] The present invention has identified a novel method for producing functional materials derived from plant proteins. By utilizing a co-solvent mixture, it is possible to exert control over the sol-gel transition, which allows for the formation of structurally robust materials derived from renewable plant protein feedstocks. The method allows for the creation of many structured materials, including hydrogels, films, microcapsules, microgels, microscale sponges, and the like. The structured materials can be reliably formed without the need for cross-linkers or any other harmful materials that would make the structured materials suitable for contact with the human body. The materials are also derived from renewable feedstocks, thus reducing the environmental impact over synthetic analogues.

[0014] In a further aspect, there is provided the use of a co-solvent mixture to modify the properties of plant proteins in solution to control sol-gel conditions, thereby forming plant materials.

[0015] It is possible to control the sol-gel conditions by selecting a solvent system that includes multiple miscible co-solvents, where a first co-solvent increases the solubility of the plant protein and a second co-solvent decreases the solubility of the plant protein.

[0016] The ratio of the first co-solvent to the second co-solvent 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.

[0017] The solvent system may contain one or more first co-solvents and / or one or more second co-solvents.

[0018] For the first time, plant-based materials can be reliably and reproducibly formed using methods that are feasible on a scalable scale. The ability to control the sol-gel conditions allows for tailoring the properties of the resulting material and / or tailoring the manufacturing process to enable the production of useful biomaterials.

[0019] The method of the present invention allows the formation of a vegetable structured material formed via a thermoreversible cold solidification gelation process. The vegetable structured material may be a vegetable protein supramolecular structure or a three-dimensional network of aggregated and entangled vegetable protein supramolecular structures.

[0020] A thermoreversible chill-set gelation method can be considered a method in which the plant protein molecules are heated above a temperature at which they form a liquid solution that can be processed into a desired configuration, and then the liquid solution can be cooled to allow a sol-gel transition that forms a network of self-assembled protein aggregates held together by non-covalent intermolecular interactions. Thus, the gelation method of the present invention does not require covalent chemical crosslinking and is therefore reversible. The present invention involves a thermoreversible cryogelation method.

[0021] Thus, in a further aspect, there is provided a vegetable-based structured material formed via a thermoreversible cool-set gelation process, which may optionally be a film, thin film, micropatterned film (or thin film), micro- or nanostructured thin film, microgel, microcapsule, microbead, bioscaffold, bio-support, sponge, microscale sponge, hard capsule, or functional coating.

[0022] In a further aspect, a vegetable-based thermoreversible gel is provided.

[0023] In a further aspect, a vegetable-based thermoreversible hydrogel is provided.

[0024] In a further aspect there is provided a composite material comprising the plant material of the invention and one or more further biopolymers, such as proteins, polysaccharides etc.

[0025] In a further aspect, there is provided a material made according to the method of the invention.

[0026] The plant-based materials and methods for producing the same of the present invention allow precise control of the sol-gel transition, thereby opening up the use of plant proteins to form biomaterials that, to date, have only been successfully produced using animal-derived proteins. Suitable biomaterials include films, microbeads, microcapsules, scaffolds, gels, sponges, etc.

[0027] In a further aspect there is provided a microbead comprising the plant material of the present invention.

[0028] In a further aspect there is provided a microcapsule comprising the plant material of the present invention.

[0029] In a further aspect, there is provided a hard capsule comprising the plant material of the present invention.

[0030] In a further aspect there is provided a sponge or microscale sponge comprising the plant material of the invention.

[0031] In a further aspect there is provided a film, preferably a thin film, comprising the plant material of the present invention.

[0032] In a further aspect, there is provided a nano- or micro-patterned film comprising the plant material of the present invention.

[0033] In a further aspect, there is provided a biological scaffold comprising the vegetable-based hydrogel of the present invention.

[0034] In a further aspect, there is provided a functional coating comprising the vegetable hydrogel of the present invention.

[0035] In a further aspect, there is provided a foodstuff, cosmetic, pharmaceutical, or medical device incorporating the plant material of the present invention.

[0036] In a further aspect, there is provided a thermoreversible method for obtaining plant material, comprising the steps of: a) forming a solution comprising one or more vegetable proteins in a solvent system, the solvent system comprising a plurality of miscible co-solvents, a first co-solvent increasing the solubility of the vegetable protein and a second co-solvent decreasing the solubility of the vegetable protein; and b) subjecting the protein in solution to a sol-gel transition to form a vegetable protein hydrogel. A method is provided, comprising:

[0037] Once the material is obtained, the thermoreversible property may be removed from the material. [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 shows soy protein isolate (SPI) hydrogels formed under different acetic acid / DI water co-solvent ratios. [Diagram 2] FIG. 1 is a SEM (scanning electron microscopy) image of a hydrogel produced using a 30% v / v co-solvent ratio. [Diagram 3] Graphs showing the estimated size distribution of SPI particles by dynamic light scattering (DLS) for SPI dispersions in water (a), NaOH pH 10 (b), HCl pH 2 (c), and 30% acetic acid (d). For each solvent, samples were prepared via either heating at 95° C. for 30 minutes (labeled as heating) or sonication for 30 minutes (labeled as sonication). All measurements were performed at 0.1% protein concentration and repeated twice. The inset graph shows the raw correlation graph for each condition. [Figure 4a] 1 is a graph showing the rheological properties of SPI hydrogels as a function of HO:acetic acid co-solvent ratio. [Figure 4b] 1 is a graph showing the shear thinning behavior of SPI hydrogels prepared at various concentrations. [Figure 4c] 1 is a graph showing the thermoreversible rheological behavior of SPI hydrogels at various temperatures. [Figure 5a] FIG. 1 is a graph showing the structural changes of SPI hydrogel secondary structure under various HO:acetic acid co-solvent ratios, calculated from the amide I band in the FTIR spectrum. [Figure 5b] FIG. 1 is a graph showing the structural changes of SPI hydrogel secondary structure under various HO:acetic acid co-solvent ratios, calculated from the amide I band in the FTIR spectrum. [Figure 5c] 1 is a graph showing the structural change of the secondary structure of SPI hydrogel at various temperatures, calculated from the amide I band in FTIR. [Figure 6] FIG. 1 is an SDS-PAGE electropherogram showing the increasing extent of protein hydrolysis with increasing amounts of acetic acid. [Figure 7] Figure 7 shows microbeads formed using hydrogels of the present invention: Figure 7a shows a schematic of microbead formation, Figure 7b shows stable microbeads suspended in an aqueous solution (pH=2), Figure 7c is an SEM image of microbeads prepared by supercritical point drying, and Figure 7d is an SEM image of the gel network on the surface of the microbeads. [Figure 8a]FIG. 8a is a schematic diagram of a multi-layer 3D microfluidic droplet generator used to generate core-shell microcapsules with both lipophilic and hydrophilic cores. [Figure 8b] FIG. 8b shows a core-shell microcapsule containing a lipophilic core suspended in an aqueous solution. [Figure 9] FIG. 1 shows core-shell microcapsules containing a hydrophilic core containing a suspension of a soluble active ingredient (riboflavin). [Figure 10a] FIG. 10a shows the results of a simulated degradation showing microcapsules in aqueous solution. [Figure 10b] FIG. 10b shows the results of simulated degradation showing microcapsules after 60 minutes in SGF (simulated gastric fluid). [Figure 10c] FIG. 10c shows the results of simulated degradation showing microcapsules after 120 minutes in SIF (simulated intestinal fluid). [Figure 11] Figure 11a shows core-shell microcapsules containing a core composed of riboflavin solution in 1% (w / w) HMP pectin, and Figure 11b is a graph showing the results of a two-stage in vitro digestibility study generated by HPLC analysis showing the cumulative release of riboflavin under simulated conditions. [Figure 12a] FIG. 12a shows the distribution of aromatic oil-loaded vegetable protein microscale sponge. [Figure 12b] FIG. 12b shows a single perfume-loaded vegetable protein microscale sponge at higher magnification (20x) where the protein microgel shell is readily observable. [Figure 13] FIG. 1 is a schematic diagram of an example of generating a stable protein film. [Figure 14] Figure 14a is a graph showing the stress-strain curves of films made according to the present invention, Figure 14b is a graph showing Young's Modulus, and Figure 14c is a graph showing % elongation to break. [Figure 15]FIG. 1 shows the FTIR spectrum of an SPI film prepared without glycerol and the spectrum of a commercial SPI dried sample. [Figure 16a] FIG. 16a is a TEM image of a dilute SPI solution dried onto a TEM grid confirming the presence of significant amounts of β-sheet crystals (scale bar: 5 nm). [Figure 16b] FIG. 16b is a close-up image of SPI film β-sheet crystals (scale bar: 2 nm). [Figure 17a] FIG. 1 shows micropatterning of equally spaced micropillar arrays onto a soy protein film surface, which significantly increases the contact angle of a water droplet (99°) compared to a non-micropatterned control sample. [Figure 17b] 1 shows the nanopatterning of regularly spaced nanochannel arrays obtained by injecting soy protein films into DVD discs. The nanostructured motifs in the soy protein films exhibit photonic properties (Mie scattering). [Figure 18] 1 shows a schematic process for making a soy protein film coating on a cardboard substrate. The soy protein film coated substrate shows a 50% reduction in water absorption when compared to the uncoated control sample. [Figure 19a] FIG. 19a shows the formation of a thin three-dimensional hydrogel layer around a 2 ml Eppendorf tube substrate. [Figure 19b] FIG. 19b shows the soy protein hard capsules obtained after the molded three-dimensional hydrogel was dried and removed from the substrate. [Figure 20a] FIG. 20a shows a pea protein hydrogel produced using a 30% v / v cosolvent ratio. [Figure 20b] FIG. 20b is a SEM (scanning electron microscopy) image of a pea protein hydrogel produced using a 30% v / v cosolvent ratio. [Figure 20c] FIG. 20c shows a pea protein isolate film. [Figure 20d]FIG. 20d shows stable pea protein microgels suspended in an aqueous solution (pH=2). [Figure 21] FIG. 2 shows potato protein hydrogels prepared from a 100 mg / ml potato protein isolate solution in a 30% (v / v) aqueous acetic acid solution. [Figure 22] 1 is a graph showing stress-strain curves of additional films made in accordance with the present invention. [Figure 23a] 1 is a photoimage of gelation using acetic acid, and HCl or NaOH of the present invention. [Figure 23b] 1 is a photoimage of gelation using acetic acid, and HCl or NaOH of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The following features apply to all aspects of the invention.

[0040] Any suitable vegetable protein may be used in the present invention. Suitable vegetable sources include soybean, pea, rice, potato, wheat, corn zein, sorghum, etc. Particular vegetable proteins include soybean protein and pea protein.

[0041] Suitable plant proteins include: - Brassica, e.g. Brassica balearica: Mallorca cabbage, Brassica carinata: Abyssinian mustard or Abyssinian cabbage, Brassica elongata: elongated mustard, Brassica fruticulosa: Mediterranean cabbage, Brassica hilarionis: St. Hilarion cabbage, Brassica juncea: Indian mustard, brown mustard, and tacana, mustard of Sarepta, Brassica napus: rapeseed, canola, rutabaga, Brassica narinosa, broadbeaked mustard, mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, Chinese broccoli, Brussels sprouts, kohlrabi, Brassica perviridis: tender greens, mustard spinach, Brassica rapa (also known as B. campestris): Chinese cabbage, turnip, rapini, komatsuna, Brassica rupestris: brown mustard, Brassica tournefortii: Asian mustard, - Solanaceae, e.g. tomato, potato, eggplant, peppers and chillies; - Cereals, such as maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, fonio, - pseudocereals, such as amaranth (red amaranth, prince-of-Wales-feather), breadfruit, buckwheat, chia, cockscomb (also called quail grass or soko), pitseed goosefoot, kaniwa, quinoa and wattleseed (also called acacia seed); - Legumes such as Acacia alata (Winged Wattle), Acacia decipiens, Acacia saligna (commonly known by various names including coojong, golden-leaved wattle, orange wattle and blue-leaf wattle), Arachis hypogaea (peanut), Astragalus galegiformis, Cytisus laburnum (common laburnum, king-salis or golden rain), Cytisus supinus, Dolichos lablab (commonly known as lablab-bean, lablab-bean, bonavist bean / pea) bean / pea, Dolichos bean, seim bean, love love bean, Egyptian bean, Indian bean, bataw, and Australian pea), Ervum lens, Genista tinctoria (common names include dyer's whin, staghorn fern, and waxen wood), Glycine max (soybean), Lathyrus clymenum (pea vine or lathyrus), Lathyrus odoratus (pea vine or lathyrus), Lathyrus sativus (pea vine or lathyrus), Lathyrus sylvestris (pea vine or lathyrus), Lotus tetragonolobus (lotus tetragonolobus), tetragonolobus (winged pea), Lupinus albus (lupine), Lupinus angustifolius (lupine), Lupinusluteus (lupine), Lupinus polyphyllus (lupine), Medicago sativa (alfalfa), Phaseolus aureus (mung bean), Phaseolus coccineus (scarlet bean), Phaseolus nanus (green bean), Phaseolus vulgaris (green bean), Pisum sativum (pea), Trifolium hybridum (clover), Trifolium pretense (red clover), Vicia faba (fava bean), Vicia sativa)(vetch), Vigna unguiculate)(cowpea), - non-leguminous plants, e.g. Acanthosicyos horrida, Acanthosicyos horrida, Aesculus hyppocastanum (horse chestnut), Anacardium occidentale (cashew nut tree), Balanites aegyptica, Bertholletia excels (Brazil nut tree), Beta vulgaris (sugar beet), Brassica napus (rapeseed), Brassica juncea (brown mustard), Brassica nigra (black mustard), Brassica hirta (Eurasian mustard), Cannabis sativa (hemp), Citrullus vulgaris (a type of watermelon), Citrus aurantiaca (citrus fruit), Cucurbita maxima (pumpkin), Fagopyrum esculentum (buckwheat), Gossypium barbadense (extra long-staple cotton), Helianthus annuus (sunflower), Nicotiana sp. (tobacco), Prunus avium (cherry), Prunus cerasus (sweet cherry), Prunus domestica (plum), Prunus amygdalus (almond), Ricinus communis(castor bean), Sesamum indicum(sesame), Sinapis alba(white mustard), Telfairia pedata(oyster nut) Further examples include:

[0042] For the avoidance of doubt, the plant-based structured material of the present invention does not encompass plants in their natural state, for example, naturally formed plant cells, organelles, or vesicles are not plant-based structured materials of the present invention.

[0043] A characteristic of plant-derived proteins is their inherent poor solubility in water. To date, this has limited the use of plant-derived proteins in generating biomaterials. However, the present invention overcomes the previous limitations associated with such proteins.

[0044] In the methods of the present invention, the material is formed by adding a vegetable protein to a solvent system, the solvent system comprising two or more miscible co-solvents as defined herein.

[0045] The first co-solvent increases the solubility of the plant protein. The first co-solvent can be considered a solubilizing co-solvent. There may be one or more solubilizing co-solvents, and the solubilizing co-solvents may fully or partially solubilize the plant protein.

[0046] Examples of solubilizing co-solvents are organic acids, which are organic compounds that have 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 allows the solubilization of vegetable proteins and also allows for gentle hydrolysis of proteins. For example, without wishing to be bound by theory, the dissolution of vegetable proteins in organic acids is possible due to the presence of an anionic solvation layer, which contributes to i) the protonation of proteins, and ii) the reduction of hydrophobic interactions. When initially dissolved in an organic acid, protonation of the vegetable protein can help stabilize the vegetable protein in its non-solvent, such as water.

[0047] In a preferred embodiment, the first co-solvent is an organic acid.

[0048] The second co-solvent has a reduced solubility of the vegetable protein compared to the first co-solvent. The second co-solvent can be considered a de-solubilising co-solvent. One or more de-solubilising co-solvents may be present.

[0049] An example of the desolubilizing second co-solvent is an aqueous buffer solution. In a further embodiment, the second co-solvent can be ethanol, methanol, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate, or hexafluoroisopropanol. In a particularly preferred embodiment, the second co-solvent is water and ethanol. In a further particularly preferred embodiment, the second co-solvent is water.

[0050] In a preferred embodiment, the concentration of the vegetable protein in the solvent system is 25-200 mg / ml, preferably 50-150 mg / ml. The ratio of organic acids may be varied depending on the protein concentration, for example using a higher organic acid ratio with increasing protein concentration.

[0051] In a preferred embodiment, the degree of protein hydrolysis is controlled to modify the properties of the resulting hydrogel. For example, increasing the acid concentration present during formation will increase the degree of protein hydrolysis. A higher degree of protein hydrolysis will result in the formation of a hydrogel that is less rigid.

[0052] To form a solution containing one or more vegetable proteins, it may be necessary to apply a physical stimulus to the protein / solvent system mixture to allow the protein to dissolve.Suitable physical stimuli include sonication, stirring, high shear mixing, or other physical techniques.A preferred technique is sonication.

[0053] In one embodiment, the solution is subjected to sonication for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or more than 30 minutes. A preferred sonication time period is about 30 minutes.

[0054] The protein solution is heated so that the liquid solution is maintained above the sol-gel transition of the protein. By modifying the solvent system (e.g., by selection from a selection of organic acids, the ratio of organic acid to additional solvent, or through additional means), it is possible to alter the sol-gel transition temperature of the protein. By appropriate selection of conditions, it is possible to carefully control the sol-gel transition of the protein, and thereby control the formation of the hydrogel.

[0055] In one embodiment, the protein solution is heated to a temperature of about or greater than 70° C. In further embodiments, the protein is heated to a temperature of about or greater than 75° C., about or greater than 80° C., about or greater than 85° C., or about 90° C. In a preferred embodiment, the protein is heated to 85° C.

[0056] The protein solution may be held at elevated temperature for a time period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, or 1 hour. A preferred time period is at least 30 minutes to allow the protein to completely solubilize. It is possible to hold the protein solution at elevated temperature for a longer period of time. This may be useful for use in commercial batch processes or fluid processing steps where it is necessary to keep the protein solution in liquid form for a longer period of time.

[0057] After heating the protein solution to a temperature above the sol-gel transition temperature of the protein, the temperature of the protein solution can be reduced to a second temperature below the sol-gel transition temperature that facilitates the formation of a hydrogel. The second temperature can be room temperature. The protein solution can be held at the reduced temperature for a time period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or about 30 minutes. A specific reduced time period is about 5 minutes. However, the method of the present invention allows the protein to remain in solution for a long period of time. Thus, if necessary, the protein solution can be held at a temperature above the sol-gel transition temperature for as long as needed to keep the protein in liquid form. This can be several hours, days, or longer. Also, because the reaction is reversible, the solution can be held at a low temperature (e.g., room temperature) where a hydrogel can form, but then heated to a temperature above the sol-gel transition temperature to return the solution to a liquid state for further processing. In this manner, the protein hydrogel remains stable for extended periods of time and may be stored for hours, days, weeks, months, or years.

[0058] The particular temperature may depend on the properties of the protein raw material, the solvent conditions used, and therefore the sol-gel transition temperature. Alternatively, the temperature ramp and ramp may be relatively fixed (e.g., about 85° C., then about room temperature), with the co-solvent mixture conditions adjusted to ensure a suitable sol-gel transition temperature for the selected plant protein.

[0059] Thus, in one embodiment, there is provided a method for forming a plant material comprising: a) forming a protein solution comprising one or more plant proteins and a solvent system, the solvent system comprising a plurality of miscible co-solvents, a first co-solvent increasing the solubility of the plant protein and a second co-solvent decreasing the solubility of the plant protein; b) subjecting the protein solution to mechanical agitation, e.g., sonication, for a period of time; c) increasing the temperature of the protein solution to a first elevated temperature above the sol-gel transition temperature for a period of time, said temperature increase being caused by the mechanical agitation of b) or from an external heat source; d) reducing the temperature of the protein solution below the sol-gel transition temperature such that the vegetable protein self-aggregates into a hydrogel, and optionally e) forming the hydrogel into a defined shape, e.g., a microgel, a microcapsule, a microscale sponge, a film, etc. The forming step may be a molding step, i.e., forming the hydrogel into a defined shape. The forming step may use a microfluidic device.

[0060] The protein solution may be held at an elevated temperature in step c) while it is allowed to conform to the desired final shape. For example, in a microfluidic device, the protein solution may be held in a reservoir of the device at an elevated temperature c), but when the solution is expelled from the device, the temperature of the solution is reduced, thereby forming a microgel or microcapsule shell. Alternatively, the protein solution may be held at an elevated temperature while it is shaped in a suitable mold, after which the temperature is reduced to allow the protein to form a hydrogel.

[0061] Without wishing to be bound by theory, it is believed that when plant proteins are added to a solvent system, the plant proteins form a highly viscous dispersion of insoluble colloidal protein aggregates.

[0062] Furthermore, the application of mechanical agitation, such as sonication, is believed to break down large colloidal protein aggregates into smaller aggregates and disrupt protein molecular interactions. Using this technique, the size of protein aggregates can be significantly reduced to particle sizes of less than 100 nm. In one embodiment, the present invention includes protein aggregates with an average size of less than 200 nm, preferably less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, or less than 30 nm.

[0063] Furthermore, when a protein solution in the presence of a co-solvent system is heated above the sol-gel temperature, it is believed that the plant protein is partially unfolded, resulting in the exposure of hydrophobic amino acids that were initially buried inside the protein native structure. Partial unfolding allows the co-solvent to interact with the unfolded protein molecule. For example, organic acids have more opportunities to protonate amino acid residues, allowing the formation of anionic salt bridges that stabilize hydrophobic interactions. Also, when heated at elevated temperatures, non-covalent intermolecular contacts between proteins are disrupted.

[0064] Furthermore, it is believed that cooling the protein solution below the sol-gel temperature allows non-covalent intermolecular contacts between proteins, thus promoting the self-organization of plant protein molecules into a network of supramolecular aggregates.

[0065] It is believed that the methods of the present invention allow plant proteins to aggregate into supramolecular structures that are held together by intermolecular hydrogen bonding interactions, particularly between beta chains.

[0066] The methods of the present invention allow for materials to be formed in which there are high levels of β-sheet intermolecular interactions, resulting in novel materials that have never been made before.

[0067] In one embodiment, the plant material of the present invention has a protein secondary structure with at least 40% intermolecular beta-sheets, at least 50% intermolecular beta-sheets, at least 60% intermolecular beta-sheets, at least 70% intermolecular beta-sheets, at least 80% intermolecular beta-sheets, or at least 90% intermolecular beta-sheets. In one embodiment, the plant material is a film. In one embodiment, the plant material is a dry material, such as a dry hydrogel. In one embodiment, the plant material is a hydrogel or other material described herein. It is believed that previous gels made from plant protein materials have a lower amount of intermolecular beta-sheets in the secondary structure, which may result in the disadvantageous properties of the prior art.

[0068] The plant material of the present invention comprises beta-sheet crystals. The plant material of the present invention may demonstrate a high degree of beta-sheet crystalline structure. The plant material may comprise at least 40% beta-sheet crystals, at least 50% beta-sheet crystals, at least 60% beta-sheet crystals, at least 70% beta-sheet crystals, at least 80% beta-sheet crystals, or at least 90% beta-sheet crystals. In one embodiment, the plant material is a film. In one embodiment, the plant material is a dry material, such as a dry hydrogel. In one embodiment, the plant material is a hydrogel, or other material as described herein.

[0069] The plant material having a highly intermolecular beta-sheet secondary structure and the plant material containing beta-sheet crystals may be any of the materials described in the present invention, including structured materials, such as microcapsules, microbeads, bioscaffolds, biosupports, sponges, microscale sponges, hard capsules, or functional coatings, etc. They may be films, membranes, micropatterned films (or membranes), micro- or nanostructured membranes, or microgels, etc.

[0070] Thus, the invention encompasses, for example, vegetable 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 of the invention.

[0071] Additionally, the present invention encompasses vegetable films comprising vegetable beta-sheet crystals, for example comprising at least 40% beta-sheet crystals, at least 50% beta-sheet crystals, at least 60% beta-sheet crystals, at least 70% beta-sheet crystals, at least 80% beta-sheet crystals, or at least 90% beta-sheet crystals. The same applies to other materials of the present invention.

[0072] The present invention provides a plant-based structured material comprising a film, a thin film, a micropatterned film (or thin film), a micro- or nanostructured thin film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a microscale sponge, a hard capsule, or a functional coating.

[0073] The materials of the present invention have advantageous mechanical properties, for example, the ability to reversibly change from a gel to a liquid upon temperature change, providing advantageous manufacturing capabilities.

[0074] In one embodiment, hydrogels produced according to the present invention have 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.

[0075] In one embodiment, the hydrogel exhibits shear thinning behavior, where the viscosity decreases when the shear rate is increased.

[0076] In one embodiment, hydrogels produced according to the invention exhibit unique thermoreversible gelation behavior. When heated at elevated temperatures and / or by applying mechanical agitation, the protein gels return to liquid form. This is a unique property not seen in previous hydrogels that did not return to a fully liquid state when heated. In contrast, gels made according to the invention can. In one embodiment, when heated at elevated temperatures and / or by applying mechanical agitation, the protein solution can have a storage modulus of less than 250 Pa, less than 100 Pa, less than 50 Pa, less than 10 Pa. This allows both the materials and methods of the invention to have unique manufacturing capabilities.

[0077] Similarly, if desired, the thermoreversibility can be removed by removing the solvent system of the present invention. For example, vegetable microcapsules can be made using the method of the present invention. Once formed, the solvent system can be washed away to stop the thermoreversible properties of the microcapsules. Thus, even if the microcapsules are subsequently heated, they remain stable and cannot remelt. This allows, for example, the microcapsules of the present invention to be subjected to high temperature processes and still remain intact and stable.

[0078] Thus, the hydrogels formed according to the present invention possess unique properties not previously seen in vegetable hydrogels, including the ability to form hydrogels from high concentrations (i.e., 5%-15% w / w) of vegetable protein from commercially available sources, and the ability of such high concentration protein solutions to remain in a liquid state upon thermal denaturation, allowing them to be molded into well-defined objects.

[0079] A feature of the material of the present invention is that there is no need to provide a crosslinker since the plant proteins are capable of self-forming hydrogels. Thus, in one embodiment of the present invention, a plant protein material (e.g., a hydrogel) is provided that is free or substantially free of crosslinkers.

[0080] However, in alternative embodiments, the hydrogels of the present invention may include a crosslinker. Suitable cross-linking agents include microbial transglutaminase, glutaraldehyde, formaldehyde, glyoxal, phenolic compounds, epoxy compounds, genipin, or dialdehyde starch.

[0081] Due to the porous network of the hydrogel, the solvent mixture within the hydrogel can be exchanged with another solvent mixture without compromising the mechanical stability of the hydrogel. A solvent exchange process can be performed to remove the organic acids from the hydrogel porous network.

[0082] In one embodiment of the invention, the method includes a step of exchanging the solvent system in which the vegetable protein hydrogel was formed with an alternative solvent system. In a further embodiment, this is carried out using a solvent exchange process. This step can be carried out after the formation of the hydrogel, but also after the structured material has been formed from the hydrogel (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 hydrogel porous network.

[0083] In one embodiment, the solvent mixture in the hydrogel is evaporated to produce a dry material, such as a thin film, a microstructured / nanostructured thin film, or a microbead. In a further embodiment, one or more co-solvents in the solvent system are volatile solvents. In a preferred embodiment, the first co-solvent is a volatile organic acid, such as acetic acid. In a preferred embodiment, the second or further co-solvent is a volatile alcohol, such as ethanol. In a further preferred embodiment, both the first co-solvent and the second or further co-solvent are volatile solvents.

[0084] In one embodiment, the material of the present invention may incorporate 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.

[0085] The amount of plasticizer incorporated may depend on the intended use of the material, such as a film. In one embodiment, the composition may include about 1% plasticizer, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or more. In further embodiments, the hydrogel may include between about 5-50% plasticizer, about 10-50%, about 20-40%, about 15-35%, or about 20% plasticizer.

[0086] The addition of plasticizers can affect the mechanical properties of a material: typically, adding a plasticizer can increase the elasticity of a material, but conversely, this typically decreases the strength of the resulting material.

[0087] The hydrogels of the present invention allow the formation of a variety of useful plant-based biomaterials. The use of plant-based materials has several advantages over the animal or petrochemical raw materials used so far. First, plant materials are renewable and can be obtained efficiently in an environmentally efficient manner. Second, plant materials are biodegradable and therefore an environmentally friendly alternative to other plastics. Third, in contrast to animal-derived proteins, plant-based proteins have the significant advantage of not introducing animal-derived proteins into humans. This has positive effects from a pharmacological and pharmaceutical point of view, where materials derived from animals must undergo rigorous inspections and processes (e.g., removing prions) to ensure that no harmful elements are present, and also because the products are suitable for vegetarians / vegans.

[0088] Because plant proteins occur naturally in the human (or other animal) diet, biomaterials made according to the present invention exhibit a high degree of digestibility compared to other biopolymers such as polysaccharides (e.g., alginate or chitosan), making them particularly suitable for pharmaceutical, food, and / or cosmetic uses.

[0089] In one embodiment, the hydrogels of the present invention can be used to form films, such as thin films. Plant protein derived films have many uses, including forming biodegradable flexible films for food packaging applications or for use with medical devices, including implantable devices.

[0090] The advantage of the plant material of the present invention over animal material (or starchy / cellulosic material) is the inherent insolubility in water of the plant material. Most biopolymer films dissolve easily in water, thus making them unusable alone for food packaging applications and requiring additional coating layers containing synthetic polymers. These problems are overcome with the present invention.

[0091] The films may have typical thicknesses of 1-1000 μm, 1-100 μm, 10-100 μm, 20-60 μm, 30-50 μm, etc.

[0092] The films may have a Young's modulus of greater than 20 MPa, greater than 50 MPa, greater than 80 MPa, greater than 100 MPa, greater than 200 MPa, greater than 300 MPa, greater than 400 MPa, greater than 500 MPa, greater than 600 MPa, or even more.

[0093] The films may have an elongation to break percentage of 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.

[0094] The Young's modulus / elongation to break percentage values ​​describe films made using the plant-based functional materials, and the properties can be replicated in all of the structured materials described herein that use the materials.

[0095] Films can be micropatterned to have features spanning 100 nm to 1000 μm that impart novel functional properties, such as superhydrophobicity (lotus leaf effect) or structural color (due to Mie scattering).

[0096] Functional composite films can be produced by embedding inorganic nanoparticles, such as gold or silver nanoparticles for the production of films with flexible electronics or antibacterial properties, respectively.

[0097] In a further embodiment, the hydrogels of the present invention can be used to form microbeads. Forming microbeads from vegetable sources overcomes the environmental concerns associated with current plastic microbeads. Microbeads are typically solid particles having a diameter of less than 1 millimeter in their largest dimension.

[0098] In one embodiment, the microbeads of the present invention have a size in their largest dimension that is 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.

[0099] In a further embodiment, the hydrogels of the present invention can be used to form microcapsules, which can be used to encapsulate a variety of substances and can find use in a variety of industrial applications, including in cosmetics, food uses, household uses, agricultural chemical uses, and pharmaceutical uses.

[0100] Microcapsules made according to the present invention offer a completely biodegradable alternative to standard synthetic polymeric microencapsulation shell materials.

[0101] The microcapsules produced according to the present invention uniquely enable microfluidic organization of plant material.

[0102] Microcapsules made according to the present invention provide, for example, superior stability against adverse storage conditions, making them a safer food or pharmaceutical grade solution for storing active ingredients, such as vitamins, essential fatty acids, or antioxidants in food applications, or pharmaceutical active agents, including both small and large molecules.

[0103] Microcapsules made according to the present invention may also have the advantage that they can be produced using microfluidic techniques that ensure high reproducibility, i.e. the ability to produce complex structures (core-shell), and / or can be made using mild processing conditions, thereby protecting the encapsulated active agent.

[0104] Robust microcapsules can also be produced from vegetable proteins by controlling the self-assembly of protein aggregates in the absence of cross-linkers or any other deleterious substances.

[0105] In one embodiment, the microcapsules of the present invention may encapsulate an inner hydrophobic composition. In a further embodiment, the microcapsules of the present invention may encapsulate an inner hydrophilic composition. In a further embodiment, the microcapsules of the present invention may encapsulate a composition of a living organism. In a further embodiment, the microcapsules of the present invention may encapsulate a powder composition. In a further embodiment, the microcapsules of the present invention may encapsulate an oil-in-water, water-in-oil, oil-in-water-in-oil, or water-in-oil-in-water emulsion, etc.

[0106] The vegetable hydrogels of the present invention can form a microcapsule shell. In one embodiment, the shell can 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. Further thicknesses include between 10 nm and 50,000 μm, between 10 μm and 100 μm, between 10 μm and 50 μm, between 1 μm and 10 μm, and the like.

[0107] The vegetable microcapsules of the present invention may encapsulate any active agent suitable for dietary supplements, cosmetics, pharmaceuticals, or agrochemicals, including vitamins, essential fatty acids, antioxidants, small molecules, hydrophilic small molecules, hydrophobic small molecules, proteins, antibodies, antibody-drug conjugates, fragrances, and other large molecules.

[0108] Suitable drugs to be encapsulated include: Crosslinkers, hardeners, organic catalysts, and metal-based catalysts (e.g., organic and inorganic complexes of platinum, palladium, titanium, molybdenum, copper, or zinc) for the polymerization of elastomer, rubber, paint, coating, adhesive, or sealant formulations; Dyes, colorants, pigments for inks, personal care products, elastomer formulations, rubber formulations, paint formulations, coating formulations, adhesive formulations, sealant formulations, or paper formulations; Perfumes for detergents, household cleaning products, personal care products, textiles (so-called smart textiles), coating formulations. Perfumes useful in the present invention are any of the compounds belonging to the list of standards published and updated by the International Fragrance Association (IFRA); aroma substances, flavors, vitamins, amino acids, proteins, essential lipids, probiotics, antioxidants, preservatives for food and foodstuffs; Fabric softeners and conditioners for detergents and personal care products; Bioactive compounds for personal care products, textiles (so-called smart textiles), such as enzymes, vitamins, proteins, vegetable extracts, moisturizers, disinfectants, antibacterial agents, sunscreens, drugs. 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, glycerin, glycine, glycolic acid, hyaluronic acid, hydroquinone, isopropyl, isostearic acid, isopropyl palmitate, oxybenzone, panthenol, proline, retinol, retinyl palmitate, salicylic acid, sorbic acid, sorbitol, triclosan, tyrosine; and Fertilizers, herbicides, insecticides, pesticides, fungicides, repellents, and bactericides for agricultural chemicals The drug may be one or more agents selected from the following:

[0109] The vegetable microcapsules of the present invention may also be useful in diagnostics and high throughput screening.

[0110] The vegetable hydrogels of the present invention may also be useful in producing microgels or microscale sponges. Microgels are microscale hydrogels. The microscale sponges can be thought of as microgels that carry a substance, for example an active ingredient.

[0111] The plant-based hydrogels of the present invention may also be useful in producing bioscaffolds and biosupports. Such materials may be useful for growing cells and tissues both in vitro and in vivo. The hydrogels may also be useful in coating medical devices and implants.

[0112] The plant proteins of the present invention may be functionalized and / or derivatized to alter the properties of the protein.

[0113] The invention will now be described with reference to the following non-limiting examples.

[0114] material - Soy Protein Isolate (SPI) (92% protein) was purchased from MP Biomedicals. Pea protein isolate (PPI) (80% protein) was purchased from Cambridge Commodities Ltd. Acetic acid (glacial), and lactic acid (natural, ≥85%), soybean oil (soya 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. - Fluorinert (FC-40) was purchased from Fluorochem. - 008-Fluorosurfactant was purchased from RAN Biotechnologies. EXAMPLES

[0115] Free-standing hydrogels were prepared according to the following process.

[0116] Glacial acetic acid was mixed with deionized water in various 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. For protein solubilization, 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 kept between 85 °C and 90 °C. After 30 minutes, a completely translucent liquid solution was obtained. The sample was allowed to cool at room temperature for 5 minutes. During this process, the liquid sample becomes a translucent free-standing hydrogel observable after vial inversion.

[0117] Hydrogels made according to Example 1 with different acetic acid / DI water ratios are shown in FIG.

[0118] Free-standing hydrogels were seen for ratios of 10% to 70% v / v acetic acid / DI water. We noted that solutions of 30% v / v or higher resulted in completely translucent solutions that rapidly formed free-standing hydrogels. The fact that the gels were completely translucent suggested that the gels were composed of small soluble aggregate structures rather than large insoluble aggregates, such as those typically found in protein chill-set gels.

[0119] The hydrogels made according to the present invention were stable and retained their structure after multiple washing steps in both water and ethanol.

[0120] Scanning electron microscopy SEM (scanning electron microscopy) was performed on the hydrogels made using a 30% v / v cosolvent ratio and is shown in Figure 2. The hydrogel microstructure confirmed the presence of a densely packed network of fine chain-like protein aggregates. The figure also shows that the gel network remains intact after removal of the acetic acid.

[0121] Thus, a thermoreversible vegetable gel was prepared for the first time.

[0122] Soy protein hydrogels and microgels were dehydrated in ethanol using 100% absolute ethanol in the final step. Samples were transferred to microporous specimen capsules (78 μm pore size, Agar Scientific) that were immersed and partially filled with 100% absolute ethanol to prevent the samples from drying out during transfer. Samples were then critical point dried using a Quorum E3100 critical point drying apparatus using 4-5 flushes with liquid CO2 with at least 15 minutes of incubation between each flush. Samples were mounted on aluminum SEM stubs using conductive carbon adhesive pads (Agar Scientific) and coated with 15 nm iridium using a Quorum K575X sputter coater. Samples were examined using an FEI Verios 460 scanning electron microscope operated at 2 keV and a probe current of 25-50 pA. Secondary electron images were acquired using either an Everhard-Thornley detector in field-free mode (low resolution) or a through-lens detector in full immersion mode (high resolution).

[0123] Particle size analysis To characterize the size of the 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 aqueous solution and treated with three different methods: (1) untreated, (2) heated in a water bath at 95°C for 30 min, or (3) sonicated at 95°C for 30 min. Particle size (hydrodynamic diameter) was measured using a Zetasizer Nano (Malvern). All samples were diluted to 0.1% (v / v) immediately after sonication before measurements were performed.

[0124] Dynamic light scattering (DLS) analysis (Figure 3) revealed that protein aggregates prepared in 30% (v / v) acetic acid solution and sonicated at 95 °C for 30 min had a significantly smaller particle size (29 ± 9.1 nm) compared to non-solubilized protein aggregates prepared under different conditions.

[0125] Rheological characterization For characterization of the rheological properties of the SPI hydrogels, the samples made via Example 1 were allowed to cool at RT for 1 h and then stored at 4° C. for 12 h.

[0126] Rheological measurements were performed using an ARES controlled strain rheometer. Tests were performed at a temperature of 20°C using 25 mm smooth parallel plates. 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).

[0127] The rheological properties of SPI hydrogels as a function of HO:acetic acid co-solvent ratio are shown in Figure 4a. It was found that the addition of small amounts of acetic acid (10% v / v) resulted in the formation of weak hydrogels, which could be attributed to incomplete solubilization of the protein and the presence of larger insoluble aggregates. On the other hand, large amounts of acetic acid also resulted in the formation of weak hydrogels (90% v / v). Hydrogels formed using co-solvent ratios of 30-70% v / v resulted in stronger hydrogels, with hydrogels prepared using 30 and 50% acetic acid (v / v) exhibiting G' in excess of 2500 Pa.

[0128] For the determination of the thermoreversible gelation properties of SPI hydrogels (75 mg / ml SPI dispersed in 30% v / v aqueous acetic acid solution), rheological measurements were performed using a Discovery HR-2 (TA Instruments) rheometer. Tests were performed using 20 mm smooth parallel plates. Tests were performed on SPI hydrogel samples at various temperatures using an angular frequency of 10 rad / s. First, the sonicated SPI solution was loaded onto a preheated rheometer probe at 90°C. After 500 seconds, the temperature was reduced to 10°C at a rate of 5°C / min. Once the temperature of the sample reached 10°C, the temperature was maintained 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 the SPI hydrogels are shown in Figure 4c.

[0129] This demonstrates the unique manufacturability of the materials of the present invention, as the gel was completely thermoreversibly transferred between the gel and liquid states. Such thermoreversibility has never been seen before.

[0130] FTIR The change in hydrogel secondary structure under various solvent ratios was investigated. The structural analysis of SPI hydrogels was carried out by using an FTIR-Equinox 55 spectrometer (Bruker). The hydrogel samples were used without further pretreatment, loaded onto an FTIR holder, and analyzed by subtracting a 50% v / v (DI water / acetic acid) standard. The atmospheric correction spectrum was subtracted from the initial FTIR spectrum, and the second derivative was applied for further analysis. Each FTIR measurement was repeated three times for every sample replicate (nine replicates per sample on average). The sensitivity of the instrument was detected as 5%. To analyze the transformation of the native structure of soy protein isolate into supramolecular aggregates, the vibrational changes of amide I, which are strictly correlated with the protein secondary structure, were tracked.

[0131] The results of FTIR measurements are shown in Figure 5a. The SPI hydrogel has a high content of α-helix (1656 cm -1 ) and intermolecular parallel β-sheet (1625cm -1 ) secondary structure is evident from second derivative analysis.

[0132] FTIR measurements of SPI hydrogel samples prepared using various solvent ratios shown in Fig. 5b reveal that the use of increasing amounts of acetic acid causes an increase in the content of α-helical structures (1654 cm-1) relative to the content of intermolecular antiparallel and parallel β-sheets (1620 cm-1).

[0133] The change in hydrogel secondary structure at different temperatures was 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 the FTIR sample holder. It can be clearly observed in Figure 5c that the amount of intermolecular parallel β-sheet structure decreases when the sample is heated at 90°C, whereas the parallel β-sheet content increases significantly when the sample is cooled to 20°C.

[0134] This is evidenced by the thermoreversibility of the present invention, which correlates this favorable property with protein secondary structure. EXAMPLES

[0135] The hydrogel was prepared according to the following process using lactic acid as a co-solvent.

[0136] Lactic acid was mixed with deionized water in various 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 / lactic acid solution at a final protein concentration of 100 mg / ml. A dispersion of insoluble protein was obtained. For protein solubilization, the mixture was exposed to ultrasonic treatment for 30 min (high frequency power output = 70 W, frequency = 20 KHz, amplitude = 90%). During this process, the sample temperature was kept between 85 °C and 90 °C. After 30 min, a completely translucent liquid solution was obtained. The sample was allowed to cool at room temperature for 5 min.

[0137] Gel electrophoresis Analysis of the various hydrolyzed protein fragments with different water:lactic acid ratios (from 0 to 90% v / v) was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4–12% gels with MES buffer.

[0138] The results are shown in Figure 6 and show that increasing the amount of lactic acid resulted in a higher degree of protein hydrolysis.

[0139] Having demonstrated that stable hydrogels can be formed using the methods of the present invention, the use of such hydrogels has been employed in microfluidic techniques to form discrete, uniform microscopic objects, such as microgels and microcapsules. EXAMPLES

[0140] Fabrication of microgels The microfluidic device (droplet generator) was fabricated using standard soft lithography techniques with a negative master photoresist (SU8 3050). The continuous oil phase (2% 008-fluorosurfactant in Fluorinert FC-40) was filled into a 2 ml tube, while the dispersed liquid SPI phase (85 mg / ml SPI in 40% v / v acetic acid held at 85 °C) was filled into a 1.5 ml tube and quickly placed on a heating block at 85 °C. To prevent gelation of the SPI solution during transport to the microfluidic device, a custom-made silicone heater (Holroyd Components) with 1 / 32 inch inner diameter stainless steel tubing was used to maintain the temperature of the PTFE tubing connecting the SPI reservoir and the inlet of the microfluidic device. The silicone heater temperature was controlled by a custom-made temperature controller. Droplets with a diameter of approximately 100 μm were generated by pumping both solutions into a standard flow-focusing droplet generator by a pressure-driven system (Elveflow OB1). Various pressure rates were tested until a uniform and continuous generation of a monodisperse population of microdroplets was achieved. The final pressure rate was 175 mbar for the dispersed aqueous phase and 200 mbar for the continuous oil phase. The droplets generated were collected in a 1 ml pipette tip and stored at room temperature for 12 hours to ensure that the gelation process was complete. 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 microgels, an equal volume of 10% PFO solution in Fluorinert FC-40 was added and mixed thoroughly for 30 seconds. The 10% PFO solution in Fluorinert FC-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, resulting in the transfer of the microgels from the oil to the aqueous phase. The supernatant containing the microgel suspension was transferred to a separate vial. A series of subsequent washing steps were performed to remove acetic acid from the microgel suspension by adding 500 μl of deionized water 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. A total of three 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 final washing step. To prepare the microgel samples for SEM analysis, the microgels were washed in a three-step procedure with 100% ethanol. First, the microgels were suspended in 25% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 3 h. After centrifugation and removal of the supernatant, the microgels were then resuspended in 50% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 3 h. Finally, after centrifugation and removal of the supernatant, the microgels were then resuspended in 100% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 12 h.

[0141] The results of this experiment are shown in Figure 7. Figure 7a shows a schematic of the microbead formation. Figure 7b shows stable microgels suspended in an aqueous solution (pH = 2). Figure 7c shows an SEM image of the microbeads prepared by supercritical point drying. Figure 7d shows an SEM image of the gel network on the surface of the microbeads.

[0142] The resulting microgels were stable in aqueous solutions under acidic conditions and in the presence of 100% ethanol. Upon self-assembly, the protein aggregates became completely insoluble in solution, thus resulting in the stability of the microgels.

[0143] These results demonstrate that mechanically robust microgels can be generated from plant proteins by simply controlling the self-organization of protein aggregates at the nanoscale and in the complete absence of cross-linkers or any deleterious substances. EXAMPLES

[0144] Fabrication of core-shell microcapsules Having demonstrated that mechanically robust microgels can be produced, the use of hydrogels for microencapsulation was explored.

[0145] For the fabrication of coaxial flow focusing microfluidic devices, a multi-step photolithography method was followed, as described in Tran, TM, Cater, S. & Abate, AR Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices. Biomicrofluidics 8, 1-7 (2014) and will be briefly described below.

[0146] Two SU8 photoresist masters were required. In the first master, a 25 μm photoresist layer for the inner channel was fabricated, followed by a 50 μm layer for the middle channel, and a 75 μm layer for the outer channel. The second master contained a 25 μm layer for the middle channel and a 50 μm layer for the outer channel. To fabricate the final coaxial flow focusing device, two different PDMS slabs were peeled off from the masters and then aligned and bonded by plasma oxidation. A small amount of sprayed water droplets were placed between the two PDMS slabs after plasma oxidation to allow alignment of the microfluidic channels. The final aligned PDMS device was baked in an oven at 65 °C overnight to complete the bonding of the two layers.

[0147] The microfluidic process for producing the core-shell microcapsules was the same as that for producing the microbeads used in Example 3. However, an additional inlet for the internal phase (core) was added to the system. For the fabrication of core-shell microcapsules containing lipophilic active agents, 450 μl of soybean oil was pre-emulsified into 550 μl of SPI protein solution (20 mg / ml, 30% v / v acetic acid). The soybean oil-in-water emulsion (internal phase), SPI solution (85 mg / ml, 40% v / v acetic acid, middle phase), and FC-40 (2% v / v 008-fluorosurfactant, outer phase) were pumped into a coaxial flow-focusing microfluidic device by a pressure-driven system (Elveflow OB1). Various pressure rates were tested until a uniform and continuous production of a monodisperse population of core-shell microcapsules with a diameter of about 120 μm was achieved (internal phase pressure (100 mbar), intermediate phase pressure (150 mbar), and external phase pressure (100 mbar)). The core-shell ratio could be adjusted simply by controlling the relative pressure between the internal and intermediate phases. The microcapsules were collected and then washed as described in the previous section.

[0148] It is noted that the inner core material remains separate from the shell material in this example, which is different from the standard microencapsulation matrix approach in which the active ingredient is premixed with the matrix material. The reason for this approach is that the pH of the starting protein solution is very low due to the large amount of acetic acid present at the beginning, and high temperatures are required to keep the protein solution in a liquid state. Instead, a 3D coaxial microfluidic device was used, which allows for the easy production of core-shell structures without compromising the stability of the encapsulated ingredients.

[0149] A schematic of a microfluidic droplet generator is shown in Figure 8a, which depicts a 3D flow-focusing microfluidic device. Core-shell microcapsules produced according to the present invention containing a lipophilic core suspended in an aqueous solution are shown in Figure 8b. The microcapsule shell contains the self-assembling SPI protein alone, and the core is a water-in-oil microemulsion (lipophilic). EXAMPLES

[0150] Fabrication of core-shell microcapsules for encapsulating surfactants The microfluidic process described in Example 4 was repeated to produce microcapsules containing a hydrophilic core. The core contained a 1% (w / w) HMP pectin solution containing a suspension of a soluble active ingredient (riboflavin).

[0151] Core-shell microcapsules made according to the present invention, containing a hydrophilic core suspended in an aqueous solution, are shown in Figure 9. The microcapsule shell contains the self-assembling SPI protein alone, and the core is a hydrophilic suspension of an active agent, in this case riboflavin. EXAMPLES

[0152] Controlled release microcapsules Since the protein shell is solely composed of protein aggregates, experiments were performed to determine whether cargo release could be triggered by degradation of the protein shell in the presence of digestive enzymes via a two-stage in vitro digestibility study.

[0153] 1 L of SGF (simulated gastric fluid) electrolyte stock 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 L of deionized water. 1 L of SIF (simulated intestinal fluid) electrolyte stock 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 L of deionized water. 1 ml of SGF was prepared by dissolving 8 mg of pepsin in SGF electrolyte, and the pH was adjusted to pH=2 by adding a small amount of 1 M HCl.

[0154] 50 μl of core-shell microcapsules made according to Example 4 were washed from the oil phase by using the method described in Example 4 (150 μl of deionized water at pH=2 was added). 200 μl of SGF was then mixed with the microcapsule suspension, followed by incubation in a thermoshaker at 37° C. and 300 rpm for 60 min. For the simulated intestinal phase, 3 mg of pancreatin was dissolved in 1 ml of SIF electrolyte. 400 μl of SIF was added to the previous microcapsules in 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.

[0155] The results of the experiment are shown in Figure 10, where Figure 10a shows the microcapsules in aqueous solution, Figure 10b shows the microcapsules still intact after 60 minutes in SGF, and Figure 10c shows the release of microcapsules after 120 minutes in SIF.

[0156] The experiments demonstrate that incubation under SGF conditions (8 mg / ml pepsin, pH=2) caused a slow degradation of the protein shell without compromising the stability of the lipophilic core that remained inside the microcapsules. Subsequent incubation in SIF (1.5 mg / ml pancreatin, pH=7) led to a significant degradation of the protein shell and complete release of the lipophilic core. Control experiments in the absence of enzymes but under the same pH conditions showed that the protein shell degradation was mainly caused by enzymatic digestion.

[0157] Thus, it was found that the microcapsules remained intact under simulated gastric conditions but rapidly disintegrated under simulated intestinal conditions, demonstrating that the microcapsules of the present invention can be used for the controlled delivery of dietary supplements or active pharmaceutical ingredients. EXAMPLES

[0158] Controlled release microcapsules containing active agents - Patent Application 20070229633 The experiment of Example 6 was repeated, but using microcapsules made according to Example 5 containing a hydrophilic core containing the active agent riboflavin.

[0159] Riboflavin microcapsules are shown in Figure 11a (i.e. microcapsules with a hydrophilic core (1% HMP pectin + riboflavin)). Figure 11b shows the results of a two-stage in vitro digestibility study generated by HPLC analysis showing the cumulative release of riboflavin under simulated conditions. It can be seen that there is a controlled release of riboflavin under simulated gastric and small intestinal conditions made possible by the encapsulation of the hydrophilic active ingredient within the vegetable protein microcapsule shell. Encapsulation is shown to prevent rapid diffusion of riboflavin and promote controlled release. EXAMPLES

[0160] Preparation of microscale sponges Soy protein microscale sponges were prepared according to the present invention. The microfluidic process described in Example 2 was repeated to produce soy protein microgels (100 μm diameter). The microgels were washed with 100% ethanol in a three-step procedure. First, the microgels were suspended in 25% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 1 hour. After centrifugation and removal of the supernatant, the microgels were then resuspended in 50% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 1 hour. Finally, after centrifugation and removal of the supernatant, the microgels were then resuspended in 100% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 1 hour. At this point, the ethanol should have completely replaced the water within the microgel porous network. The microgel suspension was centrifuged (1000 rpm, 1 min) and the ethanol supernatant was removed. Then, 250 μl of geranium oil (natural) was added to the microgels. The microgels were suspended in the oil solution by gently shaking the Eppendorf vial. At this stage, the ethanol-miscible oil phase had infiltrated the microgel porous network. After the addition of 500 μl of deionized water to the oil-microgel suspension, the oil-loaded SPI microscale sponges were then transferred to the aqueous phase. The remaining oil phase was removed from the Eppendorf tube.

[0161] Figure 12a shows the distribution of fragrance oil-loaded vegetable protein microscale sponges, and Figure 12b shows a single fragrance-loaded vegetable protein microscale sponge at higher magnification (20x) where the protein microgel shell is easily observable.

[0162] Using this approach, water-immiscible solvents such as aromatic oils can be loaded onto plant protein microscale sponges, allowing for high loading efficiency without the need to use complex microcapsule generation techniques such as core-shell construction. EXAMPLES

[0163] Preparation of films Soy protein films were prepared according to the present invention. 500 mg of soy protein isolate was dissolved in 5 mL of 30% (v / v) acetic acid (protein concentration of 100 mg / ml). The solution was then exposed to ultrasonic treatment for 30 minutes (high frequency power supply power=70 W, frequency=20 KHz, amplitude=90%). A few minutes before the end of the ultrasonic treatment process, 125 mg of glycerol was added to the SPI solution and mixed for the remaining time of the ultrasonic treatment step. Immediately after the ultrasonic treatment step, the solution was poured into a glass Petri dish to form a hydrogel (2 mm thick). The glass Petri dish was heated to around 100° C. to avoid any gelation during the pouring process. The glass dish was then removed from the heating plate and air-dried overnight. After drying, the thin film was peeled off from the glass Petri dish and stored in a 50% humidity chamber until further use. The bottom surface of the glass Petri dish was covered with a Teflon sheet to allow the film to be easily peeled off.

[0164] A schematic of the process of Example 9, along with the resulting film, is shown in Figure 13. It can be seen that mechanically robust transparent thin films are produced.

[0165] Tensile properties The tensile properties of the thin films produced in Example 9 were tested as a function of the amount of plasticizer added. Films were made using 10%, 20%, 30%, 40%, and 50% (w / w) glycerol.

[0166] The tensile properties of the films were tested using a Tinius Olson 5kN with a 10N load cell. The films were cut into 5mm wide strips and both ends of the film were adhesively attached to a paper holder with a gap length of 1mm. The holder and film were placed in the mechanical tester and the paper holder was then cut before measurements to ensure that the load was only on the film. Measurements were performed at a speed of 2mm / min. Film thickness was measured using digital calipers on individual samples. Typical film thickness was 30-50um.

[0167] The stress-strain curves of the inventive films are shown in Figure 14a. The Young's modulus of the film is shown in Figure 14b, and it can be seen that increasing the amount of plasticizer decreases the Young's modulus of the film. The percent elongation at break is shown in Figure 14c, with the greatest elongation being at 30% plasticizer.

[0168] The mechanical performance of the films obtained by this technique is superior to that of previously reported films made from commercial soy proteins due to the high degree of intermolecular interactions and subsequent self-organization of the protein molecules, in contrast to the weak intermolecular interactions typically present in soy protein films produced by injecting monomeric soy proteins in high concentrations of chaotropic agents (i.e., 8 M urea).

[0169] FTIR To investigate the secondary structure and intermolecular interactions in the films, FTIR analysis was carried out on SPI films prepared without added glycerol.

[0170] Data were collected using 128 accumulations at 4 cm-1 resolution with background subtraction. For protein structural analysis, spectra were smoothed and normalized using a quadratic 7-point windowed Savitzky-Golay filter. The second derivative at the amide I band (1600-1700 cm-1) was calculated from the smoothed data and deconvoluted to quantify secondary and quaternary structural contributions.

[0171] FT-IR analysis of the resulting films showed that a high amount of intermolecular β-sheet structure (65%) was present in the films compared to the starting soy protein isolate (FIG. 15).

[0172] Transmission electron microscopy (TEM) analysis TEM analysis was performed to study the morphology of protein self-assembled structures present in the film. For transmission electron microscopy (TEM) imaging, the SPI sample used for the formation of the film detailed in Example 9 was diluted to a concentration of 0.02%, placed on a TEM grid (C400Cu, EM resolutions) and stained with uranyl acetate. Figure 16 shows the presence of a large proportion of β-sheet crystals, which correlates with the abundance of intermolecular β-sheet structures determined by FTIR.

[0173] Beta-sheet crystals have never been seen before in plant protein materials. Such crystals share similarities with silk materials, which are known to have enhanced mechanical properties, including strength. Without wishing to be bound by theory, it is believed that said beta-sheet crystals are formed due to the method described by the present invention. Such beta-sheet crystals may contribute to the enhanced mechanical properties seen in the plant materials of the present invention in contrast to the plant gels described in the prior art. The beta-sheet crystal data, together with FTIR, clearly show that the materials made according to the present invention have a high degree of intermolecular interactions, which allows the materials to have properties (e.g. tensile strength for films) that have not been previously reported for plant materials.

[0174] Thus, the resulting vegetable material of the present invention differs from known vegetable gels, and these differences result in significantly improved properties. EXAMPLES

[0175] Micro- and Nano-Structure Patterning A film prepared according to Example 9 was micropatterned according to the following process.

[0176] To pattern microstructures such as micropillars into the protein film, a negative pattern of 20 μm×20 μm micropillar arrays was fabricated on a silicon wafer by standard photolithography techniques using SU-8 3025 as a photoresist. A mixture of polydimethylsiloxane elastomer and curing agent (Sylgard 184, Dow Corning) in a 10:1 ratio was poured onto the wafer and cured at 65° C. for 1 h. The cured PDMS was peeled off from the wafer and used as a negative pattern for the soy protein film. The soy protein solution prepared in Example 9 was poured into the PDMS and dried overnight. The SPI thin film was peeled off from the PDMS master and the resulting micropatterned structures were observed using scanning electron microscopy (SEM) (MIRA 3 FEG-SEM, TESCAN) with a 10 nm coating of platinum. The contact angle of the film was measured using a First Ten Angstroms FTA1000B.

[0177] By simply injecting the sonicated soy protein solution into the micropatterned substrate, it is possible to fabricate an array of evenly spaced micropillars on the film surface. The contact angle of the micropatterned film is 99°, which is significantly higher based on the control film sample with a non-micropatterned surface (Figure 17a). This clearly indicates that the hydrophobic surface properties can be greatly enhanced by simply patterning micropillars, such as those naturally occurring in lotus leaves, on the surface of the plant protein film.

[0178] To generate nanostructured protein films with photonic properties, DVD disks were used as molding substrates. First, the plastic outer layer was carefully removed from the DVD disk, and the remaining middle 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 in Example 9 was poured into the PDMS substrate and dried overnight at room temperature. The SPI thin film was peeled off from the PDMS master, and the resulting nanopatterned structures were observed using scanning electron microscopy (SEM) (MIRA 3 FEG-SEM, TESCAN) with a 10 nm coating of platinum. The photonic properties (Mie scattering) of the film could be easily observed with the naked eye (Figure 17b). EXAMPLES

[0179] Preparation of the coating To test the coating properties of the vegetable protein film, a small piece of cardboard (2×2 cm) was immersed in the film-forming SPI solution prepared in Example 9 in the absence of plasticizer. After immersing the cardboard in the film-forming solution for approximately 5 seconds, the cardboard was removed from the solution and air-dried at room temperature overnight. After complete drying, the protein-coated cardboard was submerged in 10 mL of deionized water to measure the water absorption of the cardboard. The water absorption was calculated by measuring the mass increase of the cardboard when submerged in deionized water for various time intervals.

[0180] As control samples, uncoated cardboard pieces and soy protein monomer coated cardboard (prepared by immersing cardboard in a soy protein solution prepared under alkaline conditions (pH=9)) were tested. Water absorption was normalized using the initial mass measured before immersing the cardboard sample in water.

[0181] As observed in Figure 18, the uncoated cardboard control sample exhibits very high water absorption after being submerged in water for 10 seconds. The amount of water absorption in the SPI-coated cardboard was reduced by almost 50% compared to the uncoated control sample or the cardboard sample coated with soy protein solution prepared under alkaline conditions, which also exhibited very poor water resistance. These results highlight that coatings produced from vegetable protein films assembled from proteins with a high degree of intermolecular interactions exhibit enhanced water-blocking properties. EXAMPLES

[0182] Hard capsule manufacturing Soy protein hard capsules were manufactured according to the present invention. First, a 100 mg / ml SPI solution was prepared by dissolving 5 g of SPI in 50 ml of an aqueous solution containing acetic acid (40% v / v). The protein dispersion was heated at 95°C for 30 min, followed by exposure to ultrasonic treatment for 5 min (RF power = 70W, frequency = 20KHz, amplitude = 90%). The solution was kept at 85°C to prevent gelation. A 2ml Eppendorf tube was immersed in the heated solution and then kept in the liquid solution for 5 seconds before being removed. A thin layer of SPI hydrogel formed around the outer surface of the 2ml Eppendorf tube within seconds of removing it from the liquid SPI solution (Figure 19a). The vial was then placed in a 45°C oven for 1 hour to ensure evaporation of the water / acetic acid solvent fraction within the hydrogel layer, generating a 3-dimensional thin film. After evaporation of the solvent, the hard capsule was removed from the Eppendorf tube (Figure 19b).

[0183] This is the first demonstration of the formation of hard capsules produced from plant proteins, a process made possible only by the thermoreversible properties of these plant materials, which allow the formation of thin, three-dimensional hydrogels onto a substrate. EXAMPLES

[0184] Pea and potato protein materials Following the procedures described in Example 1, Example 3, and Example 9, protein hydrogels, microgels, and films were produced by using pea protein isolate (80%) and potato protein isolate as starting materials.

[0185] Figure 20a shows a translucent, free-standing pea protein hydrogel visible after vial inversion.

[0186] Properties similar to those described herein are also seen for potato proteins as shown in FIG. 21, which shows a potato protein hydrogel prepared from a 100 mg / ml potato protein isolate solution in a 30% (v / v) aqueous acetic acid solution.

[0187] Example 13 demonstrates the versatility of the method of the present invention to be effective across a variety of plant protein sources. EXAMPLES

[0188] Stress-strain film analysis The stress-strain curves of the films of the invention are shown in Figure 22. Figure 22a shows the stress-strain curves of the films made according to the invention. The strain-stress curve of the self-assembled sample (blue) corresponds to an SPI film prepared from 30% (v / v) acetic acid aqueous solution in the presence of 30% glycerol (w / w, based on dry protein weight). The strain-stress curve of the unstructured sample (red) corresponds to an SPI film prepared in an alkaline aqueous solution adjusted to pH=10 using NaOH in the presence of 30% glycerol (w / w, based on dry protein weight).

[0189] FIG. 22b shows the strain-stress curves of SPI films prepared from 30% (v / v) acetic acid aqueous solution in the presence of various amounts of glycerol as a plasticizer (20%-40% w / w, relative to dry protein mass).

[0190] The self-assembled SPI films prepared with 30% (w / w) glycerol exhibit high tensile strength (15.6±2.07 MPa) and Young's modulus (209±39.1 MPa).

[0191] It can be seen that varying the amount of glycerol allows tuning of the mechanical performance of the hydrogel. For example, the mechanical performance varied from 483±58.4 MPa to 92.7±25.3 MPa for Young's modulus and from 25.0±3.49 MPa to 6.18±0.98 MPa for tensile strength when the concentration of glycerol was varied from 20 to 40% (w / w). The tensile strength and Young's modulus of the self-assembled films containing 30 w / w% glycerol were found to be higher than those of the unstructured films containing 30% (w / w) glycerol (131±22.6 MPa and 9.30±1.53 MPa for Young's modulus and tensile strength, respectively).

[0192] The samples in Example 14 were prepared as follows: 500 mg of SPI was dispersed in 5 mL of 30% (v / v) acetic acid aqueous solution and shaken thoroughly until a turbid, highly viscous dispersion was obtained. It was then sonicated for 30 min at 25% power (pulse width with 0.7 s on-time and 0.3 s off-time) using an ultrasonic homogenizer (Bandelin, HD4200). After sonication, glycerol (≧99.5%, Sigma-Aldrich) was added as a plasticizer at various concentrations (20, 30, or 40 w / w%) and the solution was sonicated for another 1 min. The hot liquid solution was immediately poured into a 7 cm glass Petri dish preheated at 90 °C. The poured solution was then dried for 3 days at room temperature (19-22 °C) and ambient humidity (typically 50-70%). The dried film was then peeled off from the mold and stored in a humidity-controlled chamber (50%) until further use.

[0193] 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 min without sonication (referred to as unstructured SPI). 30% (w / w) glycerol was then added and the hot liquid solution was poured into a glass Petri dish and allowed to dry for 3 days. EXAMPLES

[0194] Comparison of Acetic Acid vs HCl vs NaOH A comparative experiment was conducted to evaluate the difference between hydrogels made with acetic acid according to the present invention and those made using HCl or NaOH methods (which do not undergo a sol-gel transition), and the results are shown in FIG.

[0195] Figure 23 shows optical images of 100 mg / ml SPI dispersions prepared in different aqueous solutions via treatment with ultrasound at elevated temperatures. (a) SPI dispersion 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 (right) after 30 min of ultrasound treatment. (b) Inverted glass vials show that gelation is observed only for the SPI solution prepared in 30% (v / v) acetic acid. The HCl and NaOH vials show that the sol-gel transition does not occur.

[0196] In conclusion, the present invention provides a solvent system as described herein that allows a method to break intermolecular interactions at high temperatures and high shear forces. This allows selective promotion of the formation of intermolecular interactions in response to temperature changes. Such an approach allows the molding of materials into various shapes, which has not been possible with plant-derived materials until now.

[0197] Thus, the present invention can be directed to methods of shaping plant-derived materials, including the methods described herein.

[0198] In addition, the present invention creates a protein secondary structure of the self-assembling material that has a higher amount of intermolecular beta-sheet structure (compared to the protein starting material). Such a novel material has not been found in existing plant-derived materials. This novel secondary structure gives the material a unique property: higher tensile film strength.

[0199] No doubt many other effective alternatives will occur to those skilled in the art, and it will be understood that the invention is not limited to the described embodiments, but encompasses modifications that are obvious to those skilled in the art and are within the spirit and scope of the claims appended hereto.

[0200] (References) JPEG2025066108000001.jpg189163

Claims

1. A thermoreversible method for obtaining a vegetable hydrogel, comprising: a) forming a solution comprising one or more vegetable proteins in a solvent system, wherein the one or more vegetable proteins are selected from the group consisting of soybean, pea, rice, potato, wheat, and sorghum, the solvent system comprising a plurality of miscible co-solvents, a first co-solvent increasing the solubility of the vegetable protein and a second co-solvent decreasing the solubility of the vegetable protein; and b) causing a sol-gel transition of the protein in the solution to form a plant protein hydrogel; A method comprising:

2. The method described in claim 1, wherein the solvent system is then removed so that the plant protein hydrogel no longer has thermoreversible properties.

3. The method described in claim 1, wherein the solution is heated to a first temperature above the sol-gel temperature of one or more types of vegetable proteins and then reduced to a second temperature below the sol-gel temperature of the one or more types of vegetable proteins.

4. Furthermore, c) forming from said plant-based hydrogel a structured material selected from the group consisting of a film, a thin film, a micropatterned film, a micropatterned thin film film, a microstructured thin film, a nanostructured thin film, a microgel, a microcapsule, a microbead, a bioscaffold, a biosupport, a sponge, a microscale sponge, a hard capsule and a functional coating; The method of claim 1 , comprising:

5. The method of claim 1, wherein the first co-solvent is an organic acid selected from the group consisting of acetic acid, glycolic acid, lactic acid, malic acid, citric acid, and tartaric acid; and / or 2. The method of claim 1, wherein the second or further co-solvent is an aqueous buffer selected from the group consisting of water, ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate, and hexafluoroisopropanol.

6. 10. The method of claim 1, wherein the solvent system comprises a ratio of a first co-solvent to a second co-solvent of about 20-80% v / v.

7. 10. The method of claim 1, further comprising mechanical shearing the protein solution comprising the one or more plant proteins and a solvent system.

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