A heat-induced gel and its preparation method and use

Abstract

This invention discloses a thermally induced gel, its preparation method, and its application, belonging to the field of food colloids. It solves the problem that walnut meal protein obtained by existing methods cannot be used in food systems. Preparation method: Walnut meal is ground into powder, petroleum ether is added and stirred, and residual petroleum ether is removed by filtration to obtain defatted walnut meal powder; choline chloride and glycerol are mixed to form a transparent and homogeneous solution, obtaining DES solvent; defatted walnut meal powder is mixed with DES solvent, heated in a water bath and stirred, ethanol is added to the supernatant, the precipitate is refrigerated and dried to obtain DES-extracted walnut protein powder; the walnut protein powder is dispersed in water with a pH of 2.0 or 11.0; the resulting hydrated solution is heated in a water bath; and cooled to room temperature in an ice-water bath. This invention uses a DES solvent system to extract walnut meal protein under mild conditions, resulting in high protein purity, effective protection of the protein's native conformation, and gels exhibiting typical solid-state rheological characteristics with controllable mechanical strength.

Description

Technical Field

[0001] This invention belongs to the field of food colloids, specifically relating to a thermally induced gel, its preparation method, and its application. Background Technology

[0002] With the growing global demand for healthy and sustainable food, the high-value utilization of plant protein has become an important development direction for the food industry. Walnuts, as an important oilseed crop, produce walnut meal, a byproduct of oil extraction, which has a protein content of over 40%, rich in arginine, glutamic acid, and various essential amino acids, making it a high-quality plant protein resource with great development potential. However, about 70% of the protein in walnut meal is water-insoluble gluten, with a naturally compact conformation and few hydrophilic groups on its surface, resulting in extremely poor solubility and dispersibility. This severely restricts its application in food systems, and currently, it is mostly downgraded for use in animal feed, causing serious resource waste.

[0003] The gelation properties of proteins are a core functional characteristic that determines their application value in high-end fields such as plant-based meat products, gel candies, and 3D printed foods. Traditional walnut protein extraction often uses the alkali dissolution and acid precipitation method. This process, under strong acid and alkali conditions and drastic pH changes, easily leads to irreversible excessive denaturation, peptide chain breakage, and oxidative masking of active thiol groups. The resulting protein molecules are highly rigid, lack flexibility, and have missing cross-linking sites. In subsequent heat treatment, they can only form disordered aggregates or loose flocculent precipitates, failing to construct a dense, stable, elastic gel network. In addition, this process consumes a lot of acid and alkali, generates a lot of wastewater, and has low environmental and economic benefits. Deep eutectic solvent (DES), as a novel green extraction medium, shows significant advantages in the extraction of biomacromolecules due to its low toxicity, designability, and mild hydrogen-bonded network environment. DES can mildly dissolve walnut protein under near-neutral conditions, effectively avoiding damage from strong chemical stress, and better preserving the protein's natural folding framework and active groups, providing an excellent structural basis for the subsequent construction of a dense gel network. However, if damaged proteins extracted using traditional methods are placed under the same gelation conditions, their gelation ability is limited due to the thorough structural damage in the early stages. Currently, the industry lacks a systematic process that can combine the advantages of gentle DES extraction with targeted protein conformation activation and efficient gel network construction, resulting in the functional properties of walnut meal protein not being fully explored and industrialized. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing thermally induced gels to solve the problem that walnut meal protein obtained by existing methods cannot be used in food systems. This method takes the gentle extraction of DES to preserve the conformational flexibility of the protein as a necessary prerequisite, and combines extreme pH shift to promote expansion and precise thermal induction crosslinking to achieve a stable gel network that cannot be achieved by traditional methods.

[0005] Another object of the present invention is to provide a thermally induced gel.

[0006] A third objective of this invention is to provide an application of a heat-induced gel.

[0007] The technical solution of this invention is: (one) A method for preparing a heat-induced gel includes the following steps: S1. Raw material pretreatment and defatting: Grind the walnut meal into powder, add petroleum ether, stir for 4-6 hours, and change the petroleum ether 3-4 times during the process; after standing, remove the supernatant, filter to remove the residual petroleum ether, and obtain defatted walnut meal powder. S2 and DES preparation: Choline chloride and glycerol are mixed, heated and stirred to form a transparent and homogeneous solution; after cooling to room temperature, water is added and stirred evenly to obtain DES solvent; S3, DES extraction and protein recovery: Defatted walnut meal powder was mixed with DES solvent, heated in a water bath at 40-70℃ and stirred; then centrifuged at 2-8℃ and the supernatant was collected; ethanol was added to the supernatant, and the mixture was allowed to stand at 2-8℃, centrifuged to collect the precipitate, washed with distilled water and centrifuged again, and freeze-dried to obtain DES-extracted walnut protein powder. S4, pH shift dispersion and hydration: Disperse the walnut protein powder obtained in step S3 in water with a pH of 2.0 or 11.0, stir until completely dispersed, and let stand at 2~8℃ to obtain a hydrated solution. S5. Thermally induced denaturation and crosslinking: Transfer the hydrated liquid to a sealed container and heat it in a water bath at 80~100℃ for 1~2 hours, keeping it sealed. S6. Sudden cooling and gel ripening: After heating, immediately cool to room temperature in an ice-water bath at 0~4℃, and then refrigerate at 2~8℃; S7. Gel formation determination: If the sample obtained in step S6 is inverted and does not flow or break, and can be completely demolded and removed, it is determined to have formed a gel, and thus a heat-induced gel is obtained.

[0008] As a further improvement of the present invention, in step S1, the solid-liquid mass-volume ratio of walnut meal powder to petroleum ether is 1:10~20g / mL.

[0009] As a further improvement of the present invention, in step S2, the molar ratio of choline chloride to glycerol is 1:2~3.

[0010] As a further improvement of the present invention, in step S2, the heating temperature is 70~90℃.

[0011] As a further improvement of the present invention, in step S3, the solid-liquid mass-volume ratio of defatted walnut meal powder to DES solvent is 1:10~20g / mL.

[0012] As a further improvement of the present invention, in step S3, 2 to 4 times the volume of ethanol is added to the supernatant.

[0013] As a further improvement of the present invention, in step S4, the mass fraction of walnut protein powder in the solution is 10%~15%.

[0014] This method is a green and efficient preparation method that specifically preserves the conformational flexibility of walnut protein and achieves high-performance gelation. First, walnut meal is pretreated with petroleum ether to remove lipid interference. Then, the protein is gently extracted using a choline chloride / glycerol aqueous DES system at a suitable temperature (40-70℃). The hydrogen-bonded network of DES selectively dissolves the walnut protein, avoiding irreversible denaturation caused by strong acids and bases, and fully preserving free thiol groups and conformational flexibility—the structural basis for successful gelation. The extracted protein powder is pretreated with extreme pH shifts (2.0 or 11.0) to promote full extension of molecular chains through electrostatic repulsion, exposing hydrophobic microregions and cross-linking sites. Precise thermal induction at 80-100℃ triggers intermolecular hydrophobic interactions and orderly cross-linking of hydrogen and disulfide bonds. Finally, rapid cooling with ice water inhibits excessive aggregation, and low-temperature ripening promotes network rearrangement, forming a dense three-dimensional gel. All parameters are clearly defined and controllable, eliminating the need for complex purification equipment. (two) A heat-induced gel is prepared by the above-mentioned method for preparing a heat-induced gel. (three) Application of a thermally induced gel in 3D printing materials for plant-based meat products, gel candies, or food.

[0017] The beneficial effects of this invention are: 1. The method of this invention uses a choline chloride:glycerol eutectic solvent system to extract walnut meal protein under mild conditions. The protein purity reaches 86.26%±3.76%, which is 7.98 percentage points higher than that of the alkaline extraction method (78.28%±1.73%) (p<0.05); the recovery rate reaches 47.30%±3.13%, an increase of 5.62% (p<0.05); the average particle size is reduced to 753.8±72.3 nm (1015.9±214.4 nm for alkaline extraction), the fluorescence λmax is stable at 353 nm without red shift, and the disulfide bond content is significantly higher, indicating that the native conformation of the protein is effectively protected.

[0018] 2. This invention is the first to achieve the formation of self-supporting, heat-induced gels from walnut meal protein under both strongly acidic (pH 2.0) and strongly alkaline (pH 11.0) conditions, while traditional alkaline protein extraction methods only produce precipitation under the same conditions. The gels obtained by this invention exhibit typical solid-state rheological characteristics (G'>G'', 2–3 times), with controllable mechanical strength, significantly expanding the application potential of protein in various scenarios such as acidic beverages and alkaline flour products.

[0019] 3. This invention quantifies intermolecular forces using selective solvent extraction, confirming that hydrophobic interactions and disulfide bonds are the main driving forces of the gel network. The proportion of unbound proteins under acidic conditions (12.96%±1.44%) is significantly lower than that under alkaline conditions (19.28%±1.45%) (p<0.05), indicating that an acidic environment is more conducive to efficient protein network integration. The microstructure exhibits pH-dependent differences (dense clusters at pH 2, uniform network at pH 11), providing theoretical support for the targeted design of gels with different textures.

[0020] 4. This invention achieves flexible "one-material-multiple-type" production simply by adjusting the system pH: at pH 2, the gel hardness is 3.95±0.74 N and the yield stress is 646.50±200.13 Pa, suitable for products with high shape retention (such as plant-based minced meat); at pH 11, the gel elasticity is 0.92±0.07 mm, an increase of 16.5% compared to pH 2 (p<0.05), suitable for products with a soft and chewy texture (such as vegetarian ham). The process requires no additional cross-linking agents, is low-cost, easy to operate, and readily scalable for industrial production. Attached Figure Description

[0021] Figure 1 The diagram shows the effect of the conformational integrity of the walnut protein powder prepared in Comparative Example 1 and Example 1 on the raw material defatted walnut meal powder. Among them, (A) is a comparison diagram of purity, extraction rate and recovery rate, (B) is a comparison diagram of average particle size, (C) is a comparison diagram of intrinsic fluorescence spectrum, and (D) is a comparison diagram of disulfide bond content. Figure 2 The image shows a comparison of the microstructures of the walnut protein powder prepared in Example 1 and Comparative Example 1 and the raw material defatted walnut meal powder. In the image, (A) represents the raw material defatted walnut meal powder, (B) represents the walnut protein powder prepared in Example 1, and (C) represents the walnut protein powder prepared in Comparative Example 1. Figure 3 The images show the macroscopic morphology, textural properties, and rheological properties of the thermally induced gels prepared under pH 2.0 and 11.0 conditions in Example 1, respectively. (A) shows the macroscopic morphology of the thermally induced gel prepared under pH 2.0 conditions; (B) shows the macroscopic morphology of the thermally induced gel prepared under pH 11.0 conditions; (C) shows the textural properties of the thermally induced gels prepared under pH 2.0 and 11.0 conditions; and (D) shows the rheological properties of the thermally induced gels prepared under pH 2.0 and 11.0 conditions. Figure 4The images show a comparison of the internal and external microstructures of the thermally induced gels prepared under pH 2.0 and 11.0 conditions, respectively, in Example 1. (A) is a laser confocal image of the thermally induced gel prepared under pH 2.0 conditions, (B) is a 2D image of the surface three-dimensional contour of the thermally induced gel prepared under pH 2.0 conditions, (C) is a 3D image of the surface three-dimensional contour of the thermally induced gel prepared under pH 2.0 conditions, (D) is a laser confocal image of the thermally induced gel prepared under pH 11.0 conditions, (E) is a 2D image of the surface three-dimensional contour of the thermally induced gel prepared under pH 11.0 conditions, and (F) is a 3D image of the surface three-dimensional contour of the thermally induced gel prepared under pH 11.0 conditions. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 A method for preparing a heat-induced gel includes the following steps: S1. Raw material pretreatment and defatting: Grind 5.0g of walnut meal into powder, pass it through an 80-mesh sieve, add petroleum ether, the solid-liquid mass-volume ratio of walnut meal powder to petroleum ether is 1:10g / mL, stir and defatted for 5 hours, changing the petroleum ether 3 times during the process; after standing for 12 hours, remove the supernatant, filter to remove residual petroleum ether, and obtain defatted walnut meal powder. S2, DES preparation: Choline chloride and glycerol were mixed at a molar ratio of 1:3 and stirred at 80°C and 500 rpm until a transparent and homogeneous solution was formed; after cooling to room temperature, water equivalent to 10% of the solution mass was added and stirred evenly to obtain DES solvent; S3. DES Extraction and Protein Recovery: Defatted walnut meal powder was mixed with DES solvent at a solid-liquid mass-to-volume ratio of 1:15 g / mL. The mixture was heated in a 60°C water bath and magnetically stirred for 2.5 hours. Subsequently, the mixture was centrifuged at 4°C and 9000 rpm for 20 minutes, and the supernatant was collected. Four times the volume of ethanol was added to the supernatant, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation, washed twice with distilled water, centrifuged again, and freeze-dried to obtain DES-extracted walnut protein powder. S4, pH shift dispersion and hydration: Weigh 0.1g of the walnut protein powder obtained in step S3 and disperse it in 1mL of water with pH 2.0 and 11.0 respectively. Stir magnetically for 2 hours at room temperature until completely dispersed, and then let it stand at 4℃ for 12 hours to obtain the hydrated solution. S5. Thermally induced denaturation and crosslinking: Transfer the hydrated liquid to a sealed heat-resistant bottle and heat it in a 90°C water bath for 1 hour, keeping it sealed. S6. Sudden cooling and gel ripening: After heating, immediately cool to room temperature in a 0°C ice water bath, and then refrigerate at 4°C for 12 hours; S7. Gelation Determination: Invert the sample obtained in step S6. If it does not flow or break, and can be completely demolded by gently tapping the tube wall, it is considered to have successfully gelled, thus obtaining a heat-induced gel. Gels obtained at pH 2.0 and 11.0 are named D2 and D, respectively. 11 .

[0024] Example 2 A method for preparing a heat-induced gel includes the following steps: S1. Raw material pretreatment and defatting: Grind 5.0g of walnut meal into powder, pass it through an 80-mesh sieve, add petroleum ether, the solid-liquid mass-volume ratio of walnut meal powder to petroleum ether is 1:15g / mL, stir and defatted for 4 hours, changing the petroleum ether 3 times during the process; after standing for 12 hours, remove the supernatant, filter to remove residual petroleum ether, and obtain defatted walnut meal powder. S2, DES preparation: Choline chloride and glycerol were mixed at a molar ratio of 1:2 and stirred at 70°C and 500 rpm until a transparent and homogeneous solution was formed; after cooling to room temperature, water equivalent to 10% of the solution mass was added and stirred evenly to obtain DES solvent; S3. DES Extraction and Protein Recovery: Defatted walnut meal powder was mixed with DES solvent at a solid-liquid mass-to-volume ratio of 1:10 g / mL. The mixture was heated in a 40°C water bath and magnetically stirred for 1.5 hours. Subsequently, the mixture was centrifuged at 2°C and 9000 rpm for 20 minutes, and the supernatant was collected. Two volumes of ethanol were added to the supernatant, and the mixture was allowed to stand overnight at 2°C. The precipitate was collected by centrifugation, washed twice with distilled water, centrifuged again, and freeze-dried to obtain DES-extracted walnut protein powder. S4, pH shift dispersion and hydration: Weigh 0.12g of the walnut protein powder obtained in step S3 and disperse it in 1mL of water with pH 2.0 and 11.0 respectively. Stir magnetically for 2 hours at room temperature until completely dispersed, and then let it stand at 2℃ for 12 hours to obtain the hydration solution. S5, Thermally induced denaturation and crosslinking: Transfer the hydrated liquid to a sealed heat-resistant bottle and heat it in a water bath at 80°C for 1.5 hours, keeping it sealed; S6. Sudden cooling and gel ripening: Immediately after heating, cool to room temperature in an ice-water bath at 4°C, and then refrigerate at 4°C for 12 hours; S7. Gel formation determination: Invert the sample obtained in step S6. If it does not flow or break, and can be completely demolded by gently tapping the tube wall, it is determined that gel formation is successful, and the heat-induced gel is obtained.

[0025] Example 3 A method for preparing a heat-induced gel includes the following steps: S1. Raw material pretreatment and defatting: Grind 5.0g of walnut meal into powder, pass it through an 80-mesh sieve, add petroleum ether, the solid-liquid mass-volume ratio of walnut meal powder to petroleum ether is 1:20g / mL, stir and defatted for 6 hours, during which the petroleum ether is replaced 4 times; after standing for 12 hours, remove the supernatant, filter to remove residual petroleum ether, and obtain defatted walnut meal powder. S2, DES preparation: Choline chloride and glycerol were mixed at a molar ratio of 1:3 and stirred at 90°C and 500 rpm until a transparent and homogeneous solution was formed; after cooling to room temperature, water equivalent to 10% of the solution mass was added and stirred evenly to obtain DES solvent; S3. DES Extraction and Protein Recovery: Defatted walnut meal powder was mixed with DES solvent at a solid-liquid mass-to-volume ratio of 1:20 g / mL. The mixture was heated in a 70°C water bath and magnetically stirred for 1 hour. Subsequently, it was centrifuged at 8°C and 9000 rpm for 20 minutes, and the supernatant was collected. Four times the volume of ethanol was added to the supernatant, and the mixture was allowed to stand overnight at 8°C. The precipitate was collected by centrifugation, washed twice with distilled water, centrifuged again, and freeze-dried to obtain DES-extracted walnut protein powder. S4, pH shift dispersion and hydration: Weigh 0.15 g of the walnut protein powder obtained in step S3 and disperse it in 1 mL of water with pH 2.0 and 11.0 respectively. Stir magnetically for 2 hours at room temperature until completely dispersed, and then let it stand at 6℃ for 12 hours to obtain the hydrated liquid. S5. Thermally induced denaturation and crosslinking: Transfer the hydrated liquid to a sealed heat-resistant bottle and heat it in a 100°C water bath for 2 hours, keeping it sealed. S6. Sudden cooling and gel ripening: After heating, immediately cool to room temperature in an ice water bath at 2°C, and then refrigerate at 2°C for 12 hours; S7. Gel formation determination: Invert the sample obtained in step S6. If it does not flow or break, and can be completely demolded by gently tapping the tube wall, it is determined that gel formation is successful, and the heat-induced gel is obtained.

[0026] Comparative Example 1 Step A: Prepare walnut protein powder using the alkaline dissolution and acid precipitation method (ASAP); Step B: Weigh 0.1g of walnut protein powder and disperse it in 1mL of water with pH 2.0. Stir magnetically for 2 hours at room temperature until completely dispersed, and then let it stand at 4℃ for 12 hours to obtain the hydrated liquid. Step C: Transfer the hydrated solution to a sealed heat-resistant bottle and heat it in a 90°C water bath for 1 hour, keeping it sealed. Step D: Immediately after heating, cool to room temperature in a 0°C ice-water bath, then refrigerate at 4°C for 12 hours.

[0027] Macroscopic observation shows that, after the same heat treatment at pH 2.0, the protein extracted by the ASAP method in this comparative example only exhibited disordered precipitation or loose flocculent aggregation, failing to achieve effective gelation and unable to form a continuous three-dimensional network structure.

[0028] Comparative Example 2 Step A: Prepare walnut protein powder using the alkaline dissolution and acid precipitation method (ASAP); Step B: Weigh 0.1g of walnut protein powder and disperse it in 1mL of water with pH 11.0. Stir magnetically for 2 hours at room temperature until completely dispersed, and then let it stand at 4℃ for 12 hours to obtain the hydrated liquid. Step C: Transfer the hydrated solution to a sealed heat-resistant bottle and heat it in a 90°C water bath for 1 hour, keeping it sealed. Step D: Immediately after heating, cool to room temperature in a 0°C ice-water bath, then refrigerate at 4°C for 12 hours.

[0029] Macroscopic observations show that, under alkaline conditions at pH 11.0, the ASAP-extracted proteins in this comparative example only exhibited flocculent aggregation after heat treatment, failing to form a self-supporting gel. The overall structure was fluid or semi-fluid, lacking basic mechanical strength and shape retention.

[0030] (a) Evaluation of protein conformation integrity The conformational integrity of the walnut protein powders prepared in Example 1 (DES extraction) and Comparative Example 1 (alkali extraction) was evaluated using the Kjeldahl method, dynamic light scattering (DLS), intrinsic fluorescence spectroscopy, and the Ellman method. Figure 1 As shown in (A), the purity of Example 1 reached 86.26% ± 3.76%, an increase of 7.98 percentage points compared to Comparative Example 1 (78.28% ± 1.73%). Simultaneously, its protein recovery rate also increased to 47.30% ± 3.13%, an increase of approximately 5 percentage points compared to the alkaline method (41.68% ± 1.28%). It is noteworthy that despite the significant improvement in purity and recovery rate, the total extraction rate of the DES method was not significantly different from that of the alkaline method (p>0.05), indicating that this method can effectively improve product quality without sacrificing extraction efficiency.

[0031] like Figure 1As shown in (B), the average particle size of the sample prepared in Example 1 was 753.8 ± 72.3 nm, significantly lower than that of Comparative Example 1 (1015.9 ± 214.4 nm, p < 0.05), and the particle size distribution was narrower and the dispersion uniformity was better. In contrast, the average particle size of the defatted walnut meal powder was 1195 ± 82.7 nm, which was the highest among the three groups. The above differences may be attributed to the following: The eutectic solvent (DES) extraction system used in Example 1 can selectively disrupt the plant cell wall matrix under mild conditions, promoting the directional dissolution of the target protein. At the same time, its unique hydrogen bond network structure can form a dynamic solvation layer on the surface of protein molecules, effectively shielding hydrophobic interactions and inhibiting the disordered aggregation and conformational rearrangement of protein molecules during extraction, thereby obtaining protein microparticles with small particle size and uniform distribution. In contrast, the alkali dissolution and acid precipitation method used in Comparative Example 1 involves strong alkali dissolution and isoelectric point precipitation steps, which easily leads to partial denaturation of protein molecules and exposure of thiol / hydrophobic groups. Irreversible aggregation occurs under the drive of charge neutralization and van der Waals forces, resulting in increased particle size and broadened distribution. As a control, defatted walnut meal powder still forms a dense complex structure with cell wall polysaccharides, lipids and other macromolecules, and has not undergone dissociation and homogenization treatment, thus exhibiting the largest particle size.

[0032] like Figure 1 As shown in (C), the maximum emission wavelength λmax of the fluorescence in Example 1 remained stable at 353 nm without redshift, almost coinciding with the raw material defatted walnut meal, while the protein in Comparative Example 1 redshifted to 365 nm. In protein tertiary structure studies, intrinsic fluorescence is commonly used to monitor the degree to which aromatic amino acids (mainly tryptophan residues) are exposed to their surrounding hydrophilic environment. Tryptophan residues are usually embedded in the hydrophobic core of proteins. When proteins unfold, these residues are exposed to polar solvents, resulting in a redshift of the emission wavelength. The observed 12 nm redshift indicates that the tryptophan residues in Comparative Example 1 have been transferred from the hydrophobic core to a more polar microenvironment—the combined alkali-acid treatment induced significant protein unfolding, exposing the originally embedded hydrophobic region to the aqueous phase.

[0033] like Figure 1As shown in (D), the disulfide bond content of the protein sample prepared in Example 1 was 15.95±0.93 μmol / g, which was significantly higher than that of Comparative Example 1 (12.11±0.52 μmol / g, p<0.05); while the disulfide bond content of the raw material defatted walnut meal powder was only 8.47±0.15 μmol / g, which was the lowest among the three groups. The above differences may be attributed to the following: The eutectic solvent (DES) extraction system used in Example 1 can moderately promote the directional oxidation of thiol groups (-SH) to intramolecular / intermolecular disulfide bonds (-SS-) while dissolving the target protein. Its hydrogen bond network structure can effectively shield the risk of excessive oxidation and avoid irreversible loss of thiol groups, thereby retaining a higher level of crosslinkable sites in the protein particles. In contrast, the alkaline dissolution and acid precipitation method used in Comparative Example 1 is prone to non-specific oxidation or β-elimination of thiol groups due to the drastic pH fluctuations caused by strong alkaline dissolution and isoelectric point precipitation, resulting in the breakage of some disulfide bonds or thiol group blockage, thus limiting the crosslinking potential. As the raw material, defatted walnut meal powder, is an unextracted raw material, its protein components are densely wrapped by cell wall polysaccharides and lipids. In the natural conformation, most thiol groups exist in a buried state with low accessibility, so the total amount of free / oxidizable thiol groups detected is the lowest.

[0034] In summary, this invention utilizes a green extraction process with a low eutectic solvent, which effectively preserves the natural conformation of walnut protein while achieving efficient extraction, laying a structural foundation for subsequent functional applications such as gels.

[0035] (II) Evaluation of Microscopic Morphology The microstructure of the walnut protein powder and defatted walnut meal powder prepared in Example 1 and Comparative Example 1 was characterized using scanning electron microscopy (SEM). Figure 2 As shown, the defatted walnut meal powder (A) exhibits an irregular and dense agglomerated structure, composed of large, interconnected clusters with blurred boundaries and a dense overall morphology; the walnut protein powder (B) prepared in Example 1 exhibits a highly dense and uniform microstructure, characterized by uniformly distributed fine particles, a relatively smooth and continuous surface, no obvious macropores or cracks, but rich fine textures; the walnut protein powder (C) prepared in Comparative Example 1 exhibits a looser and more heterogeneous structure, with a significantly increased porosity, forming an obvious network-like architecture, and a large number of open channels and cavities were observed, indicating enhanced structural permeability.

[0036] (III) Evaluation of gel properties The thermally induced gels (labeled D2 and D3) prepared in Example 1 under acidic (pH 2.0) and alkaline (pH 11.0) conditions, respectively, were analyzed using a rotational rheometer and a texture analyzer. 11 To conduct performance evaluation, Figure 3 (A) and (B) are the thermally induced gels (D2, D) prepared respectively. 11 Macroscopic topography diagram of ).

[0037] like Figure 3 As shown in (C), in terms of textural properties, the D2 hardness (3.95±0.74N) is similar to that of D... 11 (3.84±0.36N) is equivalent, but D 11 The elasticity (0.92±0.07 mm) was 16.5% higher than that of D2 (0.79±0.02 mm), indicating that the alkaline conditional gel has superior structural recovery ability. 11 The adhesion of D2 (0.0308±0.004mJ) is slightly higher than that of D2 (0.0280±0.007mJ), which suggests that its surface may have stronger moisture retention or intermolecular forces.

[0038] like Figure 3 As shown in (D), within the linear viscoelastic region (LVE, 0.01-1%), the storage modulus (G') and loss modulus (G'') of all gel samples remained relatively constant, exhibiting a clear plateau region, indicating that the internal structure of the material was not damaged by strain. When the strain exceeded the critical value (yield point), both G' and G'' decreased significantly with increasing strain, indicating that the three-dimensional network structure of the gel was disrupted. Moreover, throughout the entire test strain range, the value of G' was always higher than that of G'', and the value of G' was approximately 2-3 times that of G'', indicating that both samples exhibited typical solid-like behavior and obvious gel characteristics. This phenomenon suggests that a stable three-dimensional network structure was formed inside the samples, with elastic response dominating.

[0039] (iv) Gel microstructure The thermally induced gels (labeled D2 and D3) prepared in Example 1 under acidic (pH 2.0) and alkaline (pH 11.0) conditions were analyzed using a confocal laser scanning microscope (CLSM) and a non-contact white light interferometer. 11 The internal and apparent microstructures were characterized. Regarding the internal microstructure (CLSM), D... 11 It exhibits a uniform and dense three-dimensional network architecture, such as Figure 4 As shown in (D), the pores are small and uniformly distributed, forming a typical continuous hydrogel network; while D2 exhibits a relatively large pore structure, such as... Figure 4 As shown in (A), the protein aggregates are densely packed in clusters and have higher fluorescence intensity, indicating that the degree of protein aggregation is higher and the network integration is tighter under acidic conditions.

[0040] In terms of apparent and microstructure, the spectral color gradation distribution of D2 in its two-dimensional height distribution map is highly uniform, such as... Figure 4As shown in (B), the color is mainly concentrated in the yellow-orange hue (corresponding to a narrow height range of 40–70 μm), with extreme color gradations such as deep blue / light white scattered sporadically. This planar mapping characteristic indicates that the microstructure spatial arrangement of the D2 surface is more regular, and the height dispersion of the micro-regions is extremely low. In contrast, D 11 The two-dimensional spectrum exhibits significant color heterogeneity, such as Figure 4 As shown in (E), the dark blue depressions (<20 μm) and the reddish-white raised areas (>50 μm) are widely interspersed, with clear boundaries and large spans, indicating that D 11 The surface microstructure has poor uniformity.

[0041] The three-dimensional surface of D2 is as follows Figure 4 As shown in (C), it only exhibits uniform and subtle undulations, and its overall shape is relatively flat; in contrast, D... 11 ,like Figure 4 As shown in (F), its surface exhibits severe non-uniform fluctuations, with large-scale peak-valley structures running through the entire observation area (spanning depths from 65.8 μm to 95.7 μm), spatially corresponding to the large areas of heterogeneous color gradations observed in the two-dimensional image. This significant deformation at the macroscopic level may be attributed to network rearrangement and uneven stress release caused by the differentiation of water migration pathways in the later stages of gelation.

[0042] This invention is simple to operate, green, safe, and environmentally friendly. Using walnut protein powder gently extracted with deep eutectic solvent (DES) as the starting material, it specifically activates the gelling potential of walnut protein through the synergistic effect of pH shift and thermal induction, achieving a highly efficient and stable gelation method. This is a healthy and environmentally friendly new method suitable for the development of plant-based foods and functional gel matrices.

Claims

1. A method for preparing a heat-induced gel, characterized in that, Includes the following steps: S1. Raw material pretreatment and defatting: Grind the walnut meal into powder, add petroleum ether, stir for 4-6 hours, and change the petroleum ether 3-4 times during the process; after standing, remove the supernatant, filter to remove the residual petroleum ether, and obtain defatted walnut meal powder. S2 and DES preparation: Choline chloride and glycerol are mixed, heated and stirred to form a transparent and homogeneous solution; after cooling to room temperature, water is added and stirred evenly to obtain DES solvent; S3, DES extraction and protein recovery: Defatted walnut meal powder was mixed with DES solvent, heated in a water bath at 40-70℃ and stirred; then centrifuged at 2-8℃ and the supernatant was collected; ethanol was added to the supernatant, and the mixture was allowed to stand at 2-8℃, centrifuged to collect the precipitate, washed with distilled water and centrifuged again, and freeze-dried to obtain DES-extracted walnut protein powder. S4, pH shift dispersion and hydration: Disperse the walnut protein powder obtained in step S3 in water with a pH of 2.0 or 11.0, stir until completely dispersed, and let stand at 2~8℃ to obtain a hydrated solution. S5. Thermally induced denaturation and crosslinking: Transfer the hydrated liquid to a sealed container and heat it in a water bath at 80~100℃ for 1~2 hours, keeping it sealed. S6. Sudden cooling and gel ripening: After heating, immediately cool to room temperature in an ice-water bath at 0~4℃, and then refrigerate at 2~8℃; S7. Gel formation determination: If the sample obtained in step S6 is inverted and does not flow or break, and can be completely demolded and removed, it is determined to have formed a gel, and thus a heat-induced gel is obtained.

2. The method for preparing a heat-induced gel according to claim 1, characterized in that: In step S1, the solid-liquid mass-volume ratio of walnut meal powder to petroleum ether is 1:10~20 g / mL.

3. The method for preparing a heat-induced gel according to claim 1, characterized in that: In step S2, the molar ratio of choline chloride to glycerol is 1:2~3.

4. The method for preparing a heat-induced gel according to claim 1, characterized in that: In step S2, the heating temperature is 70~90℃.

5. The method for preparing a heat-induced gel according to claim 1, characterized in that: In step S3, the solid-liquid mass-volume ratio of defatted walnut meal powder to DES solvent is 1:10~20g / mL.

6. The method for preparing a heat-induced gel according to claim 1, characterized in that: In step S3, 2 to 4 times the volume of ethanol is added to the supernatant.

7. The method for preparing a heat-induced gel according to claim 1, characterized in that: In step S4, the mass fraction of walnut protein powder in the solution is 10%~15%.

8. A heat-induced gel, characterized in that: It is prepared by any one of the methods for preparing a heat-induced gel according to claims 1-7.

9. The use of the thermally induced gel of claim 8 in plant-based meat products, gel candies, or food 3D printing materials.

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