Anisotropic hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of hydrogel preparation, and provides anisotropic hydrogel as well as a preparation method and application thereof. According to the invention, glutamine transaminase is added, enzymatic chemical crosslinking is introduced, and the enzymatic chemical crosslinking is combined with physical crosslinking, so that the anisotropy of the hydrogel can be stabilized, and the structure of the hydrogel is more stable. The surface of the obtained anisotropic hydrogel has an ordered orientation structure, when the anisotropic hydrogel is applied to in-vitro cell culture, cells can grow in the same direction, and specifically when the anisotropic hydrogel is used for preparing artificial meat, the taste of the obtained artificial meat is closer to the fiber property of real meat feeling. In addition, the preparation method is simple to operate. Furthermore, the fibrous material in the fibrous material dispersion liquid comprises cellulose or isolated protein amyloid fiber, and the used preparation raw materials are non-toxic and harmless, so that the fibrous material dispersion liquid can be used in the field of food.

Description

Technical Field

[0001] The 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 for 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 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 chewing experience of consumers. 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 a preparation method and application thereof. 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 glutamine transaminase 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 the first salt solution to perform salting out to obtain a salting 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 fiber, the preparation method of the isolated protein amyloid fiber dispersion comprises the following steps:

[0015] The separated protein and the ethanol-water system are mixed, the pH value is adjusted to be acidic, and the mixture is heated in a water bath to obtain the separated protein amyloid fiber dispersion;

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

[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 be acidic and before heating in a water bath, the process further includes hydration and removal of precipitation; 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 stretching degree is 1 to 6 times of 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 scheme.

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

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

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

[0033] The present invention uses a fibrous material dispersion, wherein the fibrous material can impart anisotropy to the hydrogel during subsequent stretching; the addition of transglutaminase can introduce enzymatic chemical crosslinking, which 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 taste of the obtained artificial meat is closer to the fibrous nature 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 separated protein amyloid fiber dispersion, the volume concentration of ethanol in the ethanol-water system can control the morphology of the separated protein amyloid fibers formed by the separated protein, thereby achieving the regulation of the final hydrogel micromorphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The transmission scanning electron micrographs of the separated protein amyloid fibers in each dispersion obtained in Example 1;

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

[0038] Figure 3 These are optical photographs of the anisotropic gelatin-isolate protein 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] Terminology explanation:

[0044] Anisotropy refers to the different physical or chemical properties of a material in different directions, where the physical or chemical properties include mechanical properties, expansion behavior, electrical conductivity or thermal conductivity, etc. Anisotropy is the opposite of isotropy, which means that the properties of a material are the same 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 glutamine transaminase 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 the first salt solution to perform salting out to obtain a salting 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 specifically preferably includes 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 fiber, the method for preparing the isolated protein amyloid fiber dispersion preferably comprises the following steps:

[0057] The protein isolate and the ethanol-water system are mixed, the pH value is adjusted to be acidic, and a water bath is heated 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 achieving the regulation of the final hydrogel micromorphology. In the present invention, the mixing of the protein isolate and the ethanol-water system is preferably carried out under the condition of magnetic stirring. In the present invention, the acidic pH value is preferably 1.5-3, and more preferably 2; the reagent for adjusting the pH value to be acidic is preferably a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 3 mol / L. In the present invention, the temperature of the water bath heating 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 isolated protein is preferably pea isolated protein, after the isolated protein 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 speed of the centrifugation is preferably 5000-7000 rpm, more preferably 6000 rpm, 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, before heating in a water bath, preferably, hydration and removal of precipitates are also included; the hydration temperature is preferably 4°C, the time is preferably 6 to 10 hours, and more preferably 8 hours; the hydration is preferably carried out in a refrigerator; the method of removing precipitates is preferably centrifugation, the centrifugation speed is preferably 5000 to 7000 rpm, more preferably 6000 rpm, 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, and the water is preferably deionized water.

[0061] After obtaining the fibrous material dispersion, the present invention mixes the fibrous material dispersion, gelatin solution and glutamine transaminase, 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 preparation method of the gelatin solution preferably comprises the following steps: mixing gelatin and water, and performing 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 following steps: 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, the mixing of the fibrous material dispersion, gelatin solution and transglutaminase preferably comprises the following steps: after mixing the cellulose material dispersion and gelatin solution, adjusting the pH value to 6 to 8; and then adding water and transglutaminase solution in sequence. In the present invention, the pH value can be specifically 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%; the mass ratio of glutamine aminotransferase 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 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 enables the gelatin and the fibrous material to be partially combined together by chemical crosslinking. After all the combinations are made, the molecular chains are locked and cannot be subsequently stretched. Only by achieving partial crosslinking can the stretchability be ensured while not dissolving in the subsequent process.

[0066] In the present invention, the temperature of the frozen 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 frozen 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 frozen 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 salting-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, and 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 specifically 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 application of the anisotropic hydrogel described in the above technical solution in the field of food.

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

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

[0080] The anisotropic hydrogel provided by the present invention and its preparation method and application are described in detail below in conjunction with the embodiments, 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 fibrils (PPIF) dispersion

[0083] Weigh different masses of pea 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, centrifuge at 6000 rpm for 20 minutes to remove the precipitate, adjust the pH to 2 with 3 mol / L hydrochloric acid solution, and 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%.

[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 separated protein amyloid fibers in each dispersion obtained in Example 1 are shown in FIG. Figure 1 In the figure, A from left to right are transmission scanning electron micrographs of amyloid fibers of pea protein isolate obtained from ethanol aqueous solutions with ethanol volume concentrations of 0%, 10%, 20%, 30%, 40%, and 50%, respectively, and B from left to right are transmission scanning electron micrographs of amyloid fibers of soy protein isolate obtained from ethanol aqueous solutions with ethanol volume 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 structures as the volume concentration of ethanol increases 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 of PPIF-0% and SPIF-0% are relatively straight. As the volume concentration of ethanol increases, the fibers gradually bend. When the volume concentration of ethanol continues to increase, the degree of fiber entanglement deepens, presenting a network structure. Specifically, the rigidity and flexibility of the fiber are determined by comparing the relationship between the contour length and the persistence length of the fiber; the contour length refers to the end-to-end length of the polymer along its physical contour; the persistence length is a basic property of the polymer, 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 much larger than the persistence length, it is determined to be a flexible chain, and when the contour length is smaller than the persistence length, it is determined to be a rigid chain.

[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] Weigh different masses of pea protein isolate, add 50 mL of 0% ethanol aqueous solution (i.e., water), stir magnetically to fully dissolve, centrifuge at 6000 rpm for 20 minutes to remove the precipitate, adjust the pH to 2 with 3 mol / L hydrochloric acid solution, and 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%.

[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 using 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, heat in a water bath at 80°C for 30 hours, and obtain a 10% soy protein isolate amyloid fiber dispersion.

[0092] Preparation of gelatin solution:

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

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

[0095] The pea protein isolate amyloid fiber dispersion, soybean protein isolate amyloid fiber dispersion and gelatin solution were transferred, and after magnetic stirring at room temperature for 1 hour, the pH was adjusted to 7 with 3 mol / L hydrochloric acid solution, and deionized water was added. In the final precursor solution, the mass concentration of pea / soy protein isolate amyloid fiber was 1%, and the mass concentration of gelatin was 2%. The precursor solution was placed in a 4°C refrigerator for cooling and crosslinking for 1 hour to obtain a cross-linked hydrogel. The cross-linked hydrogel was immersed in a sodium citrate solution with a mass concentration of 30% at a volume ratio of 1:100 and soaked at room temperature for 1 hour to obtain a salting-out hydrogel. The salting-out hydrogel was stretched to 5 times its original length and kept for 10 minutes to obtain a stretched hydrogel; the stretched hydrogel was immersed in a sodium citrate solution with a mass concentration of 30% at a volume ratio of 1:100 and cured for 12 hours to obtain anisotropic gelatin-pea / soy protein isolate amyloid fiber 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. The results are as follows Figure 2 As shown, Figure 2 The polarizing microscope image of the obtained anisotropic gelatin-isolate protein amyloid fiber hydrogel is shown in FIG. 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 show polarization. Anisotropic materials usually show birefringence, that is, light has different refractive indices in different directions in the material. When a beam of unpolarized light passes through an anisotropic material, it will be decomposed into two beams of polarized light, propagating along the fast axis and the slow axis respectively. This effect appears as different light and dark images under a polarizing microscope.

[0097] The obtained anisotropic gelatin-pea / soybean 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; 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 protein 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] Weigh different masses of pea protein isolate, add 30% ethanol aqueous solution by volume, stir magnetically to fully dissolve, centrifuge at 6000 rpm for 20 minutes to remove precipitate, adjust pH to 2 with 3 mol / L hydrochloric acid solution, heat in a water bath at 80°C for 30 hours, and obtain 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, heat in a water bath at 80°C for 30 hours, and 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, thereby obtaining a gelatin solution with a mass concentration of 20%.

[0105] Preparation of glutamine transaminase (TG) solution:

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

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

[0108] Take the pea protein isolate amyloid fiber dispersion or soy protein isolate amyloid fiber dispersion and gelatin solution, stir magnetically at room temperature for 1 hour, adjust the pH to 7 with 3 mol / L hydrochloric acid solution, add ionized water, add glutamine transaminase solution, and finally obtain the precursor solution with a pea / soy protein isolate amyloid fiber dispersion mass concentration of 1%, a gelatin mass concentration of 5%, and a glutamine transaminase to gelatin mass ratio of 1:10. Place the precursor solution in a 50°C water bath for pre-crosslinking for 5 minutes to obtain a pre-crosslinked hydrogel, place the pre-crosslinked hydrogel in a 4°C refrigerator for cooling and curing for 1 hour to obtain a crosslinked hydrogel. Immerse the crosslinked hydrogel in a sodium citrate solution with a mass concentration of 30%, and soak it 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 under 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 soy 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] Weigh different masses of pea protein isolate, add 10% ethanol aqueous solution by volume, stir magnetically to fully dissolve, centrifuge at 6000 rpm for 20 minutes to remove precipitate, adjust pH to 2 with 3 mol / L hydrochloric acid solution, heat in a water bath at 80°C for 30 hours, and obtain 10% pea protein isolate amyloid fiber solution by mass concentration.

[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, heat in a water bath at 80°C for 30 hours, and obtain a soy protein isolate amyloid fiber solution with a concentration of 10%.

[0115] Preparation of gelatin solution:

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

[0117] Preparation of glutamine transaminase (TG) solution:

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

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

[0120] The pea protein isolate amyloid fiber dispersion or soybean protein isolate amyloid fiber solution and gelatin solution were transferred, and after magnetic stirring at room temperature for 1 hour, the pH was adjusted to 7 with 3 mol / L hydrochloric acid solution, ionized water was added, and glutamine transaminase solution was added. The final precursor solution had a pea / soy protein isolate amyloid fiber mass concentration of 1%, a gelatin mass concentration of 5%, and a glutamine transaminase 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, and 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 sodium citrate solution with a mass concentration of 30%, and the salting-out hydrogel was obtained by soaking at room temperature for 1 hour. The salting-out hydrogel was stretched to 5 times its original length and kept for 10 minutes to obtain a stretched hydrogel; the stretched hydrogel was then immersed in a sodium citrate solution with a mass concentration of 30% and cured 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 / soybean 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: in this environment, the hydrogel after enzymatic cross-linking did not undergo obvious changes when immersed in the culture medium and was relatively stable.

[0123] The stretched anisotropic gelatin-pea / soybean protein isolate amyloid fiber hydrogel and the untreated salting-out hydrogel were used for cell culture, and the cell morphology was obtained by fluorescence microscopy. The results are shown in 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 It can be seen that the anisotropic hydrogel obtained after stretching treatment was further used for in vitro three-dimensional cell culture. The cells on the untreated hydrogel grew disorderly (see B and D), and when the cells grew on the anisotropic hydrogel constructed by stretching, their growth was 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 principle 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 glutamine transaminase 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 the first salt solution to perform salting out to obtain a salting 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.

2. The preparation method according to claim 1, characterized in that: The fibrous material in the fibrous material dispersion includes cellulose or isolated protein amyloid fibers; When the fibrous material in the fibrous material dispersion is isolated protein amyloid fiber, the preparation method of the isolated protein amyloid fiber dispersion comprises the following steps: The separated protein and the ethanol-water system are mixed, the pH value is adjusted to be acidic, and the mixture is heated in a water bath to obtain the separated protein amyloid fiber dispersion; The isolated protein is pea isolated protein or soy isolated protein; 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 be acidic, before heating in a water bath, hydration and precipitation removal are also performed; the hydration temperature is 2 to 8° C. and the time is 6 to 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 glutamine aminotransferase to gelatin is 1:1-12; The pre-crosslinking temperature is 40-60°C and the time is 2-30 minutes; The temperature of the frozen cross-linking is 4° C., and the time is 1 to 3 hours.

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 of 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 obtained 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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