Preparation method of bean curd with double-network structure

Abstract

The invention relates to the technical field of food processing, and discloses a preparation method of bean curd with a double-network structure, and the bean curd with the double-network structure is prepared from the following raw materials in parts by weight: a recombinant protein matrix and a composite coagulator, the recombinant protein matrix is an aqueous dispersion with the total protein concentration of 10% and contains 7S globulin and 11S globulin, and the mass ratio of the 7S globulin to the 11S globulin is (30: 70)-(35: 65); the final total ion concentration of the system is 0.15-0.25 mol / L due to the addition amount of the composite coagulator; the mass ratio of 7S globulin to 11S globulin in the recombinant protein matrix is 32: 68. By constructing a 7S / 11S double-network interpenetrating structure and performing variable-frequency ultrasonic coupling ion / pH gradient regulation and control, the high hardness, high elasticity and strong water binding capacity of the soybean protein gel are synergistically improved, and the dry matter utilization rate and the product homogeneity are improved.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically a method for preparing tofu with a double-network structure. Background Technology

[0002] Soy protein products are an important part of traditional Chinese food culture. Tofu, a typical soy protein gel food, owes its quality primarily to the microstructure of its gel network. The gel formation process involves the thermal denaturation and unfolding of protein molecules, exposure of hydrophobic groups, and aggregation and cross-linking induced by metal ions. In actual production and consumption, high-quality tofu needs to simultaneously possess excellent mechanical strength (hardness and elasticity) and water-holding stability. However, existing preparation technologies often struggle to balance these two properties. High-hardness products are typically accompanied by poor elasticity and significant dehydration shrinkage, while high-water-holding-capacity products are often soft and lack chewiness.

[0003] The root cause of this performance contradiction lies primarily in the limitations of the raw material components. Soy globulin is mainly composed of 11S and 7S components, which differ in molecular weight, amino acid composition, and gel behavior. 11S globulin contains more sulfur amino acids and easily forms a rigid but brittle gel network; while 7S globulin molecules are more flexible, have strong water-holding capacity but low gel strength. Traditional tofu processing mainly relies on the protein raw materials naturally composed of whole soybean milk, without targeted ratio control and structural reorganization of these two components. As a result, the two types of proteins are often randomly distributed or simply phase-separated in the gel system, failing to form an effective interpenetrating network structure to synergistically improve textural properties.

[0004] Secondly, improper control of coagulation kinetics is also a major cause of product inhomogeneity. Traditional spotting or flushing processes typically involve adding high concentrations of calcium and magnesium salt coagulants in a single step. Because the binding reaction between metal ions and protein carboxyl groups is extremely rapid, a dense flocculent layer easily forms at the local contact surface, hindering the diffusion of subsequent ions into the deeper layers of the protein micelles. This diffusion-limiting effect results in a significant ion concentration gradient within the gel, often leading to a heterogeneous gel structure with an over-hardened surface and porous interior. Macroscopically, this manifests as a rough, easily broken product surface accompanied by the precipitation of large amounts of yellowish liquid, resulting in low raw material utilization.

[0005] Although physical field-assisted techniques such as ultrasound have been applied to protein modification, existing techniques mostly employ single-frequency ultrasound treatment. While low-frequency ultrasound has a strong cavitation effect, which is helpful for initial mixing and dispersion, the strong shear forces in the later stages of gel network formation can easily damage the already formed fragile framework. High-frequency ultrasound, while gentler, is less effective at breaking down the hydration layer barrier on the protein surface in the early stages. Furthermore, existing heat-induced gelation processes typically neglect the influence of pH on protein hydrophobic interactions during cooling, lacking dynamic coupling control of environmental parameters. This makes it difficult to guide protein molecules into a highly ordered spatial arrangement, further limiting the improvement of gel performance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing tofu with a dual-network structure, which solves the problems of existing soybean protein gel products, such as disordered component structure and uncontrolled coagulation kinetics, which lead to difficulty in achieving both mechanical strength and water retention, low raw material utilization, and uneven internal texture.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a dual-network structure tofu, employing the following technical solution:

[0009] The double-network structure tofu is made from raw materials containing a recombinant protein matrix and a composite coagulant. The recombinant protein matrix is ​​an aqueous dispersion with a total protein concentration of 10%, containing 7S globulin and 11S globulin, and the mass ratio of 7S globulin to 11S globulin is controlled between 30:70 and 35:65. The amount of composite coagulant added ensures that the final total ion concentration of the system is in the range of 0.15 to 0.25 mol / L.

[0010] By employing the above technical solution and utilizing the differences in molecular structure and gel behavior between 7S globulin and 11S globulin, a dual-network interpenetrating structure combining a rigid framework and flexible filler was constructed. The specific mechanism is as follows:

[0011] Rigid framework construction: 11S globulin has a large molecular weight and contains a large number of sulfur-containing amino acids, which easily form disulfide bonds after thermal induction. 11S globulin, which accounts for 65%-70% of the system, serves as the main network framework, providing the gel with high rigidity and resistance to deformation, and forming the "rigid first network" in the double network structure.

[0012] Flexible network filling: 7S globulin has a small molecular weight and low disulfide bond content, relying mainly on non-covalent bonds (such as hydrophobic interactions and hydrogen bonds) to maintain its structure. 30%-35% of 7S globulin is dispersed within the voids of the rigid framework formed by 11S, creating a "flexible second network." This flexible network endows the product with excellent elasticity and viscoelasticity, effectively mitigating the problem of brittleness and breakage inherent in purely rigid networks.

[0013] Dual-network synergy: Strictly controlling the ratio of 7S to 11S within the range of 30:70 to 35:65 ensures a mechanical balance between rigid node density and flexible chain segment length. If the 11S ratio is too high, the gel structure becomes too dense and brittle, resulting in a rough texture; if the 7S ratio is too high, the skeletal support is insufficient, the product is prone to collapse, and water retention capacity decreases.

[0014] Ion strength regulation: By controlling the total ion concentration of the system at 0.15-0.25 mol / L, a moderate shielding of the protein surface charge was achieved. This concentration range is sufficient to compress the electric double layer to induce ordered protein assembly, while avoiding random aggregation (coarse gelation) caused by excessive ion strength, thus ensuring the uniformity and compactness of the gel microstructure.

[0015] Preferably, in the above-mentioned double-network structure tofu, the mass ratio of 7S globulin to 11S globulin in the recombinant protein matrix is ​​32:68; the composite coagulant is composed of anhydrous calcium chloride and magnesium sulfate heptahydrate in a mass ratio of 3:1.

[0016] By adopting the above technical solution, the gel properties were further optimized:

[0017] The optimal ratio is locked in: a mass ratio of 32:68, which is the thermodynamically stable point for forming a dual-network topology. At this ratio, the 7S globulin has the best encapsulation effect on the 11S globulin aggregates, minimizing the precipitation of free water and improving the water-holding capacity of the product.

[0018] Complementary dual-ion kinetics: The combination of calcium chloride and magnesium sulfate utilizes the differences in hydration radius and protein binding rate between calcium and magnesium ions. Calcium ions bind rapidly, quickly locking onto the initial protein framework; magnesium ions bind relatively slowly, allowing them to penetrate deeper into the protein aggregates for subsequent cross-linking. The 3:1 mass ratio achieves a time-difference balance between "rapid surface setting" and "deep internal cross-linking," eliminating uneven gel stress caused by excessively rapid solidification and preventing the formation of macroscopic cracks.

[0019] Preferably, the above-mentioned recombinant protein matrix is ​​prepared through specific extraction and separation steps, including: dispersing defatted soybean flour in water and extracting the protein at pH 8.0; adding sodium bisulfite to the supernatant and adjusting the pH to 6.4 to obtain crude 11S globulin; adjusting the pH of the supernatant after separation to 4.8 to obtain crude 7S globulin; and finally purifying the two proteins separately and recombining them in proportion.

[0020] By adopting the above technical solution, precise control of raw material components has been achieved:

[0021] Sodium bisulfite-assisted fractionation: Sodium bisulfite is introduced during the separation stage at pH 6.4. Its reducing properties break some intermolecular disulfide bonds, reducing the degree of polymerization of protein molecules. This allows 11S globulin to precipitate more purely, reducing the co-precipitation of 7S globulin and thus improving the purity of the separated components.

[0022] Precise recombination foundation: Through high-purity component extraction and on-demand recombination, the impact of protein component fluctuations in natural soybean raw materials on product quality is eliminated, laying the material foundation for constructing a uniform and reproducible dual-network structure.

[0023] Secondly, the present invention provides a method for preparing tofu with a dual-network structure, employing the following technical solution:

[0024] A method for preparing tofu with a dual-network structure, comprising the following steps:

[0025] S1. Raw material preparation: Prepare recombinant protein matrix and prepare composite coagulant solution and pH adjustment buffer;

[0026] S2. Stepwise thermal induction: While stirring, heat the recombinant protein matrix to 75-80℃ and hold for 5-10 minutes, then heat to 90-95℃ and hold for 3-5 minutes, and then cool to 70-75℃.

[0027] S3. Low-frequency ultrasonic-assisted gradient coagulation: Maintain the slurry temperature at 70-75℃, turn on the low-frequency ultrasonic wave with a frequency of 20-28kHz, and add the composite coagulant in three batches in pulse mode. Before each addition, adjust the pH value of the slurry to 6.8, 6.5 and 6.2 respectively.

[0028] S4. High-frequency ultrasonic coupling pH dynamic adjustment: The ultrasonic wave is switched to a high-frequency mode of 40-60kHz. In continuous wave mode, the slurry temperature is controlled to decrease linearly at a rate of 0.45-0.55℃ / min. At the same time, pH adjustment buffer is added to make the pH value of the system decrease linearly from 7.0 to 6.2 as the temperature decreases.

[0029] S5. Compression molding: Stop the ultrasonic treatment when the temperature drops to 55-60℃, break up the curd, put it into the mold, apply pressure to compress it, and cool it to obtain the final product.

[0030] By adopting the above technical solution and utilizing the three-dimensional coupling regulation mechanism of "thermal field-acoustic field-chemical field", the dynamic control of the soybean protein gel network from molecular chain unfolding to three-dimensional cross-linking was achieved. The specific mechanism of action is described below:

[0031] The graded thermal induction mechanism (step S2) employs a two-stage heating program. The first stage (75-80℃) primarily targets the quaternary structure of the protein, inducing the dissociation of 7S and 11S globulin subunits and exposing previously masked active sites. The second stage (90-95℃) primarily targets the secondary and tertiary structures of the protein, fully unfolding the molecular chains and exposing hydrophobic groups and free thiol groups. This graded treatment avoids the "pseudo-denaturation" phenomenon caused by excessively rapid heating, where surface denaturation occurs while the internal structure remains undeveloped. This provides a highly reactive protein precursor for subsequent dual-network construction.

[0032] Synergistic mechanism of low-frequency ultrasound and gradient coagulation (step S3):

[0033] Low-frequency cavitation effect: Utilizing the strong cavitation effect generated by 20-28kHz low-frequency ultrasound, microjets and shock waves are produced in the medium. This physical force disrupts the hydration layer on the surface of protein micelles, lowers the energy barrier for coagulant ions to bind to proteins, and allows ions to rapidly penetrate into the interior of protein aggregates.

[0034] Gradient coagulation kinetics: By adding the coagulant in three stages and coordinating with a stepwise decrease in pH, the kinetics of gelation rate were matched. In the initial stage, with higher pH and lower ion concentration, the reaction rate was slower, which was conducive to the formation of uniform nucleation sites. As the pH decreased and the ion concentration increased, the reaction rate accelerated, inducing the orderly growth of protein molecules on the already formed nuclei. This gradient control effectively prevented "surface hardening" and porous internal structure caused by excessively high local ion concentrations.

[0035] High-frequency ultrasound and pH dynamic coupling mechanism (step S4):

[0036] High-frequency micro-perturbation alignment: After switching to 40-60kHz high-frequency ultrasound, the cavitation effect weakens, mainly manifested as high-frequency mechanical vibration and acoustic flow effect. This micro-perturbation acts on the initially formed gel network, playing a role similar to "annealing," helping protein molecular chains adjust their spatial conformation, eliminating defects and stress concentration points within the network, and promoting close packing between molecules.

[0037] Temperature-acid synchronous coupling: controlling the linear synchronization of the cooling rate and the pH decrease rate is to coordinate the waxing and waning of hydrogen bonding and hydrophobic interactions. As the temperature decreases, hydrogen bonding strengthens; as the pH decreases, the net charge of the protein decreases, and the electrostatic repulsion weakens. The synchronous changes of these two factors cause the hydrophobic regions of the protein to contract inward, physically locking water molecules within a three-dimensional network structure, thereby improving the water retention and structural stability of the final product.

[0038] Preferably, in step S1, the preparation of the recombinant protein matrix specifically includes: raw material defatting operation, using n-hexane for multiple defatting operations; component separation operation, adding sodium bisulfite to the extraction supernatant, separating 11S globulin using pH 6.4 and 4℃ environment, and separating 7S globulin using pH 4.8; matrix recombination operation, after determining the purity, mixing and adjusting the volume according to the ratio.

[0039] By employing the above technical solution, the purity of the material basis for the dual-network construction was ensured. In particular, the addition of sodium bisulfite, as a reducing agent, opened some disulfide bonds, reduced non-specific aggregation between protein molecules, significantly improved the resolution of 11S globulin and 7S globulin during isoelectric point precipitation separation, reduced the mutual entrainment between components, and thus ensured the accuracy of subsequent recombination ratios.

[0040] Preferably, step S3 is implemented as follows: the low-frequency ultrasonic power density is set to 150-300W / L, and a pulse mode of 2s on / 1s off is adopted; the proportions of the coagulant added three times are 40%, 30% and 30% of the total amount, respectively, and the pH is adjusted to 6.8, 6.5 and 6.2 before each addition, and each is ultrasonically treated for 60 seconds.

[0041] By adopting the above technical solution, precise control over the construction of gel networks has been achieved:

[0042] Pulse mode and power control: The power density of 150-300W / L combined with the pulse mode ensures sufficient energy to break the hydration layer while avoiding excessive protein denaturation or degradation caused by the thermal effect of continuous high-intensity ultrasound.

[0043] The gel is prepared using a staged addition method: the first addition of 40% coagulant initiates the nucleation reaction and establishes the basic network framework; subsequent additions of 30% each extend and strengthen the network. Combined with a gradual decrease in pH (enhanced charge shielding), this allows the ionic cross-linking reaction to transition from the extended state of the molecular chains to the aggregated state, ensuring the homogeneity of the gel network.

[0044] Preferably, step S4 is implemented as follows: the high-frequency ultrasonic power density is set to 50-100 W / L; a buffer system consisting of 0.1 mol / L citric acid solution and 0.2 mol / L disodium hydrogen phosphate solution is used; the buffer solution is added dropwise by a peristaltic pump to control the linearity of pH change to match the rate of temperature decrease.

[0045] By employing the above technical solution, the slow-release regulation capability of the citrate-phosphate buffer system, combined with the gentle perturbation of high-frequency ultrasound, avoids the sudden drop in local pH and protein denaturation and precipitation caused by direct addition of acid. The high-frequency, low-power (50-100 W / L) ultrasound environment promotes rapid and uniform diffusion of the buffer solution in the viscous gel system, ensuring that each micro-element within the system experiences a consistent pH change during cooling, thereby obtaining an isotropic, dense microstructure.

[0046] This invention provides a method for preparing tofu with a dual-network structure. It has the following beneficial effects:

[0047] 1. This invention, through strict control of the mass ratio of 7S globulin to 11S globulin and in conjunction with the dual-kinetic cross-linking mechanism of a calcium-magnesium composite coagulant, constructs a dual-network structure at the microscopic level, with 11S globulin as the rigid framework and 7S globulin as the flexible filler. This structural design effectively overcomes the defect that the increase in hardness of traditional soybean protein gel is often accompanied by a decrease in elasticity, enabling the finished tofu to possess both high hardness and excellent elastic recovery rate, thus improving the chewy texture and deformation resistance of the product.

[0048] 2. This invention utilizes high-frequency ultrasound micromechanical perturbation to synergistically regulate pH during the cooling process, inducing the orderly inward folding of hydrophobic protein groups to form a dense hydrophobic core. This precise adjustment of spatial conformation physically binds water molecules within the gaps of a three-dimensional grid, effectively blocking the migration pathways of free water. Experimental data shows that the centrifugal water loss rate of the resulting product is reduced to below 3.5%, solving the problem of water separation and shrinkage that traditional tofu easily experiences during transportation or storage.

[0049] 3. This invention utilizes a low-frequency ultrasonic-assisted ion gradient addition strategy to eliminate diffusion barriers during gelation, promoting uniform penetration of coagulant ions into the deep layers of protein micelles. This process avoids surface hardening and internal structural porosity caused by excessively high local ion concentrations, and reduces the loss of uncrosslinked protein with the soy milk. In actual preparation, the dry matter utilization rate of soybean protein is increased to over 90%, and the finished product has a fine and uniform cross-section without visible bubbles, breaks, or hard cores. Detailed Implementation

[0050] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0051] Non-GMO soybeans from Northeast China.

[0052] Anhydrous calcium chloride, food grade, purity ≥99%, CAS No.: 10043-52-4.

[0053] Magnesium sulfate heptahydrate, food grade, purity ≥99%, CAS No.: 10034-99-8.

[0054] Anhydrous citric acid, food grade, purity ≥99.5%, CAS No.: 77-92-9.

[0055] Anhydrous disodium hydrogen phosphate, food grade, purity ≥98%, CAS No.: 7558-79-4.

[0056] Preparation Examples 1-3:

[0057] Preparation Example 1:

[0058] This preparation example provides a recombinant protein matrix with a 7S globulin to 11S globulin mass ratio of 32:68, including the following steps:

[0059] Raw material defatting: Crush non-GMO soybeans from Northeast China and pass them through a 60-mesh sieve. Add n-hexane at a material-to-liquid ratio of 1:10 (w / v), stir and defatt the soybeans at room temperature for 1 hour, filter and recover the solids, and repeat the defatting process 3 times. Place the defatted soybean powder in a ventilated place to air dry until no solvent residue remains.

[0060] Protein extraction: Defatted soybean flour was dispersed in deionized water at a solid-liquid ratio of 1:15 (w / v). The pH was adjusted to 8.0 using 2.0 mol / L sodium hydroxide solution. The mixture was stirred and extracted at room temperature for 2 hours, followed by centrifugation at 8000×g for 20 minutes. The supernatant was collected.

[0061] Component separation: Sodium bisulfite was added to the supernatant to a final concentration of 0.98 g / L. The pH was adjusted to 6.4 using 2.0 mol / L hydrochloric acid solution. The mixture was allowed to stand at 4°C for 4 hours, then centrifuged at 6000×g for 20 minutes. The precipitate was collected as crude 11S globulin, and the supernatant was collected for later use. The supernatant was further adjusted to pH 4.8 using 2.0 mol / L hydrochloric acid solution. After standing for 2 hours, the mixture was centrifuged at 6000×g for 20 minutes, and the precipitate was collected as crude 7S globulin. Both precipitates were washed twice with deionized water and dispersed in water. The mixture was neutralized to pH 7.0 with sodium hydroxide solution and freeze-dried to obtain protein powder.

[0062] Matrix recombination: After determining the protein purity by the BCA method, 320g of 7S globulin powder and 680g of 11S globulin powder were weighed, dispersed in deionized water and brought to a final volume of 10L. The mixture was mechanically stirred until homogeneous, and a recombinant protein matrix with a total protein concentration of 10% (w / v) and a 7S to 11S mass ratio of 32:68 was obtained.

[0063] Preparation Example 2:

[0064] This preparation example provides a recombinant protein matrix with a 7S globulin to 11S globulin mass ratio of 30:70, including the following steps:

[0065] Raw material defatting: Crush non-GMO soybeans from Northeast China and pass them through a 60-mesh sieve. Add n-hexane at a material-to-liquid ratio of 1:10 (w / v), stir and defatt the soybeans at room temperature for 1 hour, filter and recover the solids, and repeat the defatting process 3 times. Place the defatted soybean powder in a ventilated place to air dry until no solvent residue remains.

[0066] Protein extraction: Defatted soybean flour was dispersed in deionized water at a solid-liquid ratio of 1:15 (w / v). The pH was adjusted to 8.0 using 2.0 mol / L sodium hydroxide solution. The mixture was stirred and extracted at room temperature for 2 hours, followed by centrifugation at 8000×g for 20 minutes. The supernatant was collected.

[0067] Component separation: Sodium bisulfite was added to the supernatant to a final concentration of 0.98 g / L. The pH was adjusted to 6.4 using 2.0 mol / L hydrochloric acid solution. The mixture was allowed to stand at 4°C for 4 hours, then centrifuged at 6000×g for 20 minutes. The precipitate was collected as crude 11S globulin, and the supernatant was collected for later use. The supernatant was further adjusted to pH 4.8 using 2.0 mol / L hydrochloric acid solution. After standing for 2 hours, the mixture was centrifuged at 6000×g for 20 minutes, and the precipitate was collected as crude 7S globulin. Both precipitates were washed twice with deionized water and dispersed in water. The mixture was neutralized to pH 7.0 with sodium hydroxide solution and freeze-dried to obtain protein powder.

[0068] Matrix recombination: After determining the protein purity by the BCA method, 300g of 7S globulin powder and 700g of 11S globulin powder were weighed, dispersed in deionized water and brought to a final volume of 10L. The mixture was mechanically stirred until homogeneous, and a recombinant protein matrix with a total protein concentration of 10% (w / v) and a 7S to 11S mass ratio of 30:70 was obtained.

[0069] Preparation Example 3:

[0070] This preparation example provides a recombinant protein matrix with a 7S globulin to 11S globulin mass ratio of 32:68, including the following steps:

[0071] Raw material defatting: Crush non-GMO soybeans from Northeast China and pass them through a 60-mesh sieve. Add n-hexane at a material-to-liquid ratio of 1:10 (w / v), stir and defatt the soybeans at room temperature for 1 hour, filter and recover the solids, and repeat the defatting process 3 times. Place the defatted soybean powder in a ventilated place to air dry until no solvent residue remains.

[0072] Protein extraction: Defatted soybean flour was dispersed in deionized water at a solid-liquid ratio of 1:15 (w / v). The pH was adjusted to 8.0 using 2.0 mol / L sodium hydroxide solution. The mixture was stirred and extracted at room temperature for 2 hours, followed by centrifugation at 8000×g for 20 minutes. The supernatant was collected.

[0073] Component separation: Sodium bisulfite was added to the supernatant to a final concentration of 0.98 g / L. The pH was adjusted to 6.4 using 2.0 mol / L hydrochloric acid solution. The mixture was allowed to stand at 4°C for 4 hours, then centrifuged at 6000×g for 20 minutes. The precipitate was collected as crude 11S globulin, and the supernatant was collected for later use. The supernatant was further adjusted to pH 4.8 using 2.0 mol / L hydrochloric acid solution. After standing for 2 hours, the mixture was centrifuged at 6000×g for 20 minutes, and the precipitate was collected as crude 7S globulin. Both precipitates were washed twice with deionized water and dispersed in water. The mixture was neutralized to pH 7.0 with sodium hydroxide solution and freeze-dried to obtain protein powder.

[0074] Matrix recombination: After determining the protein purity by the BCA method, 350g of 7S globulin powder and 650g of 11S globulin powder were weighed, dispersed in deionized water and brought to a final volume of 10L. The mixture was mechanically stirred until homogeneous, and a recombinant protein matrix with a total protein concentration of 10% (w / v) and a 7S to 11S mass ratio of 35:65 was obtained.

[0075] Examples 1-3:

[0076] Example 1:

[0077] This embodiment provides a method for preparing tofu with a dual-network structure, using the recombinant protein matrix provided in Preparation Example 1, and includes the following steps:

[0078] S1. Preparation of coagulant and buffer solution: Weigh anhydrous calcium chloride and magnesium sulfate heptahydrate and mix them in a mass ratio of 3:1. Dissolve them in deionized water to prepare a coagulant stock solution with a total ion concentration of 1.0 mol / L. Prepare 0.1 mol / L citric acid solution and 0.2 mol / L disodium hydrogen phosphate solution as dynamic pH adjustment buffer systems.

[0079] S2. Stepwise thermal induction: The recombinant protein matrix (7S:11S=32:68) obtained in Preparation Example 1 was placed in a reaction vessel equipped with a jacketed temperature control and mechanical stirrer, with the stirring speed set to 120 rpm. First, the temperature was increased to 78°C at a rate of 5°C / min and held at this temperature for 8 minutes for pre-gelation; then, the temperature was rapidly increased to 93°C and held at this temperature for 4 minutes for high-temperature activation. After the heat treatment, the mixture was rapidly cooled to 72°C using a heat exchanger.

[0080] S3. Low-frequency ultrasonic-assisted gradient coagulation: Transfer the slurry to an ultrasonic treatment tank and maintain the temperature at 72℃. Turn on the low-frequency ultrasonic treatment, setting the frequency to 24kHz, the power density to 225W / L, and the pulse mode (on for 2 seconds, off for 1 second). First, adjust the pH of the slurry to 6.8, add 40% of the total coagulant (controlling the final total ion concentration of the system to 0.20mol / L), and ultrasonically treat for 60 seconds. Then, add a small amount of acid solution to adjust the pH to 6.5, add 30% of the total coagulant, and continue ultrasonic treatment for 60 seconds. Finally, adjust the pH to 6.2, add the remaining 30% of the coagulant, ultrasonically treat for 60 seconds, and then turn off the low-frequency ultrasonic treatment.

[0081] S4. High-frequency ultrasonic coupling for dynamic pH adjustment: Immediately switch the ultrasonic wave to high-frequency mode, setting the frequency to 50kHz, power density to 75W / L, and continuous wave mode. Initiate the cooling program, controlling the slurry temperature to decrease linearly at a rate of 0.5℃ / min, while simultaneously adding buffer solution via a peristaltic pump, causing the system pH to decrease linearly from 7.0 to 6.2 as the temperature decreases. When the temperature drops to 58℃ and the pH stabilizes at 6.2, stop ultrasonication and adding buffer solution.

[0082] S5. Pressing and Shaping: Break up the obtained gel curd and pour it into a mold lined with filter cloth. Apply a pressure of 25 g / cm2 and maintain the pressure for 18 minutes. After pressing, allow it to cool naturally to room temperature to obtain double-network structure tofu.

[0083] Example 2:

[0084] This embodiment provides a method for preparing tofu with a dual-network structure, including the following steps:

[0085] S1. Preparation of coagulant and buffer solution: Same as in Example 1, the total ion concentration of the coagulant mother liquor is 1.0 mol / L.

[0086] S2. Stepwise thermal induction: The recombinant protein matrix (7S:11S=30:70) obtained in Preparation Example 2 was placed in a reaction vessel and stirred at 100 rpm. The temperature was raised to 75°C and held for 5 minutes for pre-gelation; then the temperature was raised to 90°C and held for 3 minutes for high-temperature activation. After the heat treatment, the temperature was cooled to 70°C.

[0087] S3. Low-frequency ultrasonic-assisted gradient coagulation: Maintain temperature at 70℃. Turn on low-frequency ultrasound, set frequency to 20kHz, power density to 150W / L, pulse mode (on for 2 seconds / off for 1 second). Adjust the slurry pH to 6.8, add 40% of the total coagulant (controlling the final total ion concentration of the system to 0.15mol / L), and ultrasonically treat for 60 seconds; adjust the pH to 6.5, add 30% of the total coagulant, and ultrasonically treat for 60 seconds; adjust the pH to 6.2, add the remaining 30% of the coagulant, and ultrasonically treat for 60 seconds before turning off the low-frequency ultrasound.

[0088] S4. High-frequency ultrasonic coupling for dynamic pH adjustment: Switch to high-frequency mode, set the frequency to 40kHz, power density to 50W / L, and continuous wave mode. Control the cooling rate at 0.45℃ / min, while simultaneously adding buffer solution to linearly decrease the pH from 7.0 to 6.2. Stop operation when the temperature drops to 55℃.

[0089] S5. Compression molding: Crush the curd and pack it into a mold, apply a pressure of 20 g / cm2, and maintain the pressure for 15 minutes. Cool to room temperature to obtain the final product.

[0090] Example 3:

[0091] This embodiment provides a method for preparing tofu with a dual-network structure, including the following steps:

[0092] S1. Preparation of coagulant and buffer solution: Same as in Example 1, the total ion concentration of the coagulant mother liquor is 1.0 mol / L.

[0093] S2. Stepwise thermal induction: The recombinant protein matrix (7S:11S=35:65) obtained in Preparation Example 3 was placed in a reaction vessel and stirred at 150 rpm. The temperature was raised to 80°C and held for 10 minutes for pre-gelation; then the temperature was raised to 95°C and held for 5 minutes for high-temperature activation. After the heat treatment, the temperature was cooled to 75°C.

[0094] S3. Low-frequency ultrasonic-assisted gradient coagulation: Maintain temperature at 75℃. Turn on the low-frequency ultrasonic wave, setting the frequency to 28kHz, power density to 300W / L, and pulse mode (2s on / 1s off). Adjust the slurry pH to 6.8, add 40% of the total coagulant (controlling the final total ion concentration of the system to 0.25mol / L), and ultrasonically treat for 60 seconds; adjust the pH to 6.5, add 30% of the total coagulant, and ultrasonically treat for 60 seconds; adjust the pH to 6.2, add the remaining 30% of the coagulant, and ultrasonically treat for 60 seconds before turning off the low-frequency ultrasonic wave.

[0095] S4. High-frequency ultrasonic coupling for dynamic pH adjustment: Switch to high-frequency mode, set the frequency to 60kHz, power density to 100W / L, and continuous wave mode. Control the cooling rate at 0.55℃ / min, while simultaneously adding buffer solution to linearly decrease the pH from 7.0 to 6.2. Stop operation when the temperature drops to 60℃.

[0096] S5. Compression molding: Crush the curd and pack it into a mold, apply a pressure of 30 g / cm2, and maintain the pressure for 20 minutes. Cool to room temperature to obtain the final product.

[0097] Comparative Examples 1-5:

[0098] Comparative Example 1: Compared with Example 1, the difference is that in steps S3 and S4, the ultrasonic generator was not turned on at all, and only mechanical stirring and chemical / thermal operations were retained, while the rest were the same.

[0099] Comparative Example 2: Compared with Example 1, the difference is that in step S4, the high-frequency mode is not switched, but the 24kHz low-frequency ultrasonic processing in step S3 is continued. All other aspects are the same.

[0100] Comparative Example 3: Compared with Example 1, the difference is that in step S3, the low-frequency mode is not used, but a high-frequency ultrasonic wave of 50kHz is used directly for processing; all other aspects are the same.

[0101] Comparative Example 4: Compared with Example 1, the difference is that in step S3, the coagulant was not added in three parts, but the total amount of coagulant was added at once at pH 6.8, and the ultrasonic treatment was maintained for 180 seconds. All other aspects were the same.

[0102] Comparative Example 5: Compared with Example 1, the difference is that in step S4, only linear cooling is performed, no citrate-phosphate buffer is added, the pH of the system is maintained at its natural state, and everything else is the same.

[0103] Test Example 1-2:

[0104] Test Example 1:

[0105] Experimental objective:

[0106] This test aims to verify the industrial production feasibility of the preparation methods described in Examples 1 to 3, focusing on the raw material utilization efficiency and the macroscopic integrity of the molded products.

[0107] Experimental steps:

[0108] Dry matter utilization rate determination: During the preparation process, the total dry weight of soybean protein powder (7S and 11S globulin powder) added to the system was accurately weighed. After preparation, all finished tofu was collected, surface water was removed, and the wet weight of the finished product was weighed. Subsequently, a portion of the sample was placed in a 105℃ forced-air drying oven and dried to constant weight. The moisture content of the finished product was measured, and the total dry matter mass in the finished product was then deduced. The dry matter utilization rate was obtained by dividing the total dry matter mass in the finished product by the total dry weight of the added soybean protein.

[0109] Molding cycle record: Record the time span from the addition of the first batch of coagulant to the end of pressing and demolding, and evaluate the time cost of the process.

[0110] Macroscopic morphological evaluation: Visually observe the surface condition of the tofu after demolding under natural light, and record whether there are visible bubbles, cracks, breaks or free water separation; observe the smoothness of the cut surface and the density of the texture.

[0111] Experimental data (see Table 1):

[0112] Table 1

[0113] Test Project Example 1 Example 2 Example 3 Input protein dry weight (g) 1000.0 1000.0 1000.0 Wet weight of finished tofu (g) 3642.5 3418.2 3589.6 Finished product moisture content (%) 74.3 73.1 74.8 Dry matter utilization rate (%) 93.6 91.9 90.5 Total gel forming time (min) 24 22 26 Surface macroscopic state Smooth surface, free of bubbles, with sharp edges The surface is relatively smooth, with slight pores and no cracks. The surface is dense, free of bubbles and collapse. Cut surface state Uniform texture, no hard core, no layering The texture is fairly uniform, with a slightly grainy feel. Dense texture and continuous structure

[0114] in conclusion:

[0115] Analysis of the data in Table 1 shows that Examples 1 to 3 all exhibit high dry matter utilization rates, ranging from 90.5% to 93.6%. This result indicates that the "thermal-acoustic-chemical" three-field coupling system constructed in this invention has a significant material retention capacity during actual conversion processes.

[0116] From a mechanistic perspective, in conventional tofu preparation, the rapid formation of the protein network often leads to localized aggregation, with some proteins failing to cross-link effectively and being lost with the soy milk. This method utilizes the cavitation effect generated by low-frequency ultrasound to assist calcium and magnesium ions in overcoming the hydration layer barrier on the protein micelle surface, achieving deep penetration and uniform distribution of ions within the 7S-11S dual-network structure. This kinetic control significantly increases the cross-linking sites between protein molecular chains, reducing the generation of free protein and thus improving dry matter utilization.

[0117] Example 1 achieved the highest dry matter utilization rate (93.6%) and optimal macroscopic morphology under optimized parameters. This confirms the necessity of introducing high-frequency ultrasound during the pH dynamic adjustment stage. The micromechanical perturbation provided by high-frequency ultrasound effectively streamlined the protein folding pathways, inducing the orderly aggregation of hydrophobic groups to form a stable hydrophobic core. This ordered structure not only enhances the gel network's ability to encapsulate solid materials but also eliminates macroscopic cracks or faults caused by stress concentration.

[0118] The data fluctuations in Examples 2 and 3 are within a reasonable range, indicating that the dual-network construction technique can maintain a stable gelation effect within the upper and lower limits of the set process parameters. Even at the lower ultrasonic power density in Example 2, the dry matter utilization rate remained above 91%, indicating that the promoting effect of ultrasound on ion diffusion has a low threshold and strong process robustness.

[0119] Test Example 2:

[0120] Experimental objective:

[0121] This test aims to quantitatively evaluate the textural properties (hardness, elasticity, chewiness) and water-holding stability of tofu gels obtained under different preparation process conditions. By comparing the data differences between the examples and comparative examples, the specific contributions of variable frequency ultrasonic field, ion gradient coagulation, and dynamic pH adjustment to the construction of the dual-network structure and the improvement of physical properties are verified.

[0122] Experimental steps:

[0123] Sample pretreatment: Fresh tofu samples prepared in Examples 1 to 3 and Comparative Examples 1 to 5 were placed at 4°C for equilibration for 24 hours. The samples were then removed, cut into standard cubic blocks with a side length of 20 mm, and left to stand at room temperature (25°C) for 30 minutes to eliminate the effect of temperature gradient.

[0124] Texture analysis: A texture analyzer was used to perform a secondary compression test on the samples. A P / 36R cylindrical probe was selected, with the pre-test running speed set to 2.0 mm / s, the test running speed to 1.0 mm / s, and the post-test return speed to 1.0 mm / s. The compression deformation was set to 50%, and the trigger interval between the two compressions was 5 seconds. The instrument automatically collected force-time curves, and the hardness, elasticity, and chewiness values ​​of the samples were calculated using software. Each group of samples was measured in parallel six times, and the average value was taken.

[0125] Centrifugal water-holding capacity determination: Weigh approximately 5.0 g of tofu sample and accurately record its initial mass. Place the sample in a centrifuge tube with qualitative filter paper at the bottom. Set the centrifuge speed to 3000 r / min and the centrifugation time to 15 minutes. After centrifugation, carefully remove the sample, blot off surface moisture, and weigh it again. Calculate the ratio of the mass difference before and after centrifugation to the initial mass to obtain the centrifugal water loss rate. This index reflects the gel network's ability to bind internal water; the lower the water loss rate, the better the water-holding capacity. Each sample was measured in triplicate.

[0126] Experimental data (see Table 2):

[0127] Table 2

[0128] Group Hardness (g) Elasticity (Ratio) Chewable (g) Centrifugal water loss rate (%) Example 1 412.54 0.924 365.18 3.25 Example 2 389.67 0.911 342.92 3.84 Example 3 425.81 0.895 378.43 3.41 Comparative Example 1 (Traditional Process) 245.32 0.762 189.55 11.56 Comparative Example 2 (Low Frequency Throughout) 198.45 0.684 145.21 16.82 Comparative Example 3 (High Frequency Throughout) 287.19 0.815 228.64 8.43 Comparative Example 4 (No Gradient) 310.22 0.833 254.76 7.15 Comparative Example 5 (without pH adjustment) 356.91 0.856 298.37 9.22

[0129] in conclusion:

[0130] Based on the test data in Table 2, the example group outperformed the comparative groups in both mechanical strength and water-holding stability. The differences in data are analyzed below in conjunction with microscopic dynamic mechanisms:

[0131] The hardness of Example 1 reached 412.54 g, and the centrifugal water loss rate was only 3.25%, indicating that the process constructed a dense and stable gel network. Compared with Comparative Example 1 (conventional process), the hardness was significantly improved and the water loss rate was reduced. This confirms that the introduction of external field assistance (ultrasound) and component recombination technology can fundamentally change the gel stacking pattern of soybean protein, transforming it from disordered random aggregation to ordered network cross-linking.

[0132] Regarding the necessity of ultrasonic frequency control, the data from Comparative Examples 2 and 3 provide counter-evidence. Comparative Example 2, using low-frequency ultrasound throughout, exhibited the worst hardness (198.45g) and water retention (16.82% water loss) across the entire range. This is because, in the later stages of gel network formation (the setting stage), the strong cavitation effect generated by low-frequency ultrasound mechanically shears and damages the newly formed fragile network framework, leading to mesh rupture and significant water loss. Conversely, Comparative Example 3, using high-frequency ultrasound throughout, while not damaging the structure, showed a much lower hardness (287.19g) than Example 1. This is because high-frequency ultrasound has weaker penetrating power and cannot effectively drive calcium and magnesium ions to the deep layers of the protein micelles in the early stages of coagulation, resulting in a defective structure of "surface hardening and internal cavitation," failing to form a uniform mechanical support. Example 1 employed a frequency-varying strategy, namely "low-frequency penetration and anchoring in the early stage, and high-frequency combing and setting in the later stage," effectively avoiding the defects of the aforementioned single frequency and maximizing performance.

[0133] Regarding the influence of chemical field control, the hardness and water retention of Comparative Example 4 (with one-time addition of coagulant) were lower than those of Example 1. The one-time addition of high-concentration ions caused rapid and irreversible aggregation at the protein contact surface, blocking the diffusion channels for subsequent ions and resulting in uneven network cross-linking density. Gradient ion addition combined with low-frequency ultrasound ensured that the spatiotemporal distribution of ion concentration matched the protein cross-linking rate, thereby constructing a homogeneous dual-network structure.

[0134] Finally, although Comparative Example 5 (without dynamic pH adjustment) had acceptable hardness, its centrifugal water loss rate (9.22%) was significantly higher than that of Example 1. This indicates that simple thermal induction and ionic cross-linking mainly contribute to the skeletal strength, while the ability to bind water is highly dependent on the arrangement of the hydrophobic groups in the protein. In Example 1, during the cooling stage, linear pH adjustment combined with high-frequency ultrasonic perturbation induced the hydrophobic groups to fold inward in an orderly manner, forming a "cage-like" structure that physically locks water molecules in the gaps between the molecular chains. The absence of this step (Comparative Example 5) resulted in the hydrophobic groups being exposed and disordered, unable to form an effective water-locking core, thus water easily precipitated out under centrifugal force.

Claims

1. A tofu with a dual-network structure, characterized in that, Made from the following ingredients in parts by weight: Recombinant protein matrix and composite coagulant; The recombinant protein matrix is ​​an aqueous dispersion with a total protein concentration of 10%, containing 7S globulin and 11S globulin, wherein the mass ratio of 7S globulin to 11S globulin is 30:70-35:

65. The amount of the composite coagulant added results in a final total ion concentration of 0.15-0.25 mol / L.

2. The tofu with a dual-network structure according to claim 1, characterized in that, The recombinant protein matrix contains 7S globulin to 11S globulin in a mass ratio of 32:68; the composite coagulant is composed of anhydrous calcium chloride and magnesium sulfate heptahydrate in a mass ratio of 3:

1.

3. The tofu with a dual-network structure according to claim 1, characterized in that, The recombinant protein matrix was prepared through the following steps: S1. Disperse defatted soybean flour in water, adjust the pH to 8.0, extract, and collect the supernatant by centrifugation; S2. Add sodium bisulfite to the supernatant to adjust the pH to 6.

4. After standing, centrifuge to obtain crude 11S globulin precipitate. S3. Adjust the pH of the supernatant after separation to 4.8, let it stand, and then centrifuge to obtain crude 7S globulin precipitate. S4. The crude 11S globulin precipitate and the crude 7S globulin precipitate are washed with water, neutralized, and dried into powder, respectively, and then compounded and dispersed in deionized water in proportion.

4. A method for preparing tofu with a double-network structure as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Raw material preparation: Prepare recombinant protein matrix and prepare composite coagulant solution and pH adjustment buffer; S2. Stepwise thermal induction: Place the recombinant protein matrix under stirring, first heat to 75-80℃ and hold for 5-10 minutes, then heat to 90-95℃ and hold for 3-5 minutes, and then cool to 70-75℃; S3. Low-frequency ultrasonic-assisted gradient coagulation: Maintain the slurry temperature at 70-75℃, turn on the low-frequency ultrasonic wave with a frequency of 20-28kHz, and add the composite coagulant in three batches in pulse mode. Before each addition, adjust the pH value of the slurry to 6.8, 6.5 and 6.2 respectively. S4. High-frequency ultrasonic coupling pH dynamic adjustment: The ultrasonic wave is switched to a high-frequency mode of 40-60kHz. In continuous wave mode, the slurry temperature is controlled to decrease linearly at a rate of 0.45-0.55℃ / min. At the same time, the pH adjustment buffer is added dropwise to make the pH value of the system decrease linearly from 7.0 to 6.2 as the temperature decreases. S5. Compression molding: Stop the ultrasonic treatment when the temperature drops to 55-60℃, break up the curd, put it into the mold, apply pressure to compress it, and cool it to obtain the final product.

5. The method for preparing tofu with a dual-network structure according to claim 4, characterized in that, In step S1, the preparation of the recombinant protein matrix specifically includes: Raw material degreasing: Crush soybeans and sieve them, then degrease them three times with n-hexane at a material-to-liquid ratio of 1:10; Component separation: Sodium bisulfite with a final concentration of 0.98 g / L was added to the extraction supernatant, and 11S globulin was separated using an environment of pH 6.4 and 4℃, while 7S globulin was separated using pH 4.

8. Matrix recombination: After determining the protein purity, weigh 7S globulin powder and 11S globulin powder according to the mass ratio, disperse them in water and make up to a fixed volume.

6. The method for preparing tofu with a dual-network structure according to claim 4, characterized in that, In step S1, the total ion concentration of the composite coagulant solution is 1.0 mol / L; the stirring speed of the recombinant protein matrix in step S2 is 100-150 rpm.

7. The method for preparing tofu with a dual-network structure according to claim 4, characterized in that, In step S2, the first heating rate of the graded thermal induction is 5°C / min, and the heat treatment is completed by rapid cooling through a heat exchanger.

8. The method for preparing tofu with a dual-network structure according to claim 4, characterized in that, The specific implementation method of step S4 is as follows: The power density of the high-frequency ultrasound is set to 50-100W / L; The pH adjustment buffer solution comprises a 0.1 mol / L citric acid solution and a 0.2 mol / L disodium hydrogen phosphate solution; The pH-adjusting buffer solution is added dropwise using a peristaltic pump, controlling the linearity of pH changes to match the rate of temperature decrease.

9. The method for preparing tofu with a dual-network structure according to claim 4, characterized in that, The specific implementation method of step S3 is as follows: Set the low-frequency ultrasonic power density to 150-300W / L, and the pulse mode to 2s on / 1s off. After adjusting the pH to 6.8 for the first time, add 40% of the total coagulation dose and sonicate for 60 seconds; After adjusting the pH to 6.5 for the second time, add 30% of the total coagulation dose and sonicate for 60 seconds; After adjusting the pH to 6.2 for the third time, add 30% of the total coagulation dose and sonicate for 60 seconds.

10. The method for preparing tofu with a dual-network structure according to claim 4, characterized in that, In step S5, the pressing pressure is 20-30 g / cm2, and the pressure is maintained for 15-20 minutes.