Preparation method of dried orange peel and soybean protein composite fermented bean curd

Abstract

The invention relates to the technical field of food processing, and discloses a preparation method of dried orange peel and soybean protein composite fermented bean curd, which comprises the following steps: firstly, preparing an enzymolysis dried orange peel suspension and a thermal denaturation soybean protein solution; after mixing, adjusting the pH value to acidity to induce electrostatic complex coacervation, so as to prepare electrostatic complex slurry; adding a coagulating agent, and performing thermal gelation forming to obtain an acidic white body; neutralizing the surface acidity of the white body by using alkali liquor, inoculating mucor, culturing and pickling; performing deep enzymolysis on the salt billet by using a compound enzyme system containing alpha-L-rhamnosidase under a variable pressure condition; and finally, supplementing alcohol and table salt for ageing. The problem of looseness caused by poor compatibility of the pericarpium citri reticulatae fibers and protein gel is solved through an electrostatic complexing technology; the mould fermentation of an acidic matrix is realized by utilizing a surface neutralization technology; the bitter taste of the dried orange peel is effectively removed through variable-pressure assisted enzymolysis, the mass transfer efficiency is improved, the fermentation period is shortened, and the prepared composite fermented bean curd is fine and smooth in texture and coordinated in flavor.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for producing fermented bean curd made from a compound of dried tangerine peel and soybean protein. Background Technology

[0002] Fermented bean curd is a traditional fermented soybean food. In recent years, adding ingredients such as dried tangerine peel to fermented bean curd to give it specific flavors and nutritional components has become a trend in the development of fermented bean curd products.

[0003] However, current technologies for preparing tangerine peel-based fermented bean curd often involve directly mixing pulverized tangerine peel into soy milk or a gel matrix. Because tangerine peel is rich in insoluble dietary fiber, these particles have interfacial compatibility differences with the soy protein gel network. The filling of fiber particles disrupts the continuity of the protein gel, leading to weakened internal cohesion and reduced water retention in the fermented bean curd. As a result, the finished product is prone to becoming loose, crumbling, or breaking during fermentation and transportation, affecting its sensory quality and integrity.

[0004] Furthermore, dried tangerine peel contains bitter substances such as naringin. Traditional fermentation processes for fermented bean curd mainly rely on enzymes secreted by molds or natural microorganisms for decomposition. However, the enzymes produced by natural fermentation lack specificity and are difficult to effectively degrade specific bitter components in dried tangerine peel. This often results in a noticeable bitter taste remaining in the final product, masking the umami flavor of the fermented bean curd, limiting the amount of dried tangerine peel that can be added, and making it difficult to achieve a harmonious flavor profile.

[0005] Meanwhile, the post-fermentation of traditional fermented bean curd is usually carried out under normal pressure and static conditions, relying on concentration gradients to drive the diffusion of salts and enzymes into the interior of the bean curd. For composite bean curd containing tangerine peel particles, the internal matrix is ​​more complex, the mass transfer resistance is greater, and the penetration of external components into the interior is slow. This mass transfer method results in a longer production cycle and is prone to causing insufficient enzymatic hydrolysis in the center of the bean curd and uneven quality inside and outside, making it difficult to meet the efficiency and stability requirements of industrial production. Summary of the Invention

[0006] The technical problem solved by this invention is that in the process of adding fibrous auxiliary materials such as dried tangerine peel to fermented bean curd, the poor compatibility between the fiber and the soybean protein gel network leads to a loose and fragile structure of the finished product. Furthermore, the bitter substances in the dried tangerine peel are difficult to remove in traditional fermentation, and there are also problems such as slow flavor absorption and long fermentation cycle.

[0007] To address the above problems, the present invention provides the following technical solution: This invention provides a method for preparing fermented bean curd made from a mixture of dried tangerine peel and soybean protein, using the following technical solution: A method for preparing fermented bean curd made from a mixture of dried tangerine peel and soy protein includes the following steps: S1. Raw material pretreatment: Prepare functional suspension of dried tangerine peel after enzymatic hydrolysis and heat-denatured soybean protein liquid after thermal denaturation treatment. S2. Electrostatic complexation assembly: The tangerine peel functional suspension is mixed with the thermally denatured soybean protein liquid, the pH value is adjusted to acidic, and the mixture is stirred to induce the protein and pectin to re-coagulate, thus obtaining an electrostatic complexed slurry. S3. Thermal gelation molding: A coagulant is added to the electrostatic complexed slurry, and under heating conditions, protein molecules are induced to form a composite gel network structure. After cooling, pressing and cutting into pieces, acidic fermented bean curd white blank is obtained. S4. Pre-fermentation: The surface acidity of the acidic fermented bean curd blank is neutralized by using an alkaline solution, followed by inoculation with Mucor spores for cultivation to obtain Mucor fermented bean curd blank, which is then pickled to obtain salted blank. S5. First stage post-fermentation: The salt blank is immersed in a low-alcohol enzymatic hydrolysate containing a complex enzyme system and enzymatically hydrolyzed under pressure swing conditions to obtain an enzymatically reconstructed blank. S6. Second stage post-fermentation: Add edible alcohol and salt to the container containing the enzymatically reconstructed preform, and age it to obtain the finished product of tangerine peel and soybean protein compound fermented bean curd.

[0008] By adopting the above technical solution, this invention utilizes electrostatic interaction and pressure swing enzymatic hydrolysis to improve the binding state of dried tangerine peel and soy protein, as well as the flavor of the finished product. The specific principles and effects are explained below: First, regarding the binding mechanism between dried tangerine peel and soy protein. Steps S1 and S2 utilize pH adjustment to construct an electrostatic complexing system. Enzymatic hydrolysis in S1 releases pectin and other polysaccharides and flavonoids from the dried tangerine peel, while retaining some of the fibrous backbone; heat treatment unfolds the soy protein structure. In S2, the pH is adjusted to an acidic range (pH 3.8-4.2), at which point the positively charged soy protein and the negatively charged dried tangerine peel pectin are electrostatically attracted, forming a complex aggregate. This complex encapsulates the dried tangerine peel particles, preventing particle sedimentation, improving the water-holding capacity and mechanical strength of the gel network, and mitigating the problem of a loose finished product.

[0009] Second, regarding the regulation mechanism of gel structure and fermentation. Step S3 utilizes high temperature and calcium ion interaction to fix the electrostatic complex within the thermogel network, forming an acidic white substrate. Addressing the acid-sensitive nature of *Mucor*, step S4 uses an alkaline solution for rapid surface dipping treatment of the white substrate, neutralizing the surface acidity and establishing a suitable surface environment for *Mucor* growth, while maintaining an acidic interior. This structure promotes the growth of *Mucor* hyphae on the surface and the secretion of enzymes, while simultaneously inhibiting other microorganisms through the internal acidic environment, resulting in a flexible texture in the finished product.

[0010] Third, regarding mass transfer efficiency and debittering mechanism. Step S5 employs pressure swing enzymatic hydrolysis. A pressure gradient is established inside and outside the pores of the green body by cyclically changing between vacuum and positive pressure, driving liquid flow and promoting the entry of the low-alcohol enzymatic hydrolysate containing a complex enzyme system into the interior of the green body, thus improving mass transfer efficiency. α-L-rhamnosidase in the complex enzyme system decomposes bitter substances such as naringin in tangerine peel; acidic protease and low-ester pectinase work synergistically to soften the texture of the green body and release flavor compounds, shortening the fermentation cycle.

[0011] Preferably, in step S1, the preparation method of the tangerine peel functional suspension is as follows: pulverize dried tangerine peel and disperse it in water, with a material-to-liquid ratio of 1:10 to 1:15 by mass; add 0.1% to 0.3% by mass of cellulase from the tangerine peel powder; adjust the pH to 4.8 to 5.2; enzymatically hydrolyze at 45 to 50°C for 30 to 60 minutes; then raise the temperature to 90 to 95°C and hold for 5 to 10 minutes to inactivate the enzyme; and cool. The preparation method of the heat-denatured soybean protein solution is as follows: disperse soybean protein isolate in water to prepare a protein solution with a mass concentration of 10% to 12%; heat at 85 to 95°C for 25 to 35 minutes; and cool.

[0012] By adopting the above technical solutions, appropriate enzymatic hydrolysis is controlled to release pectin while retaining the supporting force of the fiber skeleton; heat treatment conditions ensure that soybean globulins are fully expanded and their binding sites are exposed.

[0013] Preferably, in step S2, the dry basis mass ratio of the tangerine peel functional suspension to the heat-denatured soybean protein solution is 1:4 to 1:5; the stirring speed is 300 to 500 rpm; the pH value is adjusted to 3.8 to 4.2; and the stirring induction time is 10 to 15 minutes.

[0014] By adopting the above technical solution, the ratio and pH range balance the formation of soluble complexes and insoluble complex aggregates, avoiding excessive protein precipitation; the shear conditions promote the uniform dispersion of the microcapsule structure.

[0015] Preferably, in step S3, the coagulant is a calcium sulfate suspension, and the amount of calcium sulfate suspension added is 0.2% to 0.4% of the total mass of the electrostatic complexing slurry; the heating conditions are: temperature 90 to 98°C, time 40 to 50 minutes.

[0016] By adopting the above technical solution, the electrostatic complex network is transformed into a more robust thermotropic gel network by utilizing high temperature and calcium ion crosslinking to withstand subsequent pressure swing treatment.

[0017] Preferably, in step S4, the alkaline solution is a sodium bicarbonate aqueous solution with a concentration of 0.5% to 1.0%; the specific operation of neutralizing the surface acidity is as follows: immersing the acidic fermented bean curd blank in the sodium bicarbonate aqueous solution for 10 to 20 seconds; the cultivation conditions are: temperature 22 to 25°C, relative humidity 90% to 95%, time 40 to 48 hours; the pickling process controls the final salt content of the salted blank to be 8% to 10%.

[0018] By adopting the above technical solutions, the quick-dip treatment adjusts the surface pH value, prevents the alkaline solution from penetrating and damaging the internal structure, and ensures the growth of the mycelium; the salinity is controlled to provide suitable osmotic pressure for subsequent enzymatic hydrolysis.

[0019] Preferably, in step S5, the salt concentration in the low-alcohol enzymatic hydrolysate is 6.0% to 8.0%, and the ethanol concentration is 0.5% to 1.5%; the complex enzyme system includes α-L-rhamnosidase, low-ester pectinase, and acidic protease; the amount of α-L-rhamnosidase added is 0.05% to 0.1% by weight / volume, the amount of low-ester pectinase added is 0.05% to 0.1% by weight / volume, and the amount of acidic protease added is 0.02% to 0.05% by weight / volume.

[0020] By adopting the above technical solution, the salinity of the enzymatic hydrolysate is controlled to be slightly lower than that of the salt blank to form a slight negative osmotic pressure, which promotes liquid penetration; low concentration of ethanol is conducive to enzyme penetration and does not inhibit activity; the three enzymes work together to achieve debittering, softening and release of flavor substances.

[0021] Preferably, in step S5, the pressure variation conditions are: temperature 35 to 40°C, pressure cyclically changing between vacuum -0.09MPa to -0.07MPa and positive pressure 0.10MPa to 0.15MPa, frequency 0.1 to 0.2Hz, and processing time 24 to 48 hours.

[0022] By adopting the above technical solution, pressure pulses are used to overcome the mass transfer resistance of the gel matrix, increase the contact between the enzyme and the substrate, and improve the enzymatic hydrolysis efficiency.

[0023] Preferably, in step S6, after adding edible alcohol and salt, the final concentration of ethanol in the broth is 10% to 12%, and the final concentration of salt is 10% to 12%; the aging time is 30 to 60 days.

[0024] By adopting the above technical solution, the concentration of alcohol and salt is increased after enzymatic hydrolysis to inhibit enzyme activity, and subsequent esterification and aroma production are carried out to form the final product.

[0025] This invention provides a method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein. It has the following beneficial effects: 1. This invention releases pectin through enzymatic pretreatment and induces the coagulation of soybean protein and pectin under acidic conditions using electrostatic effects. The resulting complex encapsulates tangerine peel microparticles, enhancing the cross-linking density of the gel network. This structure effectively prevents the sedimentation of tangerine peel particles, solves the problem of loose and brittle fermented bean curd blanks caused by insoluble fiber filling, and improves the product's molding rate and water retention.

[0026] 2. In the post-fermentation stage, this invention introduces a complex enzyme system containing α-L-rhamnosidase to specifically decompose bitter components such as naringin in tangerine peel and convert them into non-bitter substances. Combined with the synergistic effect of acidic protease and low-ester pectinase, it removes bitterness while promoting protein hydrolysis and the release of flavor precursors, thus solving the problem of bitter taste brought by tangerine peel raw materials and giving the finished product a harmonious flavor.

[0027] 3. This invention employs a pressure-switching-assisted enzymatic hydrolysis process, which utilizes the cyclical changes of vacuum and positive pressure to establish a pressure gradient inside and outside the pores of the green body, driving the enzymatic hydrolysate to quickly penetrate into the dense gel interior. This overcomes the limitation of slow natural diffusion. This physical field-assisted method accelerates the contact between the enzyme and the substrate and the biochemical reaction process, solving the problems of slow flavor infusion of macromolecular substances and long production cycles in traditional fermented bean curd. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and comparative examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Examples 1-3: Example 1

[0029] This embodiment provides a method for preparing fermented bean curd made from a mixture of dried tangerine peel and soy protein, including the following steps: S1. Dispersion and pre-modification treatment of raw materials The dried tangerine peel was pulverized by airflow and passed through a 300-mesh sieve. The tangerine peel powder was dispersed in water at a material-to-liquid ratio (w / v) of 1:10. 0.1% of cellulase by weight of the tangerine peel powder was added, the pH was adjusted to 4.8, and enzymatic hydrolysis was carried out at 45℃ for 30 minutes. Then, the temperature was raised to 90℃ and held for 10 minutes to inactivate the enzyme. The mixture was then cooled to room temperature to obtain a functional suspension of tangerine peel.

[0030] Soy protein isolate (SPI) was dispersed in deionized water to prepare a protein solution with a mass concentration of 10%; the protein was denatured by constant temperature stirring at 85°C for 25 minutes, and then cooled to 35°C to obtain a heat-denatured soy protein solution.

[0031] S2, pH-driven electrostatic complexation assembly The functional suspension of dried tangerine peel and the heat-denatured soybean protein solution were mixed evenly at a dry weight ratio of 1:5. Citric acid solution was slowly added dropwise while stirring at 300 rpm to adjust the pH of the mixture to 3.8. The mixture was stirred for 10 minutes at this pH to induce re-aggregation of the protein and pectin, resulting in an electrostatically complexed slurry containing microcapsule structures.

[0032] S3, High-temperature acid-induced thermal gelation molding Add 0.2% (by weight of calcium sulfate suspension) of the electrostatic complexing slurry to the slurry, stir rapidly until homogeneous, and then pour into a heat-resistant mold. Place the mold in a steam cabinet and heat at 90°C for 40 minutes. The high temperature induces the protein molecules under acidic conditions to form an irreversible thermogel network. After heating, allow it to cool naturally to room temperature, then press to dehydrate and cut into pieces to obtain the acidic fermented bean curd blank.

[0033] S4, Pre-fermentation A 0.5% sodium bicarbonate aqueous solution was prepared. The acidic fermented bean curd blanks were quickly dipped into the solution for 10 seconds, then removed and drained to neutralize the surface acidity. Subsequently, an acid-resistant Mucor spore suspension was sprayed onto the surface of the blanks, and they were cultured at 22℃ and 90% relative humidity for 40 hours until dense mycelia formed, yielding the Mucor fermented bean curd blanks. The Mucor fermented bean curd blanks were then pickled using a layer salt method, controlling the final salt content of the salted blanks to 8%.

[0034] S5. First-stage post-fermentation: isotonic pressure enzymatic hydrolysis Prepare an isotonic, low-alcohol compound enzymatic hydrolysate with a salt concentration of 6.0% (to form a slight negative osmotic pressure gradient with the salt blank, which is beneficial for mass transfer), an ethanol concentration of 0.5%, and add 0.05% (w / v) α-L-rhamnosidase, 0.05% (w / v) low-ester pectinase and 0.02% (w / v) acidic protease.

[0035] The pickled salt blanks were immersed in an isotonic low-alcohol compound enzymatic hydrolysate and placed in a pulsed pressure change reactor. At 35°C, the pressure was cyclically set between a vacuum of -0.07 MPa and a positive pressure of 0.10 MPa at a frequency of 0.1 Hz for 24 hours to obtain the enzymatically reconstructed blanks.

[0036] S6, Second Stage Post-Fermentation: High-Alcohol Flavor Aging Add edible alcohol and salt to the container containing the enzymatically reconstituted curd, adjusting the final concentration of ethanol and salt in the broth to 10%. Seal the container and age it at room temperature for 30 days to obtain the finished product of tangerine peel and soybean protein compound fermented bean curd. Example 2

[0037] This embodiment provides a method for preparing fermented bean curd made from a mixture of dried tangerine peel and soy protein, including the following steps: S1. Dispersion and pre-modification treatment of raw materials The dried tangerine peel was pulverized by airflow and passed through a 300-mesh sieve. The tangerine peel powder was dispersed in water at a material-to-liquid ratio (w / v) of 1:12.5. 0.2% of cellulase by weight of the tangerine peel powder was added, the pH was adjusted to 5.0, and enzymatic hydrolysis was carried out at 48℃ for 45 minutes. Then, the temperature was raised to 92℃ and held for 8 minutes to inactivate the enzyme. After cooling to room temperature, a functional suspension of tangerine peel was obtained.

[0038] Soy protein isolate (SPI) was dispersed in deionized water to prepare a protein solution with a mass concentration of 11%. The solution was heated at 90°C with constant stirring for 30 minutes to fully denature the protein. The solution was then cooled to 38°C to obtain the heat-denatured soy protein solution.

[0039] S2, pH-driven electrostatic complexation assembly The functional suspension of dried tangerine peel and the heat-denatured soybean protein solution were mixed evenly at a dry weight ratio of 1:4.5. Lactic acid solution was slowly added dropwise while stirring at 400 rpm to adjust the pH of the mixture to 4.0. The mixture was stirred for 12 minutes at this pH to induce phase separation and micro-encapsulation, yielding an electrostatically complexed slurry containing microcapsule structures.

[0040] S3, High-temperature acid-induced thermal gelation molding Add 0.3% (by weight of total mass) of calcium sulfate suspension to the electrostatic complexing slurry, stir well, and then pour into a mold. Place the mold in a water bath and heat at 94°C for 45 minutes to lock the electrostatic complex structure using acid-induced thermogelation. Cool, press, and cut into blocks to obtain acidic fermented bean curd white curd.

[0041] S4, Pre-fermentation Prepare a 0.8% sodium bicarbonate aqueous solution. Immerse the acidic fermented bean curd blanks in the solution for 15 seconds to neutralize the surface acidity to a suitable range for mold growth. Spray with an acid-resistant Mucor spore suspension and incubate at 24℃ and 92% relative humidity for 44 hours to obtain Mucor fermented bean curd blanks. Pickle the Mucor fermented bean curd blanks, controlling the final salt content of the salted blanks to 9%.

[0042] S5. First-stage post-fermentation: isotonic pressure enzymatic hydrolysis Prepare an isotonic, low-alcohol compound enzymatic hydrolysate with a salt concentration of 7.0% (to maintain an isotonic or slightly hypotonic environment), an ethanol concentration of 1.0%, and add 0.08% (w / v) α-L-rhamnosidase, 0.08% (w / v) low-ester pectinase, and 0.03% (w / v) acidic protease.

[0043] The salt blank was immersed in an isotonic, low-alcohol composite enzymatic hydrolysate. At 38°C, the pressure was cyclical between a vacuum of -0.08 MPa and a positive pressure of 0.12 MPa at a frequency of 0.15 Hz for 36 hours. The enzyme was then forced into the deep layer of the gel using a variable pressure pulse to obtain the enzymatically reconstructed blank.

[0044] S6, Second Stage Post-Fermentation: High-Alcohol Flavor Aging Add edible alcohol and salt to the container containing the enzymatically reconstituted curd, adjusting the final concentration of ethanol and salt in the broth to 11%. Seal the container and age it at room temperature for 45 days to obtain the finished product of tangerine peel and soybean protein compound fermented bean curd. Example 3

[0045] This embodiment provides a method for preparing fermented bean curd made from a mixture of dried tangerine peel and soy protein, including the following steps: S1. Dispersion and pre-modification treatment of raw materials The dried tangerine peel was pulverized by airflow and passed through a 300-mesh sieve. The tangerine peel powder was dispersed in water at a material-to-liquid ratio (w / v) of 1:15. 0.3% of cellulase by weight of the tangerine peel powder was added, the pH was adjusted to 5.2, and enzymatic hydrolysis was carried out at 50℃ for 60 minutes. Then, the temperature was raised to 95℃ and held for 5 minutes to inactivate the enzyme. The mixture was then cooled to room temperature to obtain a functional suspension of tangerine peel.

[0046] Soy protein isolate (SPI) was dispersed in deionized water to prepare a protein solution with a mass concentration of 12%; the solution was heated at a constant temperature of 95°C with stirring for 35 minutes and then cooled to 40°C to obtain a heat-denatured soy protein solution.

[0047] S2, pH-driven electrostatic complexation assembly The functional suspension of dried tangerine peel and the heat-denatured soybean protein solution were mixed evenly at a dry weight ratio of 1:4. Citric acid solution was slowly added dropwise while stirring at 500 rpm to adjust the pH of the mixture to 4.2. The mixture was stirred for 15 minutes at this pH to ensure full formation of the electrostatic complex, resulting in an electrostatically complexed slurry containing microcapsule structures.

[0048] S3, High-temperature acid-induced thermal gelation molding Add 0.4% (by weight of total mass) of calcium sulfate suspension to the electrostatically complexed slurry, stir well, and then pour into a mold. Place the mold in a steam cabinet and heat at 98°C for 50 minutes to form a high-strength, acid-resistant composite gel network structure. Cool, press, and cut into blocks to obtain acidic fermented bean curd blanks.

[0049] S4, Pre-fermentation Prepare a 1.0% sodium bicarbonate aqueous solution, and quickly dip the acidic fermented bean curd blanks into the solution for 20 seconds. Spray with an acid-resistant Mucor spore suspension and incubate at 25℃ and 95% relative humidity for 48 hours to form a thick mycelial coat, obtaining the Mucor fermented bean curd blanks. Pickle the Mucor fermented bean curd blanks, controlling the final salt content of the salted blanks to 10%.

[0050] S5. First-stage post-fermentation: isotonic pressure enzymatic hydrolysis Prepare an isotonic, low-alcohol compound enzymatic hydrolysate with a salt concentration of 8.0% (to control the slight negative osmotic pressure to prevent disintegration), an ethanol concentration of 1.5%, and add 0.1% (w / v) α-L-rhamnosidase, 0.1% (w / v) low-ester pectinase, and 0.05% (w / v) acidic protease.

[0051] The salt blanks were immersed in an isotonic, low-alcohol compound enzymatic hydrolysate. At 40°C, the pressure was cyclically set between a vacuum of -0.09 MPa and a positive pressure of 0.15 MPa, with a frequency of 0.2 Hz, for a processing time of 48 hours, to achieve deep debittering and softening of the texture, resulting in an enzymatically reconstructed blank.

[0052] S6, Second Stage Post-Fermentation: High-Alcohol Flavor Aging Add edible alcohol and salt to the container containing the enzymatically reconstituted curd, adjusting the final concentration of ethanol and salt in the broth to 12%. Seal the container and age it at room temperature for 60 days to obtain the finished product of tangerine peel and soybean protein compound fermented bean curd. Comparative Examples 1-5: Comparative Example 1:

[0053] Compared to Example 2, the difference lies in the following: In step S2, the pH of the mixing system is not adjusted to 4.0, but rather adjusted to neutral (pH 7.0) and physical mixing is performed directly; in step S3, since acid-induced thermal gel cannot be formed under neutral conditions, the amount of calcium sulfate suspension added is adjusted to 0.5% of the total mass of the slurry, and conventional brine coagulation is performed at 85°C. All other raw materials and steps are the same. Comparative Example 2:

[0054] Compared to Example 2, the difference lies in that: in step S3, the heating temperature was reduced to 55°C, the holding time was extended to 120 minutes (simulating conventional low-temperature enzyme treatment or mild gelation conditions), and the high-temperature heat treatment at 94°C was not performed. All other aspects are the same. Comparative Example 3:

[0055] Compared to Example 2, the difference lies in that: in step S4, the operation of "immersing the acidic fermented bean curd blank in a 0.8% sodium bicarbonate aqueous solution for 15 seconds" is omitted, and instead, a suspension of Mucor spores is directly sprayed onto the surface of the blank at a pH of approximately 4.0 for cultivation. All other steps are the same. Comparative Example 4:

[0056] The difference from Example 2 is that in step S5, the salt concentration in the enzymatic hydrolysate is adjusted to 1.0% (low-salt environment, non-isotonic) instead of 7.0%. All other aspects are the same. Comparative Example 5:

[0057] Compared to Example 2, the difference is that step S5 is omitted, and step S6 is performed directly. However, in step S6, the same amounts of α-L-rhamnosidase, low-ester pectinase, and acidic protease as in Example 2 are added to the broth (containing 11% ethanol and 11% salt) for a one-step post-fermentation. All other steps are the same.

[0058] Test Example 1: Feasibility Test of Process (Pre-fermentation Stage); White embryos prepared in Examples 1 to 3, Comparative Example 1 (without electrostatic complexation), and Comparative Example 3 (without surface acidity adjustment) were selected as test samples. Each group of samples was placed in a constant temperature and humidity chamber at 24±1℃ and 92±2% for pre-fermentation culture for a uniform time of 44 hours. After culture, the embryos were immediately subjected to rubbing and salting.

[0059] The testing process mainly examined two indicators: mycelial coverage and green body integrity. Mycelial coverage was determined using image analysis, whereby 20 samples were randomly selected at the end of cultivation, and surface images were taken. Gray-scale thresholding was used to segment and calculate the percentage of white mycelial area relative to the total surface area of ​​the green body. Green body integrity was determined by statistically analyzing the retention of each group of samples after mechanical rubbing and pickling / dehydration, calculating the proportion of green bodies that maintained a cubic shape without breakage relative to the initial quantity of raw materials.

[0060] Table 1. Statistics on growth and physical characteristics of different treatment groups during the pre-fermentation stage

[0061] Results Analysis and Conclusions: Experimental data show that surface acidity and the state of tangerine peel essential oil are key factors affecting the growth of Mucor and the forming of the blank.

[0062] As can be seen from the comparison between the Example 1 and Example 3, the surface pH value directly limits the colonization efficiency of *Mucor*. Example 3 omitted the sodium bicarbonate impregnation step, maintaining the surface pH of the blank at around 4.0. This acidic environment significantly inhibited the germination of *Mucor* spores, resulting in a mycelial coverage rate of only 15.3%, failing to form an effective enzyme secretion layer. Examples 1 to 3, through instantaneous surface neutralization treatment, raised the surface pH of the blank to the range of 6.2 to 6.4, meeting the physiological requirements for *Mucor* growth without altering the internal acidic environment, with mycelial coverage rates all exceeding 89%.

[0063] Comparing the examples with Comparative Example 1, it is evident that the electrostatic complex structure provides significant physical isolation for the tangerine peel essential oil. In Comparative Example 1, when mixed at pH 7.0, the soybean protein and pectin did not undergo a re-coagulation reaction, resulting in the tangerine peel essential oil containing antibacterial components such as D-limonene being distributed in a free state. Although the surface pH of Comparative Example 1 was 6.8, theoretically suitable for fungal growth, the antibacterial effect of the free essential oil limited the mycelial extension, resulting in a mycelial coverage rate of only 34.6%. The examples utilized electrostatic interactions at pH conditions ranging from 3.8 to 4.2 to encapsulate the hydrophobic essential oil components within the complex, reducing direct contact between the antibacterial components and surface mold, thereby improving the fermentation environment and promoting normal mycelial growth.

[0064] In terms of mechanical strength, the integrity rate of the blanks in both Example 1 and Comparative Example 3 was above 93%, significantly higher than the 81.2% of Comparative Example 1. This indicates that the high-temperature acid-induced thermal gelation process in step S3 promotes the formation of a dense network structure of protein molecules, which has superior resistance to mechanical shearing compared to the conventional brine gelation used in Comparative Example 1. The data confirms that the high-temperature acid-induced gelation technology can effectively improve the yield of fermented bean curd blanks during the roughening and pickling processes.

[0065] Test Example 2: Sensory quality and texture analysis of finished products (finished product stage); Fermented bean curd products prepared in Examples 1 to 3, Comparative Example 1 (without electrostatic complexation), and Comparative Example 5 (without stepwise enzymatic hydrolysis) were selected as test samples. All samples completed the second stage of post-fermentation, and the total aging time was uniformly 45 days.

[0066] The experiment combined sensory evaluation with texture profile analysis (TPA). The sensory evaluation team, consisting of 12 personnel trained in food sensory analysis, used a double-blind method to score the samples' color, tangerine peel aroma, taste, and physical appearance.

[0067] The total score is set at 100 points, with color accounting for 10 points, aroma for 30 points, taste for 40 points (focusing on bitterness residue and umami), and texture for 20 points. Texture testing was performed using a TA-XTPlus texture analyzer with a P / 36R cylindrical probe. Samples were cut into 20mm × 20mm × 20mm cubes. The speed before, during, and after testing was set to 1.0mm / s, the compression ratio to 50%, the trigger force to 5g, and the interval between two compressions to 5 seconds. Hardness and gumminess values ​​were recorded.

[0068] Table 2. Statistical data on sensory scores and textural characteristics of finished fermented bean curd products in each group.

[0069] Results Analysis and Conclusions: Table 2 shows that the enzymatic hydrolysis process parameters and raw material pretreatment methods have a significant impact on the texture and flavor of the final product.

[0070] In terms of texture, Comparative Example 5 achieved a hardness of 215.6g and an adhesiveness of 148.9g, significantly higher than the Example Group. Comparative Example 5 omitted the first stage of isotonic low-alcohol enzymatic hydrolysis, directly entering a high-concentration alcohol and salt environment. The high-temperature acid-induced thermogel formed in step S3 mainly relies on hydrophobic interactions and disulfide bonds to maintain a dense network. This structure is dense, and the endogenous enzymes have been inactivated due to the high temperature. Under a high-salt, high-alcohol environment, the activity of the exogenously added acidic protease is inhibited, failing to effectively cleave the gel backbone, resulting in the finished product maintaining the rigid structure of the thermogel. The Example Group, through step S5 in a low-alcohol environment, utilized acidic protease in conjunction with pectinase to moderately hydrolyze the gel network, reducing the hardness to the range of 55g to 73g and decreasing the adhesiveness, indicating that the gel structure had softened, forming the delicate texture characteristic of fermented bean curd.

[0071] In terms of taste, Comparative Example 5 scored only 15.6 points, with a bitter taste detected in the sensory feedback. Hesperidin is the main source of bitterness, and its degradation depends on α-L-rhamnosidase. The high-alcohol environment of Comparative Example 5 inhibited the catalytic activity of this enzyme, resulting in hesperidin residue. The Example group provided a suitable low-alcohol and pH environment for the enzymatic reaction in step S5, promoting the conversion of hesperidin into a non-bitter product, and improving the taste score to over 33 points.

[0072] Regarding aroma, Comparative Example 1 scored 12.4 points, lower than the Example Group. Comparative Example 1 used a physical mixing method and did not construct an electrostatic complex structure. During fermentation and dehydration, a large amount of volatile terpenes in the tangerine peel were lost due to the lack of encapsulation protection. The Example Group, through the protein-polysaccharide complex microcapsules formed in step S2, encapsulated volatile components. These components were slowly released during later aging, resulting in a finished product that retained a strong characteristic aroma of tangerine peel.

[0073] In summary, isotonic hydrolysis in step S5 is key to reducing the hardness of acidic gels and removing bitterness, while electrostatic complexation in step S2 is the foundation for ensuring flavor retention.

[0074] Test Example 3: Physicochemical Indicators and Residual Functional Components; Fermented bean curd products prepared in Example 2, Comparative Example 1 (without electrostatic complexation), and Comparative Example 5 (without stepwise enzymatic hydrolysis) were selected as test samples. Quantitative analysis was performed on three indicators: amino acid nitrogen, hesperidin residue, and D-limonene retention rate.

[0075] The amino acid nitrogen content was determined according to the pH meter method in GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food". The residual amount of hesperidin was determined by high performance liquid chromatography (HPLC). 5.0 g of the pulverized sample was weighed, added to 50 mL of methanol, and ultrasonically extracted for 30 minutes. The supernatant was centrifuged and filtered through a 0.45 μm filter membrane. A C18 column was used, with gradient elution of acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase. The detection wavelength was 283 nm, and quantification was performed using the external standard method. The retention rate of D-limonene was determined by gas chromatography (GC). The sample was extracted with n-hexane before injection analysis, and the ratio of the total amount of D-limonene detected in the finished product to the theoretical total amount in the tangerine peel powder at the time of feeding was calculated.

[0076] Table 3. Statistical data on physicochemical properties and functional components of finished products in each group.

[0077] Note: "Meets the standard" means that it meets the requirements for amino acid nitrogen in SB / T 10170-2007 "Fermented Bean Curd" (≥0.4g / 100g).

[0078] Results Analysis and Conclusions: Data shows that the process steps determine the biochemical indicators and flavor substance retention status of the product.

[0079] The amino acid nitrogen content reflects the degree of protein hydrolysis. The amino acid nitrogen content of Example 2 was 0.94 g / 100 g, higher than the two comparative examples. Comparative Example 1 did not undergo electrostatic complexation; the free tangerine peel essential oil inhibited early Mucor growth, resulting in insufficient secretion of the Mucor-derived complex protease system. Although this group added exogenous acidic protease in step S5, the lack of synergistic effect from the fungal enzyme system and the relatively loose structure of the preform may have led to some enzyme loss, ultimately hindering the overall protein hydrolysis process. Comparative Example 5 omitted the low-alcohol enzymatic hydrolysis step in the later stages and was directly placed in an environment with an ethanol concentration greater than 10%. The high concentration of alcohol inhibited protease activity, and the acidic thermogel backbone failed to degrade, resulting in an amino acid nitrogen release of only 0.38 g / 100 g.

[0080] The residual hesperidin content was consistent with the bitterness evaluation results in the sensory test. The residual hesperidin content in Comparative Example 5 was 925.4 mg / kg, indicating that under high-alcohol conditions, the added α-L-rhamnosidase lost its catalytic activity and could not hydrolyze hesperidin. Example 2, utilizing the low-alcohol isotonic system constructed in step S5, maintained glycosidase activity and degraded hesperidin to 58.2 mg / kg, below the bitterness threshold.

[0081] The retention rate of D-limonene reflects the effectiveness of microencapsulation. Comparative Example 1 showed a retention rate of 14.2%, indicating that most volatile terpenes were lost during heating, culturing, and pickling under physical mixing conditions. Examples 2 and 5 both showed retention rates above 75%, demonstrating that physical encapsulation structures can be formed and function effectively as long as pH-driven electrostatic complexation and high-temperature gel locking are performed. While Comparative Example 5 retained D-limonene, its texture was hard and bitter due to enzymatic failure; only Example 2 successfully retained volatile flavor compounds while achieving debittering and matrix maturation.

Claims

1. A method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein, characterized in that, Includes the following steps: S1, Raw Materials Pretreatment: A functional suspension of dried tangerine peel treated with enzymatic hydrolysis and a heat-denatured soybean protein solution were prepared separately. S2. Electrostatic complexation assembly: The tangerine peel functional suspension is mixed with the thermally denatured soybean protein liquid, the pH value is adjusted to acidic, and the mixture is stirred to induce the protein and pectin to re-coagulate, thus obtaining an electrostatic complexed slurry. S3. Thermal gelation molding: A coagulant is added to the electrostatic complexed slurry, and under heating conditions, protein molecules are induced to form a composite gel network structure. After cooling, pressing and cutting into pieces, acidic fermented bean curd white blank is obtained. S4. Pre-fermentation: The surface acidity of the acidic fermented bean curd blank is neutralized by using an alkaline solution, followed by inoculation with Mucor spores for cultivation to obtain Mucor fermented bean curd blank, which is then pickled to obtain salted blank. S5. First stage post-fermentation: The salt blank is immersed in a low-alcohol enzymatic hydrolysate containing a complex enzyme system and enzymatically hydrolyzed under pressure swing conditions to obtain an enzymatically reconstructed blank. S6. Second stage post-fermentation: Add edible alcohol and salt to the container containing the enzymatically reconstructed preform, and age it to obtain the finished product of tangerine peel and soybean protein compound fermented bean curd.

2. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S1, the preparation method of the tangerine peel functional suspension is as follows: after pulverizing the dried tangerine peel, disperse it in water with a material-to-liquid ratio of 1:10 to 1:15 by mass. Add 0.1% to 0.3% of cellulase by mass of the tangerine peel powder, adjust the pH to 4.8 to 5.2, and enzymatically hydrolyze at 45 to 50°C for 30 to 60 minutes. Then, raise the temperature to 90 to 95°C and hold for 5 to 10 minutes to inactivate the enzyme, and then cool. The method for preparing the heat-denatured soybean protein solution is as follows: soybean protein isolate is dispersed in water to prepare a protein solution with a mass concentration of 10% to 12%, heated at 85 to 95°C for 25 to 35 minutes, and then cooled.

3. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S2, the dry basis mass ratio of the tangerine peel functional suspension to the heat-denatured soybean protein solution is 1:4 to 1:5; the stirring speed is 300 to 500 rpm; the pH value is adjusted to 3.8 to 4.2; and the stirring induction time is 10 to 15 minutes.

4. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S3, the coagulant is a calcium sulfate suspension, and the amount of calcium sulfate suspension added is 0.2% to 0.4% of the total mass of the electrostatic complexing slurry; the heating conditions are: temperature 90 to 98°C, time 40 to 50 minutes.

5. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S4, the alkaline solution is a sodium bicarbonate aqueous solution with a concentration of 0.5% to 1.0%; the specific operation of neutralizing the surface acidity is to immerse the acidic fermented bean curd blank in the sodium bicarbonate aqueous solution for 10 to 20 seconds.

6. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S4, the cultivation conditions are: temperature 22 to 25°C, relative humidity 90% to 95%, and time 40 to 48 hours; the pickling process controls the final salt content of the salted material to be 8% to 10%.

7. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S5, the salt concentration in the low-alcohol enzymatic hydrolysate is 6.0% to 8.0%, and the ethanol concentration is 0.5% to 1.5%; the complex enzyme system includes α-L-rhamnosidase, low-ester pectinase, and acidic protease.

8. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 7, characterized in that, In the low-alcohol enzymatic hydrolysate, the amount of α-L-rhamnosidase added is 0.05% to 0.1% by mass / volume concentration, the amount of low-ester pectinase added is 0.05% to 0.1% by mass / volume concentration, and the amount of acidic protease added is 0.02% to 0.05% by mass / volume concentration.

9. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S5, the pressure variation conditions are: temperature 35 to 40°C, pressure cyclically changing between vacuum -0.09MPa to -0.07MPa and positive pressure 0.10MPa to 0.15MPa, frequency 0.1 to 0.2Hz, and processing time 24 to 48 hours.

10. The method for preparing a compound fermented bean curd made from dried tangerine peel and soybean protein according to claim 1, characterized in that, In step S6, after adding edible alcohol and salt, the final concentration of ethanol in the soup is 10% to 12%, and the final concentration of salt is 10% to 12%; the aging time is 30 to 60 days.

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