PROCESSO TERMORREVERSÍVEL PARA OBTER HIDROGÉIS COM BASE EM PLANTA

Description

1 / 55 “THERMO-REVERSIBLE PROCESS FOR OBTAINING PLANT-BASED HYDROGELS”

[001] Divided from BR1120210156755, deposited on 06 / 03 / 2020. FIELD OF THE INVENTION

[002] The present invention relates to plant-based materials, methods for their manufacture and biomaterials incorporating plant-based materials according to the present invention. BACKGROUND OF THE INVENTION

[003] Such synthetic polymers, such as plastic, exhibit excellent mechanical and chemical properties and have been widely used over the last 60 years. However, these polymers are not biodegradable and can accumulate in the environment, causing economic damage and likely affecting human health through the food chain and air.

[004] The development of materials that exhibit a high level of functionality, as well as a high degree of biodegradability and biocompatibility, is a fundamental objective to satisfy a social need for enhanced material performance in areas ranging from packaging to pharmaceuticals.

[005] Self-assembly has emerged as an attractive routine for the manufacture of such materials, but most of the building blocks explored so far have been of synthetic origin.

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

[007] Currently, the use of these materials for commercial application is restricted to highly soluble animal-derived proteins. Animal-derived proteins Petition 870260062218, dated 06 / 25 / 2026, page 10 / 125 2 / 55 Animal proteins commonly used in food products, such as whey protein, exhibit good biocompatibility, biodegradability, amphiphilic and functional properties, such as water solubility, emulsification capacity and foaming ability. However, there is a growing demand for the replacement of animal-derived proteins with plant-derived proteins, not only due to their lower environmental impact, but also due to their lower allergenicity and reduced cost.

[008] The formation of self-assembled materials from plant-based proteins has been reported, where hydrogels can be obtained from soy and pea proteins under a range of experimental conditions. However, the mechanical properties obtained from structured plant-based materials are generally lower compared to those obtained from animal-derived materials; and plant proteins are more difficult to process, at least in part due to their inherently low solubility in water.

[009] Thus, to date, plant-based proteins have not been successfully used as a biomaterial and it remains a challenge to generate structured protein materials from renewable and economical sources, and with environmentally sustainable methods. SUMMARY OF THE INVENTION

[0010] According to one aspect of the present invention, a method is provided for producing plant-based materials comprising: a) to form a solution comprising one or more plant-based protein(s) in a solvent system, wherein the solvent system comprises miscible cosolvents; wherein a first cosolvent increases the solubility of the plant-based protein(s) and a second cosolvent decreases the solubility of the plant-based protein(s); and b) induce the protein in solution to undergo a sol-gel transition to form Petition 870260062218, dated 06 / 25 / 2026, page 11 / 125 3 / 55 a plant-based protein hydrogel.

[0011] In another version, the method includes the step: c) to form a plant-based protein hydrogel into a structured material.

[0012] Forming plant-based protein hydrogel into a structured material allows the formation of structured materials such as gels, films, microgels, microcapsules, and the like. In a preferred embodiment, the protein hydrogel can be formed into a specific shape by molding. In another preferred embodiment, the protein hydrogel can be formed into a specified shape using a microfluidic device.

[0013] The present invention has identified a novel method for manufacturing functional materials from plant-based proteins. By using co-solvent mixtures, it is possible to control the sol-gel transition, allowing the formation of structurally robust materials from renewable plant protein sources. The process allows 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 crosslinkers or any other hazardous materials, making them suitable for contact with the human body. The materials are also derived from renewable sources, therefore reducing the environmental impact compared to synthetic analogues.

[0014] In another aspect, the use of cosolvent mixtures to modify the properties of a plant-based protein in solution is provided to control sol-gel conditions and thus form plant-based materials.

[0015] When selecting a solvent system, it comprises miscible cosolvents, where a first cosolvent increases the solubility of the protein(s) Petition 870260062218, dated 06 / 25 / 2026, page 12 / 125 4 / 55 based on plant-based and a second cosolvent decreases the solubility of the plant-based protein(s), it is possible to control the sol-gel conditions.

[0016] The ratio of the first cosolvent to the second cosolvent can vary from about 20-80% v / v, about 20-60% v / v, about 25-55% v / v, about 30-50% v / v, about 20%, about 30%, about 40%, about 50% or about 60% v / v, more preferably about 30-50% v / v. Such ratios lead to functionally useful materials.

[0017] The solvent system may contain one or more first cosolvent(s) and / or one or more second cosolvent(s).

[0018] For the first time, plant-based materials can be reliably and reproducibly formed using a scalable process. By being able to control the sol-gel conditions, it is possible to adjust the properties of the resulting material and / or adjust the manufacturing process to enable the production of useful biomaterials.

[0019] The process according to the present invention allows the formation of plant-based structured materials formed by means of a cold thermoreversible gelation process. The plant-based structured materials may be plant-based protein supramolecular structures; or may be a three-dimensional network of aggregated and entangled plant-based protein supramolecular structures.

[0020] A cold thermoreversible gelation process can be considered a process in which plant-based protein molecules can be heated above a temperature to form a liquid solution that can be processed into the desired configuration before cooling the liquid solution to allow a sol-gel transition forming a network of self-assembled protein aggregates held together by non-covalent intermolecular interactions. Thus, the gelation process according to the present invention does not Petition 870260062218, dated 06 / 25 / 2026, page 13 / 125 5 / 55 requires covalent chemical crosslinking and is therefore reversible. The present invention includes a thermoreversible cold gelation process.

[0021] In this way, in another aspect, a structured plant-based material is provided formed by means of a cold thermoreversible gelation process; wherein the structured material may optionally be a film, a thin film, a micropatterned film (or thin film), a micro- or nanostructured thin film, a microgel, a microcapsule, a microbead, a biosupport, a sponge, a microscale sponge, a hard capsule or a functional coating.

[0022] In another aspect, a thermally reversible plant-based gel is provided.

[0023] In another aspect, a thermally reversible hydrogel based on a plant is provided.

[0024] In another aspect, a composite material is provided comprising a plant-based material according to the present invention and one or more other biopolymers, for example, proteins, polysaccharides and the like.

[0025] In another aspect, materials made according to the method of the present invention are provided.

[0026] The plant-based material of the present invention and the processes for manufacturing it allow precise control of the sol-gel transition, thereby opening up the use of plant-based proteins to form biomaterials that, until now, have only been successfully manufactured using animal-derived proteins. Suitable biomaterials include films, microbeads, microcapsules, supports, gels, sponges, and the like.

[0027] In another aspect, a microbead is provided comprising a plant-based material according to the present invention.

[0028] In another aspect, a microcapsule is provided that Petition 870260062218, dated 06 / 25 / 2026, page 14 / 125 6 / 55 comprises a plant-based material according to the present invention.

[0029] In another aspect, a hard capsule comprising a plant-based material is provided according to the present invention.

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

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

[0032] In another aspect, a nanostandard or microstandard film comprising a plant-based material according to the present invention is provided.

[0033] In another aspect, a biosupport comprising a plant-based hydrogel is provided according to the present invention.

[0034] In another aspect, a functional coating comprising a plant-based hydrogel is provided according to the present invention.

[0035] In another aspect, a food product, a cosmetic, a pharmaceutical product or a medical device is provided that incorporates a plant-based material according to the present invention.

[0036] In another aspect, a thermoreversible process is provided for obtaining plant-based materials comprising: a) to form a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible cosolvents; wherein a first cosolvent increases the solubility of the plant-based protein(s) and a second cosolvent decreases the solubility of the plant-based protein(s). Petition 870260062218, dated 06 / 25 / 2026, page 15 / 125 7 / 55 solubility of the plant-based protein(s); and b) induce the protein in solution to undergo a sol-gel transition to form a plant-based protein hydrogel.

[0037] Once the material has been obtained, the thermoreversible properties can be removed from the material. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows the hydrogels of soy protein isolates (SPI) formed under various acetic acid / water DI cosolvent ratios;

[0039] Figure 2 shows a SEM (scanning electron microscopy) image of a hydrogel produced using a cosolvent ratio of 30% v / v;

[0040] Figure 3 shows the estimated particle size distributions of SPI by dynamic light scattering (DLS) for SPI dispersions in water (a), in NaOH pH10 (b), in HCl pH2 (c), and in 30% acetic acid (d). For each solvent, samples were prepared by heating at 95°C for 30 minutes (noted as heated) or sonication for 30 minutes (noted as sonicated). All measurements were performed with a protein concentration of 0.1% and repeated twice. The inset shows the crude correlogram for each condition;

[0041] Figure 4a shows the rheological properties of SPI hydrogels as a function of the H2O:acetic acid cosolvent ratios; Figure 4b shows the shear dilution behavior of SPI hydrogels prepared at different concentrations; Figure 4c shows the thermoreversible rheological behavior of SPI hydrogels at different temperatures;

[0042] Figures 5a and 5b show structural changes in the secondary structure of the SPI hydrogel under different H2O:acetic acid cosolvent ratios, calculated from the amide I bands in the FTIR spectra; Figure Petition 870260062218, dated 06 / 25 / 2026, page 16 / 125 8 / 55 Figure 5c shows the structural changes in the secondary structure of the SPI hydrogel under different temperatures, calculated from the amide I bands in the FTIR;

[0043] Figure 6 shows an SDS-PAGE electrophoretogram showing an increasing degree of protein hydrolysis with increasing amounts of acetic acid;

[0044] Figure 7 shows microspheres formed using hydrogels of the present invention. 6a shows a schematic representation of microbead formation; 6b shows stable microbeads suspended in aqueous solution (pH = 2); 6c shows SEM image of microbeads prepared by supercritical point drying; and 6d shows the SEM image of the gel network on the surface of a microbead;

[0045] Figure 8a shows a schematic representation of a multilayer 3D microfluidic droplet generator used to generate core-shell microcapsules with a lipophilic and hydrophilic core. Figure 8b shows core-shell microcapsules containing a lipophilic core suspended in aqueous solution;

[0046] Figure 9 shows core-shell microcapsules containing a hydrophilic core containing a suspension of a soluble active ingredient (Riboflavin);

[0047] Figure 10 shows the results of the simulated degradation, with Figure 10a showing the microcapsules in aqueous solution; Figure 10b showing the microcapsules after 60 minutes in SGF (simulated gastric fluid); and Figure 10c showing the microcapsules after 120 minutes in SIF (simulated intestinal fluid);

[0048] Figure 11a shows core-shell microcapsules containing a core consisting of a riboflavin solution in 1% (w / w) HMP pectin; Figure 11b shows the results of a two-stage in vitro digestibility study followed by HPLC analysis showing the cumulative release of Petition 870260062218, dated 06 / 25 / 2026, page 17 / 125 9 / 55 riboflavin under simulated conditions;

[0049] Figure 12a shows a microscale distribution of plant-based protein sponges loaded with fragrance oil. Figure 12b shows a single fragrance-loaded plant-based protein microscale sponge at higher magnification (20x), where the protein microgel shell can be easily observed;

[0050] Figure 13 shows a schematic representation of an example generating stable protein films;

[0051] Figure 14a shows stress-strain curves for films made according to the present invention; Figure 14b shows the Young's Modulus and Figure 14c shows % elongation breakage; and

[0052] Figure 15 shows the FTIR spectra of an SPI film prepared without the use of glycerol and a spectrum of a dry sample of commercial SPI;

[0053] Figure 16a shows a TEM image of a dried dilute SPI solution on a TEM grating, confirming the presence of a substantial amount of β-sheet crystals (scale bar: 5 nm). Figure 16b shows a close-up image of β-sheet crystals from an SPI film (scale bar: 2 nm);

[0054] Figure 17a shows the micropattern of a uniform arrangement of micropillars on a soy protein film surface, which significantly increases the contact angle of a water droplet (99°) compared to a non-micropattern control sample. Figure 17b shows the nanopatterning of a uniform array of nanochannels obtained by molding a soy protein film onto a DVD disc. Nanostructured motifs in the soy protein film exhibit photonic properties (Mie scattering);

[0055] Figure 18 shows a schematic process for producing Petition 870260062218, dated 06 / 25 / 2026, page 18 / 125 10 / 55 coatings of soy protein film on a cardboard substrate. The substrate coated with soy protein film shows a 50% decrease in water uptake when compared to an uncoated control sample;

[0056] Figure 19a shows the formation of a thin three-dimensional hydrogel layer around a 2 ml Eppendorf tube substrate. Figure 19b shows a hard soy protein capsule obtained after drying the molded three-dimensional hydrogel and removing it from the substrate;

[0057] Figure 20a shows a pea protein hydrogel produced using a cosolvent ratio of 30% v / v; Figure 20b shows a SEM (scanning electron microscopy) image of a pea protein hydrogel produced using a cosolvent ratio of 30% v / v; Figure 20c shows an isolated pea protein film. Figure 20d shows stable pea protein microgels suspended in aqueous solution (pH = 2);

[0058] Figure 21 shows a potato protein hydrogel prepared from a solution of 100 mg / ml potato protein isolate in a 30% (v / v) aqueous acetic acid solution;

[0059] Figure 22 shows stress-strain curves for other films made according to the present invention;

[0060] Figure 23 shows optical images of gelation using acetic acid according to the present invention and HCl or NaOH. DETAILED DESCRIPTION

[0061] The following characteristics apply to all aspects of the invention.

[0062] Any suitable plant-based proteins can be used in the present invention. Suitable plant sources include soybean, pea, rice, potato, wheat, corn zein, sorghum, and the like. Specific plant proteins include soybean proteins and pea proteins. Petition 870260062218, dated 06 / 25 / 2026, p. 19 / 125 11 / 55

[0063] Suitable plant-based proteins also include: - Brassicas: including 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 and leaf mustard, Sarepta mustard, Brassica napus: rapeseed, canola, rutabaga, Brassica narinosa: broad-beaked mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, kai-lan, Brussels sprouts, kohlrabi, Brassica perviridis: tender green, mustard spinach, Brassica rapa (syn. B. campestris): Chinese cabbage, turnip, rapini, komatsuna, Brassica rupestris: brown mustard, Brassica tournefortii: Asian mustard - Solanaceae: including tomato, potato, eggplant, bell pepper and chili pepper; - Cereals: including corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, fonio - Pseudocereals: including amaranth (love-lies-bleeding, red amaranth, pince-of-Wales-feather), bread knots, buckwheat, chia, celosia (also called quail grass or soko), pitseed, goosefoot, qaniwa, quinoa, and wattleseed (also called acacia seed); - Legumes: including Acacia alata (winged acacia), Acacia decipiens, Acacia saligna (commonly known by various names, including coojong, golden acacia, orange acacia, blue-leaf acacia), Arachis hypogaea (peanut), Astragalus galegiformis, Cytisus laburnum (common laburnum, golden chain or golden rain), Cytisus supinus, Dolichos lablab (common names include hyacinth bean, bonavista bean / pea, dolichos bean, seim bean, lablab bean, Egyptian bean, Indian bean, bataw bean and Australian pea), Ervun lens (lentil), Genista tinctorial (common names include dyer's whin, waxen woad and waxen wood), Glycine max (soybean), Lathyrus clymenum (peas or vetch), Lathyrus odoratus (peas or Petition 870260062218, dated 06 / 25 / 2026, p. 20 / 125 12 / 55 vetches) Lathyrus staivus (peas or vetches), Lathyrus siltris (peas or vetches), Lotus tetragonolobus (asparagus pea or winged pea), Lupinus albus (Lupinus), Lupinus angustifolius (lupin), Lupinus leteus (Lupinus), Lupinus polyphyllus (Lupinus), Medicago sativa (Alfalfa), Phaseolus aureus (mung bean), Phaseolus coccineus (Runner bean), Phaseolus nanus (green bean / French bean), Phaseolus vulgaris (green bean / French bean), Pisum sativum (pea), Trifolium hybridum (clover), Trifolium pretense (red clover), Vicia faba (broad bean), Vicia sativa (vetch), Vigna unguiculata (black-eyed pea) - Non-legumes: including: Acanshosicyos horrida (Horse chestnut), Aesculus hyppocastanum (Horse chestnut), Anacardium occidentale (Cashew tree), Balanites aegyptica (Aegyptian palm), Bertholletia excelsa (Brazil nut), Beta vulgaris (Sugar beet), Brassica napus (Rapeseed), Brassica juncea (Brown mustard), Brassica nigra (Black mustard), Brassica hirta (Eurasian mustard), Cannabis sativa (Marijuana), Citrullus vulgaris (Watermelon species), Citrus aurantiaca (Citrus), Cucurbita maxima (Pumpkin), Fagopyrum esculentum (Knotweed), Gossypium barbadense (Extra long cotton), Heianthus annuus (Sunflower), Nicotiana sp. (Tobacco), Prunus avium (cherry), Prunus cerasus (sour cherry), Prunus domestica (plum), Prunus amygdalus (almond), Ricinus communis (bean / castor oil plant), Sasamum indicum (sesame), Sinapis alba (white mustard), Terfalrea pedata (oyster nut).

[0064] For the avoidance of doubt, the plant-based structured materials according to the present invention do not include plants in their natural state. For example, naturally formed plant cells, organelles or vesicles are not plant-based structured materials of the present invention.

[0065] A characteristic of plant-derived proteins is their inherently low solubility in water. Until now, this has limited their use in the generation of biomaterials. However, the present invention has overcome these previous limitations. Petition 870260062218, dated 06 / 25 / 2026, page 21 / 125 13 / 55 associated with such proteins.

[0066] In the methods according to the present invention, the material is formed by adding the plant-based protein to a solvent system, wherein the solvent system comprises two or more miscible cosolvents, as defined herein.

[0067] The first cosolvent increases the solubility of the plant-based protein(s). The first cosolvent can be considered a solubilizing cosolvent. There may be one or more solubilizing cosolvent(s), and the solubilizing cosolvent(s) may totally or partially solubilize the plant-based protein(s).

[0068] Examples of solubilizing cosolvents are organic acids. An organic acid is an organic compound with acidic properties. Suitable organic acids include acetic acid or an α-hydroxy acid. Suitable α-hydroxy acids include glycolic acid, lactic acid, malic acid, citric acid, and tartaric acid. Preferred organic acids are acetic acid and lactic acid. Using an organic acid allows for the solubilization of plant protein and similarly allows for the mild hydrolysis of the protein. For example, without wanting to be limited by theory, the solubility of plant-based proteins in organic acid is possible due to: i) protein protonation and ii) the presence of an anion solvation layer that contributes to a reduction in hydrophobic interactions. Once initially dissolved in organic acid, protonation of plant-based proteins can help stabilize them in their non-solvent, e.g., water.

[0069] In a preferred embodiment, the first cosolvent is an organic acid.

[0070] The second cosolvent decreased the solubility of the plant-based protein(s) compared to the first cosolvent. The second Petition 870260062218, dated 06 / 25 / 2026, page 22 / 125 14 / 55 cosolvent can be considered a desolubilizing cosolvent. There may be one or more desolubilizing cosolvent(s).

[0071] Examples of second desolubilization cosolvent(s) are an aqueous buffer solution. In another embodiment, the second cosolvent 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 cosolvent is water and ethanol. In another particularly preferred embodiment, the second cosolvent is water.

[0072] In a preferred embodiment, the concentration of plant-based protein(s) in the solvent system is 25-200 mg / ml, preferably 50-150 mg / ml. The organic acid ratio may vary depending on the protein concentration, for example using a higher organic acid ratio with increasing protein concentration.

[0073] In a preferred embodiment, the degree of protein hydrolysis is controlled to modify the properties of the resulting hydrogel. For example, increasing the concentration of acid present during formation will increase the degree of protein hydrolysis. A higher degree of protein hydrolysis leads to the formation of less rigid hydrogels.

[0074] In order to form a solution comprising one or more plant-based proteins, it may be necessary to apply physical stimulus to the protein / solvent system mixture to enable protein dissolution. Suitable physical stimulus includes ultrasonication, agitation, high shear mixing, or other physical techniques. A preferred technique is ultrasonication.

[0075] In one embodiment, the solution is subjected to ultrasonication for a period of approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or more than 30 minutes. A preferred ultrasonication period is Petition 870260062218, dated 06 / 25 / 2026, p. 23 / 125 15 / 55 approximately 30 minutes.

[0076] The protein solution is heated so that the liquid solution is maintained above the sol-gel transition temperature for the protein(s). By modifying the solvent system (e.g., by selecting the choice of organic acid, the ratio of organic acid to another solvent, or by other means), it is possible to modify the sol-gel transition temperature for the protein(s). Through the appropriate selection of conditions, it is possible to carefully control the sol-gel transition of the protein, thereby controlling hydrogel formation.

[0077] In one embodiment, the protein solution is heated to about or above 70°C. In another embodiment, the protein is heated to about or above 75°C, about or above 80°C, about or above 85°C, or about 90°C. In a preferred embodiment, the protein is heated to 85°C.

[0078] The protein solution can be maintained at an elevated temperature for a period of time of approximately 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 proteins to fully solubilize. It is possible to maintain the protein solution at an elevated temperature for a longer period of time. This can be useful for a commercial batch process or for use in a fluid processing step where it is necessary to retain the protein solution in liquid form for longer periods of time.

[0079] Having heated the protein solution above the protein sol-gel transition temperature, the protein solution temperature can be reduced to a second temperature below the sol-gel transition temperature to facilitate hydrogel formation. The second temperature can be room temperature. The protein solution can be maintained at the reduced temperature for a period of time of approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes. Petition 870260062218, dated 06 / 25 / 2026, page 24 / 125 16 / 55 minutes or approximately 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 extended periods of time. As such, depending on the need, the protein solution can be maintained above the sol-gel transition temperature for the time required to retain the protein in liquid form. This can take hours, days, or longer. Furthermore, since the reaction is reversible, a solution could, for example, be maintained at a lower temperature (e.g., room temperature), where a hydrogel will form, but subsequently heated above the sol-gel transition temperature to return the solution to the liquid state for further processing. Protein hydrogels in this manner can be stored for hours, days, weeks, months, or years, as the hydrogel remains stable for a long time.

[0080] The specific temperatures will depend on the properties of the protein source, the solvent conditions used, and therefore the sol-gel transition temperature. Alternatively, the elevated and reduced temperatures can be relatively fixed (e.g., around 85°C, then around room temperature) and the cosolvent mixture conditions are adjusted to ensure a suitable sol-gel transition temperature for the selected plant-based protein.

[0081] Thus, in one embodiment, a method is provided for forming a plant-based material comprising: a) to form a protein solution comprising one or more plant-based proteins and a solvent system, wherein the solvent system comprises miscible cosolvents; wherein a first cosolvent increases the solubility of the plant-based protein(s) and a second cosolvent decreases the solubility of the plant-based protein(s); b) subject the protein solution to mechanical agitation for a Petition 870260062218, dated 06 / 25 / 2026, page 25 / 125 17 / 55 time period, for example, ultrasonication; c) raise the temperature of the protein solution to an initial temperature elevated above the sol-gel transition temperature for a period of time. This temperature increase may be caused by mechanical agitation in b) or by an external heat source; d) reduce the temperature of the protein solution to below the sol-gel transition temperature so that the plant-based proteins self-assemble into a hydrogel; and, optionally e) To form the hydrogel into a defined shape, for example microgels, microcapsules, microscale sponges, films, and the like. The forming step can be a molding step, i.e., shaping the hydrogel into a defined form. The forming step can use a microfluidic device.

[0082] The protein solution can be maintained at an elevated temperature in step c) while the solution is shaped into the desired final form. For example, within a microfluidic device, the protein solution can be maintained at an elevated temperature c) within the device's reservoir, but when the solution is expelled from the device, its temperature decreases, thus forming a microgel or a microcapsule shell. Alternatively, the protein solution can be maintained at an elevated temperature while being molded into a suitable mold where, after the temperature decreases, the proteins are allowed to form into a hydrogel.

[0083] Without wishing to be limited by theory, it is believed that when plant protein is added to the solvent system, the plant protein forms a highly viscous dispersion of insoluble colloidal protein aggregates.

[0084] Furthermore, it is believed that the application of mechanical agitation, for example ultrasonication, disrupts large aggregates of colloidal proteins into smaller ones, as well as disrupting the intermolecular interactions of proteins. Petition 870260062218, dated 06 / 25 / 2026, page 26 / 125 18 / 55 Using this approach, the size of protein aggregates can be significantly reduced to particle sizes below 100 nm. In one embodiment, the present invention comprises protein aggregates with an average size of less than 200 nm, preferably less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, or less than 30 nm.

[0085] Furthermore, it is believed that by heating the protein solution in the presence of a cosolvent system above the sol-gel temperature, plant-based proteins partially unfold, resulting in the exposure of hydrophobic amino acids initially buried within the native protein structure. Once partially unfolded, the cosolvents are able to interact with the unfolded protein molecules. For example, an organic acid has greater access to protonated amino acid residues, as well as enabling the formation of anionic salt bridges that stabilize hydrophobic interactions. In addition, after heating at elevated temperatures, non-covalent protein-protein intermolecular contacts are disrupted.Furthermore, it is believed that after cooling the protein solution below the sol-gel temperature, non-covalent protein-protein intermolecular contacts are activated, thereby promoting the self-assembly of plant protein molecules into a network of supramolecular aggregates.

[0086] It is believed that the process of the present invention allows plant-based proteins to aggregate into supramolecular structures maintained by intermolecular hydrogen bond interactions and, in particular, between β strands.

[0087] The process of the present invention allows materials to be formed in which there are high levels of intermolecular β-sheet interactions. This leads to new materials that have not been made before. Petition 870260062218, dated 06 / 25 / 2026, page 27 / 125 19 / 55

[0088] In one embodiment, the plant-based materials according to the present invention have a protein secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet. In one embodiment, the plant-based material is a film. In one embodiment, the plant-based material is a dry material, for example, a dry hydrogel. In one embodiment, the plant-based material is a hydrogel or other material described herein. It is believed that prior gels made from plant-based protein sources may have lower amounts of intermolecular β-sheets in the secondary structure, leading to disadvantageous properties of the prior art.

[0089] The plant-based materials according to the present invention comprise β-sheet crystals. The plant-based materials according to the present invention may exhibit a high degree of β-sheet crystal structures. The plant-based materials may comprise at least 40% β-sheet crystals, at least 50% β-sheet crystals, at least 60% β-sheet crystals, at least 70% β-sheet crystals, at least 80% β-sheet crystals, or at least 90% β-sheet crystals. In one embodiment, the plant-based material is a film. In one embodiment, the plant-based material is a dry material, for example, a dry hydrogel. In one embodiment, the plant-based material is a hydrogel or other material described herein.

[0090] Plant-based materials having said high intermolecular β-sheet secondary structures and said plant-based materials comprising β-sheet crystals may be any of the materials described in the present invention, including structured materials such as Petition 870260062218, dated 06 / 25 / 2026, page 28 / 125 20 / 55 microcapsules, microbeads, bioscaffolds, biosupports, sponges, microscale sponges, hard capsules or functional coatings and the like. They may also be films, thin films, micropatterned films (or thin films), micro- or nanostructured thin films or microgels and the like.

[0091] Thus, the present invention encompasses, for example, plant-based films having a protein secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet. The same applies to other materials according to the present invention.

[0092] In addition, the present invention encompasses, for example, plant-based films comprising plant-based β-sheet crystals, including at least 40% β-sheet crystals, at least 50% β-sheet crystals, at least 60% β-sheet crystals, at least 70% β-sheet crystals, at least 80% β-sheet crystals, or at least 90% β-sheet crystals. The same applies to other materials according to the present invention.

[0093] 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 biosupport, a sponge, a microscale sponge, a hard capsule or a functional coating.

[0094] The materials according to the present invention have advantageous mechanical properties. For example, the ability to reversibly change from gel to liquid until the temperature change allows for advantageous manufacturing capabilities. Petition 870260062218, dated 06 / 25 / 2026, page 29 / 125 21 / 55

[0095] In one embodiment, the hydrogels produced according to the present invention have a storage modulus (G') at 10rad / s greater than 500Pa, greater than 1000Pa, greater than 2500Pa, greater than 3000Pa, greater than 4000Pa.

[0096] In one embodiment, hydrogels exhibit shear dilution behavior, whereby viscosity decreases with increasing shear rates.

[0097] In one embodiment, the hydrogels produced according to the present invention exhibit a unique thermoreversible gelling behavior. Upon heating at elevated temperatures and / or by applying mechanical agitation, the protein gels return to a liquid form. This is a unique property not observed with previous hydrogels, which would not return to a fully liquid state after heating. In contrast, the gels made according to the present invention can. In one embodiment, upon heating at elevated temperatures and / or by applying mechanical agitation, the protein solutions 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 present invention to have unique manufacturing capabilities.

[0098] Similarly, if desired, thermoreversibility can be removed by removing the solvent system of the present invention. For example, plant-based microcapsules can be made using the methods of the present invention. Once formed, the solvent system can be removed by washing, which will interrupt the thermoreversible properties of the microcapsules. Thus, if the microcapsules are subsequently heated, they will remain stable and will not melt again. This allows, for example, the microcapsules according to the present invention to be subjected to high-temperature processes while remaining intact and stable. Petition 870260062218, dated 06 / 25 / 2026, p. 30 / 125 22 / 55

[0099] Thus, the hydrogels formed according to the present invention have unique properties not seen in previous plant-based hydrogels. This includes the ability to form hydrogels from plant-based proteins at high concentrations (i.e., 5%–15% w / w) from commercially available sources and the ability to maintain these highly concentrated protein solutions in the liquid state after heat denaturation, which allows them to be molded into well-defined objects.

[00100] A feature of the materials of the present invention is that it is not necessary to provide crosslinking agents, since the plant-based proteins self-form hydrogels. In one embodiment of the present invention, a plant protein material (e.g., a hydrogel) is therefore provided that does not contain or substantially does not contain a crosslinking agent.

[00101] However, in an alternative embodiment, the hydrogels according to the present invention may comprise a crosslinking agent. Suitable crosslinking agents include microbial transglutaminase, glutaraldehyde, formaldehyde, glyoxal, phenolic compounds, epoxy compounds, genipin, or dialdehyde starch.

[00102] 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 porous hydrogel network.

[00103] In one embodiment of the present invention, the method includes the step of changing the solvent system in which the plant-based protein hydrogel was formed to an alternative solvent system. In another embodiment, this is accomplished using a solvent exchange process. This step can be performed after the hydrogel has formed, but it can also be performed after the hydrogel has been formed into a structured material (for example, after step b) or c) of the method. Petition 870260062218, dated 06 / 25 / 2026, page 31 / 125 23 / 55 of the present invention). In a preferred embodiment, an aqueous buffer is used to replace an organic acid cosolvent mixture within the porous hydrogel network.

[00104] In one embodiment, the solvent mixture within the hydrogel is allowed to evaporate to generate dry materials, such as thin films, microstructured / nanostructured thin films, or microbeads. In another embodiment, one or more cosolvent(s) in the solvent system is a volatile solvent. In a preferred embodiment, the first cosolvent is a volatile organic acid, such as acetic acid. In a preferred embodiment, the second or other cosolvent(s) is / are a volatile alcohol, such as ethanol. In yet another preferred embodiment, both the first cosolvent and the second or other cosolvent(s) are volatile solvents.

[00105] 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.

[00106] The amount of plasticizer to be incorporated will depend on the use of the material, for example, a film. In one embodiment, the composition may comprise about 1% plasticizer, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or more. In another embodiment, the hydrogel may comprise between about 550% plasticizer, about 10-50%, about 20-40%, about 15-35% or about 20% plasticizer.

[00107] Adding a plasticizer can influence the mechanical properties of the material. Typically, adding a plasticizer will increase the elasticity of the material, but this usually reduces the material's strength. Petition 870260062218, dated 06 / 25 / 2026, page 32 / 125 24 / 55 result.

[00108] The hydrogels of the present invention allow the formation of a variety of useful plant-based biomaterials. The use of plant-based materials has a number of advantages over previously used animal or petrochemical sources. Firstly, plant sources are renewable and can be obtained in an environmentally efficient manner. Secondly, plant sources are biodegradable and therefore an environmentally sound alternative to other plastics. Thirdly, in contrast to animal-derived proteins, plant-based proteins have the significant advantage of not introducing animal-derived proteins into a human being.This has positive impacts from both a pharmacological and pharmaceutical perspective, where animal-derived material must undergo rigorous checks and processes to ensure that no adverse elements are present (e.g., removal of prions and similar substances); but also because the products are suitable for vegetarians / vegans.

[00109] Since plant-based proteins are naturally present in a human's (or other animal's) diet, biomaterials made according to the present invention exhibit a higher degree of digestibility compared to other biopolymers, such as polysaccharides (e.g., alginates or chitosan). This makes them particularly suitable for pharmaceutical, food, and / or cosmetic use.

[00110] In one embodiment, the hydrogels of the present invention can be used to form films, for example, thin films. Plant-based protein-derived films have many applications, including the formation of flexible biodegradable films for applications in food packaging or for use with medical devices, including implantable devices.

[00111] One advantage of the plant-based materials of the present Petition 870260062218, dated 06 / 25 / 2026, page 33 / 125 25 / 55 The invention concerning animal-based materials (or starch / cellulose-based materials) has an inherent insolubility in water. Most biopolymer films would dissolve easily in water, making them unusable for food packaging applications on their own, requiring an extra coating layer with a synthetic polymer. These problems are overcome with the present invention.

[00112] The films can have a typical thickness of 1-1000 μm, 1-100 μm, 10-100 μm, 20-60 μm, 30-50 μm and similar.

[00113] The films may have a Young's modulus above 20 MPa, above 50 MPa, above 80 MPa, above 100 MPa, above 200 MPa, above 300 MPa, above 400 MPa, above 500 MPa, above 600 MPa or more.

[00114] The films may have a breakage rate of elongation above 10%, above 20%, above 30%, above 40%, above 50%, above 60%, above 70%, above 80%, above 90%, above 100% or more.

[00115] The Young's modulus / elongation percentage values ​​describe films made with plant-based functional materials. These properties can be replicated in all structured materials, as described herein, using said materials.

[00116] The films can be micropatterned with features ranging from 100 nm to 1000 μm, allowing for new functional properties, such as: superhydrophobicity (lotus leaf effect) or structural color (attributed to Mie scattering).

[00117] Functional composite films can be produced by incorporating inorganic nanoparticles, such as gold nanoparticles or silver nanoparticles, to generate flexible electronics or films with antibacterial properties, respectively. Petition 870260062218, dated 06 / 25 / 2026, page 34 / 125 26 / 55

[00118] In another embodiment, the hydrogels of the present invention can be used to form microbeads. Forming microbeads from a plant source overcomes the environmental problems with current plastic-based microbeads. Microbeads are typically solid particles with a diameter of less than one millimeter in their largest dimension.

[00119] In one embodiment, the microbeads according to the present invention have a size of less than 1 mm in the largest dimension, 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.

[00120] In another embodiment, the hydrogels of the present invention can be used to form microcapsules. The microcapsules can be used to encapsulate a variety of substances and find various industrial applications, including in cosmetics, food, household products, agrochemicals, and pharmaceuticals.

[00121] Microcapsules made according to the present invention provide a completely biodegradable alternative to standard synthetic polymer microencapsulation coating materials.

[00122] Microcapsules made according to the present invention uniquely allow plant-based materials to assemble microfluidically.

[00123] Microcapsules made according to the present invention provide, for example, excellent stability against adverse storage conditions and become a safer food-grade or pharmaceutical-grade solution for preserving active ingredients, for example vitamins, essential fatty acids or antioxidants in food or pharmaceutical use, active agents including small and large molecules.

[00124] Microcapsules made according to the present invention of Petition 870260062218, dated 06 / 25 / 2026, p. 35 / 125 27 / 55 similar forms may have the advantage of being generated using microfluidic technologies, ensuring high reproducibility, the ability to generate complex structures (core-shell) and / or they can be made using moderate processing conditions, thus protecting the active agent they encapsulate.

[00125] Robust microcapsules can likewise be generated from plant-based proteins by controlling the self-assembly of protein aggregates in the absence of crosslinkers or any other hazardous substances.

[00126] In one embodiment, the microcapsules according to the present invention can encapsulate an internal hydrophobic composition. In another embodiment, the microcapsules according to the present invention can encapsulate an internal hydrophilic composition. In another embodiment, the microcapsules according to the present invention can encapsulate a living organism composition. In another embodiment, the microcapsules according to the present invention can encapsulate a powder composition. In yet another embodiment, the microcapsules according to the present invention can encapsulate an oil-in-water, water-in-oil, oil-in-water-in-oil, or water-in-oil-in-water emulsion and the like.

[00127] Plant-based hydrogels according to the present invention can form the microcapsule shell. In one embodiment, the shell may have a thickness of about 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 25μm, 30μm, 30 μm, 35 μm, 40 μm or 50 μm and to taste. Other thicknesses include between 10nm-50000μm, between 10μm-100μm, between 10μm-50μm, between 10μm-10μm and similar. Petition 870260062218, dated 06 / 25 / 2026, page 36 / 125 28 / 55

[00128] The plant-based microcapsules according to the present invention can encapsulate any suitable nutraceutical, cosmetic, pharmaceutical or agrochemical active agent, including vitamins, essential fatty acids, antioxidants, small molecules, hydrophilic small molecules, hydrophobic small molecules, proteins, antibodies, drug-antibody conjugates, fragrances and other large molecules.

[00129] Suitable encapsulated agents include one or more agents selected from: Crosslinking agents, hardeners, organic catalysts and metal-based catalysts (e.g., organo-complexes and inorgano-complexes of platinum, palladium, titanium, molybdenum, copper or zinc) for polymerization of elastomer formulations, rubber formulations, paint formulations, coating formulations, adhesive formulations or sealant formulations; inks, 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, cleaning products, personal care products, textiles (so-called smart textiles), coating formulations. The fragrances useful for the invention are any of the compounds belonging to the list of standards published and updated by the International Fragrance Association (IFRA); Aromas, flavors, vitamins, amino acids, proteins, essential lipids, probiotics, antioxidants, preservatives for animal feed and food products; Fabric softeners and conditioners for laundry, detergents, and personal hygiene products; bioactive compounds, such as enzymes, vitamins, proteins, extracts Petition 870260062218, dated 06 / 25 / 2026, page 37 / 125 29 / 55 vegetable oils, moisturizers, disinfectants, antibacterial agents, sunscreens, medicines, personal care products, textiles (so-called smart textiles). These compounds include, but are not limited to, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, para-aminobenzoic acid, alpha hydroxy acid, camphor, ceramides, ellagic acid, glycerin, glycine, glycolic acid, hyaluronic acid, hydroquinone, isopropyl isostearate, isopropyl palmitate, oxybenzone, panthenol, proline, retinol, retinyl palmitate, salicylic acid, sorbic acid, sorbitol, triclosan, tyrosine; and fertilizers, herbicides, insecticides, pesticides, fungicides, repellents and disinfectants for agrochemicals.

[00130] Plant-based microcapsules according to the present invention can likewise be useful in high-throughput diagnostics and assessment.

[00131] Plant-based hydrogels according to the present invention can likewise be useful in the manufacture of microgels or microscale sponges. Microgels are hydrogels on a microscale. Micro-sponges can be considered microgels loaded with a substance, for example, an active ingredient.

[00132] Plant-based hydrogels according to the present invention can likewise be useful in the manufacture of bioscaffolds and biosupports. These materials can be useful in vitro and in vivo for the growth of cells and tissues. The hydrogels can likewise be useful in coating medical devices and implants.

[00133] Plant-based proteins according to the present invention can be functionalized and / or derivatized to alter the protein properties.

[00134] The invention will now be described with reference to the following Petition 870260062218, dated 06 / 25 / 2026, p. 38 / 125 30 / 55 non-limiting examples. Materials - 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 (Glycine max soybean oil), (-)-Riboflavin, glycerol, and PPO (1H,1H,2H,2H-Perfluoro-1-octanol) were purchased from Sigma Aldrich. Pectin was kindly provided by Cargill. - Fluorinert (FC-40) was acquired from Fluorochem. - 008-FluoroSurfactant was acquired from RAN Biotechnologies. Example 1

[00135] The self-sustaining hydrogels were prepared according to the following process.

[00136] Glacial acetic acid was mixed with deionized water in different proportions (10% v / v, 30% v / v, 50% v / v, 70% v / v, 90% v / v). Soy protein isolate was added to the DL water / acetic acid solution at a final protein concentration of 100 mg / ml. A non-soluble protein dispersion was obtained. To solubilize the protein, the mixture was exposed to ultrasound for 30 min (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 min, 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 transforms into a translucent, self-supporting hydrogel observable after inverting the vial.

[00137] Hydrogels made according to Example 1 for the different Petition 870260062218, dated 06 / 25 / 2026, page 39 / 125 31 / 55 acetic acid / water DI ratios are shown in Figure 1.

[00138] Self-supporting hydrogels were observed with acetic acid / water DI ratios of 10%–70% v / v. It was noted that for solutions of 30% v / v and above, a completely translucent solution was produced that rapidly formed a self-supporting hydrogel. The fact that the gel was completely translucent suggested that it was composed of small structures of soluble aggregates rather than large insoluble aggregates, such as those normally found in cold-solidified protein gels. The hydrogels made according to the invention were stable and maintained their structure after several washing steps in water and ethanol. Scanning Electron Microscopy

[00139] SEM (scanning electron microscopy) was performed on the hydrogel made using a cosolvent ratio of 30% v / v is shown in Figure 2. The microstructure of the hydrogel confirmed the presence of a densely packed network of fine filament protein aggregates. The figure similarly shows that the gel network remains intact after removal of the acetic acid.

[00140] A plant-based thermoreversible gel was therefore prepared for the first time.

[00141] Soy protein hydrogels and microgels were dehydrated in ethanol using 100% dry ethanol in the final step. Samples were transferred to microporous specimen capsules (78 μm pore size, Agar Scientific) soaked and partially filled with 100% dry ethanol to prevent the sample from drying out during transfer. Samples were then critical point dried using a Quorum E3100 critical point dryer using 4-5 discharges with liquid CO2 and at least 15 minutes of incubation between each discharge. Samples were mounted on aluminum SEM stubs using conductive carbon adhesive pads (Agar Scientific) and coated with Petition 870260062218, dated 06 / 25 / 2026, page 40 / 125 32 / 55 iridium of 15 nm using a Quorum K575X spray applicator. Samples were visualized using a FEI Verios 460 scanning electron microscope conducted at 2 keV and a probe current of 25-50 pA. Secondary electron images were acquired using an Everhard-Thornley detector in field-free mode (low resolution) or a Through-Lens detector in total immersion mode (high resolution). Particle size analysis

[00142] In order to characterize the size of SPI aggregates, SPI dispersions were prepared in several 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) aqueous acetic acid solution) and treated with three different methods: (1) untreated, (2) heated for 30 minutes at 95°C in a water bath, or (3) sonicated for 30 minutes at 95°C. Particle size (hydrodynamic diameter) was measured using Zetasizer Nano (Malvern). All samples were diluted to 0.1% (v / v) immediately after sonication treatment and measurements were performed subsequently.

[00143] Dynamic dispersive scan (DLS) analysis (Figure 3) revealed that protein aggregates prepared in a 30% (v / v) acetic acid solution sonicated for 30 minutes at 95°C have a significantly smaller particle size (29 ± 9.1 nm) compared to non-solubilized aggregates prepared under different conditions. Rheological Characterization

[00144] To characterize the rheological properties of SPI hydrogels, samples produced using Example 1 were allowed to cool at room temperature for 1 hour and then kept at 4°C for 12 hours.

[00145] Rheological measurements were performed using an ARES controlled strain rheometer. Tests were performed on smooth parallel plates. Petition 870260062218, dated 06 / 25 / 2026, page 41 / 125 33 / 55 of 25 mm at a temperature of 20°C. Strain and frequency scans were performed on SPI hydrogel samples containing increasing amounts of acetic acid. Strain scans were performed using a frequency of 10 rad / s from 0.01 to 100% strain. Frequency scans were performed using a 1% strain (within the linear viscoelastic region) from 0.1 to 100 rad / s. All rheological measurements were performed under temperature control (20 ± 0.25°C).

[00146] The rheological properties of SPI hydrogels as a function of H2O:acetic acid cosolvent ratios are shown in Figure 4a. It can be observed that the addition of low amounts of acetic acid (10% v / v) results in the formation of weak hydrogels, which can be attributed to incomplete protein solubilization and the presence of larger insoluble aggregates. On the other hand, large amounts of acetic acid similarly resulted in the formation of weak hydrogels (90% v / v). Hydrogels formed with a cosolvent ratio of 30-70% v / v resulted in stronger hydrogels, with hydrogels prepared with 30 and 50% acetic acid (v / v) showing a G' greater than 2500 Pa.

[00147] To determine the thermoreversible gelation properties of an SPI hydrogel (75 mg / ml SPI dispersed in a 30% v / v aqueous acetic acid solution), rheological measurements were performed using a Discovery HR-2 rheometer (TA Instruments). The tests were performed using 20 mm smooth parallel plates. The tests were carried out on an SPI hydrogel sample at different temperatures using an angular frequency of 10 rad / s. Initially, a sonicated SPI solution was loaded onto a rheometer probe preheated to 90°C. After 500 seconds, the temperature was lowered to 10°C at a rate of 5°C / min. Once the sample temperature reached 10°C, it was held at 10°C for 500 seconds. Finally, the sample was heated to 90°C at a rate of 5°C / min. The results for the thermoreversible rheological properties Petition 870260062218, dated 06 / 25 / 2026, page 42 / 125 34 / 55 of the SPI hydrogels are shown in Fig. 4c.

[00148] This shows the unique manufacturing capabilities of the materials of the present invention, since the thermoreversibly gel moved completely between the gel and liquid states. This thermoreversibility has not been observed previously. FTIR

[00149] Changes in the secondary structure of the hydrogel under different solvent ratios were investigated. Structural analysis of SPI hydrogels was performed using an Equinox 55 FTIR spectrometer (Bruker). Hydrogel samples were used without any other pretreatment and were loaded onto the FTIR support and analyzed by subtracting a 50% v / v reference (DI water / acetic acid). The atmospheric compensation spectrum was subtracted from the original FTIR spectra, and a secondary derivative was applied for further analysis. Each FTIR measurement was repeated 3 times for each sample replicate (an average of 9 replicates per sample). The instrument sensitivity was detected at 5%. To resolve the transformation of the native structure of the soy protein isolate into supramolecular aggregates, vibrational changes in amide I, which is strictly correlated with the secondary structure of the protein, were followed.

[00150] The results of the FTIR measurements are shown in Figure 5a. It is clear from the analysis of the 2nd derivative that the SPI hydrogels have a high content of α-helix (1656 cm-1) and parallel β-sheet intermolecular secondary structure (1625 cm-1).

[00151] FTIR measurements of SPI hydrogel samples prepared using different solvent ratios shown in Figure 5b reveal that the use of increasing amounts of acetic acid causes an increase in αhelical structure (1654 cm-1) versus the content of antiparallel and intermolecular parallel β-sheet (1620 cm-1). Petition 870260062218, dated 06 / 25 / 2026, page 43 / 125 35 / 55

[00152] Changes in the secondary structure of the hydrogel at different temperatures were similarly investigated. FTIR measurements of an SPI hydrogel sample (SPI 100 mg / ml dispersed in 30% v / v acetic acid) were performed by incubating the sample at different temperatures in the FTIR sample holder (90°C, 55°C and 20°C). It can be clearly observed in Figure 5c that, upon heating the sample to 90°C, the amount of intermolecular parallel β-sheet structure is decreased, while after cooling the sample to 20°C, the content of the parallel β-sheet is significantly increased.

[00153] This is evidenced by the thermoreversibility of the present invention, correlating this advantageous property with the secondary structure of the protein. Example 2

[00154] The hydrogels were prepared using lactic acid as a co-solvent according to the following process.

[00155] Lactic acid was mixed with deionized water in different proportions (10% v / v, 30% v / v, 50% v / v, 70% v / v, 90% v / v). Soy protein isolate was added to the DL water / lactic acid solution at a final protein concentration of 100 mg / ml. A non-soluble protein dispersion was obtained. To solubilize the protein, the mixture was exposed to ultrasound for 30 min (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 min, a completely translucent liquid solution was obtained. The sample was allowed to cool at room temperature for 5 min. Gel electrophoresis

[00156] The analysis of different hydrolyzed protein fragments at different water:lactic acid ratios (from 0 to 90% v / v) was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4 - 12% gels with MES buffer. Petition 870260062218, dated 06 / 25 / 2026, page 44 / 125 36 / 55

[00157] The results are shown in Figure 6 and it can be seen that increasing amounts of lactic acid led to a greater degree of protein hydrolysis.

[00158] Having established that stable hydrogels can be formed using the methods of the present invention, the use of such hydrogels has been employed in microfluidic approaches to form discrete and uniform microscopic objects, such as microgels and microcapsules. Example 3 - microgel manufacturing

[00159] Microfluidic devices (droplet generator) were fabricated using standard soft lithography techniques with photoresist negative master (SU8 3050). The continuous oil phase (2% 008-FluoroSurfactant in Fluorinert FC-40) was loaded into a 2 ml tube, while the dispersed liquid SPI phase (85 mg / ml SPI in 40% v / v acetic acid, maintained at 85°C) was loaded into a 1.5 ml tube and rapidly placed in a heating block at 85°C. In order to prevent gelation of the SPI solution during transport to the microfluidic device, a custom-made silicone heater (Holroyd Components), comprising a 1 / 32 ID stainless steel tube, was used to maintain the temperature of the PTFE tube connecting the SPI reservoir and the inlet to the microfluidic device. The temperature of the silicone heater was controlled by a custom temperature controller.Droplets of approximately 100 μm in diameter were generated by pumping both solutions into a standard flow-focus droplet generator using a pressure-driven system (Elveflow OB1). Different pressure rates were tested until a uniform and continuous generation of a monodisperse population of microdroplets was achieved. The final pressure rates were 175 mbar for the aqueous dispersed phase and 200 mbar for the continuous oil phase. The generated droplets were collected in a 1 ml pipette tip and kept at room temperature for 12 hours to ensure completion of the gelation process. Petition 870260062218, dated 06 / 25 / 2026, page 45 / 125 37 / 55 The formed microgels were then washed using a standard demulsification procedure: the continuous oil phase containing fluorosurfactant was first removed from the vial. For 500 µL of microgels, an equal volume of 10% PFO solution in Fluorinert FC-40 was added and thoroughly mixed 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 were 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 3 washing steps were performed to remove acetic acid from the microgel suspension. Mechanically stable microgels suspended in deionized water (pH = 2) were obtained after the final washing step. In order to prepare the microgel samples for SEM analysis, the microgels were washed with 100% ethanol in a 3-step procedure: the microgels were first suspended in a 25% v / v aqueous ethanol solution and left under constant stirring (100 rpm) for 3 h. After centrifugation and removal of the supernatant, the microgels were then resuspended in a 50% v / v aqueous ethanol 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 a 100% v / v aqueous ethanol solution and left under constant stirring (100 rpm) for 12 hours.

[00160] The results of this experiment are shown in Figure 7. A Petition 870260062218, dated 06 / 25 / 2026, p. 46 / 125 38 / 55 Figure 7a shows a schematic representation of microbead formation. Figure 6b shows stable microgels suspended in aqueous solution (pH = 2). Figure 7d shows the SEM image of microbeads prepared by supercritical point drying. Figure 7e shows the SEM image of the gel network on the surface of a microbead.

[00161] The resulting microgels were stable in aqueous solution under acidic conditions, as well as in the presence of 100% ethanol. Once self-assembled, the protein aggregates are completely insoluble in solution, hence the stability of the microgels.

[00162] These results demonstrate that mechanically robust microgels can be generated from plant-based proteins simply by controlling the self-assembly of protein aggregates at the nanoscale and in the complete absence of crosslinkers or any hazardous substances. Example 4 - core-shell microcapsule fabrication

[00163] Having established that mechanically robust microgels can be generated, the use of hydrogels for microencapsulation was explored.

[00164] For the fabrication of a coaxial flow focusing microfluidic device, a multi-step photolithographic process was followed. The photolithographic process is explained in Coaxial Flow Focusing in Poly(dimethylsiloxane) Microfluidic Devices Tran, TM, Cater, S. & Abate, AR, but is briefly explained below.

[00165] Two SU8 photoresist masters were required. In the first master, a 25 μm photoresist layer was fabricated for the inner channel, 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 plates were Petition 870260062218, dated 06 / 25 / 2026, page 47 / 125 39 / 55 masters were removed and subsequently 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 in an oven at 65°C overnight to complete the bonding between the two layers.

[00166] The microfluidic process for generating core-shell microcapsules was equivalent to that employed in Example 3 for generating microbeads. However, an additional input for the internal phase (core) was added to the system. For the fabrication of core-shell microcapsules containing a lipophilic active ingredient, 450 μl of soybean oil were pre-emulsified in 550 μl of SPI protein solution (20 mg / ml, 30% v / v acetic acid). The oil-in-water emulsion (internal phase), SPI solution (85 mg / ml, 40% v / v acetic acid, intermediate phase) and FC-40 (2% v / v O08-fluorosurfactant, external phase) were pumped into a microfluidic device with a coaxial flow focus by a pressure-driven system (Elveflow OB1).Different pressure rates were tested until a uniform and continuous generation of a monodisperse population of core-shell microcapsules with ~120 μm diameter was achieved (internal pressure phase (100 mbar), intermediate pressure phase (150 mbar), and external pressure phase (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 subsequently washed as described in the previous section.

[00167] It can be seen that the inner core materials are kept separate in this example from the shell materials, which is different from a standard microencapsulation matrix approach where the active ingredients are pre-mixed with the matrix materials. The reason for this approach was the pH of the initial protein solution being very low due to the large amount of acetic acid initially present and similarly the high temperatures. Petition 870260062218, dated 06 / 25 / 2026, page 48 / 125 40 / 55 required to maintain the protein solution in a liquid state. Instead, a 3-D coaxial microfluidic device was used, which allowed for the easy production of core-shell structures without compromising the stability of the encapsulated ingredients.

[00168] A schematic representation of the microfluidic droplet generator is shown in Figure 8a, showing a 3D flow-focusing microfluidic device. The 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 comprises only self-assembled SPI proteins and the core is an oil-in-water (lipophilic) microemulsion. Example 5 - Manufacturing a core-shell microcapsule encapsulating an active agent.

[00169] The microfluidic process described in Example 4 was repeated to generate a microcapsule comprising a hydrophilic core. The core comprised a 1% (w / w) HMP pectin solution containing a suspension of a soluble active ingredient (Riboflavin).

[00170] 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 comprises only self-assembled SPI proteins and the core is a hydrophilic suspension of an active agent, in this case riboflavin. Example 6 - Controlled-release microcapsules

[00171] Since protein shells are composed exclusively of protein aggregates, an experiment was conducted to confirm that cargo release can be triggered by protein shell degradation in the presence of digestive enzymes, through a test of Petition 870260062218, dated 06 / 25 / 2026, page 49 / 125 41 / 55 two-phase in vitro digestibility.

[00172] A 1L raw material solution of SGF (simulated gastric fluid) electrolyte solution was prepared by dissolving 0.257g of KCl, 0.061g of KH2PO4, 1.05g of NaHCO3, 1.38g of NaCl, 0.122g of MgCl2 (H2O) 6 and 0.024g of (NH4)2CO3 in 1L of deionized water. A 1 L raw material solution of SIF (simulated intestinal fluid) electrolyte solution was prepared by dissolving 0.253 g of KCl, 0.054 g of KH2PO4, 3.57 g of NaHCO3, 1.12 g of NaCl, 0.335 g of MgCl2(H2O)6, 0.44 g of CaCl2^2H2O, and 0.23 g of bile extract in 1 L of deionized water. 1 mL of SGF was prepared by dissolving 8 mg of pepsin in the SGF electrolyte solution, and the pH was adjusted to pH = 2 by adding a small amount of 1 M HCl.

[00173] 50 μL of core-shell microcapsules made according to Example 4 were washed from the oil phase using the method described in Example 4 (150 μL of deionized water at pH = 2 was added). Then, 200 μL of SGF were mixed with the microcapsule suspension, followed by incubation at 37°C and 300 rpm for 60 min in a Thermo Shaker. For the simulated intestinal phase, 3 mg of pancreatin were dissolved in 1 ml of SIF electrolyte solution. 400 μL of SIF were added to the previous microcapsule in SGF solution. The pH was adjusted to 7 by adding a small amount of 1 M NaOH. The microcapsules were incubated at 37°C for 120 min.

[00174] The results of the experiment are shown in Figure 10, with Figure 10a showing the microcapsules in aqueous solution; Figure 10b shows the microcapsules still intact after 60 minutes in SGF; and Figure 10c showing the release of the microcapsules after 120 minutes in SIF.

[00175] Experiments show that incubation under SGF conditions (8 mg / ml Pepsin, pH = 2) triggered a slow degradation of the protein shell without compromising the stability of the lipophilic core, which remained Petition 870260062218, dated 06 / 25 / 2026, page 50 / 125 42 / 55 within the microcapsule. 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 enzymes, but under the same pH conditions, indicated that protein shell degradation was primarily triggered by enzymatic digestion.

[00176] It can therefore be seen that the microcapsules remained intact under simulated gastric conditions, but rapidly disintegrated under simulated intestinal conditions, demonstrating that the microcapsules according to the present invention can be used for controlled delivery of nutraceutical or pharmaceutical active ingredients. Example 7 - Controlled-release microcapsules containing an active agent

[00177] The experiment from Example 6 was repeated, but using microcapsules containing a hydrophilic core containing the active agent riboflavin, made according to Example 5.

[00178] 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 followed 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 gastric and small intestine conditions, made possible by the encapsulation of the hydrophilic active ingredient within a plant-based protein microcapsule shell. Encapsulation prevents rapid diffusion of riboflavin and promotes controlled release. Example 8 - Preparation of sponges on a microscale

[00179] Microscale sponges of soy protein were prepared according to the present invention. The microfluidic process described in Example 2 was Petition 870260062218, dated 06 / 25 / 2026, page 51 / 125 43 / 55 repeated to generate soy protein microgels (100 μm in diameter). The microgels were washed with 100% ethanol in a 3-step procedure: the microgels were first suspended in a 25% v / v aqueous ethanol solution and left under constant stirring (100 rpm) for 1 h. After centrifugation and removal of the supernatant, the microgels were then resuspended in a 50% v / v aqueous ethanol solution and left under constant stirring (100 rpm) for 1 h. Finally, after centrifugation and removal of the supernatant, the microgels were then resuspended in a 100% v / v aqueous ethanol solution and left under constant stirring (100 rpm) for 1 h. At this point, the ethanol should have completely replaced the water in the porous network of the microgel. The microgel suspension was centrifuged (1000 rpm, 1 min) and the ethanol supernatant was removed. Next, 250μl of geranium oil (natural) were added to the microgels.The microgels were suspended in the oil solution by gently shaking the Eppendorf tube. At this stage, the ethanol-miscible oil phase permeated the porous network of the microgel. After adding 500 μl of deionized water to the oil microgel suspension, the oil-laden SPI microscale sponges were then transferred to the aqueous phase. The remaining oil phase was removed from the Eppendorf tube.

[00180] Figure 12a shows a microscale distribution of plant-based protein sponges loaded with fragrance oil. Figure 12b shows a single fragrance-loaded plant-based protein microscale sponge at higher magnification (20x), where the protein microgel shell can be easily observed.

[00181] Using this approach, water-immiscible solvents can be loaded into microscale sponges of plant-based proteins, such as fragrance oils. This method allows for high loading efficiencies without the need to use complex microcapsule generation approaches, such as Petition 870260062218, dated 06 / 25 / 2026, page 52 / 125 44 / 55 core-shell architectures. Example 9 - Film preparation

[00182] Soy protein films were prepared according to the present invention. 500 mg of soy protein isolate were dissolved in 5 mL of 30% (v / v) acetic acid (protein concentration of 100 mg / mL). The solution was then exposed to ultrasound (High-Frequency Power Output = 70W, Frequency = 20KHz, Amplitude = 90%) for 30 min. A few minutes before the end of the ultrasound process, 125 mg of glycerol were added to the SPI solution and mixed with the remaining time of the ultrasound step. Immediately after the ultrasound step, the solution was melted in a glass Petri dish to form a hydrogel (2 mm thick). The glass Petri dish was heated to approximately 100°C to prevent any gelation during the melting process. The glass dish was then removed from the heating plate and air-dried overnight. After drying, the thin film was removed from the glass petri dish and stored in a chamber with 50% humidity until use.The underside of the glass Petri dish was coated with Teflon foil so that the films could be easily removed.

[00183] A schematic representation of the process according to Example 9 together with the resulting films is shown in Figure 13. It can be seen that mechanically robust transparent thin films are generated. Tensile properties

[00184] The tensile properties of the thin films generated in Example 9 were tested as a function of the amount of plasticizer added. The films were manufactured with glycerol at 10%, 20%, 30%, 40% and 50% (w / w).

[00185] The tensile properties of the film were tested using a 5 kN Tinius Olson load cell with 10 N. The film was cut into strips 5 mm wide and both ends of the films were glued together with a Petition 870260062218, dated 06 / 25 / 2026, page 53 / 125 45 / 55 paper support with a 1 mm gap length. The support and film were mounted on the mechanical testing machine, and then the paper folder was cut before measurement to ensure that the load was applied only to the film. The measurement was performed at a speed of 2 mm / min. The film thickness was measured for individual samples using a digital caliper. The typical film thickness was 30-50 µm.

[00186] The stress-strain curves for the films of the present invention are shown in Figure 14a. The Young's modulus of the films is shown in Figure 14a, where it can be seen that increasing the amount of plasticizer reduces the Young's modulus for the films. The percent elongation at break is shown in Figure 14b with the greatest elongation shown at 30% plasticizer.

[00187] The mechanical performance of films obtained by this approach is superior to that of previously reported films made from commercial soy protein. This is due to the high degree of intermolecular interactions and subsequent self-assembly of protein molecules, as opposed to the weak intermolecular interactions normally present in soy protein films produced by fusing monomeric soy protein in high concentrations of chaotropic agents (i.e., 8M urea). FTIR

[00188] To investigate the secondary structure and intermolecular interactions in the film, FTIR analysis was performed on the SPI film prepared without the addition of glycerol.

[00189] Data were collected using 128 scans with a resolution of 4 cm-1 with antecedent subtractions. For the structural analysis of the proteins, the spectra were smoothed with a 7-point, 2nd-order Savitzky-Golay window filter and normalized. The second derivative in the Amide I band (1600 - 1700 cm-1) Petition 870260062218, dated 06 / 25 / 2026, page 54 / 125 46 / 55 was calculated from the smoothed data to deconvolve and quantify the secondary and quaternary structural contributions.

[00190] FT-IR analysis of the generated film showed that a greater amount of intermolecular β-sheet structures (65%) are present in the film compared to the initial soy protein isolate (Figure 15) Transmission Electron Microscopy (TEM) Analysis

[00191] TEM analysis was performed to investigate the morphology of the self-assembled protein structures present in the films. For transmission electron microscopy (TEM) imaging, a sample of SPI used for film formation as detailed in Example 9 was diluted to a concentration of 0.02% and deposited on a TEM grid (C400Cu, EM resolutions), stained with uranyl acetate. Figure 16 shows the presence of a large proportion of β-sheet crystals, which correlates with the large number of intermolecular β-sheet structures determined by FTIR.

[00192] Beta-sheet crystals have not been previously observed with plant protein materials. These crystals share similarities with silk materials, which are known to have enhanced mechanical properties, including strength. Without wishing to be limited by theory, it is believed that said beta-sheet crystals are formed due to the process described by the present invention. Such beta-sheet crystals may contribute to the enhanced mechanical properties seen with the plant-based materials of the present invention in contrast to the plant-based gels described in the prior art. 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 them to have properties that have not been previously reported for plant-based materials (such as tensile strength for the films).

[00193] Plant-based materials resulting from the present invention Petition 870260062218, dated 06 / 25 / 2026, page 55 / 125 47 / 55 are therefore different from known plant-based gels, and these differences result in markedly improved properties. Example 10 - Microstructure and nanostructure standardization

[00194] The films made according to Example 9 were micro-standardized according to the process below.

[00195] In order to standardize microstructures in a protein film, such as micropillars, a negative pattern of a 20 μm x 20 μm micropillar array was fabricated on a silicon wafer using standard photolithographic techniques with 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 hour. The cured PDMS was removed from the wafer and used as a negative pattern for the soy protein film. A soy protein solution, as prepared in Example 9, was fused onto the PDMS and dried overnight. The thin SPI film was removed from the master PDMS and the resulting micropatterned structures were observed using scanning electron microscopy (SEM) with a 10 nm platinum coating (MIRA 3 FEG-SEM, TESCAN). The film contact angle was measured using an FTA1000B, First Ten Angstroms.

[00196] By simply casting the sonicated soy protein solution onto a micropatterned substrate, it is possible to fabricate a uniform array of micropillars on the film surface. The contact angle of the micropatterned films was 99°, which is significantly greater than a control film sample with a non-micropatterned surface (Figure 17a). This clearly shows that by simply patterning micropillars such as those naturally present in lotus leaves onto the surface of a plant-based protein film, the hydrophobic surface properties can be greatly enhanced. Petition 870260062218, dated 06 / 25 / 2026, page 56 / 125 48 / 55

[00197] To generate nanostructured protein films with photonic properties, a DVD disc was used as a template substrate. First, the outer plastic layer was carefully removed from the DVD disc, and the remaining intermediate layer was used. PDMS was poured onto the DVD substrate and cured for 1 hour at 65°C. The standardized PDMS was removed from the DVD substrate, and a film-forming soy protein solution, as prepared in Example 9, was fused onto the PDMS substrate and dried at room temperature overnight. The thin SPI film was removed from the master PDMS, and the resulting nanopatterned structures were observed using scanning electron microscopy (SEM) with a 10 nm platinum coating (MIRA 3 FEG-SEM, TESCAN). The photonic properties (Mie scattering) of the film can be easily observed with the naked eye (Figure 17b). Example 11 - Coating preparation

[00198] In order to test the coating properties of plant-based protein films, a small piece of cardboard (2x2 cm) was immersed in a film-forming SPI solution, as prepared in Example 9, in the absence of plasticizers. 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 immersed in 10 mL of deionized water to measure the water uptake of the cardboard. Water uptake was calculated by measuring the weight increase of the cardboard when immersed in deionized water at different time intervals.

[00199] As control samples, a piece of uncoated cardboard and a piece of cardboard coated with soy protein monomer (prepared by immersing the cardboard in a soy protein solution prepared under alkaline conditions (pH = 9)) were tested. Water uptake was normalized to the initial weight. Petition 870260062218, dated 06 / 25 / 2026, page 57 / 125 49 / 55 measured before soaking the cardboard samples in water.

[00200] As observed in Figure 18, the uncoated cardboard control sample exhibits very high water absorption after immersion for 10 seconds in water. 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 a soy protein solution prepared under alkaline conditions, which similarly exhibited very low water resistance. These results highlight that coatings generated from plant-based protein films assembled from proteins with a high degree of intermolecular interactions exhibit enhanced water barrier properties. Example 12 - Hard capsule manufacturing

[00201] Soy protein capsules were manufactured according to the present invention. A 100 mg / ml SPI solution was first 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 to 95°C for 30 min, followed by ultrasound exposure for 5 min (High frequency power output = 70W, frequency = 20KHz, amplitude = 90%). The solution was maintained at 85°C to prevent gelation. A 2 ml Eppendorf tube was immersed in the heated solution, then held in the liquid solution for 5 seconds and subsequently removed. A thin layer of SPI hydrogel formed around the outer surface of the 2 ml Eppendorf tube within seconds of removing it from the liquid SPI solution (Figure 19a). The vial was then placed in an oven at 45°C for one hour to ensure the evaporation of the water / acetic acid solvent fraction within the hydrogel layer to generate a thin three-dimensional film.After the solvent evaporated, the capsule was removed from the Eppendorf tube (Figure 19b).

[00202] This is the first example showing capsule formation. Petition 870260062218, dated 06 / 25 / 2026, page 58 / 125 50 / 55 hard materials are produced from plant-based proteins. This process is only possible due to the thermally reversible properties of these plant-based materials, which allow the formation of a thin, three-dimensional hydrogel on the substrate. Example 13 - Pea and potato protein materials

[00203] The procedures described in Example 1, Example 3, and Example 9 were followed to produce protein hydrogels, microgels, and films using pea protein isolate (80%) and potato protein isolate as starting materials.

[00204] Figure 20a shows a translucent, self-supporting Pea Protein hydrogel observable after inversion of the vial.

[00205] Properties similar to those described here were similarly observed with potato protein as shown in Figure 21, which shows a potato protein hydrogel prepared from a solution of 100 mg / ml potato protein isolate in a 30% (v / v) aqueous acetic acid solution.

[00206] Example 13 shows the versatility of the processes of the present invention working with a variety of plant-based protein sources. Example 14 - Stress-strain film analysis

[00207] Stress-strain curves are shown for the films of the present invention in Figure 22. Figure 22a shows stress-strain curves for films made according to the present invention. Stress-strain curves for a self-assembled sample (blue) correspond to an SPI film prepared from a 30% (v / v) aqueous acetic acid solution in the presence of 30% (w / w, relative to dry protein weight) glycerol. Stress-strain curves for an unstructured sample (red) correspond to an SPI film prepared in an alkaline aqueous solution adjusted to pH = 10 using NaOH in the presence of 30% glycerol (w / w, relative to protein weight). Petition 870260062218, dated 06 / 25 / 2026, page 59 / 125 51 / 55 dry).

[00208] Figure 22b shows stress-strain curves for SPI films prepared from a 30% (v / v) aqueous acetic acid solution in the presence of different amounts of glycerol as a plasticizer (20% to 40% w / w, relative to the dry protein weight).

[00209] 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).

[00210] It can be observed that varying the amount of glycerol allows adjusting the mechanical performance of the hydrogel. For example, the mechanical performance changed 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 glycerol concentration varied from 20-40% (w / w). The tensile strength and Young's modulus of self-assembled films with 30% w / w glycerol were found to be higher than the values ​​for unstructured films with 30% (w / w) glycerol (131 ± 22.6 MPa and 9.30 ± 1.53 MPa for Young's modulus and tensile strength, respectively).

[00211] The samples in Example 14 were prepared as follows:

[00212] 500 mg of SPI were dispersed in 5 mL of 30% (v / v) aqueous acetic acid solution and stirred well until a cloudy, highly viscous dispersion was obtained. This was then sonicated using ultrasonic homogenizers (Bandelin, HD4200) for 30 minutes at 25% power (pulse durations of 0.7 seconds on time and 0.3 seconds off time). 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 a further 1 minute. The hot liquid solution was immediately melted in a 7 cm glass Petri dish preheated to 90°C. The melted solution was then dried for 3 days in Petition 870260062218, dated 06 / 25 / 2026, page 60 / 125 52 / 55 ambient temperature (19-22°C) and ambient humidity (typically 50-70%). The dried film was then removed from the mold and stored in a humidity-controlled chamber (50%) until use.

[00213] As a control experiment, films were similarly prepared in an aqueous alkaline solution, where a dispersion of 100 mg / ml SPI was prepared in an aqueous solution of pH = 10 (adjusted with NaOH) and heated for 30 minutes at 95°C without sonication (referred to as unstructured SPI). 30% (w / w) glycerol was then added, the hot liquid solution was melted in a glass petri dish and dried for 3 days. Example 15 - comparison of acetic acid vs HCl vs NaOH

[00214] A comparative experiment was conducted to evaluate the difference between hydrogels made according to the present invention with acetic acid and methods using HCl or NaOH (which do not undergo a sol-gel transition). The results are shown in Figure 23.

[00215] Figure 23 shows optical images of a 100 mg / ml SPI dispersion prepared in different aqueous solutions by means of high-temperature ultrasonication treatment. (a) SPI dispersion in 30% (v / v) aqueous acetic acid solution (left), acidic aqueous solution adjusted to pH = 2 using HCl (middle), and alkaline aqueous solution adjusted to pH = 10 using NaOH after 30 minutes of sonication (right). (b) The glass vials are inverted to show that gelation is only observed for the SPI solution prepared in 30% (v / v) acetic acid. The HCl and NaOH vials show that no sol-gel transition occurs.

[00216] In conclusion, the present invention provides solvent systems as described herein that enable a method of disrupting intermolecular interactions at high temperature and high shear. This allows for the selective promotion of the formation of intermolecular interactions after a change in Petition 870260062218, dated 06 / 25 / 2026, page 61 / 125 53 / 55 temperature. This approach allows the material to be molded into different shapes. Until now, this has not been possible for materials of plant origin.

[00217] As such, the present invention can be directed to a method of molding plant-derived material, comprising the methods described herein.

[00218] Furthermore, the present invention creates a secondary protein structure of the self-assembled material with a greater number of intermolecular β-sheet structures (compared to the protein starting material). These novel materials have not been seen in existing plant-derived materials. This novel secondary structure gives the materials unique properties, such as increased film tensile strength.

[00219] Undoubtedly, many other effective alternatives will occur to the skilled person. It will be understood that the invention is not limited to the embodiments described and includes apparent modifications to those skilled in the art and which fall within the spirit and scope of the appended claims. References 1. Ahnert, SE, Marsh, JA, Hernández, H., Robinson, CV & Teichmann, SA. Assembly principles reveal a periodic table of protein complexes. Science (80-). 350, (2015). 2. Mezzenga, R. & Fischer, P. 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). Petição 870260062218, de 25 / 06 / 2026, pág. 62 / 125 54 / 55 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. e outro 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 CrossLinked 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. e outro Soy protein nano-aggregates with improved functional properties prepared by sequential pH treatment and ultrasonication. Food Hydrocoll. 55, 200-209 (2016). 13. Jiang, S. e outro Modifying the physicochemical properties of pea protein by pH-shifting and ultrasound combined treatments. Ultrason. Sonochem. 38, 835842 (2017). 14. Mason, T. O. e outro Expanding the solvent chemical space for selfassembly of dipeptide nanostructures. ACS Nano 8, 1243-1253 (2014). 15. Li, Y. e outro Understanding the dissolution of α-zein in aqueous ethanol Petition 870260062218, dated 06 / 25 / 2026, page 63 / 125 55 / 55 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. FoodChem. 55, 9877-9882 (2007). Petition 870260062218, dated 06 / 25 / 2026, page 64 / 125

Claims

1 / 2 CLAIMS 1. Thermoreversible process for obtaining plant-based hydrogels CHARACTERIZED in that it comprises: a) forming a solution comprising one or more plant-based protein(s) in a solvent system, wherein the one or more plant-based protein(s) are selected from the group consisting of soybean, pea, rice, potato, wheat or sorghum, and the solvent system comprises miscible cosolvents; wherein a first cosolvent increases the solubility of the plant-based protein(s), and a second cosolvent decreases the solubility of the plant-based protein(s); wherein the first cosolvent is an organic acid selected from the group consisting of acetic acid, glycolic acid, lactic acid, malic acid, citric acid and tartaric acid; and wherein a second or other cosolvent(s) is an aqueous buffer;and wherein a physical stimulus is applied to the protein(s) / solvent system mixture to enable the dissolution of the protein(s), wherein the physical stimulus is selected from ultrasonication, agitation and high shear mixing; and b) induce the protein in solution to undergo a sol-gel transition to form a plant-based protein hydrogel.

2. Process according to claim 1, CHARACTERIZED in that the solvent system is subsequently removed so that the plant-based protein hydrogel no longer exhibits thermoreversible properties.

3. Process according to claim 1, CHARACTERIZED in that the solution is heated to a first temperature above the sol-gel temperature of one or more plant-based protein(s), then reduced to a second temperature below the sol-gel temperature of one or more plant-based protein(s).

4. Process, according to claim 1, CHARACTERIZED in that it further comprises: Petition 870260062218, dated 06 / 25 / 2026, page 65 / 125 2 / 2 c) forming the plant-based protein hydrogel into a structured material selected from the group consisting of a film, a thin film, a micropatterned film, a micropatterned thin film, a microstructured thin film, a nanostructured thin film, a microgel, a microcapsule, a microbead, a biosupport, a sponge, a microscale sponge, a hard capsule, or a functional coating.

5. Process according to claim 1, CHARACTERIZED in that a second or other co-solvent(s) is an aqueous buffer selected from the group consisting of water, ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, hexanol, t-butanol, ethyl acetate or hexafluoroisopropanol.

6. Process according to claim 1, CHARACTERIZED in that the solvent system comprises a ratio of the first cosolvent to the second cosolvent of 20 to 80% v / v.

7. Process, according to claim 1, CHARACTERIZED in that it further comprises mechanical shearing of said protein solution comprising one or more plant-based protein(s) and a solvent system. Petition 870260062218, dated 06 / 25 / 2026, p. 66 / 125

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