Purine-reducing composite non-phosphorus water-retaining agent and application thereof in aquatic products

Abstract

The application discloses a kind of purine-reducing composite phosphorus-free water-retaining agents and its application in aquatic products, belong to food additive technical field.Water-retaining agent includes Ca 2+ -EGCG metal-phenolic network and water-retaining functional component;The Ca 2+ -EGCG metal-phenolic network is formed by calcium ion and epigallocatechin gallate through ultrasonic-assisted coordination self-assembly;The water-retaining functional component includes sorbitol, trehalose, sodium chloride, sodium bicarbonate and potassium bicarbonate.The Ca 2+ -EGCG metal-phenolic network is constructed by covalent-coordination double crosslinking mechanism, which significantly enhances the shrimp meat protein gel network structure, realizes the synergistic improvement of water-retaining performance.On the one hand, EGCG in MPNs is grafted on shrimp meat protein through covalent bond, and the purine-reducing functional unit is embedded in protein molecules in a chemical bond manner;On the other hand, Ca 2+ As a natural crosslinking agent involved in the formation of protein gel network, it enhances the compactness and orderliness of the gel network, significantly improves the water holding capacity of the product.

Description

Technical Field

[0001] This invention belongs to the field of aquatic product processing technology, specifically relating to a purine-reducing compound phosphorus-free water-retaining agent and its application in aquatic products. Background Technology

[0002] Aquatic products are rich in high-quality protein, various amino acids, and polyunsaturated fatty acids, possessing high nutritional value. However, during processing, transportation, and freezing, aquatic products commonly suffer from moisture loss, protein denaturation, color deterioration, and alterations in texture, severely impacting their edible quality and commercial value. Water retention treatment is a crucial step in aquatic product processing to improve product quality. Adding water-retaining agents can significantly reduce the loss of moisture and nutrients, maintain the tenderness of the product, and increase yield.

[0003] Polyphosphates are currently the most widely used water-retaining agents in aquatic product processing. However, the excessive use of phosphates has many drawbacks. On the one hand, excessive phosphate use can lead to a deterioration in product flavor and a coarse texture. Furthermore, phosphates can be hydrolyzed by enzymes in muscle, significantly reducing their water-retaining effect. On the other hand, when dietary phosphate content is too high, it can bind with calcium in the intestines to form insoluble calcium orthophosphate, thereby reducing calcium absorption and harming human health. Excessive intake may also increase the risk of bone loss and cardiovascular disease.

[0004] Currently, research has begun both domestically and internationally on using starch, modified starch, soy protein and soy protein isolate, casein, sodium alginate and its lysates to replace traditional phosphate-based water-retaining agents. Studies have shown that different types of phosphate-free water-retaining agents, when combined, can not only produce synergistic effects but also reduce dosage and save costs. Trehalose can form a new continuous phase through glass transition, hindering water molecule movement and thus improving water retention; alkaline salts can further stabilize the spatial structure of proteins by raising the pH value of aquatic products, improving water retention; sorbitol, sodium citrate, and other components are also widely used in phosphate-free water-retaining systems. However, the water retention effect of existing phosphate-free water-retaining agents is generally lower than or only equivalent to that of traditional phosphate-containing water-retaining agents, making it difficult to achieve a truly superior performance. How to achieve a synergistic improvement in water retention performance while completely replacing phosphates remains a pressing technical challenge in this field.

[0005] On the other hand, aquatic products are high in purines, and long-term excessive intake can lead to purine metabolism disorders, increasing serum uric acid levels and inducing hyperuricemia and gout. With the increasing prevalence of hyperuricemia, consumers' demand for low-purine aquatic products is becoming increasingly urgent. Xanthine oxidase (XOD) is a key enzyme in the purine metabolism pathway, catalyzing the oxidation of hypoxanthine to xanthine, and then xanthine to uric acid. Inhibiting XOD activity is an effective strategy to reduce uric acid production. Epigallocatechin gallate (EGCG) is one of the main active components of tea polyphenols. Studies have shown that EGCG has significant inhibitory activity against XOD and is an effective natural XOD inhibitor. However, EGCG is extremely unstable in alkaline environments and is easily oxidized and degraded, which greatly limits its application in alkaline water-retaining systems such as aquatic product processing.

[0006] In summary, existing technologies lack a composite water-retaining agent that can effectively replace phosphates to achieve excellent water retention while simultaneously reducing the purine content in aquatic products. This invention addresses this technological gap by providing a purine-reducing composite phosphate-free water-retaining agent and its application in aquatic products. Summary of the Invention

[0007] Technical Problem to be Solved: To address the aforementioned technical problems, the objective of this invention is to provide a purine-reducing, phosphorus-free water-retaining agent and its application in aquatic products. This invention constructs a Ca... 2+ -EGCG metal-phenolic networks significantly enhance the gel network structure of shrimp meat protein through a covalent-coordination dual crosslinking mechanism, achieving a synergistic improvement in water retention performance. On the one hand, EGCG in MPNs is covalently grafted onto shrimp meat protein, embedding the purine-lowering functional unit into the protein molecule via chemical bonds; on the other hand, incompletely grafted EGCG, through Ca... 2+ Coordination bonds anchor in the intermolecular spaces of the protein network, Ca 2+ As a natural cross-linking agent, it participates in the formation of protein gel networks, enhancing the density and orderliness of the gel network. The covalent-coordination double cross-linking network strengthens the protein gel structure at the molecular level, significantly improving the water-holding capacity of the product. Measurements showed that the shrimp prepared with the water-holding agent of this invention (Example 3) achieved a water-holding capacity of 91.2%, significantly better than Comparative Example 2 (water-holding capacity of 85.6% with only the water-holding functional component), and the water-holding capacity was 4.0 percentage points higher than that of the traditional phosphorus-containing water-holding agent (Comparative Example 6, water-holding capacity of 87.2%). This achieves phosphorus-free substitution with water-holding performance reaching or even exceeding the level of traditional phosphorus-containing water-holding agents.

[0008] Technical solution: A purine-lowering compound phosphorus-free water-retaining agent, wherein the water-retaining agent contains Ca at a mass ratio of 1:50 to 1:200. 2+ -EGCG metal-phenolic network and water-retaining functional components; The Ca2+ -EGCG metal-phenolic network is formed by the ultrasound-assisted coordination self-assembly of calcium ions and epigallocatechin gallate ester; The water-retaining functional components include sorbitol, trehalose, sodium chloride, sodium bicarbonate, and potassium bicarbonate.

[0009] Furthermore, the Ca 2+ The preparation method of the EGCG metal-phenolic network includes the following steps: Step 1: Dissolve epigallocatechin gallate (EGCG) in deionized water to prepare an EGCG solution with a concentration of 2-5 mmol / L, and protect it with nitrogen gas. Step 2: Dissolve food-grade calcium salt in deionized water to prepare a calcium ion solution with an equimolar ratio to EGCG; Step 3: Under stirring conditions, calcium ion solution is slowly added dropwise to EGCG solution, and coordination self-assembly reaction is carried out under ultrasonic treatment; Step 4: Centrifuge the reaction solution, collect the precipitate, wash with deionized water, and freeze-dry to obtain Ca. 2+ -EGCG metal-phenolic network powder.

[0010] Furthermore, the food-grade calcium salt mentioned in step two is calcium lactate or calcium gluconate.

[0011] Furthermore, in step three, the volume ratio of the calcium ion solution to the EGCG solution is 1:1 to 1:2; the ultrasonic treatment parameters are: power 100 to 300 W, frequency 20 to 40 kHz, temperature 25 to 35℃, and time 10 to 30 min.

[0012] Furthermore, in step four, the centrifugation speed is 8000–12000 r / min, the centrifugation time is 10–15 min, and the number of washing cycles is 2–3.

[0013] Furthermore, the concentration of sorbitol in the water-retaining functional component is 7 g / L, the concentration of trehalose is 5 g / L, the concentration of sodium chloride is 13 g / L, the total concentration of sodium bicarbonate and potassium bicarbonate is 8-15 g / L, and the mass ratio of sodium bicarbonate to potassium bicarbonate is 1:1-2:1.

[0014] This invention also provides the application of the above-mentioned composite phosphate-free water-retaining agent in aquatic products.

[0015] Furthermore, the application includes the following steps: Step 1: Immerse the aquatic products in the composite phosphate-free water-retaining agent at 0-4°C; Step 2: Place the soaked aquatic products in an ultrasonic processor for ultrasonic treatment; Step 3: Remove and drain, then quick-freeze or freeze.

[0016] Furthermore, in step one, the ratio of aquatic products to the compound phosphate-free water-retaining agent is 1:1.2 to 1:1.5 (g / mL); the soaking time is 1 to 3 hours.

[0017] Furthermore, the ultrasonic treatment parameters in step two are: power 100-500 W, frequency 20-50 kHz, temperature 30-35℃, and time 10-60 min.

[0018] Furthermore, the quick-freezing temperature in step three is below -35℃, and the frozen storage temperature is -18℃. Beneficial effects

[0019] 1. This invention uses Ca 2+ Using its empty orbital as a coordination center, it forms a coordination bond with the lone pair electrons of the oxygen atom of the phenolic hydroxyl group in EGCG, locking EGCG in the metal-phenolic network framework, forming a structurally stable metal-phenolic network (MPNs), which effectively solves the technical problem of easy oxidation and degradation of EGCG in alkaline water-retaining environments.

[0020] 2. The Ca constructed in this invention 2+ -EGCG metal-phenolic networks significantly enhance the gel network structure of shrimp meat protein through a covalent-coordination dual crosslinking mechanism, achieving a synergistic improvement in water retention performance. On the one hand, EGCG in MPNs is covalently grafted onto shrimp meat protein, embedding the purine-lowering functional unit into the protein molecule via chemical bonds; on the other hand, incompletely grafted EGCG, through Ca... 2+ Coordination bonds anchor in the intermolecular spaces of the protein network, Ca 2+ As a natural cross-linking agent, it participates in the formation of protein gel networks, enhancing the density and orderliness of the gel network. The covalent-coordination double cross-linking network strengthens the protein gel structure at the molecular level, significantly improving the water-holding capacity of the product. Measurements showed that the shrimp prepared with the water-holding agent of this invention (Example 3) achieved a water-holding capacity of 91.2%, significantly better than Comparative Example 2 (water-holding capacity of 85.6% with only the water-holding functional component), and the water-holding capacity was 4.0 percentage points higher than that of the traditional phosphorus-containing water-holding agent (Comparative Example 6, water-holding capacity of 87.2%). This achieves phosphorus-free substitution with water-holding performance reaching or even exceeding the level of traditional phosphorus-containing water-holding agents.

[0021] 3. In this invention, EGCG exists in shrimp meat in the form of MPNs (Multiple Ventilation Networks). During processing and freezing, it is protected by coordination bonds and released responsively in the acidic environment of the stomach after consumption, achieving targeted delivery and effective dose release of EGCG. The coordination bonds of MPNs are stable under alkaline / neutral conditions, locking EGCG within the network framework; upon entering the acidic environment of the stomach (pH 1-3), H... + With Ca2+ Competitive coordination leads to the dissociation of the metal-phenolic coordination bond, triggering the release of EGCG.

[0022] 4. This invention utilizes the alkaline environment provided by the water-retaining agent (sodium bicarbonate and potassium bicarbonate in the water-retaining agent) to trigger protein structural changes, while simultaneously using ultrasonic energy to drive covalent bond formation. The two work synergistically in the same process sequence—sodium bicarbonate and potassium bicarbonate in the water-retaining agent complete protein unfolding and structural activation during the 1-3h soaking process, and the ultrasonic grafting process (≤30min) covalently anchors EGCG to the protein before degradation occurs. Attached Figure Description

[0023] Figure 1 For Ca in Example 1 2+ SEM image of EGCG metal-phenolic network (MPNs-A) (scale bar 2 μm); Figure 2 This is an HPLC chromatogram of the xanthine content in the sample of Example 3; Figure 3 The image shows the HPLC chromatogram of xanthine content in sample 6 (Comparative Example). Detailed Implementation

[0024] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0025] Example 1 Ca 2+ The preparation method of the EGCG metal-phenolic network includes the following steps: Step 1: Dissolve epigallocatechin gallate in deionized water to prepare an EGCG solution with a concentration of 4 mmol / L, and protect it with nitrogen gas. Step 2: Dissolve calcium lactate in deionized water to prepare a calcium ion solution with a concentration of 4 mmol / L; Step 3: Under stirring conditions, calcium ion solution is slowly added dropwise to EGCG solution at a volume ratio of 1:1.5. The mixture is then treated with ultrasound at 200 W, 30 kHz, and 30 °C for 20 min to carry out coordination self-assembly reaction. Step 4: Centrifuge the reaction solution at 10000 r / min for 13 min, collect the precipitate, wash twice with deionized water, and freeze-dry to obtain Ca. 2+-EGCG metal-phenolic network powder (MPNs-A).

[0026] Measurements showed that: (1) The average hydrated particle size of MPNs-A was determined by DLS to be 135.2 ± 3.1 nm. SEM images (e.g.) Figure 1 (As shown) reveals that these nanounits are further cross-linked to form a porous network structure with micron-sized pores; the PDI is 0.19, the Zeta potential is -34.6±3.8 mV, the MPNs-A particle size distribution is uniform, and it has good dispersion stability.

[0027] (2) FTIR spectroscopy showed that, compared with pure EGCG, MPNs-A had a lower concentration in the range of 3350–3560 cm⁻¹. -1 The stretching vibration peak of the phenolic hydroxyl group (OH) at that position was significantly broadened and redshifted by approximately 20 cm⁻¹. -1 At 1690 cm -1 The intensity of the C=O stretching vibration peak at that location decreased significantly.

[0028] Example 2 Ca 2+ The preparation method of the EGCG metal-phenolic network includes the following steps: Step 1: Dissolve epigallocatechin gallate in deionized water to prepare an EGCG solution with a concentration of 4 mmol / L, and protect it with nitrogen gas. Step 2: Dissolve calcium gluconate in deionized water to prepare a calcium ion solution with a concentration of 4 mmol / L; Step 3: Under stirring conditions, calcium ion solution is slowly added dropwise to EGCG solution at a volume ratio of 1:1.5. The mixture is then treated with ultrasound at 200 W, 30 kHz, and 30 °C for 20 min to carry out coordination self-assembly reaction. Step 4: Centrifuge the reaction solution at 10000 r / min for 13 min, collect the precipitate, wash twice with deionized water, and freeze-dry to obtain Ca. 2+ -EGCG metal-phenolic network powder (MPNs-B).

[0029] Measurements showed that: (1) According to DLS measurements, the average hydrated particle size of MPNs-B is 148.5±5.2 nm, the PDI is 0.23, and the Zeta potential is -31.5±4.3 mV. MPNs-B has a uniform particle size distribution and good dispersion stability.

[0030] (2) FTIR spectroscopy showed that, compared with pure EGCG, MPNs-B had a lower concentration in the range of 3350–3560 cm⁻¹. -1The stretching vibration peak of the phenolic hydroxyl group (OH) at that position was significantly broadened and redshifted by approximately 20 cm⁻¹. -1 At 1690 cm -1 The intensity of the C=O stretching vibration peak at the position was significantly reduced. This result is consistent with the FTIR spectrum characteristics of Example 1 (calcium lactate), indicating that calcium gluconate and calcium lactate can also form a stable metal-phenolic network structure with EGCG through coordination with phenolic hydroxyl groups.

[0031] Comparative Example 1 Ca 2+ The preparation method of the EGCG metal-phenolic network includes the following steps: Step 1: Dissolve epigallocatechin gallate in deionized water to prepare an EGCG solution with a concentration of 4 mmol / L, and protect it with nitrogen gas. Step 2: Dissolve calcium lactate in deionized water to prepare a calcium ion solution with a concentration of 4 mmol / L; Step 3: Under stirring conditions, the calcium ion solution is slowly added dropwise to the EGCG solution, and the mixture is stirred at 300 rpm for 20 min to carry out the coordination self-assembly reaction; Step 4: Centrifuge the reaction solution at 10000 r / min for 13 min, collect the precipitate, wash twice with deionized water, and freeze-dry to obtain Ca. 2+ -EGCG metal-phenolic network powder (MPNs-C).

[0032] Measurements showed that: (1) DLS measurements showed that the average hydrated particle size of MPNs-C was 286.5 ± 12.7 nm, the PDI was 0.38, and the Zeta potential was -18.6 ± 5.3 mV. Compared with Example 1, the particle size of MPNs-C was significantly increased, the particle size distribution was significantly widened (PDI > 0.3), and the absolute value of the Zeta potential decreased. This indicates that the particle size uniformity of MPNs-C without ultrasonic treatment was poor, and the electrostatic repulsion between particles was weakened, resulting in a significant decrease in dispersion stability.

[0033] (2) FTIR spectroscopy showed that, compared with pure EGCG, MPNs-C was significantly reduced in the range of 3350–3560 cm⁻¹. -1 The stretching vibration peak of the phenolic hydroxyl group (OH) at that position only broadened slightly, with a red shift of approximately 5–8 cm. -1 ; at 1690 cm -1 The intensity of the C=O stretching vibration peak at the point decreased to some extent, but the change was significantly smaller than in Example 1. This indicates that, without ultrasonic assistance, Ca... 2+ The coordination reaction with EGCG is incomplete, resulting in low coordination efficiency.

[0034] Example 3 Preparation of a purine-lowering, phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.8g MPNs-A, stir and disperse evenly to obtain the final product.

[0035] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0036] Example 4 Preparation of a purine-lowering, phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.4g MPNs-A, stir and disperse evenly to obtain the final product.

[0037] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0038] Example 5 Preparation of a purine-lowering, phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.2g MPNs-A, stir and disperse evenly to obtain the final product.

[0039] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0040] Example 6 Preparation of a purine-lowering, phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.4g MPNs-B, stir and disperse evenly to obtain the final product.

[0041] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0042] Example 7 Preparation of a purine-lowering, phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.4g MPNs-A, stir and disperse evenly to obtain the final product.

[0043] Applied to cod: Step 1: Take 1 kg of fresh cod meat and immerse it in the above water-retaining agent at a material-to-liquid ratio of 1:1.3, and soak it at 4℃ for 2 hours; Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0044] Comparative Example 2 Preparation of a composite phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve to obtain the product.

[0045] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0046] Comparative Example 3 Preparation of a composite phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.4g EGCG, stir and disperse evenly to obtain the product.

[0047] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0048] Comparative Example 4 Preparation of a composite phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.3g EGCG and 0.1g calcium lactate, stir and disperse evenly to obtain the final product.

[0049] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0050] Comparative Example 5 Preparation of a purine-lowering, phosphorus-free water-retaining agent: (1) Take 7 g of sorbitol, 5 g of trehalose, 13 g of sodium chloride, 7.5 g of sodium bicarbonate and 7.5 g of potassium bicarbonate, add 1 L of deionized water and dissolve; (2) Add 0.4g MPNs-C, stir and disperse evenly to obtain the final product.

[0051] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0052] Comparative Example 6 (Traditional Phosphorus-Containing Water Retention Agent) Water-retaining agent preparation: Formulate a traditional phosphorus-containing water-retaining agent: sodium tripolyphosphate 2.5 g / L, sodium pyrophosphate 2.5 g / L, sodium chloride 13 g / L.

[0053] Application in shrimp: Step 1: Take 1 kg of fresh whiteleg shrimp meat and soak it in the above water-retaining agent at a ratio of 1:1.3 (material to liquid) for 2 hours at 4°C. Step 2: After removal, place it in an ultrasonic processor and ultrasonically treat it for 25 minutes at 35℃, 300 W, and 40 kHz. Step 3: Remove and drain, then quick-freeze at -35℃ or below, and store at -18℃.

[0054] Performance testing: (1) Determination of water holding capacity: Shrimp samples treated with water-retaining agent and quick-frozen (frozen for 28 days) were thawed naturally at room temperature for 2 hours, and weighed (m1) after the surface moisture was absorbed with absorbent paper. The water holding capacity was calculated according to the following formula: Water holding capacity (%) = (m1 / m0) × 100% Where m0 is the initial mass of the shrimp. Each sample was measured in triplicate, and the average value was taken.

[0055] Table 1 Water Holding Rate

[0056]

[0057] As shown in Table 1, the water holding capacity of Comparative Example 2 was 85.6%, indicating that the phosphorus-free water-retaining functional component itself also has a good basic water-retaining effect. The water holding capacity of Comparative Example 3 was 86.2%, and that of Comparative Example 4 was 86.5%, both of which were not significantly different from Comparative Example 2 (85.6%) (p>0.05), indicating that free EGCG and its simple mixture with calcium lactate cannot form an effective covalent-coordination double crosslinked gel reinforcement network like intact MPNs. The water holding capacity of Comparative Example 5 was only 86.3%, significantly lower than that of Example 4 (88.6%) with the same amount of added EGCG (p<0.05), and comparable to that of Example 5 (86.8%) with only 0.2g of intact MPNs added, indicating that MPNs-C prepared without ultrasonic assistance, due to incomplete coordination and loose structure, cannot form an effective gel reinforcement network even with increased addition. The water holding capacity of Example 5 was 86.8%, comparable to Comparative Example 6 (87.2%), indicating that even at low addition levels, MPNs can compensate for the water retention effect of phosphorus-containing water-retaining agents. The water holding capacity of Example 4 was 88.6%, significantly better than Comparative Example 6 (87.2%) (p<0.05); the water holding capacity of Example 3 reached 91.2%, significantly better than the traditional phosphorus-containing water-retaining agent Comparative Example 6 (87.2%), achieving phosphorus-free substitution and water retention performance reaching or even exceeding the level of traditional phosphorus-containing water-retaining agents. From Example 5 to Example 4 to Example 3, it can be seen that when the MPN addition amount increases from 0.2 g / L to 0.8 g / L, the water holding capacity increases significantly (86.8%→88.6%→91.2%), indicating that the water retention enhancement effect of MPNs has obvious concentration dependence. The water holding capacity of Example 6 (88.4%) was not significantly different from that of Example 4, proving that MPNs constructed from calcium gluconate can also effectively perform water retention functions. The water holding capacity of Example 7 was 87.9%, which was slightly lower than that of the shrimp system, but still significantly better than that of Comparative Example 2 and Comparative Example 6.

[0058] (2) XOD activity determination (spectrophotometry) XOD activity assays typically use xanthine as a substrate to detect the amount of uric acid produced, and are performed using ultraviolet spectrophotometry (295 nm).

[0059] Sample pretreatment: Take 2.00 g of shrimp meat, add 8 mL of 0.1 mol / L PBS (pH 7.4), and extract by sonication at 25℃ and 300 W for 25 min with stirring during extraction. Centrifuge at 8000 r / min and 4℃ for 15 min, and use the supernatant as the test solution.

[0060] Reaction system (96-well plate, total volume 250 μL): Sample group: 50 μL test solution + 50 μL XOD (0.03 U / mL) + 150 μL xanthine (0.46 mmol / L) Blank control group: 50 μL test solution + 50 μL PBS + 150 μL xanthine Negative control group: 50 μL PBS + 50 μL XOD + 150 μL xanthine Negative control group: 50 μL PBS + 50 μL PBS + 150 μL xanthine Reaction conditions: After incubation at 37℃ for 20 min, absorbance at 295 nm was measured using an ELISA reader.

[0061] Inhibition rate calculation: .

[0062] (3) Determination of uric acid content Use a commercially available uric acid assay kit (uric acid enzyme method) and follow the instructions: 1. Add the following to a 96-well plate in sequence: 10 μL of the test solution + 100 μL of reagent one + 80 μL of reagent two. 2. Incubate at 37℃ in the dark for 5 min, and read the absorbance value A1 at 520 nm. 3. Add 10 μL of reagent three, mix well, and react at 37°C in the dark for 10 min. Read the absorbance value at 520 nm (A2). 4. ΔA = A2 - A1 ; Where D is the dilution factor.

[0063] (4) Determination of xanthine content (HPLC method) Sample preparation: Purine compounds in shrimp were treated with a mixed acid of CF3COOH / HCOOH / H2O, shaken in a 90℃ water bath for 12 min, and then evaporated to dryness in a 65℃ water bath by rotary evaporation.

[0064] Resolution and filtration: The residue was resolution with KH2PO4-H3PO4 mobile phase and filtered through a 0.22 μm filter membrane.

[0065] Chromatographic conditions: Column: Waters Atlantis T3 (250 mm × 4.6 mm, 5 μm) Mobile phase: 0.01 mol / L KH2PO4-H3PO4 (pH 3.70) Flow rate: 1.0 mL / min Detection wavelength: 254 nm Column temperature: 25℃ Injection volume: 10 μL Quantitative analysis: External standard method; a standard curve was plotted based on xanthine standards (good linearity in the range of 0.5–250.0 mg / L, r...). 2 ≈1.0000).

[0066] Method validation: The limit of detection of this method is 0.042–0.096 mg / L, the limit of quantitation is 0.139–0.320 mg / L, the recovery rate is 97.83%–102.21%, and the precision RSD is between 0.04% and 0.30%.

[0067] Table 2 Functional Indicators

[0068] Note: Uric acid content refers to the concentration in the extract (supernatant after extraction with 2 g shrimp meat + 8 mL PBS), used to reflect the relative differences in XOD inhibition effects among different treatment groups.

[0069] As shown in Table 2, the XOD inhibition rates of all embodiments of the present invention were significantly higher than those of the comparative examples (p<0.05). Example 3 showed an XOD inhibition rate of 74.7%, with the uric acid content in the extract decreasing to 0.71 μmol / mL, a reduction of approximately 27.6% compared to Comparative Example 2; the xanthine content was 25.1 mg / kg, an increase of approximately 52.1% compared to Comparative Example 2 (16.5 mg / kg). The increase in xanthine content is due to the inhibition of xanthine metabolism after XOD activity was suppressed. This result is consistent with the increasing trend of the XOD inhibition rate, demonstrating the effective inhibition of endogenous XOD activity in shrimp by the present invention. Examples 4-5 show that when the amount of MPNs added decreased from 0.8 g / L to 0.4 g / L and 0.2 g / L, the XOD inhibition rate decreased sequentially (74.7%→62.3%→48.6%), the uric acid content increased sequentially (0.71→0.82→0.89 μmol / mL), and the xanthine content decreased sequentially (25.1→21.6→18.7 mg / kg, all significantly higher than the 16.5 mg / kg in Comparative Example 2). This is because the stronger the XOD activity, the more the substrate xanthine is metabolized and consumed. Therefore, the xanthine residue and the XOD inhibition rate show an inverse relationship, indicating that the XOD inhibition effect is positively correlated with the amount of MPNs added, and the trend of metabolite changes is highly consistent with the enzyme activity inhibition. The XOD inhibition rate of Example 6 (60.8%) was not significantly different from that of Example 4, indicating that MPNs constructed from different calcium sources can effectively inhibit XOD activity. The XOD inhibition rate of Example 7 was 58.2%, slightly lower than that of the shrimp system, but still significantly better than the comparative examples, indicating that the water-retaining agent has good XOD inhibition activity in different aquatic product matrices. Comparative Example 2 had an XOD inhibition rate of only 2.4%, with the highest uric acid content (0.98 μmol / mL) and the lowest xanthine content (16.5 mg / kg). The XOD inhibition rates of Comparative Example 3 were 38.8%, Comparative Example 4 was 32.4%, and Comparative Example 5 was 22.5%, all three significantly lower than that of Example 4 (62.3%) with the same amount of additive (p<0.05). Although Comparative Examples 3-5 showed some XOD inhibitory activity, it was significantly lower than that of Example 4 (62.3%) (p<0.05). This indicates that although free EGCG retained some XOD inhibitory activity (38.8% in Comparative Example 3), the intact MPNs structure could more effectively protect the inhibitory activity of EGCG and promote its covalent grafting with shrimp meat protein. Furthermore, ungrafted EGCG was largely lost during sample pretreatment. MPNs-C without ultrasound-assisted coordination also failed to effectively protect EGCG. In contrast, the intact MPNs structure (MPNs-A / MPNs-B) formed by ultrasound-assisted coordination self-assembly in this invention effectively maintained the structural integrity of EGCG and drove its covalent grafting with shrimp meat protein, thus better preserving the XOD inhibitory activity.The XOD inhibition rate of Comparative Example 6 was only 3.1%, which was not significantly different from that of Comparative Example 2 (2.4%). This shows that traditional phosphorus-containing water-retaining agents do not have XOD inhibition function.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A purine-lowering, phosphorus-free water-retaining agent, characterized in that, The water-retaining agent contains Ca at a mass ratio of 1:50 to 1:

200. 2+ -EGCG metal-phenolic network and water-retaining functional components; The Ca 2+ -EGCG metal-phenolic network is formed by the ultrasound-assisted coordination self-assembly of calcium ions and epigallocatechin gallate ester; The water-retaining functional components include sorbitol, trehalose, sodium chloride, sodium bicarbonate, and potassium bicarbonate.

2. The composite phosphorus-free water-retaining agent according to claim 1, characterized in that, The Ca 2+ The preparation method of the EGCG metal-phenolic network includes the following steps: Step 1: Dissolve epigallocatechin gallate in deionized water to prepare an EGCG solution with a concentration of 2-5 mmol / L, and protect it with nitrogen gas. Step 2: Dissolve food-grade calcium salt in deionized water to prepare a calcium ion solution with an equimolar ratio to EGCG; Step 3: Under stirring conditions, the calcium ion solution is slowly added dropwise to the EGCG solution, and the coordination self-assembly reaction is carried out under ultrasonic treatment; Step 4: Centrifuge the reaction solution, collect the precipitate, wash with deionized water, and freeze-dry to obtain Ca. 2+ -EGCG metal-phenolic network powder.

3. The composite phosphorus-free water-retaining agent according to claim 2, characterized in that, In step three, the volume ratio of the calcium ion solution to the EGCG solution is 1:1 to 1:2; the ultrasonic treatment parameters are: power 100 to 300 W, frequency 20 to 40 kHz, temperature 25 to 35℃, and time 10 to 30 min.

4. The composite phosphorus-free water-retaining agent according to claim 2, characterized in that, In step four, the centrifugation speed is 8000–12000 r / min, the centrifugation time is 10–15 min, and the number of washing cycles is 2–3.

5. The composite phosphorus-free water-retaining agent according to claim 1, characterized in that, The water-retaining functional components contain sorbitol at a concentration of 7 g / L, trehalose at a concentration of 5 g / L, and sodium chloride at a concentration of 13 g / L. The total concentration of sodium bicarbonate and potassium bicarbonate is 8–15 g / L, and the mass ratio of sodium bicarbonate to potassium bicarbonate is 1:1–2:

1.

6. The application of the composite phosphate-free water-retaining agent according to any one of claims 1-5 in aquatic products.

7. The application according to claim 6, characterized in that, Includes the following steps: Step 1: Immerse the aquatic products in the composite phosphate-free water-retaining agent at 0-4°C; Step 2: Place the soaked aquatic products in an ultrasonic processor for ultrasonic treatment. Step 3: Remove and drain, then quick-freeze or freeze.

8. The application according to claim 7, characterized in that, In step one, the ratio of aquatic products to compound phosphate-free water-retaining agent is 1:1.2 to 1:1.5; the soaking time is 1 to 3 hours.

9. The application according to claim 7, characterized in that, The ultrasonic treatment parameters in step two are: power 100-500 W, frequency 20-50 kHz, temperature 30-35℃, and time 10-60 min.

10. The application according to claim 7, characterized in that, The quick-freezing temperature in step three is below -35℃, and the frozen storage temperature is -18℃.

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