Biomimetic hydrogel-based connective tissue and preparation method and application thereof

Abstract

The application relates to a biomimetic hydrogel-based connective tissue and a preparation method and application thereof, and belongs to the technical field of plant-based food processing. In view of the defects of single taste and lack of real meat connective tissue feeling of existing plain meat products, a continuous phase component (containing plant protein, water-soluble dietary fiber and ferulic acid) is mixed with a pH-activated plant polysaccharide colloidal solution (containing a glucan gum, a galactomannan gum and a fucoidan gum); then, calcium ions and transglutaminase are introduced in sequence to trigger ion crosslinking and enzymatic crosslinking in sequence; and finally, a heat curing treatment and cold storage curing are carried out to obtain a biomimetic hydrogel-based connective tissue which is excellent in mechanical property, uniform in texture, high in water holding property and stable in heat. The biomimetic hydrogel-based connective tissue prepared by the application can be used for developing a new generation of high-end plant-based meat products.

Description

Technical Field

[0001] This invention belongs to the field of plant-based food processing technology, and relates to a biomimetic hydrogel-based connective tissue, its preparation method and application, specifically to a plant-based hydrogel with biomimetic animal connective tissue texture and structure and its preparation method, as well as the application of this biomimetic connective tissue in the production of vegetarian beef products with multi-layered realistic meat texture. Background Technology

[0002] Plant-based meat (vegan meat) prepared using high-humidity extrusion technology can already mimic the fibrous structure of animal muscle quite well. However, existing products generally suffer from a uniform texture and a lack of depth in chewing, especially lacking the unique elasticity, juiciness, and multi-layered chewing experience provided by connective tissues such as fascia and tendons in real meat. This lack of "connective tissue feel" has become a core technological bottleneck restricting the sensory quality of high-end plant-based meat products from approaching that of real meat.

[0003] To address these issues, existing technologies employ methods to control product texture by adding a single colloid (such as carrageenan) or simple compound colloids. However, the gel networks formed by these methods struggle to achieve a good balance between mechanical strength, water retention, and thermal stability. Specifically, excessively hard gels are prone to brittleness, while excessively soft gels lack chewiness; poor water retention leads to dryness; and insufficient thermal stability causes structural collapse after cooking. Therefore, existing methods cannot realistically simulate the complex, stable, and functionally synergistic three-dimensional network structure of natural connective tissue. Based on this, developing a plant-based component that comprehensively mimics natural connective tissue from molecular structure to macroscopic properties, and structurally composites it with plant-based meat substrates, holds significant industrial value. Summary of the Invention

[0004] To address the shortcomings of existing plant-based meat products, such as monotonous texture and lack of realistic meat connective tissue, this invention provides a method for preparing biomimetic hydrogel-based connective tissue with excellent mechanical properties, high water retention, and thermal stability. This method involves compounding and homogenizing a continuous phase component (containing plant protein, water-soluble dietary fiber, and ferulic acid) with a pH-activated plant polysaccharide colloidal solution (containing glucomannan colloids, galactomannan colloids, and seaweed sulfated polysaccharide colloids) to form a uniform initial reaction system. Subsequently, calcium ions and transglutaminase are sequentially introduced to trigger ionic and enzymatic cross-linking, constructing a primary gel network with both strength and elasticity at the molecular level. The network stability is further enhanced and the enzyme preparation is inactivated through thermal curing. Finally, a cold-curing process allows the gel network to fully relax and solidify, resulting in a biomimetic hydrogel-based connective tissue with uniform texture and stable properties. Furthermore, this invention utilizes the prepared biomimetic hydrogel-based connective tissue to process plant-based beef using structured composite technology, resulting in a final product that highly approximates real beef in texture and taste.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a method for preparing biomimetic hydrogel-based connective tissue, the method comprising the following steps: S1. Add plant protein, water-soluble dietary fiber and ferulic acid to water, stir evenly to form a continuous phase solution. This step fully hydrates the protein and provides a uniform reaction system for subsequent cross-linking. S2. Add glucomannan colloid, galactomannan colloid and seaweed sulfate polysaccharide colloid to water, heat to boiling to fully dissolve the colloids, and adjust the pH to 7.5-9.0 to obtain a polysaccharide colloidal solution. Heating to boiling is to form a homogeneous colloidal solution, and adjusting the pH is to fully dissolve and activate the polysaccharides, which is beneficial for subsequent ionic cross-linking. S3. Mix the continuous phase solution obtained in S1 with the polysaccharide colloidal solution obtained in S2 until homogeneous. Add calcium ion solution to the mixed material to perform ion crosslinking. The mixing process ensures that the continuous phase solution and the polysaccharide colloidal solution are fully mixed to form a homogeneous and stable system. The mixing process can be carried out by manually stirring first and then homogenizing. S4. Add transglutaminase to the ionically cross-linked material, stir well, and then carry out enzymatic cross-linking. This step utilizes calcium ions to undergo ion cross-linking with the carboxyl or sulfate groups of polysaccharides to form a preliminary gel network. This step utilizes transglutaminase to catalyze covalent cross-linking between protein molecules, thereby enhancing the strength and elasticity of the gel network. S5. The enzymatically cross-linked material is subjected to heat curing and cold curing in sequence to obtain biomimetic hydrogel-based connective tissue; the stability of the gel network is further enhanced by heat treatment, and the texture and taste of the gel are improved by cold curing.

[0006] In one embodiment of the present invention, the plant protein in S1 includes any one or a mixture of any two or more of the following: soy protein isolate, pea protein, chickpea protein, peanut protein isolate, wheat gluten protein, mung bean protein, quinoa protein, and nut protein; the water-soluble dietary fiber includes any one or a mixture of any two or more of the following: oat β-glucan, barley β-glucan, konjac glucomannan, pectin, and inulin.

[0007] Further specifying, the plant protein is soy protein isolate, because soy protein isolate has better solubility and gelling properties; the water-soluble dietary fiber is oat β-glucan, because oat β-glucan has high viscosity, good gel-forming ability, excellent water-holding and emulsifying stability, and also has physiological activities such as regulating intestinal function and anti-oxidation.

[0008] In one embodiment of the present invention, the mass ratio of the plant protein, water-soluble dietary fiber and ferulic acid in S1 is (8-12):(4-6):(1-3).

[0009] Further specifying, based on the total mass of the biomimetic hydrogel-based connective tissue product as 100%, the mass percentages of plant protein, water-soluble dietary fiber, and ferulic acid mentioned in S1 are 1%, 0.5%, and 0.2%, respectively, and the mass ratio of the three components is 10:5:2. Under this ratio, the best gel performance and taste can be obtained, and the nutritional value of the product can be effectively improved.

[0010] In one embodiment of the present invention, the glucomannan colloid in S2 includes any one or a mixture of any two or more of konjac gum, konjac flour, and congee gum; the galactomannan colloid includes any one or a mixture of any two or more of locust bean gum, guar gum, and tara gum; and the seaweed sulfated polysaccharide colloid includes any one or a mixture of any two or more of carrageenan, sodium alginate, agar, and fucoidan.

[0011] Further specifying, the glucomannan colloid is konjac gum, because konjac gum has high viscosity, excellent water retention and gel-enhancing effect, which can significantly improve gel strength and structural stability; the galactomannan colloid is locust bean gum, because locust bean gum has good synergistic effect with various colloids, which can improve gel elasticity and water retention and inhibit water separation; the seaweed sulfated polysaccharide colloid is carrageenan, because carrageenan gels rapidly, has high gel strength and transparency, and excellent thermal reversibility and compatibility, which can give the system good formability and chewy texture.

[0012] In one embodiment of the present invention, based on the total mass of the biomimetic hydrogel-based connective tissue product as 100%, the mass percentage of glucomannan colloid in S2 is 0%-5%, the total mass percentage of galactomannan colloid and seaweed sulfated polysaccharide colloid is 3%-5%, and the mass ratio of galactomannan colloid to seaweed sulfated polysaccharide colloid is 2:8-4:6. By limiting the amount of polysaccharides added as described above, the structure and taste of the gel can be guaranteed.

[0013] Further specifying, the mass ratio of the galactomannan colloid to the seaweed sulfated polysaccharide colloid is 4:6.

[0014] Preferably, based on the total mass of the biomimetic hydrogel-based connective tissue product as 100%, the mass percentage of glucomannan colloid in S2 is 1%, the mass percentage of galactomannan colloid is 2%, and the mass percentage of seaweed sulfate polysaccharide colloid is 3%.

[0015] In one embodiment of the present invention, S2 uses an alkaline solution to adjust the pH, wherein the alkaline solution includes, but is not limited to, sodium bicarbonate solution, sodium carbonate solution, and sodium hydroxide solution; the alkaline solution is a baking soda (sodium bicarbonate) solution, because baking soda has better safety; the mass fraction of the baking soda solution is 0.5%-5% to control the concentration of the baking soda solution and ensure the accuracy of pH adjustment; the amount of baking soda added is 0.05%-0.1% of the weight of the connective tissue on a pure substance basis, to precisely control the amount of baking soda added and avoid the pH value being too high.

[0016] In one embodiment of the present invention, the pH in step S2 is adjusted to 8.5.

[0017] In one embodiment of the present invention, the boiling in step S2 is maintained for 10-20 minutes.

[0018] In one embodiment of the present invention, the calcium ion solution in S3 includes any one or a mixture of any two or more of calcium chloride solution, calcium lactate solution, and calcium gluconate solution.

[0019] Furthermore, the calcium ion solution is a calcium chloride solution. Because calcium chloride has high solubility and stable ion release, it can efficiently promote the cross-linking and formation of colloidal molecules, improve the density and structural strength of the gel, and has high purity and few impurities, and has no adverse effect on the flavor and color of the system.

[0020] In one embodiment of the present invention, the homogenization in S3 is performed at 8000-13000 rpm for 2-6 minutes.

[0021] In one embodiment of the present invention, the amount of calcium ions added in S3 accounts for 0.018%-0.181% of the total mass of the biomimetic hydrogel-based connective tissue product: the ionic crosslinking is performed at 8000-13000 rpm for 1-3 min.

[0022] In one embodiment of the present invention, the amount of transglutaminase added in S4 is 0.05%-0.5% of the plant protein content, and the enzymatic cross-linking is carried out at 45-55°C for 1-2 hours.

[0023] Further specified, the amount of transglutaminase added in S4 is 0.15% of the plant protein content.

[0024] In one embodiment of the present invention, the thermosetting treatment in S5 is heating at 85-95°C for 20-40 minutes, and the cold curing is refrigerating at 2-8°C for at least 12 hours.

[0025] In one embodiment of the present invention, the method further includes adding trace elements, vitamins, amino acids and other nutrients to the connective tissue to improve the nutritional value of the plant-based meat product.

[0026] In one embodiment of the present invention, the method further includes: adding natural flavorings, sweeteners, acidulants and other taste improvers to the connective tissue to improve the flavor of the plant-based meat product and make it closer to the taste of real animal meat.

[0027] A second objective of this invention is to provide biomimetic hydrogel-based connective tissue prepared by the above-described preparation method.

[0028] A third objective of this invention is to provide a food containing the aforementioned biomimetic hydrogel-based connective tissue.

[0029] A fourth objective of this invention is to provide the application of the above-mentioned biomimetic hydrogel-based connective tissue in the processing of plant-based meat.

[0030] In one embodiment of the present invention, a method for preparing a bouncy and juicy vegetarian beef product is provided. The method involves cutting the biomimetic hydrogel-based connective tissue obtained above into strips, sheets, or blocks, then combining it with a high-moisture extrusion plant-based meat component with a fibrous structure in a mold, then pouring in a 5%-8% soy protein isolate solution for bonding, then pressing, steaming at 90-100℃ for 1-2 hours, cooling to room temperature, and then refrigerating at 2-8℃ for more than 6 hours to form a vegetarian beef blank. This method combines biomimetic connective tissue with plant-based meat to produce a vegetarian beef product with a realistic meat texture, solving the technical problem that existing vegetarian beef products lack the connective tissue texture of real meat.

[0031] In one embodiment of the present invention, the high-moisture extruded plant-based meat substrate is prepared by a twin-screw extrusion-low-temperature desiccation combined technology: the plant-based meat substrate is prepared by a 36-twin-screw extrusion with extrusion parameters of 9 kg / h solid feed rate, 12 kg / h liquid feed rate, 220 rpm screw speed, and 162 N·m torque, to provide specific extrusion parameters and ensure the fibrous structure of the plant-based meat.

[0032] In one embodiment of the present invention, the biomimetic hydrogel-based connective tissue accounts for 20%-60% of the mass of the vegetarian beef, preferably 30%-50%, in order to optimize the amount of connective tissue added and balance the taste and cost of the vegetarian beef.

[0033] In one embodiment of the present invention, the mass ratio of the soy protein isolate solution, the biomimetic hydrogel-based connective tissue, and the plant-based meat is 30:40:60.

[0034] In one embodiment of the present invention, the 6% soy protein isolate solution is prepared by mixing 6 g of soy protein isolate and 100 g of deionized water evenly at a stirring speed of 400-600 rpm for 40-60 min, thereby providing a method for preparing the soy protein isolate solution and ensuring the adhesive effect.

[0035] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following significant technical effects: This invention successfully constructed a dense and stable three-dimensional gel network by compounding a continuous phase component (containing plant protein, water-soluble dietary fiber, and ferulic acid) with a plant-based polysaccharide colloidal solution (containing glucomannan colloids, galactomannan colloids, and seaweed sulfated polysaccharide colloids) and sequentially performing ionic crosslinking, enzymatic crosslinking, thermal curing, and cold-curing processes. The plant protein provides excellent water solubility and emulsifying properties, the water-soluble dietary fiber increases the viscosity of the system, and ferulic acid, as a natural antioxidant, improves the product's stability. The synergistic effect of the multiple crosslinking processes achieves a good balance between mechanical strength, water retention, and thermal stability in the biomimetic connective tissue.

[0036] The biomimetic connective tissue prepared by this invention can approximately replicate the texture and chewiness of natural animal connective tissue, specifically in the following aspects: Excellent textural properties: By optimizing the compounding ratio and cross-linking process of konjac gum, locust bean gum, and carrageenan, the gel achieves an optimal balance between hardness, elasticity, and chewiness. Textural profile analysis shows that all its indicators are close to or reach the level of natural connective tissue, effectively simulating the elasticity and chewiness during meat chewing. Superior water-holding capacity: The dense gel network structure and the synergistic effect between protein-polysaccharide molecules endow this biomimetic connective tissue with an extremely high water-holding capacity (up to 80%-90% or more), ensuring that the product effectively locks in moisture during processing, storage, and cooking, resulting in a juicy texture and preventing dryness. Good thermal stability: The gel network has thermal irreversibility, maintaining structural stability within the normal cooking temperature range (such as around 100℃), and is not easily melted or collapsed, ensuring the morphological integrity and consistent taste of the product during heat processing. Excellent mechanical properties: Mechanical indicators such as gel strength and fracture strain show that the biomimetic connective tissue prepared by this invention is significantly superior to the control sample of single cross-linking or single polysaccharide system in terms of mechanical properties, and achieves a synergistic improvement in high strength and high toughness.

[0037] The biomimetic connective tissue prepared by this invention possesses elastic and malleable physical properties. Through cutting and recombination, it can be structurally composited with a high-moisture extruded plant-based meat substrate with a fibrous structure in a mold. Interface fusion is achieved using a protein adhesive solution, followed by steaming and final cold-freezing to form a multi-layered plant-based meat structure with an internal biomimetic connective tissue layer, and an appearance and texture highly similar to real beef. Specifically: **Structurally Realistic:** This tissue can form clear and independent structural layers with plant-based meat fibers, accurately simulating the natural texture of alternating "muscle bundles-connective tissue" in real muscle, completely changing the current situation of monolithic internal structures in plant-based meat products. **Accurate Sensory Reproduction:** By precisely controlling the mechanical properties and water-holding capacity of the gel network, a synergistic effect of elastic chewiness and juicy texture is achieved, giving the product a complex and dynamic experience in the mouth that closely resembles real meat. **Stable Processing Performance:** The gel network has good mechanical stability and structural retention, maintaining its intended shape and layers during subsequent processing, making it suitable for industrial production processes.

[0038] This invention constructs biomimetic connective tissue based on natural raw materials such as soy protein and water-soluble dietary fiber. While maintaining structural function, it also enhances the nutritional value of the product, aligning with the trend towards clean labeling and achieving a fusion of "structure and nutrition." This technology overcomes the limitations of existing plant-based meat products, which suffer from a single texture and lack of chewiness, providing a reliable technical solution for developing a new generation of high-end plant-based meat products and possessing significant industrial application value. Attached Figure Description

[0039] Figure 1 The images shown are SEM microscopic morphological images of the biomimetic connective tissues prepared in the embodiments and comparative examples of this invention; wherein, Figure 1 A, B, C, D, E, F, and G in the figure correspond to biomimetic hydrogel connective tissue samples S1, S2, S3, D1, D2, D3, and D4, respectively. Figure 2 These are macroscopic morphological diagrams of the biomimetic connective tissues prepared in the embodiments and comparative examples of the present invention; Figure 3 This is a bar chart comparing the water holding capacity of biomimetic connective tissues prepared in the embodiments and comparative examples of the present invention; Figure 4 The above are DSC thermal analysis curves of the biomimetic connective tissues prepared in the embodiments and comparative examples of the present invention. Figure 5 This is a schematic diagram of the macroscopic cross-sectional structure of a vegetarian beef product. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0042] In the following embodiments, unless otherwise specified, all proportions are mass percentages.

[0043] Example 1: Preparation of biomimetic connective tissue (locust bean gum: carrageenan = 4:6) Soy protein isolate, oat β-glucan, and ferulic acid were added to water and stirred at 500 rpm using an electronic stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water, and the mixture was heated to boiling and held for 15 minutes. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and homogenized at 10,000 rpm for 5 minutes. Calcium chloride solution was added to the homogenized material, and the mixture was stirred at 10,000 rpm for 1 minute to perform ionic crosslinking. Transglutaminase (TG enzyme) was then added, and the mixture was manually stirred and reacted in a 50°C water bath for 1 hour to perform enzymatic crosslinking. The crosslinked material was poured into a mold and heated in a 90°C water bath for 30 minutes to thermally solidify and inactivate the enzyme. After cooling, the mixture was refrigerated at 4°C for 24 hours to obtain the biomimetic hydrogel connective tissue sample S1. Soy protein isolate, oat β-glucan, and ferulic acid accounted for 1%, 0.5%, and 0.2% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 2%, and 3% of the prepared hydrogel connective tissue, respectively; calcium chloride was added at 0.15% of the mass of the prepared hydrogel connective tissue; and TG enzyme was added at 0.15% of the mass of soy protein isolate.

[0044] Example 2: Preparation of biomimetic connective tissue (Locust bean gum: carrageenan = 2:8) Soy protein isolate, oat β-glucan, and ferulic acid were added to water and stirred at 500 rpm using an electronic stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water, and the mixture was heated to boiling and held for 15 minutes. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and homogenized at 10,000 rpm for 5 minutes. Calcium chloride solution was added to the homogenized material, and the mixture was stirred at 10,000 rpm for 1 minute to perform ionic crosslinking. Transglutaminase (TG enzyme) was then added, and the mixture was manually stirred and reacted in a 50°C water bath for 1 hour to perform enzymatic crosslinking. The crosslinked material was poured into a mold and heated in a 90°C water bath for 30 minutes to thermally solidify and inactivate the enzyme. After cooling, the mixture was refrigerated at 4°C for 24 hours to obtain the biomimetic hydrogel connective tissue sample S2. Soy protein isolate, oat β-glucan, and ferulic acid accounted for 1%, 0.5%, and 0.2% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 1%, and 4% of the prepared hydrogel connective tissue, respectively; calcium chloride was added at 0.15% of the mass of the prepared hydrogel connective tissue; and TG enzyme was added at 0.15% of the mass of soy protein isolate.

[0045] Example 3: Preparation of biomimetic connective tissue (Locust bean gum: carrageenan = 8:2) Soy protein isolate, oat β-glucan, and ferulic acid were added to water and stirred at 500 rpm using an electronic stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water, and the mixture was heated to boiling and held for 15 min. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and homogenized at 10,000 rpm for 5 min. Calcium chloride solution was added to the homogenized material, and the mixture was stirred at 10,000 rpm for 1 min to perform ionic crosslinking. Transglutaminase (TG enzyme) was then added, and after manual stirring, the mixture was reacted in a 50℃ water bath for 1 h to perform enzymatic crosslinking. The crosslinked material was poured into a mold and heated in a 90℃ water bath for 30 min to perform thermosetting and enzyme inactivation. After cooling, the mixture was refrigerated at 4℃ for 24 h to obtain the biomimetic hydrogel connective tissue sample S3. Soy protein isolate, oat β-glucan, and ferulic acid accounted for 1%, 0.5%, and 0.2% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 4%, and 1% of the prepared hydrogel connective tissue, respectively; calcium chloride was added at 0.15% of the mass of the prepared hydrogel connective tissue; and TG enzyme was added at 0.15% of the mass of soy protein isolate.

[0046] Comparative Example 1: Preparation of biomimetic connective tissue (without enzymatic cross-linking) Soy protein isolate, oat β-glucan, and ferulic acid were added to water and stirred at 500 rpm using an electric stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water, and the mixture was heated to boiling and held for 15 min. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and homogenized at 10,000 rpm for 5 min. Calcium chloride solution was added to the homogenized material, and the mixture was mixed at 10,000 rpm for 1 min to perform ionic crosslinking. The mixture was reacted in a 50°C water bath for 1 h without the addition of transglutaminase. The material was poured into a mold and heated in a 90°C water bath for 30 min for thermosetting. After cooling, the mixture was refrigerated at 4°C for 24 h to obtain the biomimetic hydrogel connective tissue sample D1. Soy protein isolate, oat β-glucan, and ferulic acid accounted for 1%, 0.5%, and 0.2% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 2%, and 3% of the prepared hydrogel connective tissue, respectively; and calcium chloride was added at 0.15% of the mass of the prepared hydrogel connective tissue.

[0047] Comparative Example 2: Preparation of biomimetic connective tissue (without ion crosslinking) Soy protein isolate, oat β-glucan, and ferulic acid were added to water and stirred at 500 rpm using an electric stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water and heated to boiling, held for 15 minutes. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and homogenized at 10,000 rpm for 5 minutes. Transglutaminase (TG enzyme) was added, and after manual stirring, the mixture was reacted in a 50°C water bath for 1 hour for enzymatic cross-linking. The cross-linked material was poured into a mold and heated in a 90°C water bath for 30 minutes for thermosetting and enzyme inactivation. After cooling, the mixture was refrigerated at 4°C for 24 hours to obtain the biomimetic hydrogel connective tissue sample D2. Soy protein isolate, oat β-glucan, and ferulic acid accounted for 1%, 0.5%, and 0.2% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 2%, and 3% of the prepared hydrogel connective tissue, respectively; and the amount of TG enzyme added was 0.15% of the amount of soy protein isolate.

[0048] Comparative Example 3: Preparation of biomimetic connective tissue (without enzymatic or ionic cross-linking) Soy protein isolate, oat β-glucan, and ferulic acid were added to water and stirred at 500 rpm using an electric stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water, and the mixture was heated to boiling and held for 15 minutes. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and then homogenized at 10,000 rpm for 5 minutes. The resulting material was poured into a mold and thermosetting in a 90°C water bath for 30 minutes. After cooling, it was refrigerated at 4°C for 24 hours to obtain the biomimetic hydrogel connective tissue sample D3. Soy protein isolate, oat β-glucan, and ferulic acid accounted for 1%, 0.5%, and 0.2% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 2%, and 3% of the prepared hydrogel connective tissue, respectively.

[0049] Comparative Example 4: Preparation of biomimetic connective tissue (without ferulic acid) Soy protein isolate and oat β-glucan were added to water and stirred at 500 rpm using an electronic stirrer to form a continuous phase solution. Konjac gum, locust bean gum, and carrageenan were added to water and heated to boiling, held for 15 min. Then, 7% sodium bicarbonate solution was added to adjust the pH to 8.5, yielding a polysaccharide colloidal solution. The continuous phase solution and the polysaccharide colloidal solution were mixed and homogenized at 10,000 rpm for 5 min. Calcium chloride solution was added to the homogenized material, and the mixture was stirred at 10,000 rpm for 1 min to perform ionic crosslinking. Transglutaminase (TG enzyme) was then added, manually stirred, and reacted in a 50℃ water bath for 1 h to perform enzymatic crosslinking. The crosslinked material was poured into a mold and heated in a 90℃ water bath for 30 min to thermally solidify and inactivate the enzyme. After cooling, the material was refrigerated at 4℃ for 24 h to obtain the biomimetic hydrogel connective tissue sample D4. Soy protein isolate and oat β-glucan accounted for 1% and 0.5% of the prepared hydrogel connective tissue, respectively; konjac gum, locust bean gum, and carrageenan accounted for 1%, 2%, and 3% of the prepared hydrogel connective tissue, respectively; the amount of calcium chloride added was 0.15% of the mass of the prepared hydrogel connective tissue; and the amount of TG enzyme added was 0.15% of the mass of soy protein isolate.

[0050] The biomimetic connective tissues prepared in Examples 1-3 and Comparative Examples 1-4 were observed in terms of macroscopic and microscopic morphology. Their water retention, texture profile (hardness, elasticity, chewiness, cohesion, and adhesiveness), thermal stability, thermal transition temperature, and mechanical properties (gel strength and fracture strain) were measured and analyzed.

[0051] Macroscopic morphological observation: Each prepared gel sample was placed on a black background and photographed under the same lighting conditions. By visual observation and image comparison, the uniformity of appearance, the presence of obvious bubbles or cracks, the appropriateness of transparency, and the goodness of moldability (ease of demolding and plasticity) were evaluated.

[0052] Microscopic morphological observation: Each sample was pre-frozen at -80°C for 12 h, and then freeze-dried in a vacuum freeze dryer for 48 h until completely dry. The dried samples were then fixed on a double-sided conductive copper sample holder and sputter-coated with gold. The pre-treated samples were then observed at 500x magnification, and different samples were photographed to observe their microstructure and evaluate network continuity and pore uniformity.

[0053] Water-holding capacity determination: Water holding capacity (WHC) was determined by centrifugation. Approximately 5 g of gel sample (W1) was weighed and placed in a centrifuge tube of known mass (W0). The sample was centrifuged at 5000 rpm for 10 min. After centrifugation, the supernatant water was poured off, and excess water on the surface was blotted dry with filter paper. The total mass of the sample and centrifuge tube after centrifugation was weighed (W2).

[0054] Analysis of the texture profile: Hardness, elasticity, and chewiness were measured using a TA-10 probe. An appropriate amount of sample was placed on the texture analyzer platform and axially compressed through the probe at a speed of 1 mm / s to 55% of the deformation. The allowable recovery time between two compression cycles was 4 s, and the trigger point load was 5 g.

[0055] Determination of thermal stability and thermal transition temperature: Differential scanning calorimetry (DSC) was used to determine the thermal stability and thermal transition temperature of the gel. The specific method involved placing 10 mg of sample in a solid crucible, pressing it into a tablet and sealing it. The initial temperature was 40 °C, and the temperature was increased to 200 °C at a rate of 10 °C / min, which was considered a complete heating process.

[0056] Determination of mechanical properties: The gel strength and fracture strain were determined using a texture analyzer. Specifically, the gel sample underwent uniaxial compression testing using the texture analyzer, and the gel strength and fracture strain were measured. Specific parameters were set as follows: test speed of 1 mm / s, and compression deformation of 85% of the original sample height.

[0057] The macroscopic and microscopic observation results of the biomimetic connective tissues prepared in each embodiment and comparative example are as follows: Figure 1 and Figure 2 As shown. By Figure 1It can be seen that S1 prepared in Example 1 exhibits a uniform, dense, and continuous three-dimensional porous network structure with complete and interconnected pore walls, and the most regular microstructure; while the network structures of samples prepared in other examples and comparative examples have defects such as looseness, breakage, or fragmentation. Figure 2 It can be seen that the S1 prepared in Example 1 has a full and regular macroscopic morphology, a smooth surface, and can stably maintain the preset shape, exhibiting the best formability and structural integrity; while the samples prepared in other examples and comparative examples show varying degrees of deformation, collapse, or damage. The above results indicate that the S1 prepared in Example 1 has the best microstructure and macroscopic morphology, which ensures its good mechanical properties and thermal stability.

[0058] The water-holding capacity test results of the biomimetic connective tissues prepared in each embodiment and comparative example are as follows: Figure 3 As shown. By Figure 3 It can be seen that there are significant differences in water holding capacity between the samples prepared in each embodiment and the comparative example. The water holding capacity of S3 prepared in Example 3 is the highest, about 88%; the water holding capacities of samples S1, D2, and D3 are similar, about 85%; and the water holding capacity of sample D4 is the lowest, about 80%. S1 prepared in Example 1 exhibits good water holding capacity, which is significantly better than some embodiments and comparative examples.

[0059] The textural profile analysis results of the biomimetic connective tissues prepared in each embodiment and comparative example are shown in Table 1, and the mechanical property test results are shown in Table 2.

[0060] Table 1. Texture profile analysis results of biomimetic connective tissues prepared in each embodiment and comparative example.

[0061] Table 2. Results of mechanical property testing of biomimetic connective tissues prepared in each embodiment and comparative example.

[0062] As shown in Table 1, when the mass ratio of locust bean gum to carrageenan is between 2:8 (S2) and 4:6 (S1), the resulting gel exhibits optimal hardness (>51 N) and chewiness (>96 mJ), with small standard deviation, good repeatability, and a uniform and stable texture. In contrast, when the proportion of locust bean gum is too high (S3, the mass ratio of locust bean gum to carrageenan is 8:2), the gel's hardness and chewiness decrease sharply, resulting in a soft and brittle texture that fails to provide the lasting elasticity and resilience required by connective tissue. Therefore, a specific blending ratio of locust bean gum to carrageenan is crucial for precisely controlling the connective tissue texture of the biomimetic hydrogel and simulating a realistic chewy feel. Controlling the dry basis mass ratio of locust bean gum to carrageenan between approximately 2:8 and 4:6 is a necessary condition for obtaining elastic and chewy properties that closely resemble those of real meat connective tissue.

[0063] As shown in Table 2, the fracture strain (76.00±1.26%) of Example S1 (complete process) was significantly higher than that of other samples, and the gel strength (2.22±0.16 KPa) was at a relatively high level. Its mechanical properties better meet the requirements of natural connective tissue for "flexibility and tear resistance," indicating that its gel network can resist large destructive stress and undergo significant deformation without easily breaking, achieving a balance between "strength" and "toughness." The comparative data provides direct counter-evidence: the absence of enzymatic crosslinking (D1) or ionic crosslinking (D2) both lead to a significant decrease in one or two key mechanical indicators, proving that multiple synergistic crosslinking is the core of constructing a high-strength, high-toughness gel network. The D4 sample, lacking ferulic acid, had the weakest performance, indicating that the intermolecular interactions involving ferulic acid are crucial to network stability. This proves that calcium ions, transglutaminase, and ferulic acid play an indispensable synergistic role in the system of this invention, jointly constructing a dense and elastic three-dimensional network structure.

[0064] The results of thermal stability and thermal transition temperature measurements of the biomimetic connective tissues prepared in each embodiment and comparative example are as follows: Figure 4 As shown. By Figure 4 It can be seen that the biomimetic hydrogel-based connective tissues of each embodiment and comparative example all exhibit typical endothermic transition peaks. These peaks correspond to the thermal denaturation or decrosslinking process of the gel network and can characterize its thermal stability. Among them, S1 prepared in Example 1 has the highest endothermic peak temperature, approximately 160-170℃, which is significantly higher than that of the samples prepared in other embodiments (S2, S3) and the samples prepared in the comparative examples (D1-D4). Moreover, the heat flow changes slowly in the high-temperature range above 140℃, indicating that its gel network structure is more stable at high temperatures and is not prone to rapid degradation or collapse, exhibiting the best thermal stability.

[0065] In summary, the present invention, through a multi-synergistic strategy of "ionic crosslinking-enzymatic crosslinking-thermal curing" and a specific polysaccharide compound system, has prepared a biomimetic hydrogel-based connective tissue that achieves excellent biomimicry of natural connective tissue in terms of mechanical properties, textural characteristics and functions, and exhibits a synergistic enhancement effect.

[0066] Example 4: Preparation of vegetarian beef The biomimetic connective tissue (sample S1) obtained in Example 1 was cut into strips of 3 mm × 3 mm × 10 mm. 60 parts by weight of the high-moisture extrusion-prepared soy protein-based plant-based meat (with a fibrous structure) and 40 parts by weight of the above connective tissue strips were mixed in a mold. A 6% (w / w) soy protein isolate binder solution was poured in, and the mixture was lightly compacted. The amount of soy protein isolate binder solution added was 30% of the total mixture. The pressed blank was placed in a steamer and steamed at 100°C for 1 hour. After cooling to room temperature, it was refrigerated overnight (12 hours) at 4°C. After demolding, the final vegetarian beef product P1 was obtained (see...). Figure 5 ).

[0067] The biomimetic connective tissue prepared in Example 1 of this invention successfully endows plant-based beef products with a multi-layered, near-realistic sensory experience. The excellent water-holding capacity of this connective tissue ensures that it effectively locks in moisture within the composite product, releasing it during chewing, overcoming the "dryness" problem of traditional plant-based meat and achieving a "juicy" texture. The high thermal stability of this connective tissue ensures that the product maintains its shape and is not easily disintegrated during heat processing such as steaming and boiling. More importantly, as... Figure 5 As shown, after the biomimetic connective tissue is structurally composited with a fibrous plant-based meat substrate, a clear, biomimetic alternating layer of "muscle bundles-connective tissue" can be formed on the cross-section of the product. Combined with the superior mechanical properties (high fracture strain) of this connective tissue, the final product allows users to experience the "tearing sensation" of plant protein fibers and the "elasticity" of the biomimetic connective tissue in succession during chewing, accompanied by the release of juices. This achieves a high degree of simulation of real beef in terms of texture, mouthfeel, and flavor.

[0068] In summary, the biomimetic hydrogel-based connective tissue of this invention and its application in plant-based beef processing, through innovative design of component formulation and processes, achieves systematic performance enhancement and functional biomimicry at multiple levels, including molecular crosslinking, network structure, macroscopic texture, and the sensory characteristics of the final product. It not only significantly improves the textural complexity and edible pleasure of plant-based meat products but also possesses good processing stability and industrial production potential, providing a reliable and effective solution for developing next-generation high-end plant-based meat products.

[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for preparing biomimetic hydrogel-based connective tissue, characterized in that, Includes the following steps: S1. Add plant protein, water-soluble dietary fiber and ferulic acid to water, stir evenly to form a continuous phase solution; S2. Add glucomannan colloid, galactomannan colloid and seaweed sulfate polysaccharide colloid to water, heat to boiling to fully dissolve the colloids, and adjust the pH to 7.5-9.0 to obtain a polysaccharide colloidal solution. S3. Mix the continuous phase solution obtained in S1 with the polysaccharide colloidal solution obtained in S2 until homogeneous, and add calcium ion solution to the mixed material to carry out ion crosslinking. S4. Add transglutaminase to the ion-crosslinked material, stir well, and then carry out enzymatic crosslinking. S5. The enzymatically cross-linked material is subjected to heat curing and cold refrigeration in sequence to obtain biomimetic hydrogel-based connective tissue.

2. The preparation method according to claim 1, characterized in that, The plant protein mentioned in S1 includes any one or a mixture of two or more of the following: soy protein isolate, pea protein, chickpea protein, peanut protein isolate, wheat gluten protein, mung bean protein, quinoa protein, and nut protein; the water-soluble dietary fiber includes any one or a mixture of two or more of the following: oat β-glucan, barley β-glucan, konjac glucomannan, pectin, and inulin.

3. The preparation method according to claim 1, characterized in that, The mass ratio of plant protein, water-soluble dietary fiber and ferulic acid in S1 is (8-12):(4-6):(1-3).

4. The preparation method according to claim 1, characterized in that, The glucomannan colloids in S2 include any one or a mixture of two or more of konjac gum, konjac flour, and congee gum; the galactomannan colloids include any one or a mixture of two or more of locust bean gum, guar gum, and tara gum; and the seaweed sulfated polysaccharide colloids include any one or a mixture of two or more of carrageenan, sodium alginate, agar, and fucoidan. Based on 100% of the total mass of the biomimetic hydrogel-based connective tissue product, the glucomannan colloids comprise 0%-5% of the total mass, the galactomannan colloids and seaweed sulfated polysaccharide colloids comprise 3%-5% of the total mass, and the mass ratio of galactomannan colloids to seaweed sulfated polysaccharide colloids is 2:8-4:

6.

5. The preparation method according to claim 1, characterized in that, The calcium ion solution mentioned in S3 includes any one or a mixture of two or more of calcium chloride solution, calcium lactate solution, and calcium gluconate solution. The amount of calcium ions added accounts for 0.018%-0.181% of the total mass of the biomimetic hydrogel-based connective tissue product. The ionic crosslinking is carried out at 8000-13000 rpm for 1-3 min.

6. The preparation method according to claim 1, characterized in that, The amount of transglutaminase added in S4 is 0.05%-0.5% of the plant protein content, and the enzymatic cross-linking is carried out at 45-55℃ for 1-2 hours.

7. The preparation method according to claim 1, characterized in that, The thermosetting process described in S5 involves heating at 85-95°C for 20-40 minutes, and the cold curing process involves refrigerating at 2-8°C for at least 12 hours.

8. The biomimetic hydrogel-based connective tissue prepared by any of the preparation methods described in claims 1-7.

9. A food containing the biomimetic hydrogel-based connective tissue of claim 8.

10. The application of the biomimetic hydrogel-based connective tissue of claim 8 in the processing of plant-based meat.

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