Anisotropic hydrogel and its preparation method and application

Abstract

The present invention belongs to the technical field of hydrogel preparation, and provides an anisotropic hydrogel, a preparation method thereof, and an application thereof. The present invention adds transglutaminase and introduces enzymatic chemical cross-linking, which, combined with physical cross-linking, can stabilize the anisotropy of the hydrogel, making the structure of the hydrogel more stable. The surface of the anisotropic hydrogel obtained by the present invention has an ordered orientation structure. When applied to in vitro cell culture, it can enable cells to grow in the same direction. Specifically, when used to prepare artificial meat, the taste of the obtained artificial meat is closer to the fibrous texture of real meat. In addition, the preparation method of the present invention is simple to operate. Furthermore, the fibrous material in the fibrous material dispersion includes cellulose or isolated protein amyloid fibers, and the raw materials used for preparation are non-toxic and harmless, and can be used in the food field.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel preparation, and in particular to an anisotropic hydrogel and a preparation method and application thereof. Background Art

[0002] In recent years, hydrogels have been widely used in food science, tissue engineering, drug delivery, and cell culture due to their good biocompatibility and high water content. Hydrogels can be used as functional materials in food processing, texture regulation, and nutrient delivery. For example, hydrogels can be designed to encapsulate active ingredients or regulate the texture and taste of food, thereby improving the stability and flavor of the food. In addition, hydrogels are also widely used to simulate food structures to study the physical and chemical properties of food during processing and storage. In recent years, innovative research based on hydrogel materials has focused on how to develop structures with anisotropic properties to simulate the physical properties of food in different directions. In particular, in food texture design, anisotropic hydrogels can achieve the mechanical response of food in different chewing directions, thereby improving the consumer's chewing experience. This type of anisotropic hydrogel material not only has important application prospects in the development of new foods, but also provides new solutions for functional foods and personalized nutrition design.

[0003] Traditional methods for constructing anisotropic hydrogels include freezing, templating, and electrospinning, but these methods often have the disadvantage of complex processes. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an anisotropic hydrogel and its preparation method and application. The preparation method provided by the present invention is simple to operate.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing anisotropic hydrogel, comprising the following steps:

[0007] providing a dispersion of a fibrous material;

[0008] The fibrous material dispersion, gelatin solution and transglutaminase are mixed, and the obtained precursor solution is pre-crosslinked and freeze-crosslinked in sequence to obtain a cross-linked hydrogel;

[0009] mixing the cross-linked hydrogel and a first salt solution, and performing salting-out to obtain a salted-out hydrogel;

[0010] stretching the salting-out hydrogel to obtain a stretched hydrogel;

[0011] mixing the stretched hydrogel with a second salt solution and curing the mixture to obtain the anisotropic hydrogel;

[0012] There is physical cross-linking in the fibrous material dispersion.

[0013] Preferably, the fibrous material in the fibrous material dispersion comprises cellulose or isolated protein amyloid fibers;

[0014] When the fibrous material in the fibrous material dispersion is isolated protein amyloid fibers, the preparation method of the isolated protein amyloid fiber dispersion comprises the following steps:

[0015] Mixing the protein isolate and an ethanol-water system, adjusting the pH to be acidic, and heating the system in a water bath to obtain the protein isolate amyloid fiber dispersion;

[0016] The isolated protein is pea protein isolate or soy protein isolate;

[0017] The volume concentration of ethanol in the ethanol-water system is 0 to 50%;

[0018] The acidic pH value is 1.5 to 3;

[0019] The water bath heating temperature is 70-90° C. and the heating time is 20-40 hours.

[0020] Preferably, when the isolated protein is soy protein isolate, after adjusting the pH value to acidic and before heating in a water bath, the process further includes hydration and precipitate removal; the hydration temperature is 2 to 8° C. and the time is 6 to 10 hours.

[0021] Preferably, in the precursor solution, the mass concentration of the fibrous material is 0.5-8%, the mass concentration of gelatin is 3-10%, and the mass ratio of transglutaminase to gelatin is 1:1-12;

[0022] The pre-crosslinking temperature is 40-60°C and the time is 2-30 minutes;

[0023] The temperature of the frozen cross-linking is 4° C., and the time is 1 to 3 hours.

[0024] Preferably, the salt in the first salt solution includes one or more of sodium chloride, sodium sulfate and sodium citrate, and the mass concentration of the first salt solution is 15-40%;

[0025] The salting-out time is 0.5 to 2 hours.

[0026] Preferably, the degree of stretching is 1 to 6 times the original length.

[0027] Preferably, the salt in the second salt solution includes sodium citrate, and the mass concentration of the second salt solution is 15-40%;

[0028] The curing time is 10 to 20 hours.

[0029] The present invention also provides an anisotropic hydrogel prepared by the preparation method described in the above technical solution.

[0030] The present invention also provides the application of the anisotropic hydrogel described in the above technical solution in the food field.

[0031] Preferably, the food sector includes the preparation of artificial meat.

[0032] The invention provides a method for preparing anisotropic hydrogel.

[0033] The present invention uses a fibrous material dispersion, in which the fibrous material can impart anisotropy to the hydrogel during subsequent stretching; by adding transglutaminase, enzymatic chemical crosslinking can be introduced, and enzymatic chemical crosslinking is combined with physical crosslinking (from salting out) to stabilize the anisotropy of the hydrogel, making the structure of the hydrogel more stable and having excellent mechanical properties. The surface of the anisotropic hydrogel obtained by the present invention has an ordered orientation structure, and when applied to in vitro cell culture, cells can be grown in the same direction. When specifically used to prepare artificial meat, the mouthfeel of the obtained artificial meat is closer to the fibrousness of real meat. Finally, the preparation method provided by the present invention is simple to operate.

[0034] Furthermore, the fibrous material in the fibrous material dispersion includes cellulose or isolated protein amyloid fibers. The raw materials used in the present invention are non-toxic and harmless and can be used in the food field.

[0035] Furthermore, when preparing the protein isolate amyloid fiber dispersion, the volume concentration of ethanol in the ethanol-water system can control the morphology of the protein isolate amyloid fibers formed by the protein isolate, thereby achieving the regulation of the final hydrogel micromorphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Transmission scanning electron micrographs of the isolated protein amyloid fibers in each dispersion obtained in Example 1;

[0037] Figure 2 This is a polarizing microscope image of the anisotropic gelatin-isolate amyloid fiber hydrogel obtained in Example 2;

[0038] Figure 3 These are optical photographs of the anisotropic gelatin-isolate amyloid fiber hydrogel obtained in Example 2 at different incubation times at 27° C. and 37° C.;

[0039] Figure 4 This is the stress-strain curve of the soy protein isolate amyloid fiber-salting-out hydrogel obtained in Example 3;

[0040] Figure 5 This is a scanning electron micrograph of the anisotropic gelatin-pea protein isolate amyloid fiber hydrogel obtained in Example 4;

[0041] Figure 6 These are optical photographs of the anisotropic gelatin-isolate amyloid fiber hydrogel obtained in Example 4 at different incubation times in an incubator at 27° C. and 37° C.;

[0042] Figure 7 These are cell morphologies obtained after cell growth on unstretched and stretched hydrogels in Example 4. DETAILED DESCRIPTION

[0043] Explanation of terms:

[0044] Anisotropy refers to the fact that a material has different physical or chemical properties in different directions. Physical or chemical properties include mechanical properties, expansion behavior, electrical or thermal conductivity, etc. Anisotropy is the opposite of isotropy, which means that the material has the same properties in all directions.

[0045] The present invention provides a method for preparing anisotropic hydrogel, comprising the following steps:

[0046] providing a dispersion of a fibrous material;

[0047] The fibrous material dispersion, gelatin solution and transglutaminase are mixed, and the obtained precursor solution is pre-crosslinked and freeze-crosslinked in sequence to obtain a cross-linked hydrogel;

[0048] mixing the cross-linked hydrogel and a first salt solution, and performing salting-out to obtain a salted-out hydrogel;

[0049] stretching the salting-out hydrogel to obtain a stretched hydrogel;

[0050] mixing the stretched hydrogel with a second salt solution and curing the mixture to obtain the anisotropic hydrogel;

[0051] There is physical cross-linking in the fibrous material dispersion.

[0052] Unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0053] The present invention provides a fibrous material dispersion.

[0054] In the present invention, there is a physical cross-linking effect in the fibrous material dispersion, and the physical cross-linking effect preferably includes but is not limited to hydrophobic association, hydrogen bond, ionic bond and host-guest interaction.

[0055] In the present invention, the mass concentration of the fibrous material dispersion is preferably 1-10%, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In the present invention, the fibrous material in the fibrous material dispersion preferably comprises cellulose or isolated protein amyloid fibers.

[0056] In the present invention, when the fibrous material in the fibrous material dispersion is preferably isolated protein amyloid fibers, the method for preparing the isolated protein amyloid fiber dispersion preferably comprises the following steps:

[0057] The protein isolate is mixed with an ethanol-water system, the pH is adjusted to an acidic value, and the mixture is heated in a water bath to obtain the protein isolate amyloid fiber dispersion. In the present invention, the protein isolate is preferably pea protein isolate or soy protein isolate. In the present invention, the volume concentration of ethanol in the ethanol-water system is preferably 0-50%, specifically 0%, 10%, 20%, 30%, 40%, or 50%. In the present invention, the volume concentration of ethanol in the ethanol-water system can control the morphology of the protein isolate amyloid fibers formed by the protein isolate, thereby regulating the microscopic morphology of the final hydrogel. In the present invention, the mixing of the protein isolate and the ethanol-water system is preferably performed under magnetic stirring. In the present invention, the acidic pH is preferably 1.5-3, more preferably 2; the reagent for adjusting the pH to an acidic value is preferably a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 3 mol / L. In the present invention, the water bath heating temperature is preferably 70-90°C, specifically 70°C, 75°C, 80°C, 85°C or 90°C; the time is preferably 20-40 hours, more preferably 30 hours, specifically 20 hours, 24 hours, 30 hours, 36 hours or 40 hours.

[0058] In the present invention, when the protein isolate is preferably pea protein isolate, after the protein isolate and the ethanol-water system are mixed and before the pH value is adjusted to be acidic, it is preferably further included to remove the precipitate, and the method for removing the precipitate is preferably centrifugation. The centrifugal speed is preferably 5000-7000 rpm, more preferably 6000 rpm, and the time is preferably 10-30 minutes, more preferably 20 minutes.

[0059] In the present invention, when the isolated protein is soy protein isolate, after adjusting the pH value to acidic and before heating in a water bath, preferably, hydration and precipitate removal are also performed; the hydration temperature is preferably 4°C, the time is preferably 6 to 10 hours, and more preferably 8 hours; the hydration is preferably performed in a refrigerator; the method of removing the precipitate is preferably centrifugation, the centrifugal speed is preferably 5000 to 7000 rpm, more preferably 6000 rpm, and the time is preferably 10 to 30 minutes, and more preferably 20 minutes.

[0060] In the present invention, when the fibrous material is cellulose, the method for preparing the cellulose dispersion preferably comprises the following steps: dispersing cellulose in water to obtain the cellulose dispersion, wherein the water is preferably deionized water.

[0061] After obtaining the fibrous material dispersion, the present invention mixes the fibrous material dispersion, gelatin solution and transglutaminase, and sequentially performs pre-crosslinking and freeze-crosslinking on the obtained precursor solution to obtain a crosslinked hydrogel.

[0062] In the present invention, the mass concentration of the gelatin solution is preferably 5-20%, specifically 5%, 10%, 15% or 20%. In the present invention, the method for preparing the gelatin solution preferably comprises the following steps: mixing gelatin and water and subjecting the mixture to magnetic stirring to obtain the gelatin solution. In the present invention, the water is preferably deionized water.

[0063] In the present invention, the transglutaminase is preferably used in the form of a transglutaminase solution, and the mass concentration of the transglutaminase solution is preferably 10%. In the present invention, the method for preparing the transglutaminase solution preferably comprises the steps of mixing the transglutaminase and water and performing magnetic stirring to obtain the transglutaminase solution. In the present invention, the water is preferably deionized water.

[0064] In the present invention, mixing the fibrous material dispersion, gelatin solution, and transglutaminase preferably includes the following steps: after mixing the fibrous material dispersion and gelatin solution, adjusting the pH to 6 to 8; and then sequentially adding water and the transglutaminase solution. In the present invention, the pH value can specifically be 6, 7, or 8. In the present invention, in the precursor solution, the mass concentration of the fibrous material is preferably 0.5-8%, more preferably 1-5%, specifically 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5% or 8%; the mass concentration of gelatin is preferably 3-10%, specifically 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, and the mass ratio of transglutaminase to gelatin is preferably 1:1-12, specifically 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11 or 1:12.

[0065] In the present invention, the pre-crosslinking temperature is preferably 40-60°C, specifically 40°C, 45°C, 50°C, 55°C, or 60°C; the pre-crosslinking time is preferably 2-30 minutes, specifically 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. In the present invention, the pre-crosslinking allows the gelatin and fibrous material to be partially bonded together through chemical crosslinking. After complete bonding, the molecular chains are locked and cannot be subsequently stretched. Only by achieving partial crosslinking can stretchability be maintained while preventing subsequent dissolution.

[0066] In the present invention, the temperature of the freeze cross-linking is preferably 4°C, and the time is preferably 1 to 3 hours, specifically 1 hour, 2 hours or 3 hours. In the present invention, the freeze cross-linking is preferably carried out in a refrigerator. In the present invention, gelatin is a temperature-sensitive macromolecule. As the temperature decreases, the gelatin solution will change from liquid to solid, and this change is reversible and can become liquid again as the temperature increases. In the present invention, the freeze cross-linking can solidify the solution to facilitate the subsequent salting-out operation.

[0067] After obtaining the cross-linked hydrogel, the present invention mixes the cross-linked hydrogel with a first salt solution and performs salting out to obtain the salted-out hydrogel.

[0068] In the present invention, the salt in the first salt solution preferably includes one or more of sodium chloride, sodium sulfate, and sodium citrate, more preferably sodium citrate. In the present invention, the mass concentration of the first salt solution is preferably 15-40%, more preferably 20-35%, and specifically can be 15%, 20%, 25%, 30%, 35%, or 40%. In a specific embodiment of the present invention, the first salt solution is preferably a sodium citrate aqueous solution with a mass concentration of 30%.

[0069] In the present invention, the volume ratio of the cross-linked hydrogel to the first salt solution is preferably 1:50-500.

[0070] In the present invention, the salting-out time is preferably 0.5 to 2 hours, more preferably 1 hour, specifically 0.5 hours, 1 hour, 1.5 hours or 2 hours; the salting-out temperature is preferably room temperature, i.e., no additional heating or cooling is required.

[0071] After obtaining the salting-out hydrogel, the present invention stretches the salting-out hydrogel to obtain a stretched hydrogel.

[0072] In the present invention, the stretching degree is preferably 1 to 6 times of the original length, specifically 1, 2, 3, 4, 5 or 6 times. In the present invention, the holding time of the stretching is preferably 5 to 15 minutes, specifically 5 minutes, 10 minutes or 15 minutes.

[0073] After obtaining the stretched hydrogel, the present invention solidifies the stretched hydrogel and the second salt solution to obtain the anisotropic hydrogel.

[0074] In the present invention, the salt in the second salt solution preferably includes sodium citrate, and the mass concentration of the second salt solution is preferably 15-40%, more preferably 20-35%, specifically 15%, 20%, 25%, 30%, 35% or 40%.

[0075] In the present invention, the curing time is preferably 10 to 20 hours, specifically 10 hours, 12 hours, 15 hours, 18 hours or 20 hours, and the temperature is preferably room temperature, that is, no additional heating or cooling is required.

[0076] The present invention also provides an anisotropic hydrogel prepared by the preparation method described in the above technical solution. In the present invention, the surface of the anisotropic hydrogel presents a directional groove shape.

[0077] The present invention also provides the application of the anisotropic hydrogel described in the above technical solution in the food field.

[0078] In the present invention, the food field preferably includes artificial meat preparation.

[0079] In the present invention, the surface of the anisotropic hydrogel is directional and groove-shaped, so that cells can grow in a directional manner on its surface, thereby forming fibrous artificial meat. The resulting artificial meat tastes closer to real meat.

[0080] The anisotropic hydrogel provided by the present invention, its preparation method and application are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0081] Example 1 (Preparation of Pea / Soybean Protein Isolate Amyloid Fiber Dispersion)

[0082] Preparation of Pea Protein Isolate Amyloid Fiber (PPIF) Dispersion

[0083] Different masses of pea protein isolate were weighed, 50 mL of deionized water or ethanol aqueous solutions of different volume concentrations (10%, 20%, 30%, 40%, 50%) were added, and after being fully dissolved by magnetic stirring, the mixture was centrifuged at 6000 rpm for 20 minutes to remove the precipitate. The pH was adjusted to 2 with 3 mol / L hydrochloric acid solution, and the mixture was heated in a water bath at 80°C for 30 hours to obtain a pea protein isolate amyloid fiber dispersion with a mass concentration of 10%.

[0084] Preparation of soy protein isolate amyloid fibrils (SPIF) dispersion:

[0085] Weigh different masses of soy protein isolate, add 50 mL of deionized water or ethanol aqueous solution of different volume concentrations (10%, 20%, 30%, 40%, 50%), stir magnetically to fully dissolve, adjust the pH to 2 with 3 mol / L hydrochloric acid solution, place in a 4°C refrigerator for hydration for 8 hours, then centrifuge at 6000 rpm for 20 minutes to remove the precipitate, and then heat in a water bath at 80°C for 30 hours to obtain a pea protein isolate amyloid fiber dispersion with a mass concentration of 10%.

[0086] The morphology of the separated protein amyloid fibers in the dispersions obtained under different conditions was observed using a transmission scanning electron microscope (JEM-1400flash). Figure 1 As shown, Figure 1 The transmission scanning electron micrographs of the isolated protein amyloid fibers in each dispersion obtained in Example 1 are shown. Figure 1 In the figure, A from left to right are transmission electron micrographs of amyloid fibers of pea protein isolate obtained from aqueous ethanol solutions with ethanol concentrations of 0%, 10%, 20%, 30%, 40%, and 50%, respectively; B from left to right are transmission electron micrographs of amyloid fibers of soy protein isolate obtained from aqueous ethanol solutions with ethanol concentrations of 0%, 10%, 20%, 30%, 40%, and 50%. Figure 1 It can be seen that the morphology of pea protein isolate amyloid fibers and soy protein isolate amyloid fibers gradually changes from rigid to flexible and network-like structure with the increase of ethanol volume concentration during preparation. Among them, straight fibers can be considered as rigid fibers, and curved and soft fibers are flexible fibers. Figure 1 As shown, the fibers with PPIF-0% and SPIF-0% are relatively straight. As the ethanol volume concentration increases, the fibers gradually bend. As the ethanol volume concentration continues to rise, the fiber entanglement deepens, presenting a network structure. Specifically, the rigidity and flexibility of a fiber are determined by comparing its contour length with its persistence length. Contour length refers to the end-to-end length of a polymer along its physical contour. Persistence length is a fundamental property of polymers, and one of the most practical and widely used methods for estimating it is to calculate the end-to-end mean square distance between contour segments. When the contour length is significantly greater than the persistence length, the chain is considered flexible, and when the contour length is less than the persistence length, the chain is considered rigid.

[0087] Example 2 (Construction of stretchable pea / soybean protein isolate amyloid fiber-gelatin hydrogel by salting out)

[0088] Preparation of pea protein isolate amyloid fibrils (PPIF) dispersion:

[0089] Different masses of pea protein isolate were weighed, 50 mL of 0% ethanol aqueous solution (i.e., water) was added, magnetic stirring was used to fully dissolve, and the precipitate was removed by centrifugation at 6000 rpm for 20 minutes. The pH was adjusted to 2 with 3 mol / L hydrochloric acid solution and heated in a water bath at 80°C for 30 hours to obtain a pea protein isolate amyloid fiber dispersion with a mass concentration of 10%.

[0090] Preparation of soy protein isolate amyloid fibrils (SPIF) dispersion:

[0091] Weigh different masses of soy protein isolate, add 50 mL of 0% ethanol aqueous solution (i.e., water), stir magnetically to fully dissolve, adjust the pH to 2 with 3 mol / L hydrochloric acid solution, place in a 4°C refrigerator for hydration for 8 hours, centrifuge at 6000 rpm for 20 minutes to remove the precipitate, and heat in an 80°C water bath for 30 hours to obtain a soy protein isolate amyloid fiber dispersion with a concentration of 10%.

[0092] Preparation of gelatin solution:

[0093] Weigh gelatin, add 50 mL of deionized water, and stir magnetically to fully dissolve it to obtain a gelatin solution with a mass concentration of 20%.

[0094] Preparation of stretchable pea / soybean protein isolate amyloid fiber-gelatin hydrogel:

[0095] A dispersion of pea protein isolate amyloid fibrils, a dispersion of soy protein isolate amyloid fibrils, and a gelatin solution were pipetted and magnetically stirred at room temperature for 1 hour. The pH was then adjusted to 7 with 3 mol / L hydrochloric acid solution, and deionized water was added, resulting in a precursor solution with a 1% pea / soy protein isolate amyloid fibril concentration and a 2% gelatin concentration. The precursor solution was cooled in a refrigerator at 4°C for 1 hour to crosslink and obtain a cross-linked hydrogel. The cross-linked hydrogel was immersed in a 30% sodium citrate solution at a volume ratio of 1:100 for 1 hour at room temperature to obtain a salted-out hydrogel. The salted-out hydrogel was stretched to 5 times its original length and held for 10 minutes to obtain a stretched hydrogel. The stretched hydrogel was then immersed in a 30% sodium citrate solution at a volume ratio of 1:100 and cured for 12 hours to obtain anisotropic gelatin-pea / soy protein isolate amyloid fibril hydrogels.

[0096] Polarization microscopy was used to observe the polarization phenomenon of the anisotropic gelatin-pea / soybean protein isolate amyloid fiber hydrogel obtained after stretching. Figure 2 As shown, Figure 2 is a polarizing microscope image of the obtained anisotropic gelatin-isolate protein amyloid fiber hydrogel. Figure 2In the figure, A is a polarizing microscope image of anisotropic gelatin-pea protein isolate amyloid fiber hydrogel, and B is a polarizing microscope image of anisotropic gelatin-soy protein isolate amyloid fiber hydrogel. Figure 2 It can be seen that the stretched hydrogels all exhibit polarization. Anisotropic materials often exhibit birefringence, meaning that light has different refractive indices in different directions within the material. When a beam of unpolarized light passes through an anisotropic material, it is split into two polarized beams, propagating along the fast and slow axes respectively. This effect manifests as different brightness and darkness in a polarizing microscope.

[0097] The obtained anisotropic gelatin-pea / soy protein isolate amyloid fiber hydrogel was immersed in culture medium and placed in incubators at 27°C and 37°C, respectively. The gel state at different incubation times was recorded. The results are as follows: Figure 3 As shown, Figure 3 The optical photographs of the obtained anisotropic gelatin-isolate protein amyloid fiber hydrogel at different incubation times at 27°C and 37°C are shown; wherein, A is the optical photograph of the anisotropic gelatin-pea protein isolate amyloid fiber hydrogel at different incubation times at 27°C and 37°C, and B is the optical photograph of the anisotropic gelatin-soy protein isolate amyloid fiber hydrogel at different incubation times at 27°C and 37°C. Figure 3 It can be seen that under this environment, the anisotropic gelatin-isolate amyloid fiber hydrogel dissolved after being immersed in the culture medium.

[0098] Example 3 (Enzymatic-salting-out synergistic construction of anisotropic pea / beta-amyloid fiber-gelatin hydrogel)

[0099] Preparation of pea protein isolate amyloid fibrils (PPIF) dispersion:

[0100] Different masses of pea protein isolate were weighed, added to a 30% volume concentration of ethanol aqueous solution, and fully dissolved by magnetic stirring. The mixture was then centrifuged at 6000 rpm for 20 minutes to remove the precipitate. The pH was adjusted to 2 using 3 mol / L hydrochloric acid solution and heated in a water bath at 80°C for 30 hours to obtain a pea protein isolate amyloid fiber dispersion with a mass concentration of 10%.

[0101] Preparation of soy protein isolate amyloid fibrils (SPIF) dispersion:

[0102] Weigh different masses of soy protein isolate, add 30 mL of 30% ethanol aqueous solution, stir magnetically to fully dissolve, adjust the pH to 2 with 3 mol / L hydrochloric acid solution, place in a 4°C refrigerator for hydration for 8 hours, centrifuge at 6000 rpm for 20 minutes to remove the precipitate, and heat in an 80°C water bath for 30 hours to obtain a soy protein isolate amyloid fiber dispersion with a concentration of 10%.

[0103] Preparation of gelatin solution:

[0104] Weigh gelatin, add 50 mL of deionized water, and stir magnetically to fully dissolve it to obtain a gelatin solution with a mass concentration of 20%.

[0105] Preparation of transglutaminase (TG) solution:

[0106] Weigh 1 g of transglutaminase, add 10 mL of deionized water, and stir magnetically to fully dissolve, thereby obtaining a transglutaminase solution with a mass concentration of 10%.

[0107] Preparation of stretchable pea / soybean protein isolate amyloid fiber-gelatin hydrogel:

[0108] The pea protein isolate amyloid fiber dispersion or soy protein isolate amyloid fiber dispersion and gelatin solution were transferred and magnetically stirred at room temperature for 1 hour. The pH was adjusted to 7 using a 3 mol / L hydrochloric acid solution. Deionized water was added, and a transglutaminase solution was added. The resulting precursor solution had a pea / soy protein isolate amyloid fiber dispersion concentration of 1%, a gelatin concentration of 5%, and a transglutaminase to gelatin mass ratio of 1:10. The precursor solution was placed in a 50°C water bath for pre-crosslinking for 5 minutes to obtain a pre-crosslinked hydrogel. The pre-crosslinked hydrogel was placed in a 4°C refrigerator for cooling and curing for 1 hour to obtain a cross-linked hydrogel. The cross-linked hydrogel was immersed in a 30% sodium citrate solution and soaked at room temperature for 1 hour to obtain a salting-out hydrogel.

[0109] The salting-out hydrogel prepared from the soy protein isolate amyloid fibers obtained at 30% ethanol concentration was stretched to 3 times of its original length using a texture analyzer, and the stress-strain curve was recorded as follows: Figure 4 As shown, Figure 4 The stress-strain curve of soybean protein isolate amyloid fiber-salting out hydrogel is shown in Figure 2. Figure 4 It can be seen that when stretched to 3 times, the stress of the salting-out hydrogel can reach 62.29 kPa.

[0110] Example 4 (Enzymatic-salting-out synergistic construction of anisotropic pea / beta-amyloid fiber-gelatin hydrogel)

[0111] Preparation of pea amyloid fiber (PPIF) dispersion:

[0112] Different masses of pea protein isolate were weighed, added to a 10% ethanol aqueous solution by volume, and fully dissolved by magnetic stirring. The mixture was then centrifuged at 6000 rpm for 20 minutes to remove the precipitate. The pH was adjusted to 2 using a 3 mol / L hydrochloric acid solution, and heated in a water bath at 80°C for 30 hours to obtain a pea protein isolate amyloid fiber solution with a mass concentration of 10%.

[0113] Preparation of soybean amyloid fiber (SPIF) dispersion:

[0114] Weigh different masses of soy protein isolate, add 50 mL of 10% ethanol aqueous solution, stir magnetically to fully dissolve, adjust the pH to 2 with 3 mol / L hydrochloric acid solution, place in a 4°C refrigerator for hydration for 8 hours, centrifuge at 6000 rpm for 20 minutes to remove the precipitate, and heat in an 80°C water bath for 30 hours to obtain a 10% soy protein isolate amyloid fiber solution.

[0115] Preparation of gelatin solution:

[0116] Weigh gelatin, add 50 mL of deionized water, and stir magnetically to fully dissolve it to obtain a gelatin solution with a mass concentration of 20%.

[0117] Preparation of transglutaminase (TG) solution:

[0118] Weigh 1 g of transglutaminase, add 10 mL of deionized water, and stir magnetically to fully dissolve, thereby obtaining a transglutaminase solution with a mass concentration of 10%.

[0119] Preparation of stretchable pea / soybean protein isolate amyloid fiber-gelatin hydrogel:

[0120] A pea protein isolate amyloid fiber dispersion or soy protein isolate amyloid fiber solution and a gelatin solution were transferred and magnetically stirred at room temperature for 1 hour. The pH was then adjusted to 7 with 3 mol / L hydrochloric acid solution. Deionized water and transglutaminase solution were then added. The resulting precursor solution contained 1% pea / soy protein isolate amyloid fiber concentration, 5% gelatin concentration, and a transglutaminase to gelatin ratio of 1:10. The precursor solution was pre-crosslinked in a 50°C water bath for 5 minutes to obtain a pre-crosslinked hydrogel. The pre-crosslinked hydrogel was then cooled and cured in a 4°C refrigerator for 1 hour to obtain a cross-linked hydrogel. The cross-linked hydrogel was immersed in a 30% sodium citrate solution at room temperature for 1 hour to obtain a salted-out hydrogel. The salted-out hydrogel was stretched to 5 times its original length and held for 10 minutes to obtain a stretched hydrogel. The stretched hydrogel was then immersed in a 30% sodium citrate solution to cure for 12 hours to obtain an anisotropic gelatin-pea / soy protein isolate amyloid fiber hydrogel.

[0121] Scanning electron microscopy was used to obtain the surface morphology of anisotropic gelatin-pea protein isolate amyloid fiber hydrogel. Figure 5 As shown, Figure 5 The scanning electron micrograph of the obtained anisotropic gelatin-pea protein isolate amyloid fiber hydrogel is shown in FIG. Figure 5It can be seen that the surface of the stretched hydrogel presents directional grooves, which is a typical anisotropic hydrogel morphology.

[0122] The anisotropic gelatin-pea / soy protein isolate amyloid fiber hydrogel was immersed in culture medium and placed in incubators at 27°C and 37°C, respectively. The gel state at different incubation times was recorded. The results are as follows. Figure 6 As shown, Figure 6 Optical photographs of the obtained anisotropic gelatin-isolate amyloid fiber hydrogel at different incubation times at 27°C and 37°C; Figure 6 In the figure, A is an optical photograph of anisotropic gelatin-pea protein isolate amyloid fiber hydrogel at different incubation times at 27°C and 37°C, and B is an optical photograph of anisotropic gelatin-soy protein isolate amyloid fiber hydrogel at different incubation times at 27°C and 37°C. Figure 6 It can be seen that: under this environment, the enzymatically cross-linked hydrogel did not undergo significant changes when immersed in the culture medium and was relatively stable.

[0123] The stretched anisotropic gelatin-pea / soy protein isolate amyloid fiber hydrogel and the untreated salting-out hydrogel were used for cell culture, and the cell morphology was observed by fluorescence microscopy. Figure 7 As shown, Figure 7 The cell morphology obtained after cell growth on unstretched and stretched hydrogels. Figure 7 In the figure, A is the cell morphology grown on the stretched anisotropic gelatin-pea protein isolate amyloid fiber hydrogel, B is the cell morphology grown on the unstretched salted-out pea protein isolate amyloid fiber-gelatin hydrogel, C is the cell morphology grown on the stretched anisotropic gelatin-soy protein isolate amyloid fiber hydrogel, and D is the cell morphology grown on the unstretched salted-out soy protein isolate amyloid fiber-gelatin hydrogel. Figure 7 As can be seen, the anisotropic hydrogels obtained after stretching were further used for in vitro three-dimensional cell culture. While cells on untreated hydrogels grew in a disordered manner (see B and D), when cells were grown on the stretched anisotropic hydrogels, their growth became directional (see A and C).

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing anisotropic hydrogel, characterized in that: The following steps are involved: providing a dispersion of a fibrous material; The fibrous material dispersion, gelatin solution and transglutaminase are mixed, and the obtained precursor solution is pre-crosslinked and freeze-crosslinked in sequence to obtain a cross-linked hydrogel; mixing the cross-linked hydrogel and a first salt solution, and performing salting-out to obtain a salted-out hydrogel; stretching the salting-out hydrogel to obtain a stretched hydrogel; mixing the stretched hydrogel with a second salt solution and curing the mixture to obtain the anisotropic hydrogel; There is physical cross-linking in the fibrous material dispersion; The pre-crosslinking temperature is 40-60°C and the time is 2-30 minutes; The freezing cross-linking temperature is 4°C and the time is 1 to 3 hours; The fibrous material in the fibrous material dispersion is isolated protein amyloid fiber.

2. The preparation method according to claim 1, characterized in that When the fibrous material in the fibrous material dispersion is isolated protein amyloid fibers, the preparation method of the isolated protein amyloid fiber dispersion comprises the following steps: Mixing the protein isolate and an ethanol-water system, adjusting the pH to be acidic, and heating the system in a water bath to obtain the protein isolate amyloid fiber dispersion; The isolated protein is pea protein isolate or soy protein isolate; The volume concentration of ethanol in the ethanol-water system is 0 to 50%; The acidic pH value is 1.5 to 3; The water bath heating temperature is 70-90° C. and the heating time is 20-40 hours.

3. The preparation method according to claim 2, characterized in that When the isolated protein is soy protein isolate, after adjusting the pH value to acidic, before heating in a water bath, hydration and precipitate removal are further performed; the hydration temperature is 2-8° C. and the time is 6-10 hours.

4. The preparation method according to claim 1, characterized in that In the precursor solution, the mass concentration of the fibrous material is 0.5-8%, the mass concentration of gelatin is 3-10%, and the mass ratio of transglutaminase to gelatin is 1:1-12.

5. The preparation method according to claim 1, characterized in that The salt in the first salt solution includes one or more of sodium chloride, sodium sulfate and sodium citrate, and the mass concentration of the first salt solution is 15-40%; The salting-out time is 0.5 to 2 hours.

6. The preparation method according to claim 1, characterized in that The degree of stretching is 1 to 6 times the original length.

7. The preparation method according to claim 1, characterized in that The salt in the second salt solution includes sodium citrate, and the mass concentration of the second salt solution is 15-40%; The curing time is 10 to 20 hours.

8. Anisotropic hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the anisotropic hydrogel according to claim 8 in the food field.

10. The use according to claim 9, characterized in that The food sector includes the preparation of artificial meat.

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