Botanical functional material

Abstract

The present invention relates to plant-based materials, to a process for the preparation thereof and to biomaterials incorporating the plant-based materials of the invention.SOLUTION: A) forming a solution comprising one or more vegetable proteins in a solvent system, wherein the solvent system comprises a plurality of miscible co-solvents, wherein a first co-solvent increases the solubility of the vegetable protein and a second co-solvent decreases the solubility of the vegetable protein; and b) allowing the protein in the solution to undergo a sol-gel transition to form a vegetable protein hydrogel.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 an important objective in meeting societal needs for improved material 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 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 proteins, 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 than 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 the production of structured protein materials from renewable and cost-effective feedstocks using environmentally sustainable methods remains a challenge. [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) causing a sol-gel transition of the protein in the solution to form a plant protein hydrogel; A method is provided, comprising:

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

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

[0013] The present invention has identified a novel method for producing functional materials derived from plant proteins. By utilizing a cosolvent mixture, it is possible to exert control over the sol-gel transition, enabling the formation of structurally robust materials derived from renewable plant protein feedstocks. The method enables the creation of many structured materials, including hydrogels, films, microcapsules, microgels, microscale sponges, and more. The structured materials can be reliably formed without the need for crosslinkers 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 their environmental impact compared to synthetic analogs.

[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] Sol-gel conditions can be controlled 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 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 for the formation of a plant-based structured material formed via a thermoreversible, cool-set gelation process. The plant-based structured material may be a plant protein supramolecular structure or a three-dimensional network of aggregated and entangled plant protein supramolecular structures.

[0020] A thermoreversible chill-set gelation method can be considered a method in which plant protein molecules are heated to a temperature above 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.

[0021] Thus, in a further aspect, there is provided a plant-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 plant-based thermoreversible hydrogel is provided.

[0024] In a further aspect, there is provided a composite material comprising the plant material of the present 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 present invention.

[0026] The plant-based materials and methods for producing the same of the present invention allow for 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, and the like.

[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 present 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 plant-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. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows soy protein isolate (SPI) hydrogels formed under various acetic acid / DI water co-solvent ratios. [Figure 2] 1 is a SEM (scanning electron microscopy) image of a hydrogel produced using a 30% v / v co-solvent ratio. [Figure 3] Figure 3a is a graph showing the rheological properties of SPI hydrogels as a function of HO:acetic acid co-solvent ratio, and Figure 3b is a graph showing the shear thinning behavior of SPI hydrogels prepared at various concentrations. [Figure 4] 1 is a graph showing the structural changes of SPI hydrogel secondary structure under various H2O:acetic acid co-solvent ratios, calculated from the amide I band in the FTIR spectrum. [Figure 5] FIG. 1 is an SDS-PAGE electropherogram showing the increasing extent of protein hydrolysis with increasing amounts of acetic acid. [Figure 6]Figure 6 shows microbeads formed using the hydrogel of the present invention: Figure 6a shows a schematic diagram of microbead formation, Figure 6b shows stable microbeads suspended in an aqueous solution (pH=2), Figure 6c shows Tht-dyed microbeads suspended in a 50 μm Tht aqueous solution, Figure 6d is an SEM image of microbeads prepared by supercritical point drying, and Figure 6e is an SEM image of the gel network on the surface of the microbeads. [Figure 7] Figure 7a is a schematic diagram of the multilayer 3D microfluidic droplet generator used to generate core-shell microcapsules with both lipophilic and hydrophilic cores, and Figure 7b shows core-shell microcapsules containing lipophilic cores suspended in an aqueous solution. [Figure 8] FIG. 1 shows core-shell microcapsules containing a hydrophilic core containing a suspension of a soluble active ingredient (riboflavin). [Figure 9] Figure 9a shows simulated degradation results showing microcapsules in aqueous solution, Figure 9b shows simulated degradation results showing microcapsules after 60 minutes in SGF (simulated gastric fluid), and Figure 9c shows simulated degradation results showing microcapsules after 120 minutes in SIF (simulated intestinal fluid). [Figure 10] Figure 10a shows core-shell microcapsules containing a core composed of riboflavin solution in 1% (w / w) HMP pectin, and Figure 10b 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 11] Figure 11a shows the distribution of fragrance oil-loaded vegetable protein microscale sponges, and Figure 11b shows a single fragrance-loaded vegetable protein microscale sponge at higher magnification (20x), where the protein microgel shell is easily observable. [Figure 12] FIG. 1 is a schematic diagram of an example of producing a stable protein film. [Figure 13]Figure 13a is a graph showing stress-strain curves for films made in accordance with the present invention, Figure 13b is a graph showing Young's modulus, and Figure 13c is a graph showing % elongation to break. [Figure 14a] FIG. 1 shows the micropatterning of a regularly spaced micropillar array 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 14b] 1 shows the nanopatterning of regularly spaced nanochannel arrays obtained by injecting soy protein films into DVD discs. The nanostructured motifs in soy protein films exhibit photonic properties (Mie scattering). [Figure 15] 1 shows a schematic process for producing 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 an uncoated control sample. [Figure 16] Figure 16a shows the formation of a thin 3D hydrogel layer around a 2 ml Eppendorf tube substrate, and Figure 16b shows the soy protein hard capsule obtained after the extruded 3D hydrogel was dried and removed from the substrate. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following features apply to all aspects of the invention.

[0038] Any suitable vegetable protein can 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.

[0039] A characteristic of plant-derived proteins is their inherent poor solubility in water. To date, this has limited their use in generating biomaterials. However, the present invention overcomes the previous limitations associated with such proteins.

[0040] 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.

[0041] The first co-solvent increases the solubility of the vegetable protein. The first co-solvent can be considered a solubilizing co-solvent. One or more solubilizing co-solvents may be present, and the solubilizing co-solvents may fully or partially solubilize the vegetable protein.

[0042] An example of a solubilizing cosolvent is an organic acid. An organic acid is an organic compound with acidic properties. Suitable organic acids include acetic acid or alpha-hydroxy acids. Suitable alpha-hydroxy acids include glycolic acid, lactic acid, malic acid, citric acid, and tartaric acid. Preferred organic acids are acetic acid and lactic acid. The use of organic acids allows for the solubilization of plant proteins and also allows for gentle hydrolysis of proteins. For example, without wishing to be bound by theory, the dissolution of plant proteins in organic acids is possible due to i) the protonation of the protein and ii) the presence of an anionic solvation layer that contributes to the reduction of hydrophobic interactions. When initially dissolved in an organic acid, the protonation of the plant protein can help stabilize the plant protein in its non-solvent, such as water.

[0043] In a preferred embodiment, the first co-solvent is an organic acid.

[0044] The second co-solvent has a reduced plant protein solubility compared to the first co-solvent. The second co-solvent can be considered a desolubilizing co-solvent. One or more desolubilizing co-solvents may be present.

[0045] An example of a desolubilizing second co-solvent is an aqueous buffer solution. In a further embodiment, the second co-solvent may be ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, 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.

[0046] In a preferred embodiment, the concentration of the vegetable protein in the solvent system is 25 to 200 mg / ml, preferably 50 to 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.

[0047] In a preferred embodiment, the degree of proteolysis is controlled to modify the properties of the resulting hydrogel. For example, increasing the acid concentration present during formation will increase the degree of proteolysis. A higher degree of proteolysis results in the formation of a hydrogel with less rigidity.

[0048] 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 proteins to dissolve. Suitable physical stimuli include sonication, stirring, high shear mixing, or other physical techniques. A preferred technique is sonication.

[0049] 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.

[0050] The protein solution is heated to maintain a liquid solution above the sol-gel transition of the protein. By modifying the solvent system (e.g., by selecting 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 a hydrogel.

[0051] In one embodiment, the protein solution is heated to about or above 70° C. In further embodiments, the protein is heated to about or above 75° C., about or above 80° C., about or above 85° C., or about 90° C. In a preferred embodiment, the protein is heated to 85° C.

[0052] The protein solution may be held at the 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 fully solubilize. It is possible to hold the protein solution at the elevated temperature for longer periods of time. This may be useful for use in commercial batch processes or fluid processing steps where it is necessary to maintain the protein solution in liquid form for longer periods of time.

[0053] 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 to facilitate 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 reduction time period is about 5 minutes. However, the methods of the present invention allow the protein to remain in solution for longer periods. Thus, if desired, the protein solution can be held above the sol-gel transition temperature for as long as needed to maintain 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 the hydrogel can form, but then heated 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 and can be stored for hours, days, weeks, months, or years.

[0054] The particular temperature may depend on the properties of the protein 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.

[0055] Thus, in one embodiment, there is provided a method for forming plant material, the method comprising: a) forming a protein solution comprising one or more vegetable proteins and 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; 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, such as a microgel, microcapsule, microscale sponge, 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.

[0056] The protein solution can be held at an elevated temperature in step c) while it conforms to the desired final shape. For example, in a microfluidic device, the protein solution can 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 can 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.

[0057] Without wishing to be bound by theory, it is believed that when the plant protein is added to a solvent system, the plant protein forms a highly viscous dispersion of insoluble colloidal protein aggregates.

[0058] Furthermore, the application of mechanical agitation, such as sonication, is believed to break down large colloidal protein aggregates into smaller aggregates and disrupt protein intermolecular interactions.

[0059] Furthermore, when a protein solution in the presence of a cosolvent system is heated above the sol-gel temperature, the plant protein is thought to partially unfold, resulting in the exposure of hydrophobic amino acids that were initially buried within the protein's native structure. Partial unfolding allows the cosolvent 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. Furthermore, heating at elevated temperatures disrupts non-covalent intermolecular contacts between proteins.

[0060] Furthermore, cooling the protein solution below the sol-gel temperature is believed to allow non-covalent intermolecular contacts between proteins, thus promoting the self-organization of plant protein molecules into a network of supramolecular aggregates.

[0061] 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.

[0062] The methods of the present invention allow materials to be formed in which there is a high level of β-sheet intermolecular interactions, resulting in novel materials that have never been made before.

[0063] 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.

[0064] 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, which provides advantageous manufacturing capabilities.

[0065] In one embodiment, the hydrogels produced according to the present invention have a storage modulus (G') at 10 rad / s greater than 500 Pa, greater than 1000 Pa, greater than 2500 Pa, greater than 3000 Pa, greater than 4000 Pa.

[0066] In one embodiment, the hydrogel exhibits shear thinning behavior, where the viscosity decreases when the shear rate is increased.

[0067] Thus, the hydrogels formed according to the present invention possess unique properties not previously found in vegetable-based 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 liquid upon thermal denaturation, allowing them to be molded into well-defined objects.

[0068] A feature of the materials of the present invention is that there is no need to provide a cross-linking agent because 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 cross-linking agents.

[0069] However, in alternative embodiments, the hydrogels of the present invention may comprise a cross-linking agent, suitable cross-linking agents include microbial transglutaminase, glutaraldehyde, formaldehyde, glyoxal, phenolic compounds, epoxy compounds, genipin, or dialdehyde starch.

[0070] Due to the porous network of the hydrogel, the solvent mixture within the hydrogel can be exchanged for another solvent mixture without compromising the mechanical stability of the hydrogel. A solvent exchange process can be performed to remove the organic acid from the hydrogel porous network.

[0071] In one embodiment of the present 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 hydrogel is formed, but it can also be carried out after the structured material is formed from the hydrogel (e.g., after step b) or c) of the method of the present invention). In a preferred embodiment, an aqueous buffer is used to replace the organic acid co-solvent mixture within the hydrogel porous network.

[0072] In one embodiment, the solvent mixture within the hydrogel is evaporated to produce a dry material, such as a thin film, a microstructured / nanostructured thin film, or microbeads. 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.

[0073] In one embodiment, the materials 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.

[0074] The amount of plasticizer incorporated can depend on the intended use of the material, such as a film. In one embodiment, the composition can contain 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 can contain between about 5-50% plasticizer, about 10-50%, about 20-40%, about 15-35%, or about 20% plasticizer.

[0075] The addition of plasticizers can affect the mechanical properties of a material: typically, adding a plasticizer can increase the elasticity of the material, but conversely, this typically decreases the strength of the resulting material.

[0076] The hydrogels of the present invention enable the formation of a variety of useful plant-based biomaterials. The use of plant-based materials has several advantages over previously used animal or petrochemical raw materials. First, plant materials are renewable and can be efficiently obtained in an environmentally efficient manner. Second, plant materials are biodegradable, thus providing an environmentally friendly alternative to other plastics. Third, in contrast to animal-derived proteins, plant proteins have the significant advantage of not introducing animal-derived proteins into humans. This has positive implications from a pharmacological and pharmaceutical standpoint, where animal-derived materials must undergo rigorous inspections and processes (e.g., prion removal) to ensure the absence of harmful elements, and also because the products are suitable for vegetarians / vegans.

[0077] 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.

[0078] In one embodiment, the hydrogels of the present invention can be used to form films, e.g., 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.

[0079] The advantage of the plant materials of the present invention over animal-based materials (or starchy / cellulosic materials) is their inherent insolubility in water. Most biopolymer films dissolve readily in water, making them unusable for food packaging applications alone and requiring an additional coating layer containing a synthetic polymer. These problems are overcome using the present invention.

[0080] The film may have a typical thickness of 1 to 1000 μm, 1 to 100 μm, 10 to 100 μm, 20 to 60 μm, 30 to 50 μm, and the like.

[0081] The film 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 more.

[0082] The film may have a percent elongation to break 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.

[0083] Films can be micropatterned to have features ranging from 100 nm to 1000 μm that impart novel functional properties, such as superhydrophobicity (lotus leaf effect) or structural color (due to Mie scattering).

[0084] 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.

[0085] In a further embodiment, the hydrogels of the present invention can be used to form microbeads. Forming microbeads from plant-based materials 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.

[0086] In one embodiment, the microbeads of the present invention have a size in the largest dimension of less than 1 mm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, or less than 100 μm.

[0087] 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 industries, including in cosmetics, food applications, household product applications, agricultural chemical applications, and pharmaceutical applications.

[0088] Microcapsules made according to the present invention offer a completely biodegradable alternative to standard synthetic polymer microencapsulation shell materials.

[0089] Microcapsules made in accordance with the present invention uniquely enable microfluidic organization of plant material.

[0090] 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 for food use, or pharmaceutical active agents, including both small and large molecules.

[0091] 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.

[0092] Robust microcapsules can also be produced from plant proteins by controlling the self-assembly of protein aggregates in the absence of cross-linkers or any other deleterious substances.

[0093] 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.

[0094] The vegetable hydrogels of the present invention can form microcapsule shells. In one embodiment, the shells can have thicknesses 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, 35 μm, 40 μm, or 50 μm. Additional thicknesses include those between 10 nm and 50,000 μm, between 10 μm and 100 μm, between 10 μm and 50 μm, and between 10 μm and 10 μm.

[0095] The vegetable microcapsules of the present invention may encapsulate any active agent suitable for dietary supplements, cosmetics, pharmaceuticals, or agricultural chemicals, including vitamins, essential fatty acids, antioxidants, small molecules, hydrophilic small molecules, hydrophobic small molecules, proteins, antibodies, antibody-drug conjugates, fragrances, and other large molecules.

[0096] 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; Fragrances for detergents, household cleaning products, personal care products, textiles (so-called smart textiles), coating formulations. The fragrances 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); Aromatic substances, flavorings, 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, and drugs, including, but 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, and tyrosine; Agricultural chemicals for fertilizers, herbicides, insecticides, pesticides, fungicides, repellents, and bactericides The present invention includes one or more drugs selected from the following:

[0097] The vegetable microcapsules of the present invention may also be useful in diagnostics and high-throughput screening.

[0098] The vegetable hydrogels of the present invention may also be useful in producing microgels or microscale sponges. A microgel is a microscale hydrogel. A microscale sponge can be thought of as a microgel carrying a substance, such as an active ingredient.

[0099] 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 for coating medical devices and implants.

[0100] The plant proteins of the present invention may be functionalized and / or derivatized to alter the properties of the protein.

[0101] The invention will now be described with reference to the following non-limiting examples.

[0102] material - Soy protein isolate (SPI) (92% protein) was purchased from MP Biomedicals. Acetic acid (glacial), lactic acid (natural, ≥85%), soybean oil (soybean oil from Glycine max), (-)-riboflavin, glycerol, and PFO (1H,1H,2H,2H-perfluoro-1-octanol) were purchased from Sigma-Aldrich. Pectin was kindly provided by Cargill. - Fluorinert (FC-40) was purchased from Fluorochem. - 008-Fluorosurfactant was purchased from RAN Biotechnologies. [Example]

[0103] Free-standing hydrogels were prepared according to the following process.

[0104] 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. To solubilize the protein, the mixture was exposed to ultrasonic treatment for 30 minutes (high-frequency power output = 70 W, frequency = 20 kHz, amplitude = 90%). During this process, the sample temperature was maintained 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 became a translucent, free-standing hydrogel, observable after inversion of the vial.

[0105] Hydrogels made according to Example 1 with different acetic acid / DI water ratios are shown in FIG.

[0106] Freestanding hydrogels were observed for acetic acid / DI water ratios ranging from 10% to 70% v / v. We noted that solutions above 30% v / v resulted in completely translucent solutions that rapidly formed freestanding 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.

[0107] The hydrogels made according to the present invention were stable and retained their structure after multiple washing steps in both water and ethanol.

[0108] 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.

[0109] Thus, a thermoreversible vegetable gel was prepared for the first time.

[0110] 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 partially filled with 100% absolute ethanol to prevent the samples from drying out during transfer. The samples were then critical point dried using a Quorum E3100 critical point dryer using four to five 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 of 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).

[0111] 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 hour and then stored at 4°C for 12 hours.

[0112] 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).

[0113] The rheological properties of SPI hydrogels as a function of HO:acetic acid cosolvent ratio are shown in Figure 3. 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 (90% v / v) also resulted in the formation of weak hydrogels. Hydrogels formed using cosolvent ratios of 30–70% v / v resulted in stronger hydrogels, with hydrogels prepared using 30 and 50% acetic acid (v / v) exhibiting G' values ​​exceeding 2500 Pa.

[0114] FTIR The changes in the secondary structure of the hydrogel under various solvent ratios were investigated. Structural analysis of the SPI hydrogel was performed using an FTIR-Equinox 55 spectrometer (Bruker). The hydrogel samples were used without further pretreatment, mounted on an FTIR holder, and analyzed by subtracting a 50% v / v (DI water / acetic acid) standard. The atmospherically corrected 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 (an average of nine replicates per sample). The instrument sensitivity 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 closely correlated with protein secondary structure, were tracked.

[0115] The results of FTIR measurements are shown in Figure 4. The SPI hydrogel has a high content of α-helix (1656 cm -1 ) intermolecular parallel β-sheet (1625cm -1 ) secondary structure is evident from second derivative analysis. [Example]

[0116] The hydrogel was prepared according to the following process using lactic acid as a cosolvent.

[0117] 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. To solubilize the protein, the mixture was subjected to ultrasonic treatment for 30 minutes (high-frequency power output = 70 W, frequency = 20 kHz, amplitude = 90%). During this process, the sample temperature was maintained 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.

[0118] 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.

[0119] The results are shown in Figure 5 and show that increasing the amount of lactic acid resulted in a higher degree of protein hydrolysis.

[0120] 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. [Example]

[0121] 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, maintained at 85 °C) was filled into a 1.5 ml tube and quickly placed on an 85 °C heating block. To prevent gelation of the SPI solution during transport to the microfluidic device, a custom-made silicone heater (Holroyd Components) with a 1 / 32-inch inner diameter stainless steel tube 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 using a pressure-driven system (Elveflow OB1). Various pressure rates were tested until uniform and continuous production 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 resulting droplets were collected in a 1 ml pipette tip and stored at room temperature for 12 hours to ensure completion of the gelation process. The formed microgels were then washed using a standard demulsification procedure. First, the continuous oil phase containing the fluorosurfactant was removed from the vial. For 500 μl 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 subsequent series of washing steps was 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 microgel samples for SEM analysis, the microgels were washed with 100% ethanol in a three-step procedure. First, the microgels were suspended in a 25% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 3 hours. After centrifugation and removal of the supernatant, the microgels were then resuspended in a 50% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 3 hours. Finally, after centrifugation and removal of the supernatant, the microgels were then resuspended in a 100% v / v ethanol-aqueous solution and left under constant stirring (100 rpm) for 12 hours.

[0122] The results of this experiment are shown in Figure 6. Figure 6a shows a schematic of microbead formation. Figure 6b shows stable microgels suspended in an aqueous solution (pH = 2). Figure 6d shows an SEM image of microbeads prepared by supercritical point drying. Figure 6e shows an SEM image of the gel network on the surface of the microbeads.

[0123] The resulting microgels were stable in aqueous solution 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.

[0124] These results demonstrate that mechanically robust microgels can be produced from plant proteins by simply controlling the self-assembly of protein aggregates at the nanoscale and in the complete absence of cross-linkers or any deleterious substances. [Example]

[0125] Fabrication of core-shell microcapsules Having demonstrated that mechanically robust microgels can be produced, the use of hydrogels for microencapsulation was explored.

[0126] For the fabrication of coaxial flow-focusing microfluidic devices, we followed a multi-step photolithography method described in Tran, T. M., Cater, S. & Abate, A. R., "Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices." Biomicrofluidics 8, 1–7 (2014), but will be briefly described below.

[0127] 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 separate PDMS plates were peeled from the masters and then aligned and bonded by plasma oxidation. A small amount of sprayed water droplets was placed between the two PDMS plates after plasma oxidation to allow alignment of the microfluidic channels. The final aligned PDMS device was baked overnight in an oven at 65 °C to complete the bonding of the two layers.

[0128] The microfluidic process for producing core-shell microcapsules was identical to 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 approximately 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.

[0129] It is noted that the inner core material remains separate from the shell material in this example, which differs 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 initially present, 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.

[0130] A schematic diagram of a microfluidic droplet generator is shown in Figure 7a, which depicts a 3D flow-focusing microfluidic device. Core-shell microcapsules produced in accordance with the present invention, containing a lipophilic core suspended in an aqueous solution, are shown in Figure 7b. The microcapsule shell contains solely self-assembling SPI proteins, and the core is a water-in-oil microemulsion (lipophilic). [Example]

[0131] Fabrication of core-shell microcapsules 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).

[0132] Core-shell microcapsules made in accordance with the present invention, containing a hydrophilic core suspended in an aqueous solution, are shown in Figure 8. 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. [Example]

[0133] Controlled-release microcapsules Since the protein shell is solely composed of protein aggregates, experiments to determine whether cargo release can be triggered by degradation of the protein shell in the presence of digestive enzymes were performed via a two-stage in vitro digestibility test.

[0134] One liter 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. One liter 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.

[0135] 50 μl of core-shell microcapsules prepared 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 minutes. For the simulated intestinal phase, 3 mg of pancreatin was dissolved in 1 ml of SIF electrolyte solution. 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 minutes.

[0136] The results of the experiment are shown in Figure 9, where Figure 9a shows the microcapsules in aqueous solution, Figure 9b shows the microcapsules still intact after 60 minutes in SGF, and Figure 9c shows the release of microcapsules after 120 minutes in SIF.

[0137] The experiments demonstrate that incubation under SGF conditions (8 mg / ml pepsin, pH = 2) caused 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) resulted in significant degradation of the protein shell and complete release of the lipophilic core. Control experiments in the absence of enzyme but under the same pH conditions showed that protein shell degradation was primarily caused by enzymatic digestion.

[0138] 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. [Example]

[0139] Controlled-release microcapsules containing active agents The experiment of Example 6 was repeated, but using microcapsules made according to Example 5 containing a hydrophilic core containing the active agent riboflavin.

[0140] Riboflavin microcapsules are shown in Figure 10a (i.e., microcapsules with a hydrophilic core (1% HMP pectin + riboflavin)). Figure 10b shows the results of a two-stage in vitro digestibility study generated by HPLC analysis showing the cumulative release of riboflavin under simulated conditions. Under simulated gastric and small intestinal conditions, it can be seen that there is a controlled release of riboflavin 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. [Example]

[0141] 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 a 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 a 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 a 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 minute), and the ethanol supernatant was removed. Next, 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 penetrated the microgel porous network. After adding 500 μl of deionized water to the oil-microgel suspension, the oil-loaded SPI microscale sponge was then transferred to the aqueous phase. The remaining oil phase was removed from the Eppendorf tube.

[0142] Figure 11a shows the distribution of fragrance oil-loaded vegetable protein microscale sponges, and Figure 11b shows a single fragrance-loaded vegetable protein microscale sponge at higher magnification (20x), where the protein microgel shell is easily observable.

[0143] 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 production techniques such as core-shell construction. [Example]

[0144] Film preparation 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: 100 mg / ml). The solution was then subjected to ultrasonic treatment for 30 minutes (high-frequency power output: 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 remainder 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 of the glass Petri dish was covered with a Teflon sheet to facilitate easy peeling of the film.

[0145] A schematic of the process of Example 9, along with the resulting film, is shown in Figure 12. It can be seen that mechanically robust transparent thin films are produced.

[0146] 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 produced using 10%, 20%, 30%, 40%, and 50% (w / w) glycerol.

[0147] 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 1mm gap length. The holder and film were placed in the mechanical tester, and the paper holder was then cut before measurement to ensure that the load was applied only to the film. Measurements were performed at a speed of 2mm / min. Film thickness was measured on each sample using a digital caliper. Typical film thicknesses were 30-50µm.

[0148] The stress-strain curves of the films of the present invention are shown in Figure 13a. The Young's modulus of the film is shown in Figure 13a, 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 13b, with the maximum elongation being at 30% plasticizer.

[0149] 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-assembly 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). [Example]

[0150] Microstructure and Nanostructure Patterning The film prepared according to Example 9 was micropatterned according to the following process.

[0151] To pattern microstructures such as micropillars into the protein film, a negative pattern of a 20 μm × 20 μm micropillar array was fabricated on a silicon wafer by standard photolithography techniques using SU-8 3025 as a photoresist. A 10:1 mixture of polydimethylsiloxane elastomer and curing agent (Sylgard 184, Dow Corning) was poured onto the wafer and cured at 65°C for 1 hour. The cured PDMS was peeled off from the wafer and used as a negative pattern for the soy protein film. The soy protein solution prepared in Example 9 was poured into the PDMS and allowed to dry overnight. The SPI thin film was peeled off from the PDMS master, and the resulting micropatterned structure was observed using scanning electron microscopy (SEM) (MIRA 3 FEG-SEM, TESCAN) with a 10 nm platinum coating. The contact angle of the film was measured using a First Ten Angstroms FTA1000B.

[0152] By simply injecting 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 than that of the control film sample with a non-micropatterned surface (Figure 14a). This clearly demonstrates that the hydrophobic surface properties can be greatly enhanced by simply patterning micropillars, such as those naturally found in lotus leaves, on the surface of a plant protein film.

[0153] To generate nanostructured protein films with photonic properties, DVD discs were used as molding substrates. First, the plastic outer layer was carefully removed from the DVD disc, and the remaining intermediate layer was used. PDMS was poured onto the DVD substrate and cured at 65°C for 1 hour. The patterned PDMS was peeled off from the DVD substrate, and the film-forming soy protein solution prepared in Example 9 was injected into the PDMS substrate and allowed to dry overnight at room temperature. The SPI thin film was peeled off from the PDMS master, and the resulting nanopatterned structure was observed using scanning electron microscopy (SEM) (MIRA 3 FEG-SEM, TESCAN) with a 10 nm platinum coating. The photonic properties (Mie scattering) of the film could be easily observed with the naked eye (Figure 14b). [Example]

[0154] Preparation of the coating To test the coating properties of the vegetable protein film, small pieces of cardboard (2 x 2 cm) were immersed in the film-forming SPI solution prepared in Example 9 in the absence of plasticizer. After immersion in the film-forming solution for approximately 5 seconds, the cardboard was removed from the solution and air-dried overnight at room temperature. 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.

[0155] As control samples, uncoated cardboard pieces and soy protein monomer-coated cardboard (prepared by immersing the cardboard in a soy protein solution prepared under alkaline conditions (pH = 9)) were tested. The water absorption was normalized using the initial mass measured before immersing the cardboard sample in water.

[0156] As observed in Figure 15, 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 nearly 50% compared to the uncoated control sample or the cardboard sample coated with the 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 interaction exhibit enhanced water-blocking properties. [Example]

[0157] Hard capsule manufacturing Soy protein hard capsules were fabricated 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 minutes and then exposed to ultrasonic treatment for 5 minutes (radio frequency power output = 70 W, frequency = 20 kHz, amplitude = 90%). The solution was maintained at 85°C to prevent gelation. A 2 ml Eppendorf tube was immersed in the heated solution, then kept in the liquid solution for 5 seconds and then removed. A thin layer of SPI hydrogel formed around the outer surface of the 2 ml Eppendorf tube within a few seconds of removing it from the liquid SPI solution (Figure 16a). 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, producing a three-dimensional thin film. After evaporation of the solvent, the hard capsule was removed from the Eppendorf tube (Figure 16b).

[0158] This is the first demonstration of the formation of hard capsules produced from plant proteins, a process made possible by the thermoreversible properties of these plant materials, which allow the formation of thin, three-dimensional hydrogels onto a substrate.

[0159] 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.

[0160] (References) 1. Ahnert, SE, Marsh, JA, Hernandez, H., Robinson, CV & Teichmann, SA Principles of assembly reveal a periodic table of protein complexes. Science (80-. ). 350, (2015). 2. Mezzenga, R. & Fischer, P. The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions. Reports Prog. Phys. 76, (2013). 3. Knowles, TPJ & Mezzenga, R. Amyloid Fibrils as Building Blocks for Natural and Artificial Functional Materials. Adv. Mater. 6546-6561 (2016). doi:10.1002 / adma.201505961 4. Shimanovich, U. et al. Protein Microgels from Amyloid Fibril Networks. ACS Nano 9, 43-51 (2014). 5. Zhou, X. M. et al. Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry. ACS Nano 9, 5772-5781 (2015). 6. Shen, Y. et al. Amyloid fibril systems reduce, stabilize and deliver bioavailable nanosized iron. Nat. Nanotechnol. 12, 642-647 (2017). 7. Nystrom, G., Fong, W. K. & Mezzenga, R. Ice-Templated and Cross-Linked Amyloid Fibril Aerogel Scaffolds for Cell Growth. Biomacromolecules 18, 2858-2865 (2017). 8. Amagliani, L. & Schmitt, C. Globular plant protein aggregates for stabilization of food foams and emulsions. Trends Food Sci. Technol. 67, 248-259 (2017). 9. Munialo, C. D., Martin, A. H., Van Der Linden, E. & De Jongh, H. H. J. Fibril formation from pea protein and subsequent gel formation. J. Agric. Food Chem. 62, 2418-2427 (2014). 10. Munialo, C. D., van der Linden, E. & de Jongh, H. H. J. The ability to store energy in pea protein gels is set by network dimensions smaller than 50nm. Food Res. Int. 64, 482-491 (2014). 11. Lee, K. H., Ryu, H. S. & Rhee, K. C. Protein solubility characteristics of commercial soy protein products. JAOCS, J. Am. Oil Chem. Soc. 80, 85-90 (2003). 12. Lee, H. et al. Soy protein nano-aggregates with improved functional properties prepared by sequential pH treatment and ultrasonication. Food Hydrocoll. 55, 200-209 (2016). 13. Jiang, S. et al. Modifying the physicochemical properties of pea protein by pH-shifting and ultrasound combined treatments. Ultrason. Sonochem. 38, 835-842 (2017). 14. Mason, T. O. et al. Expanding the solvent chemical space for self-assembly of dipeptide nanostructures. ACS Nano 8, 1243-1253 (2014). 15. Li, Y. et al. Understanding the dissolution of α-zein in aqueous ethanol and acetic acid solutions. J. Phys. Chem. B 116, 12057-12064 (2012). 16. Akkermans, C. et al. Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate. J. Agric. Food Chem. 55, 9877-9882 (2007).

Claims

1. 1. A method for producing plant material, comprising: 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) causing a sol-gel transition of the protein in the solution to form a plant protein hydrogel; A method comprising:

2. c) forming a structured material from the plant protein hydrogel 2. The method of claim 1, comprising:

3. 3. The method of claim 2, wherein the structured material is formed from the plant protein hydrogel before, during, or after the sol-gel transition.

4. 4. The method of any one of claims 1 to 3, wherein the vegetable protein hydrogel is molded into a structured material or a microfluidic device is used to form a structured material from the vegetable protein hydrogel.

5. 5. The method according to any one of claims 1 to 4, used to produce structured materials, such as films, thin films, micropatterned films (or thin films), micro- or nanostructured thin films, microgels, microcapsules, microbeads, biological scaffolds, biological supports, sponges, microscale sponges, hard capsules, or functional coatings.

6. 6. The method according to any one of claims 1 to 5, wherein the plant protein is obtained from soybean, pea, rice, potato, wheat, maize zein, or sorghum, preferably the plant protein is selected from soybean protein, pea protein, potato protein, and / or rice protein.

7. 7. The method according to claim 1, wherein the first co-solvent is an organic acid, preferably acetic acid and / or an alpha-hydroxy acid, which may preferably be selected from glycolic acid, lactic acid, malic acid, citric acid, and / or tartaric acid, with particularly preferred organic acids being acetic acid and / or lactic acid.

8. 8. The method according to claim 1, wherein the second or further co-solvent is an aqueous buffer solution, preferably selected from water, ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate or hexafluoroisopropanol, particularly preferably water and / or ethanol, more particularly preferably water.

9. 9. The method of any one of claims 1 to 8, wherein the solvent system comprises a co-solvent ratio of about 20-80% v / v, preferably about 20-60% v / v, about 25-55% v / v, about 30-50% v / v, about 20%, about 30%, about 40%, about 50%, or about 60% v / v, most preferably about 30-50% v / v.

10. 10. The method of any one of claims 1 to 9, further comprising mechanical shearing, preferably sonication, of the protein solution comprising one or more plant proteins and a solvent system.

11. 11. The method of claim 1, wherein the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant proteins and then reduced to a second temperature below the sol-gel temperature of the one or more plant proteins to form a hydrogel.

12. 12. A vegetable hydrogel formed using the method of any one of claims 1 to 11.

13. A plant-based structured material that 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.

14. A plant-based structured material formed via a thermoreversible cool-set gelation process, which can optionally be a film, thin film, micropatterned film (or thin film), micro- or nanostructured thin film, microgel, microcapsule, microbead, bioscaffold, biosupport, sponge, microscale sponge, hard capsule, or functional coating.

15. Plant-based thermoreversible hydrogel.

16. 16. A composite material comprising plant material as defined in any one of claims 1 to 15 and one or more further biopolymers, such as proteins and / or polysaccharides.

17. A food, cosmetic, pharmaceutical, medical device, or biological material incorporating the plant-based material of claim 12, the plant-based structuring material of claim 13 or 14, the plant-based thermoreversible hydrogel of claim 15, or the composite material of claim 16.

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