Frozen bullfrog composite non-phosphorus water-retaining agent and preparation method and application thereof

Abstract

The application provides a frozen bullfrog composite non-phosphorus water-retaining agent and a preparation method and application thereof. The water-retaining agent is prepared by self-assembly of silk fibroin nanofiber bundles and microcrystalline cellulose / modified oat beta-glucan micro support columns driven by honey dry powder, and is prepared in cooperation with functional components such as blood powder and aldehyde-based pectin. The water-retaining agent can be effectively embedded into bullfrog muscle tissue, and can inhibit damage caused by large ice crystals through physical support and anti-freezing protection during the frozen storage stage. Through elastic filling, gel network and covalent anchoring during the thawing and cooking stages, the water-retaining agent can significantly reduce juice loss, prevent protein oxidative denaturation and maintain meat tenderness, achieve water-retaining and color-protecting effects, and solve the technical problems of high health risk of traditional phosphate water-retaining agents and quality deterioration caused by conventional freezing.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a compound phosphorus-free water-retaining agent for frozen bullfrogs, its preparation method, and its application. Background Technology

[0002] Bullfrogs belong to the class Amphibia, order Anura, family Ranidae, and are a traditional meat-producing frog species. Bullfrogs grow quickly, have tender and flavorful meat, are rich in nutrients, and possess certain medicinal value. Their high protein and low fat content makes them popular with consumers. After 60 years of aquaculture development, they have become one of my country's important specialty aquatic products. Statistics show that my country's bullfrog production reached 215,000 tons in 2022 and continues to rise annually. Due to the seasonality of live bullfrogs, significant losses during transport, and the high risk of spoilage and short shelf life after slaughter and refrigeration, frozen storage is an indispensable step in bullfrog processing.

[0003] However, bullfrog meat is prone to forming large ice crystals during normal freezing, causing significant mechanical damage to its muscle tissue and cells. This leads to substantial juice loss, textural degradation, and fat and protein oxidation during storage, resulting in a severe decline in the product's flavor, quality, and nutritional value. Furthermore, the desiccation and thawing losses during freezing not only cause economic losses for businesses but also reduce consumer confidence in repurchase due to significant quality loss after thawing and cooking. Therefore, using water-retaining agents is one of the most effective methods to reduce quality deterioration of aquatic products during freezing and storage. Compound phosphates, as the most widely used water-retaining agent for aquatic products, can effectively reduce moisture loss during processing, transportation, and storage, maintain the tenderness of the product, and increase yield. However, when the addition of compound phosphates exceeds 0.4%, it produces an unpleasant metallic or soapy taste. Excessive consumption can not only disrupt the body's calcium-phosphorus balance but also cause blood clotting, increasing the risk of rickets, osteoporosis, goiter, and cardiovascular diseases. Patent CN 113973900 A discloses a method for freezing and preserving bullfrogs, using a compound water-retaining agent composed of sodium hexametaphosphate, sodium tripolyphosphate, sodium pyrophosphate, trehalose, sorbitol, sodium chloride, and glycerin. The addition of sodium hexametaphosphate and sodium tripolyphosphate at 1-3% exceeds the optimal consumption level of 0.4%, seriously impacting health.

[0004] Given the negative effects of phosphorus-containing water-retaining agents during use, the development and application of phosphorus-free water-retaining agents in frozen (pre-processed) aquatic products has become a new trend. Patent CN116195618 B discloses a phosphorus-free frozen storage water-retaining agent for tilapia fillets and its method. The composition of the agent is as follows (by mass fraction): sorbitol 0.3-0.7%, sodium citrate 0.1-0.5%, soy protein isolate 0.5-1.3%, and the remainder is distilled water. This technology improves the textural properties of fish fillets and enhances their antifreeze and water-retaining properties. However, there is a significant difference between the texture of tilapia fillets and bullfrog meat, so this method is not applicable to bullfrog meat. Patent CN 115568495 A discloses a quick-freezing method for improving the quality of frozen bullfrogs. This method primarily controls the concentration of gamma-glutamyl transferase (TG enzyme), vacuum tumbling time, and quick-freezing temperature to enhance the water-holding capacity of the bullfrogs during quick-freezing, storage, thawing, and cooking. However, the optimal activity temperature of TG enzyme is typically 40-50℃, while tumbling is usually performed at 0-4℃ to inhibit microorganisms. Furthermore, a settling and maturation step is lacking after vacuum tumbling and before quick-freezing. The cross-linking reaction of TG enzyme is a relatively slow process; immediate quick-freezing forcibly stops the enzymatic reaction, resulting in low and incomplete cross-linking efficiency. Given these process contradictions, the water-holding capacity, stability, and reproducibility of the final product are difficult to guarantee, lacking the reliability for industrial application. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a composite phosphate-free water-retaining agent for frozen bullfrogs and its preparation method. The water-retaining agent is prepared by self-assembling silk fibroin nanofiber bundles with microcrystalline cellulose / modified oat β-glucan micron-sized support pillars driven by honey powder, and synergistically incorporating functional components such as blood cell powder and aldehyde-modified pectin. This water-retaining agent can effectively embed itself into bullfrog muscle tissue, inhibiting damage from large ice crystals during frozen storage through physical support and antifreeze protection; during thawing and cooking, it significantly reduces juice loss, prevents protein oxidation and denaturation, and maintains meat tenderness through elastic filling, gel network, and covalent anchoring, achieving water retention and color protection effects. This solves the technical problems of high health risks associated with traditional phosphate water-retaining agents and quality deterioration caused by conventional freezing.

[0006] Technical solution: A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs, comprising the following steps: S1. After dissolving silk fibroin in water, a silk fibroin solution with a concentration of 8-12% is obtained. The solution is electrospun into nanofiber membranes and dispersed in a composite solution containing trehalose and sorbitol. After sonicating at 200-400W for 10-20 minutes, 0.5-2% of blood cell powder is added to the composite solution and sonicated at 200-400W for 5-10 minutes to obtain a nanofiber bundle dispersion. S2. Dissolve 1-3% microcrystalline cellulose whiskers and 2-6% modified oat β-glucan in water, add 1-3% plasma powder, stir evenly to obtain a micron-supported column dispersion; S3. Mix the nanofiber bundle dispersion with the micron support column dispersion, then add 3-8% honey powder and 0.5-1.5% sodium citrate, adjust the pH to 6.0, stir evenly, add 0.5-2% aldehyde-modified pectin, stir at 200-300 rpm for 15-30 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0007] Furthermore, the electrospinning conditions in step S1 are: applied voltage 15-25 kV, receiving distance 10-20 cm, and spinning solution flow rate 0.5-1.5 mL / h.

[0008] Furthermore, in step S1, the concentration of trehalose in the composite solution is 2-5%, and the concentration of sorbitol is 1-3%; the mass-to-volume ratio of the nanofiber membrane to the composite solution is (0.5-2) g: 100 mL.

[0009] Furthermore, the specific steps for modifying oat β-glucan in step S2 are as follows: Step 1. Disperse oat β-glucan in an ethanol-water mixed solvent with a volume ratio of (3-4):1, add a 10-20% NaOH solution, and alkalize at 10-25℃ for 1-3 hours to obtain alkalized oat β-glucan. Step 2. Transfer the alkalized oat β-glucan to a sealed pressure reactor, and under nitrogen protection at 40-60℃, introduce propylene oxide and react for 4-8 hours; Step 3. After the reaction is complete, cool to room temperature, adjust the pH to 6.5-7.5, filter, wash with ethanol, and vacuum dry at 40-50℃ to obtain modified oat β-glucan.

[0010] Furthermore, in step 2, the amount of propylene oxide introduced is 20-30% of the dry weight of oat β-glucan.

[0011] Furthermore, in step S3, the volume ratio of the nanofiber bundle dispersion to the micron support column dispersion is 1:(1-2).

[0012] Furthermore, the specific preparation method of aldehyde-modified pectin in step S3 is as follows: Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (the molar ratio of sodium periodate to pectin galacturonic acid units is 0.3:1) and react for 10 hours. Then, add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze-dry.

[0013] The above preparation method yields a composite phosphorus-free water-retaining agent for frozen bullfrogs.

[0014] The application of the above-mentioned frozen bullfrog compound phosphate-free water-retaining agent in bullfrog meat processing.

[0015] Furthermore, this includes the following steps: 1) After removing the skin and scraps from the fresh bullfrogs, wash them to remove the blood, dirt, and mud from the surface, and you will get fresh bullfrog meat. 2) Lay the fresh bullfrog meat flat and punch holes in both the back and thigh sides. The diameter of the punch needle is 1mm. When punching, only pierce the bullfrog meat 0.5-1cm to obtain pre-treated bullfrog meat. 3) Immerse the pretreated bullfrog meat in a frozen bullfrog compound phosphate-free water-retaining agent at a mass-to-volume ratio of 1:(1.25-1.5) for 3-15 hours at 4℃; 4) Drain the soaked bullfrog meat and flash-freeze it using liquid nitrogen; 5) After the bullfrog meat is frozen, it is packaged and then stored in a -18℃ cold storage.

[0016] Beneficial effects: This invention prepares a frozen bullfrog composite phosphate-free water-retaining agent with a micron-sized support column as the skeleton, and its surface is tightly wrapped with nanofiber bundles to form a composite structure. This overall structure can be embedded in the bullfrog muscle fiber bundles and myofibril gaps to achieve water retention and color protection effects, thereby improving the quality of bullfrog meat. The preparation of the composite phosphorus-free water-retaining agent in this invention can be roughly divided into three steps: 1) First, the preparation of nanofiber bundles: a silk fibroin solution is electrospun into nanofiber membranes, dispersed in an aqueous solution containing trehalose and sorbitol, and ultrasonically dispersed to ensure uniform loading of each component onto the fiber surface. Then, blood cell powder is added for further ultrasonic dispersion, resulting in a nanofiber bundle dispersion; 2) Next, the preparation of micron-supported columns: microcrystalline cellulose whiskers are mixed with modified oat β-glucan, and blood plasma powder is added to form a rigid-elastic composite micron-supported column dispersion; wherein, due to the unmodified... Oat β-glucan is prone to losing elasticity due to intermolecular hydrogen bond coagulation when frozen at -18℃, and is easily dissolved after thawing. It will also cause excessive swelling and structural collapse during cooking. Therefore, oat β-glucan was modified by hydroxypropylation. 3) Finally, the preparation of the composite phosphorus-free water-retaining agent is carried out. The nanofiber bundle dispersion is mixed with the micron support column dispersion, and honey powder and sodium citrate are added. The hydrogen bonds of the honey powder drive the nanofiber bundles to tightly wrap around the surface of the micron support column and self-assemble to form a composite structure. Then, aldehyde-modified pectin is added to make the aldehyde-modified pectin uniformly dispersed in the mixture. After obtaining the composite phosphate-free water-retaining agent, this invention is applied to the processing of bullfrog meat. The bullfrog meat is soaked in the composite phosphate-free water-retaining agent. During the frozen storage stage, it can play the following roles: (1) Micron support column: The rigid rod-shaped structure of microcrystalline cellulose whiskers is embedded in the gap of bullfrog muscle fiber bundles to form a physical support skeleton, resist the compressive stress of ice crystal growth, and prevent muscle fiber bundles from collapsing and breaking; Modified oat β-glucan gives the support column elasticity, buffers the mechanical impact of ice crystal expansion, reduces the probability of muscle cell membrane being punctured by ice crystals, and maintains the integrity of muscle tissue; After plasma powder is dissolved in the system, it begins to form a preliminary protein network at low temperature, which physically intertwines with the micron support column, enhances the toughness of the overall structure and the wrapping of muscle fibers, and lays the foundation for the heat-resistant gel network in the subsequent cooking stage; (2) Nanofiber bundles are wrapped around the surface of the micron support column and embedded in the gap of myofibrils to form a nanoscale physical barrier, locking in the muscle bound water. Trehalose and sorbitol are loaded together on the fiber surface, replacing water molecules and binding with the polar groups of myofibrillar protein to form a hydration protective film, preventing myoprotein denaturation at low temperature, while lowering the freezing point of the system, inhibiting ice crystal growth and recrystallization, and reducing the damage to cells and water loss from large ice crystals from the root; (3) Blood cell powder is evenly dispersed in the system, and its natural heme can effectively inhibit the oxidative denaturation of myoglobin under frozen storage conditions, achieving color protection; The fructose, glucose and other small molecule sugars in honey powder further enhance the antifreeze protection, stabilize proteins and water molecules through hydrogen bonding, and the natural polyphenols and organic acid components play a preliminary antioxidant role, protecting lipids and myoglobin; (4) The aldehyde group of aldehyde-modified pectin can directly react with the amino group of bullfrog myoprotein in a Schiff base covalent reaction, firmly anchoring the water-retaining agent structure on the surface of the muscle fiber, avoiding the loss of the water-retaining agent with the freeze-thaw liquid, and ensuring that the structural components continue to play a role; In this invention, after the frozen bullfrog meat treated with a composite phosphorus-free water-retaining agent is thawed, the water-retaining agent can play the following roles during the thawing stage: (1) Micron support pillars: The rigidity of microcrystalline cellulose whiskers maintains the spatial morphology of muscle fiber bundles and prevents muscle tissue from collapsing due to water seepage; Modified oat β-glucan has strong hydrophilicity and quickly adsorbs the meat juice seeping out during the thawing process, reducing water loss. At the same time, its viscosity enhances the binding force between the water-retaining agent and muscle fibers, and the covalent bond further reduces the solubility rate; (2) Silk fibroin nanofiber bundles swell when exposed to water, filling the tiny gaps in myofibrils after thawing and reducing the seepage channels of meat juice; Trehalose and sorbitol are slowly released with swelling and continue to bind with myofibrils. Combined, it prevents secondary denaturation of myoglobin during thawing, maintains the hydration capacity of protein, and avoids the meat becoming tough; blood cell powder continues to play a synergistic antioxidant and color-protecting role under the oxidative stress environment of thawing, preventing myoglobin from browning rapidly due to contact with oxygen; (3) after the honey powder is completely dissolved, it increases the viscosity and sweetness of the liquid phase on the surface of the meat, reduces water activity, delays possible microbial growth during thawing, and the osmotic pressure formed by its sugar helps to reduce excessive leakage of juice; (4) the aldehyde-based pectin covalent bond ensures that the water-retaining agent does not fall off with the thawing meat juice, and continuously provides structural support for muscle tissue. At the same time, its pectin molecules can wrap myoglobin, reduce myoglobin contact with oxygen, and indirectly achieve the color-protecting effect; In this invention, the water-retaining agent can play the following roles during steaming of frozen bullfrog meat soaked in a compound phosphate-free water-retaining agent: (1) Micron support pillars: Microcrystalline cellulose whiskers are heat-resistant and rigid, which can disperse the local stress generated by the thermal contraction of muscle fibers, avoid muscle fiber breakage due to excessive contraction, and maintain the looseness of muscle tissue; Modified oat β-glucan swells moderately when heated, accurately fills the gaps generated by the contraction of muscle fibers, and at the same time, its elasticity neutralizes the rigid contraction of muscle fibers, improves the tenderness of meat, and avoids dryness; Plasma powder rapidly denatures and cross-links at high temperature to form a strong, dense and heat-resistant three-dimensional gel network, which holds the muscle fibers, micron support pillars, and nano The fiber and other components are wrapped and bonded to form a strong ultimate water-locking barrier, which greatly blocks and holds the juice that is extracted during the cooking process, which is the key to significantly reducing cooking loss; (2) After being heated, the silk fibroin nanofiber bundles undergo slight molecular entanglement with myoprotein, further locking in the bound water extracted by the myoprotein during the cooking process and maintaining the juiciness of the meat; (3) The covalent bonds between aldehyde-modified pectin and myoprotein are partially and slowly broken at high temperature, avoiding stiffness of muscle tissue due to excessive bonding. At the same time, the broken pectin molecules are gel-like, which can adsorb the free water extracted during cooking, further improving water retention. The colloidal properties of pectin can also make the meat more tender. Attached image description: Figure 1 Appearance images of frozen bullfrog products prepared as blank, Examples 7 and 8 and Comparative Examples 6-12. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Example 1

[0018] A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 0.5 g: 100 mL. After sonication at 300 W for 20 min, 0.5% of blood cell powder was added to the composite solution, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 1% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 1% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 3% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0019] Example 2 A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 0.5% of blood cell powder was added to the composite solution, and the membrane was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 1% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 1% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 3% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0020] Example 3 A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 2 g: 100 mL. After sonication at 300 W for 20 min, 0.5% of blood cell powder was added to the composite solution, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 1% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 1% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 3% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0021] Example 4 A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 1% of blood cell powder was added to the composite solution, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 1% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 1% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 3% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0022] Example 5 A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 2% of the composite solution of blood cell powder was added, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 3% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 3% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 3% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0023] Example 6 A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 2% of the composite solution of blood cell powder was added, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 3% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 2% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 6% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0024] Comparative Example 1 The difference between this comparative example and Example 6 is that no blood cell powder was added, as detailed below: A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-to-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, a nanofiber bundle dispersion was obtained. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 3% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 2% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 6% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0025] Comparative Example 2 The difference between this comparative example and Example 6 is the addition of unmodified oat β-glucan, as detailed below: A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 2% of the composite solution of blood cell powder was added, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Dissolve 3% microcrystalline cellulose whiskers and 4% oat β-glucan in water, add 2% plasma powder, stir evenly to obtain a micron-supported column dispersion; S3. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 hours. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S4. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 6% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0026] Comparative Example 3 The difference between this comparative example and Example 6 is that no plasma powder was added, as detailed below: A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 2% of the composite solution of blood cell powder was added, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 3% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water and stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 6% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, stir at 300 rpm for 20 min, and the frozen bullfrog composite phosphorus-free water-retaining agent is obtained.

[0027] Comparative Example 4 The difference between this comparative example and Example 6 is that no honey powder was added, as detailed below: A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 2% of the composite solution of blood cell powder was added, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 3% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 2% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Dissolve low-methoxylated citrus pectin in water to prepare a 2% solution. Under light-protected conditions at 4°C, add sodium periodate (molar ratio of sodium periodate to pectin galacturonic acid unit is 0.3:1) and react for 10 h. Then add excess ethylene glycol to quench the reaction, dialyze for 2 days, and freeze dry to obtain aldehyde-modified pectin. S7. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 1.0% sodium citrate, adjust the pH to 6.0, stir evenly, add 1% aldehyde-modified pectin, and stir at 300 rpm for 20 min to obtain the frozen bullfrog composite phosphorus-free water-retaining agent.

[0028] Comparative Example 5 The difference between this comparative example and Example 6 is that aldehyde-modified pectin was not added, as detailed below: A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs includes the following steps: S1. After dissolving silk fibroin in water, a 10% silk fibroin solution was obtained. The solution was electrospun into a nanofiber membrane. A voltage of 20 kV was applied, the receiving distance was 20 cm, the spinning solution flow rate was 1.0 mL / h, and the nanofiber membrane was dispersed in a composite solution containing 4% trehalose and 2% sorbitol. The mass-volume ratio of the nanofiber membrane to the composite solution was 1 g: 100 mL. After sonication at 300 W for 20 min, 2% of the composite solution of blood cell powder was added, and the solution was sonicated at 300 W for 10 min to obtain a nanofiber bundle dispersion. S2. Disperse oat β-glucan in a 3:1 volume ratio ethanol-water mixed solvent, add 10% NaOH solution, and alkalize at 20℃ for 2 hours to obtain alkalized oat β-glucan; S3. Transfer the alkalized oat β-glucan to a closed pressure reactor, and under nitrogen protection at 50°C, introduce 25% of the dry weight of oat β-glucan propylene oxide and react for 6 hours. S4. After the reaction is complete, cool to room temperature, adjust pH to 7.0, filter, wash with ethanol, and dry under vacuum at 50°C to obtain modified oat β-glucan; S5. Dissolve 3% microcrystalline cellulose whiskers and 4% modified oat β-glucan in water, add 2% plasma powder, stir evenly to obtain a micron-supported column dispersion; S6. Mix the nanofiber bundle dispersion with the micron support column dispersion at a volume ratio of 1:1, then add 6% honey powder and 1.0% sodium citrate, adjust the pH to 6.0, and stir evenly to obtain the frozen bullfrog composite phosphorus-free water-retaining agent.

[0029] Performance testing: (1) Zeta potential The zeta potentials of the composite phosphorus-free water-retaining agents prepared in Examples 1-6 and Comparative Examples 1-5 were determined using a dynamic light scattering instrument.

[0030] Table 1. Zeta potential of composite phosphorus-free water-retaining agent

[0031] A higher absolute value of the Zeta potential indicates a stronger electrostatic repulsion and better physical stability of the system. Table 1 shows that the composite water-retaining agents prepared in the examples all have relatively high absolute values ​​of Zeta potential. Compared with the best example 6, the absolute values ​​of the Zeta potential in each comparative example decreased, indicating reduced system stability. Comparative Example 1 (without blood cell powder) had a smaller negative impact on the potential due to the lack of amphiphilic protein components. Comparative Example 2 (using unmodified β-glucan) had a lower potential because the molecules easily condensed through hydrogen bonds in the system, resulting in uneven particle distribution and poor dispersion. Comparative Examples 3 (without blood plasma powder) and 4 (without honey powder) lacked protein components that could participate in network construction and key hydrogen bond donors that drove self-assembly, respectively, resulting in a loose composite structure and reduced particle surface charge density. Comparative Example 5 (without aldehyde-modified pectin) lacked "anchoring" molecules that could covalently cross-link with various components, especially proteins, leading to the most unstable composite structure, the easiest particle aggregation, and therefore the lowest absolute value of the Zeta potential and the worst stability.

[0032] (2) Antioxidant properties DPPH free radical scavenging rate determination: DPPH working solution (0.1 mM): Accurately weigh 3.94 mg of DPPH reagent, dissolve it in anhydrous ethanol and dilute to 100 mL in a brown volumetric flask, shake well and prepare fresh before use.

[0033] Sample test solution: Accurately measure 1.0 mL of the composite water-retaining agent stock solution prepared in each example and comparative example, and dilute with water to a suitable concentration gradient.

[0034] Positive control: Prepare a 0.1 mg / mL aqueous solution of ascorbic acid (vitamin C) as a reference.

[0035] Add samples to test tubes, with three replicates for each sample, and take the average value. The sample assay group consists of 2 mL of sample assay solution + 2 mL of DPPH working solution; the sample control group consists of 2 mL of sample assay solution + 2 mL of anhydrous ethanol; the DPPH control group consists of 2 mL of water + 2 mL of DPPH working solution; and the blank group consists of 2 mL of water + 2 mL of anhydrous ethanol. Vortex all systems for 30 seconds to ensure complete reaction, then incubate at room temperature in the dark for 30 minutes. After the reaction, immediately measure the absorbance at 517 nm using a UV-Vis spectrophotometer, zeroing with the blank group. The DPPH free radical scavenging rate is calculated using the following formula: DPPH free radical scavenging rate (%) = [1 - (A 样品测定组 -A 样品对照组 ) / A DPPH对照组 ]×100% Table 2. DPPH free radical scavenging rate of composite phosphate-free water-retaining agent

[0036] As shown in Table 2, the DPPH free radical scavenging rate of the composite phosphorus-free water-retaining agent prepared in the examples ranged from 52.3% to 84.5%. However, the DPPH free radical scavenging rates of Comparative Example 1 (without blood cell powder) and Comparative Example 4 (without honey powder) decreased significantly. This was attributed to the absence of the core antioxidant components—heme iron in blood cell powder and polyphenols and organic acids in honey powder, respectively. These components are key substances that provide electrons or hydrogen atoms to neutralize DPPH free radicals. Although the scavenging rates of Comparative Examples 2, 3, and 5 decreased to varying degrees, the decrease was relatively small.

[0037] Therefore, the composite phosphorus-free water-retaining agent prepared in Example 6 was selected for subsequent freezing treatment of bullfrog meat.

[0038] Example 7 The steps for using a compound phosphate-free water-retaining agent to process bullfrog meat are as follows: 1) After removing the skin and scraps from the fresh bullfrogs, wash them to remove the blood, dirt, and mud from the surface, and you will get fresh bullfrog meat. 2) Lay the fresh bullfrog meat flat and punch holes in both the back and thigh sides. The diameter of the punch needle is 1mm. When punching, only pierce the bullfrog meat 0.5-1cm to obtain pre-treated bullfrog meat. 3) Immerse the pre-treated bullfrog meat in a frozen bullfrog compound phosphate-free water-retaining agent at a mass-to-volume ratio of 1:1.25 and soak at 4°C for 10 hours; 4) Drain the soaked bullfrog meat and flash-freeze it using liquid nitrogen; 5) After the bullfrog meat is frozen, it is packaged and stored in a -18℃ freezer. It should be washed before consumption.

[0039] Example 8 The steps for using a compound phosphate-free water-retaining agent to process bullfrog meat are as follows: 1) After removing the skin and scraps from the fresh bullfrogs, wash them to remove the blood, dirt, and mud from the surface, and you will get fresh bullfrog meat. 2) Lay the fresh bullfrog meat flat and punch holes in both the back and thigh sides. The diameter of the punch needle is 1mm. When punching, only pierce the bullfrog meat 0.5-1cm to obtain pre-treated bullfrog meat. 3) Soak the pre-treated bullfrog meat in a frozen bullfrog compound phosphate-free water-retaining agent at a mass-to-volume ratio of 1:1.5 for 15 hours at 4°C. 4) Drain the soaked bullfrog meat and flash-freeze it using liquid nitrogen; 5) After the bullfrog meat is frozen, it is packaged and stored in a -18℃ freezer. It should be washed before consumption.

[0040] Comparative Example 6 The difference between this comparative example and Example 7 is that the fresh bullfrog meat was not perforated, as detailed below: The steps for using a compound phosphate-free water-retaining agent to process bullfrog meat are as follows: 1) After removing the skin and scraps from the fresh bullfrogs, wash them to remove the blood, dirt, and mud from the surface, and you will get fresh bullfrog meat. 2) Soak fresh bullfrog meat in a frozen bullfrog compound phosphate-free water-retaining agent at a mass-to-volume ratio of 1:1.25 for 10 hours at 4°C; 4) Drain the soaked bullfrog meat and flash-freeze it using liquid nitrogen; 5) After the bullfrog meat is frozen, it is packaged and stored in a -18℃ freezer. It should be washed before consumption.

[0041] Comparative Example 7 The difference between this comparative example and Example 7 is that it uses the frozen bullfrog composite phosphorus-free water-retaining agent prepared in Comparative Example 1.

[0042] Comparative Example 8 The difference between this comparative example and Example 7 is that the frozen bullfrog composite phosphorus-free water-retaining agent prepared in Comparative Example 2 is used.

[0043] Comparative Example 9 The difference between this comparative example and Example 7 is that it uses the frozen bullfrog composite phosphorus-free water-retaining agent prepared in Comparative Example 3.

[0044] Comparative Example 10 The difference between this comparative example and Example 7 is that it uses the frozen bullfrog composite phosphorus-free water-retaining agent prepared in Comparative Example 4.

[0045] Comparative Example 11 The difference between this comparative example and Example 7 is that it uses the frozen bullfrog composite phosphorus-free water-retaining agent prepared in Comparative Example 5.

[0046] Comparative Example 12 The difference between this comparative example and Example 7 is that a commercially available phosphorus-containing water-retaining agent was used.

[0047] Performance testing: (1) Product appearance comparison Bullfrogs that were not soaked in the composite phosphate-free water-retaining agent were used as blanks, and the frozen bullfrogs prepared in Examples 7 and 8 and Comparative Examples 6-12 were compared and observed.

[0048] Depend on Figure 1 As can be seen, after soaking in the compound phosphorus-free water-retaining agent of this invention, the color of the bullfrog meat surface and the inside of the muscle is significantly improved, exhibiting a uniform, bright reddish luster, and the translucency of the meat is significantly enhanced.

[0049] (2) Soaking weight gain rate Accurately weigh the fresh bullfrog meat from Examples 7-8 and Comparative Examples 6-12 as M, soak it in a compound phosphate-free water-retaining agent, remove the sample after treatment, place it on a filter screen to drain the surface moisture, weigh it again and record the weight M1. The soaking weight gain rate is calculated according to the following formula: Soaking weight gain rate (%) = [(M1-M) / M] × 100 In the formula: M1—the mass of bullfrog meat after soaking (g); M—the mass of bullfrog meat after slaughter (g).

[0050] (3) Thawing loss rate Before thawing, weigh the bullfrogs. After thawing, wipe the surface of the bullfrog meat dry and weigh it again. Each group is repeated three times. Calculate the thawing loss rate using the following formula: Thawing loss rate (%) = [(M2-M3) / M2]×100 In the formula: M2—mass of bullfrog meat before thawing (g); M3—mass of bullfrog meat after thawing (g).

[0051] (4) Cooking loss rate After thawing, wipe the surface of the bullfrog meat dry and steam it in an 85℃ water bath for 20 minutes. Let the steamed bullfrog meat cool, wipe it dry again, and weigh it. Determine the cooking loss rate using the following formula: Cooking loss rate (%) = [(M4-M5) / M4]×100 In the formula: M4— the mass of bullfrog meat before steaming (g); M5— the mass of bullfrog meat after steaming (g).

[0052] (5) Total loss rate After steaming or boiling, wipe the surface moisture of the bullfrog meat dry and weigh it. Determine the total loss rate using the following formula: Total loss rate (%) = [(M5-M)] × 100 In the formula: M5 — mass of bullfrog meat after steaming (g); M — mass of bullfrog meat after slaughter (g).

[0053] Table 3

[0054] Table 3 shows that Examples 7-8 have better water retention effects, while the weight gain rate of bullfrog meat in Comparative Example 6, which was not perforated, is lower than that in Example 1. This is because the water-retaining agent in bullfrog meat that was not perforated has low penetration efficiency and cannot quickly reach the microchannels inside the muscle tissue, resulting in limited improvement in water retention inside the muscle and significant juice loss during subsequent thawing and cooking. Comparative Examples 7 and 10 lacked blood cell powder or honey powder in their water-retaining agents, resulting in a slight decrease in water retention performance. Comparative Example 8 used unmodified β-glucan. The loss rate was significantly higher than that of the examples, demonstrating the necessity of hydroxypropylation modification for maintaining the elasticity of β-glucan during frozen storage and preventing excessive swelling and collapse during cooking. Comparative Example 9 had the highest cooking loss rate, indicating that the heat-resistant gel network formed by plasma powder is the most critical barrier to resist heat shrinkage during cooking and achieve ultimate water retention. Comparative Example 11 lacked aldehyde-modified pectin in its water-retaining agent, and the water-retaining agent structure was easily detached from the muscle during freeze-thaw cycles, resulting in significant juice loss during thawing. Comparative Example 12 used a commercially available phosphorus-containing water-retaining agent, which had a relatively poor water retention effect.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a composite phosphorus-free water-retaining agent for frozen bullfrogs, characterized in that, Includes the following steps: S1. After dissolving silk fibroin in water, a silk fibroin solution with a concentration of 8-12% is obtained. The solution is electrospun into nanofiber membranes and dispersed in a composite solution containing trehalose and sorbitol. After sonicating at 200-400W for 10-20 minutes, 0.5-2% of blood cell powder is added to the composite solution and sonicated at 200-400W for 5-10 minutes to obtain a nanofiber bundle dispersion. S2. Dissolve 1-3% microcrystalline cellulose whiskers and 2-6% modified oat β-glucan in water, add 1-3% plasma powder, stir evenly to obtain a micron-supported column dispersion; S3. Mix the nanofiber bundle dispersion with the micron support column dispersion, then add 3-8% honey powder and 0.5-1.5% sodium citrate, adjust the pH to 6.0, stir evenly, add 0.5-2% aldehyde-modified pectin, stir at 200-300 rpm for 15-30 min to obtain the frozen bullfrog composite phosphorus-free water-retaining agent. The specific steps for modifying oat β-glucan in step S2 are as follows: Step 1. Disperse oat β-glucan in an ethanol-water mixed solvent with a volume ratio of (3-4):1, add a 10-20% NaOH solution, and alkalize at 10-25℃ for 1-3 hours to obtain alkalized oat β-glucan. Step 2. Under nitrogen protection, alkalized oat β-glucan is purged with propylene oxide and reacted for 4-8 hours. Step 3. After the reaction is complete, cool to room temperature, adjust the pH to 6.5-7.5, filter, wash with ethanol, and vacuum dry to obtain modified oat β-glucan.

2. The preparation method of the frozen bullfrog composite phosphorus-free water-retaining agent according to claim 1, characterized in that, The electrospinning conditions in step S1 are: applied voltage 15-25 kV, receiving distance 10-20 cm, and spinning solution flow rate 0.5-1.5 mL / h.

3. The preparation method of a composite phosphorus-free water-retaining agent for frozen bullfrogs according to claim 1, characterized in that: In step S1, the concentration of trehalose in the composite solution is 2-5%, and the concentration of sorbitol is 1-3%; the mass-volume ratio of the nanofiber membrane to the composite solution is (0.5-2) g: 100 mL.

4. The preparation method of a composite phosphorus-free water-retaining agent for frozen bullfrogs according to claim 1, characterized in that: In step 2, the amount of propylene oxide introduced is 20-30% of the dry weight of oat β-glucan.

5. The preparation method of a composite phosphorus-free water-retaining agent for frozen bullfrogs according to claim 1, characterized in that, In step S3, the volume ratio of the nanofiber bundle dispersion to the micron support column dispersion is 1:(1-2).

6. The frozen bullfrog composite phosphorus-free water-retaining agent prepared by the preparation method according to any one of claims 1-5.

7. The application of the frozen bullfrog composite phosphate-free water-retaining agent according to claim 6 in the processing of bullfrog meat.

8. The application according to claim 7, characterized in that, Includes the following steps: 1) After removing the skin and scraps from the fresh bullfrogs, wash them to remove the blood, dirt, and mud from the surface, and you will get fresh bullfrog meat. 2) Lay the fresh bullfrog meat flat and punch holes in both sides of its back and thighs to obtain pre-treated bullfrog meat; 3) Immerse the pretreated bullfrog meat in a frozen bullfrog compound phosphate-free water-retaining agent at a mass-to-volume ratio of 1:(1.25-1.5) for 3-15 hours at 4℃; 4) Drain the soaked bullfrog meat and flash-freeze it using liquid nitrogen; 5) After the bullfrog meat is frozen, it is packaged and then stored in a -18℃ cold storage.

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