A product, method and use thereof for retarding the hardening of the carapace of soft-shell crabs

Abstract

This invention belongs to the field of aquaculture technology, specifically relating to a product, method, and application for delaying the hardening of soft-shell crab shells. This invention provides a product for delaying the hardening of soft-shell crab shells, formulated from linseed gum, dipotassium glycyrrhizate, inositol hexaphosphate, EGCG, and an emulsifier, and develops a method for delaying the hardening of soft-shell crab shells based on this product. First, the molting soft-shell crab is placed in an environment of 0–4℃ for 3–5 minutes to shrink the micropores of its shell. Then, it is placed on a sterile substrate with a moisture content of 60–70%, and a film-forming blocking agent formulated from linseed gum, dipotassium glycyrrhizate, inositol hexaphosphate, EGCG, and polyglycerol fatty acid esters is evenly sprayed on. After drying for 5–8 minutes, an edible film forms on the body surface, blocking calcium ion penetration. This method, with its synergistic effect of low temperature and film formation, delays the hardening of the soft-shell crab shell for 72–96 hours, with a survival rate ≥95%. The method is simple to operate, suitable for conventional aquaculture environments, and can significantly improve the quality and economic benefits of soft-shell crabs.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a product, method, and application for delaying the hardening of the shell of soft-shell crabs. Background Technology

[0002] Crabs undergo periodic molting during their growth. Newly molted soft-shell crabs are highly valued for their soft shells, tender texture, and white appearance, making them particularly popular in the high-end catering market. However, under natural conditions, the shells of soft-shell crabs harden relatively quickly, typically completing the mineralization process within 4 to 8 hours after molting. This results in a very limited market sales window, severely restricting their commercial potential and the enhancement of their added value.

[0003] Currently, research on technologies to delay shell hardening in soft-shell crabs is scarce, and there are no systematic solutions that effectively intervene by regulating chitin synthesis, mineral transport, and competitive absorption of minerals in water. Traditional methods mainly rely on the regulation of environmental factors, such as controlling physical conditions like water temperature, salinity, and light intensity. However, these methods have limited effects, unclear mechanisms of action, and are greatly affected by environmental fluctuations, making it difficult to achieve stable and controllable application results.

[0004] Therefore, there is an urgent need to develop a new, safe, efficient, environmentally friendly, and highly operable method to synergistically intervene in the hardening process of soft-shell crabs from both physiological metabolism and external environmental perspectives, thereby prolonging the duration of their soft-shell state. Summary of the Invention

[0005] Based on the principle of biomineralization regulation, this invention proposes for the first time an intervention strategy that combines natural plant colloids, metal ion chelators, and antioxidant active ingredients to act on the surface of soft-shell crab carapace. By blocking the transport pathways of chitin and calcium and magnesium ions in the body and water, the mineralization process of the carapace is effectively inhibited.

[0006] The first aspect of the present invention is to provide a composition for delaying the hardening of the shell of soft-shell crabs.

[0007] A second aspect of the present invention is to provide a film-forming inhibitor for soft-shell crabs.

[0008] The third objective of this invention is to provide a method for preparing the film-forming blocking agent of the second aspect of this invention.

[0009] The fourth aspect of this invention aims to provide the application of the composition of the first aspect of this invention and the film-forming blocking agent of the second aspect of this invention.

[0010] The fifth aspect of this invention aims to provide a method for delaying the hardening of the shell of soft-shell crabs.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0012] In a first aspect, the present invention provides a composition for delaying the hardening of the shell of soft-shell crabs, comprising natural plant colloids, metal ion chelating agents, antioxidant active ingredients, antibacterial stabilizers, and emulsifiers.

[0013] In some embodiments of the present invention, the natural plant colloid includes flaxseed gum.

[0014] In some embodiments of the present invention, the metal ion chelating agent includes inositol hexaphosphate.

[0015] In some embodiments of the present invention, the antioxidant active ingredient includes epigallocatechin gallate.

[0016] In some embodiments of the present invention, the antibacterial stabilizer includes dipotassium glycyrrhizate.

[0017] In some embodiments of the present invention, the emulsifier includes polyglycerol fatty acid ester (PGFE), polyglycerol-2 dihydroxystearate (PGPH), polyethylene glycol (PEG), polyglycerol-10 laurate (PG10L), alkyl glycoside (APG), sodium stearoyl glutamate, and lecithin.

[0018] In some embodiments of the present invention, the composition comprises, by weight:

[0019] Flaxseed gum 6-8 parts, dipotassium glycyrrhizate 1-1.5 parts, inositol hexaphosphate 3-4 parts, epigallocatechin gallate (EGCG) 0.8-1.2 parts, emulsifier 2.5-3 parts.

[0020] In some embodiments of the present invention, the viscosity of the flaxseed gum is 10,000 to 14,000 mPa·s, with the viscosity of a 1 wt% flaxseed gum aqueous solution at 25°C as the standard.

[0021] In some embodiments of the present invention, the galactose to xylose molar ratio of the flaxseed gum is ≥2:1.

[0022] In some embodiments of the present invention, when flaxseed gum has high viscosity and a galactose to xylose molar ratio ≥2:1, the intermolecular forces are strong, easily forming a tight network structure, giving the film-forming inhibitor film good elasticity and toughness. Its molecular chain arrangement and ratio affect the spatial structure and interactions; high viscosity increases the frictional force between molecular chains, improving film strength and stability. This makes the protective film robust and durable, effectively blocking external substances and delaying the hardening of soft-shell crabs. However, this does not mean that flaxseed gum with galactose to xylose molar ratios in other ranges cannot achieve the technical effects of the present invention. In some embodiments of the present invention, flaxseed gum has good film-forming properties, forming a physical barrier on the surface of soft-shell crabs. Dipotassium glycyrrhizate has certain antibacterial and membrane-stabilizing effects, preventing the membrane from being damaged by microorganisms. Inositol hexaphosphate can bind with metal ions such as calcium ions, reducing the concentration of calcium ions in the water that can be used for shell hardening. EGCG has antioxidant properties, protecting the soft-shell crab surface cells from oxidative damage and maintaining normal cellular physiological functions. Emulsifiers help to mix the components evenly, making the film formation more uniform.

[0023] A second aspect of the present invention provides a crab film-forming inhibitor, comprising: the composition of the first aspect of the present invention and a buffer solution.

[0024] In some embodiments of the present invention, the buffer solution comprises a phosphate buffer solution.

[0025] In some embodiments of the present invention, the pH of the buffer solution is 7.0 to 7.5.

[0026] In some embodiments of the present invention, the osmotic pressure of the buffer solution is adjusted to 300±20 mOsm / kg.

[0027] In some embodiments of the present invention, phosphate buffer provides a suitable pH and osmotic pressure environment for the entire system, ensuring the stability and biocompatibility of the film-forming inhibitor. When the film-forming inhibitor is sprayed or applied to the surface of the soft-shell crab, flaxseed gum molecules intertwine to form a network structure, encapsulating other components and forming a membrane with a certain degree of elasticity and toughness. Inositol hexaphosphate combines with calcium ions in water to form a complex, reducing the concentration of free calcium ions and decreasing the formation of calcium carbonate in the shell. EGCG protects the cell membranes and intracellular biomolecules on the surface of the soft-shell crab by scavenging free radicals, maintaining normal cellular metabolism. After surface blocking treatment, the protective film on the surface of the soft-shell crab effectively blocks external substances, further slowing down the hardening of the shell, prolonging the soft-shell state, and improving the quality and usability of the soft-shell crab.

[0028] In some embodiments of the present invention, the film-forming blocking agent comprises, by mass fraction:

[0029] Flaxseed gum 0.6-0.8 wt%, dipotassium glycyrrhizate 0.1-0.15 wt%, inositol hexaphosphate 0.3-0.4 wt%, epigallocatechin gallate 0.08-0.12 wt%, emulsifier 0.25-0.3 wt%, PBS buffer solution balance.

[0030] A third aspect of the present invention provides a method for preparing the soft-shell crab film-forming inhibitor described in the second aspect of the present invention, comprising the following steps:

[0031] 1) Add natural plant colloids to a buffer solution and stir to obtain a colloidal solution;

[0032] 2) Add metal ion chelating agents, antioxidant active ingredients, antibacterial stabilizers and emulsifiers to the colloidal solution, mix, and form an emulsion;

[0033] 3) Add buffer solution to a final volume of 100 wt%.

[0034] In some embodiments of the present invention, the stirring temperature in step 1) is 48~52°C.

[0035] In some embodiments of the present invention, the mixing temperature in step 2) is 38~40°C.

[0036] In some embodiments of the present invention, flaxseed gum is readily soluble in phosphate buffer at 50±2℃, and high-speed stirring accelerates the dissolution. The remaining components are added after cooling to 38~40℃, a temperature that ensures component stability while facilitating thorough dispersion. At this temperature, the emulsifier exhibits good emulsifying properties, allowing the oil phase and water to mix and preventing stratification. Throughout the stirring process, flaxseed gum molecules gradually and uniformly disperse, forming a stable mixture with the other components. The resulting film-forming inhibitor has uniform composition and excellent film-forming properties, forming a uniform and continuous protective film on the surface of soft-shell crabs, effectively delaying shell hardening.

[0037] A fourth aspect of the present invention provides the use of the composition of the first aspect of the present invention and the soft-shell crab film-forming inhibitor of the second aspect of the present invention in delaying the hardening of the soft-shell crab carapace.

[0038] A fifth aspect of the present invention provides a method for delaying hardening in soft-shell crabs, comprising the following steps:

[0039] 1) Pre-cooling treatment of soft-shell crabs;

[0040] 2) Blocking of film formation on the body surface: Treat soft-shell crabs with the film-forming blocking agent described in the second aspect of the present invention.

[0041] In some embodiments of the present invention, the steps further include:

[0042] 3) Temporary holding management: The soft-shell crabs treated in step (2) are transferred to a temporary holding tank with dissolved oxygen ≥6 mg / L, and the pH of the water is corrected to 7.8–8.2 and ammonia nitrogen ≤0.1 mg / L every 2 hours by online monitoring.

[0043] Because crabs are relatively weak after molting, prolonged exposure to dryness can cause physiological damage and lead to death. Therefore, applying a film-forming inhibitor and then placing soft-shell crabs in a temporary holding tank can improve their survival rate.

[0044] In some embodiments of the present invention, the soft-shell crabs undergoing the pre-cooling treatment in step 1) are soft-shell crabs that have molted within 1 hour.

[0045] In some embodiments of the present invention, the temperature of the pre-cooling treatment is 0~4°C.

[0046] In some embodiments of the present invention, the pre-cooling time is 3 to 5 minutes.

[0047] In some embodiments of the present invention, the temperature of the crab body after pre-cooling treatment is 5~8℃.

[0048] In some embodiments of the present invention, the pre-cooling treatment is carried out in a refrigerator or cold storage. By precisely controlling the temperature at 0-4°C using a refrigeration system, the soft-shell crab is placed in a low-temperature environment, allowing heat to dissipate to the surroundings and lowering the surface temperature to 5-8°C. At this temperature, the rate of physiological and biochemical reactions within the crab decreases, the movement of biomolecules slows down, and enzyme activity is inhibited, thereby affecting the physiological processes related to shell hardening and effectively slowing down the hardening rate of the soft-shell crab.

[0049] In some embodiments of the present invention, the body surface film-forming barrier is treated on a wet substrate.

[0050] In some embodiments of the present invention, the moisture content of the wetted substrate is 60-70%.

[0051] In some embodiments of the present invention, the substrate includes nonwoven fabric and sponge.

[0052] In some embodiments of the present invention, a nonwoven fabric or sponge with a moisture content of 60-70% is used as a moistening substrate. This material is water-retaining and breathable, maintaining the surface humidity of the soft-shell crab and facilitating its gas exchange. When placed on the substrate, the moisture evaporates slowly, keeping the surface moist and allowing the film-forming inhibitor to diffuse and penetrate, forming a uniform protective film. This improves the treatment effect and delays the hardening of the soft-shell crab's shell.

[0053] In some embodiments of the present invention, after spraying the film-forming inhibitor onto the crab body, it is left to dry (exposed to air for 3 to 10 minutes, preferably 5 to 8 minutes) to allow the film to form.

[0054] In some embodiments of the present invention, during the temporary holding period in step (3), the pH is monitored and maintained at 7.8-8.2 every 2 hours (adjuster: sodium bicarbonate). Soft-shell crabs are sensitive to the pH value of the water. A suitable pH range helps maintain the acid-base balance in the soft-shell crabs and ensure their normal physiological functions. In a weakly alkaline environment with a pH of 7.8-8.2, the physiological processes of soft-shell crabs such as respiration and digestion can proceed normally. At the same time, it is also beneficial to inhibit the growth and reproduction of harmful microorganisms, reduce the occurrence of diseases, and improve the survival rate and quality of soft-shell crabs. The ammonia nitrogen concentration is ≤0.1 mg / L (detection method: Nessler's reagent spectrophotometric method HJ 535-2009). Ammonia nitrogen is one of the common pollutants in water bodies, and it has strong toxicity to soft-shell crabs. When the ammonia nitrogen concentration is too high, it will affect the respiratory function of soft-shell crabs, leading to metabolic disorders in their bodies, and even causing poisoning and death. Maintaining an ammonia nitrogen concentration of ≤0.1 mg / L can provide a clean and safe living environment for soft-shell crabs, reduce the harm of ammonia nitrogen to soft-shell crabs, and ensure their healthy growth.

[0055] In some embodiments of the present invention, the temporary holding pond (a rearing pond made of polypropylene material, with a volume of 4m×5m×1.2m, equipped with a temperature control system of 16-25℃ and dissolved oxygen ≥6 mg / L) is used. Polypropylene material has advantages such as corrosion resistance, high strength, and non-toxicity, making it suitable for the construction of rearing ponds. The temperature control system can regulate the water temperature through heating or cooling equipment, and the dissolved oxygen control system can increase the dissolved oxygen content in the water through aeration equipment. The temporary holding pond for soft-shell crabs provides a suitable living space for them. The water temperature of 16-25℃ can meet the growth needs of soft-shell crabs and promote their metabolism; dissolved oxygen ≥6 mg / L can ensure the respiratory needs of soft-shell crabs and maintain their normal physiological functions. During the temporary holding period, by monitoring and controlling water parameters, such as pH value and ammonia nitrogen concentration, a good living environment can be created for soft-shell crabs, improving their survival rate and quality.

[0056] In the above aspects of the present invention, soft-shell crab is a morphological state of crab, and the crab species include, but are not limited to: mud crab, swimming crab, and Chinese mitten crab.

[0057] The beneficial effects of this invention are:

[0058] 1. Significantly prolongs the soft-shell state: Through pre-cooling treatment to reduce the metabolic rate, the use of film-forming inhibitors on the body surface to prevent water loss and the invasion of external substances, and precise temporary holding management, the soft-shell state of crabs can be extended to 72-96 hours, which is significantly higher than the 12-24 hours of existing technologies.

[0059] 2. Improved survival rate: Mild treatment conditions reduce stress on crabs, and the survival rate of soft-shell crabs can reach over 95% within 48 hours after treatment.

[0060] 3. Safe and environmentally friendly: The film-forming blocking agents used are natural or food-grade substances that meet food safety requirements and have no risk of chemical residues.

[0061] 4. Easy to operate: Each step is easy to implement in a farm or processing plant, without the need for complicated equipment and technology.

[0062] 5. Low cost: The raw materials used, such as flaxseed gum, are low-cost and require small quantities, which is conducive to large-scale promotion and application. Attached Figure Description

[0063] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0064] Figure 1 The results show the comparison of water vapor transmission rate (WVTR), film thickness, curing time, and 96-hour survival rate of natural plant colloids with different viscosities. A represents the water vapor transmission rate (WVTR) result; B represents the film thickness result; C represents the curing time result; and D represents the 96-hour survival rate result.

[0065] Figure 2 This section compares the water vapor transmission rate (WVTR), film thickness, curing time, and 96-hour survival rate of different colloid types. A represents the water vapor transmission rate (WVTR) result; B represents the film thickness result; C represents the curing time result; and D represents the 96-hour survival rate result.

[0066] Figure 3 The results show the comparison of different chelating substances in terms of calcium ion chelating ability, hardening time, and 96-hour survival rate. A represents the calcium ion chelating ability; B represents the hardening time; and C represents the 96-hour survival rate.

[0067] Figure 4 The results show the comparison of different types of antioxidants in terms of total antioxidant capacity (TEAC), curing time, and 96-hour survival rate. A represents the total antioxidant capacity (TEAC) result; B represents the curing time result; and C represents the 96-hour survival rate result. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Terminology Explanation:

[0069] Soft-shell crab: A crab that has just molted and whose shell has not yet hardened and remains soft (refer to Zhejiang Provincial Local Standard DB33 / T2312-2012). In this invention, a crab is defined as a soft-shell crab when the hardness of the central area of ​​its carapace is below 20HA. 20HA is the critical value for an edible soft shell, and the stable hardness of the central area of ​​the carapace after natural hardening is approximately 35HA.

[0070] Experimental materials:

[0071] Healthy soft-shelled mud crabs, weighing 120±20g, were selected from indoor factory-style recirculating aquaculture boxes within 10-30 minutes after molting, with no broken limbs or injuries.

[0072] Flaxseed gum (viscosity 12000 mPa·s, 1% aqueous solution at 25℃, galactose to xylose molar ratio 2.3:1, BASF (China) Co., Ltd.), dipotassium glycyrrhizate (food grade, BASF (China) Co., Ltd.), inositol hexaphosphate (purity ≥98%, BASF (China) Co., Ltd.), EGCG (purity ≥95%, BASF (China) Co., Ltd.), polyglycerol fatty acid ester (HLB=12.2, BASF (China) Co., Ltd.), phosphate buffer (0.1M Na2HPO4 / NaH2PO4, pH 7.2±0.1, osmotic pressure 305 mOsm / kg, Beijing Solarbio Science & Technology Co., Ltd.).

[0073] Substrate: Non-woven fabric (sterilized by high-pressure steam at 121℃ for 20min, and the moisture content was adjusted to 65±2%).

[0074] Instruments and equipment:

[0075] Temperature control and precooling device: laboratory refrigerator (temperature control accuracy ±0.5℃, Beijing Fuyi Electric Appliance Co., Ltd.); Body surface treatment platform: ultra-clean workbench (equipped with thermometer and hygrometer, Qingdao Haier Biomedical Co., Ltd.); Temporary holding tank: polypropylene material (4m×5m×1.2m), equipped with circulating water constant temperature system (temperature control accuracy ±0.5℃), microporous aerator (dissolved oxygen monitor real-time display), pH online monitor (accuracy ±0.1), ammonia nitrogen test kit (compliant with HJ 535-2009 standard); Auxiliary instruments: magnetic stirrer (adjustable speed, Shanghai Sile Instrument Co., Ltd.), handheld sprayer (capacity 500mL, nozzle orifice diameter 0.5mm, Zhejiang Wenxin Electromechanical Manufacturing Co., Ltd.), LD-A digital display Shore C hardness tester (accuracy ±1HA, Dongguan Zhongte Precision Instrument Technology Co., Ltd.), electronic thermometer (accuracy ±0.1℃, Shenzhen Delixi Group Co., Ltd.).

[0076] Example 1

[0077] A composition for delaying the hardening of the soft-shell crab's carapace, comprising the following components:

[0078] 7 parts of flaxseed gum (viscosity of 12,000 mPa·s based on 1wt% aqueous solution at 25℃, galactose:xylose = 2.3:1), 1.2 parts of dipotassium glycyrrhizate, 3.5 parts of inositol hexaphosphate, 1 part of epigallocatechin gallate (EGCG), and 2.8 parts of polyglycerol fatty acid ester.

[0079] Preparation of film-forming inhibitors:

[0080] 1) Measure 800 mL of phosphate buffer, heat to 50±2℃, add 7.0 g of flaxseed gum (0.7 wt%), place on a magnetic stirrer and stir at 500±50 rpm for 10 min until the gum is completely swollen and becomes a transparent colloid.

[0081] 2) Cool the colloid to 39±1℃, and add 1.2g dipotassium glycyrrhizate (0.12wt%), 3.5g inositol hexaphosphate (0.35wt%), 1.0g EGCG (0.10wt%), and 2.8g polyglycerol fatty acid ester (0.28wt%) in sequence. Adjust the stirring speed to 400±50rpm and continue stirring for 15min to form a uniform emulsion.

[0082] 3) Add phosphate buffer to a total weight of 1000g, stir for 5 minutes, and then dispense into sprayers.

[0083] Example 2

[0084] A composition for delaying the hardening of the soft-shell crab's carapace, comprising the following components:

[0085] Flaxseed gum 8 parts (viscosity 12,000 mPa·s, galactose:xylose = 2.3:1), dipotassium glycyrrhizate 1.5 parts, inositol hexaphosphate 4 parts, epigallocatechin gallate (EGCG) 1.2 parts, polyglycerol fatty acid ester 3 parts.

[0086] Preparation of film-forming inhibitors:

[0087] 1) Measure 800 mL of phosphate buffer, heat to 50±2℃, add 8.0 g of flaxseed gum (0.8 wt%), place on a magnetic stirrer and stir at 500±50 rpm for 10 min until the gum is completely swollen and becomes a transparent colloid.

[0088] 2) Cool the colloid to 39±1℃, and add 1.5g dipotassium glycyrrhizate (0.15wt%), 4g inositol hexaphosphate (0.4wt%), 1.2g EGCG (0.12wt%), and 3g polyglycerol fatty acid ester (0.3wt%) in sequence. Adjust the stirring speed to 400±50rpm and continue stirring for 15min to form a uniform emulsion.

[0089] 3) Add phosphate buffer to a total weight of 1000g, stir for 5 minutes, and then dispense into sprayers.

[0090] Example 3

[0091] A composition for delaying the hardening of the soft-shell crab's carapace, comprising the following components:

[0092] Flaxseed gum 6 parts (viscosity 12,000 mPa·s, galactose:xylose = 2.3:1), dipotassium glycyrrhizate 1.0 part, inositol hexaphosphate 3 parts, epigallocatechin gallate (EGCG) 0.8 parts, polyglycerol fatty acid ester 2.5 parts.

[0093] Preparation of film-forming inhibitors:

[0094] 1) Measure 800 mL of phosphate buffer, heat to 50±2℃, add 6.0 g of flaxseed gum (0.6 wt%), place on a magnetic stirrer and stir at 500±50 rpm for 10 min until the gum is completely swollen and becomes a transparent colloid.

[0095] 2) Cool the colloid to 39±1℃, and add 1.0g dipotassium glycyrrhizate (0.1wt%), 3g inositol hexaphosphate (0.3wt%), 0.8g EGCG (0.8wt%), and 2.5g polyglycerol fatty acid ester (0.25wt%) in sequence. Adjust the stirring speed to 400±50rpm and continue stirring for 15min to form a uniform emulsion.

[0096] 3) Add phosphate buffer to a total weight of 1000g, stir for 5 minutes, and then dispense into sprayers.

[0097] Example 4

[0098] A film-forming inhibitor that delays the hardening of the soft-shell crab's carapace, differing from Example 1 in that the emulsifier is replaced with PEG (BASF (China) Co., Ltd.).

[0099] Example 5

[0100] A film-forming inhibitor that delays the hardening of the soft-shell crab's shell, differing from Example 1 in that the emulsifier is replaced with PGPH (New Japan Rika Co., Ltd.).

[0101] Example 6

[0102] A film-forming inhibitor that delays the hardening of the soft-shell crab's shell, differing from Example 1 in that the emulsifier is replaced with PG10L (Kao Corporation).

[0103] Comparative Example 1

[0104] Similar to Example 1, except that the viscosity of the flaxseed gum was adjusted to 8000 mPa·s, calculated as a 1% aqueous solution at 25°C.

[0105] Comparative Example 2

[0106] Similar to Example 1, except that the viscosity of the flaxseed gum was adjusted to 15000 mPa·s, calculated as a 1% aqueous solution at 25°C.

[0107] Comparative Example 3

[0108] Similar to Example 1, except that flaxseed gum was replaced with guar gum (Jiangsu Jiujia Biotechnology Co., Ltd.) of the same viscosity.

[0109] Comparative Example 4

[0110] Similar to Example 1, except that the flaxseed gum was replaced with xanthan gum of the same viscosity (DEOSENBiochemical (Ordos) Ltd.).

[0111] Comparative Example 5

[0112] Similar to Example 1, except that flaxseed gum was replaced with carrageenan (Hunan Ercon Pharmaceutical Co., Ltd.) of the same viscosity.

[0113] Comparative Example 6

[0114] Similar to Example 1, except that flaxseed gum was replaced with locust bean gum (Jiangsu Jiujia Biotechnology Co., Ltd.) of the same viscosity.

[0115] Comparative Example 7

[0116] Similar to Example 1, except that dipotassium glycyrrhizate is replaced with sodium benzoate (Nanjing Songguan Biotechnology Co., Ltd.).

[0117] Comparative Example 8

[0118] Similar to Example 1, except that dipotassium glycyrrhizate is replaced with potassium sorbate (Jiangsu Jiujia Biotechnology Co., Ltd.).

[0119] Comparative Example 9

[0120] Similar to Example 1, except that dipotassium glycyrrhizate is replaced with sodium dehydroacetate (Zhengzhou Yuhe Food Additives Co., Ltd.).

[0121] Comparative Example 10

[0122] Similar to Example 1, except that dipotassium glycyrrhizate is replaced with methylparaben (Nanjing Songguan Biotechnology Co., Ltd.).

[0123] Comparative Example 11

[0124] Similar to Example 1, except that inositol hexaphosphate is replaced with ethylenediaminetetraacetic acid (EDTA) (Shandong Qiangsen Chemical Co., Ltd.).

[0125] Comparative Example 12

[0126] Similar to Example 1, except that inositol hexaphosphate was replaced with citric acid (Shandong Lemon Biochemical Co., Ltd.).

[0127] Comparative Example 13

[0128] Similar to Example 1, except that inositol hexaphosphate is replaced with tartaric acid (BASF (China) Co., Ltd.).

[0129] Comparative Example 14

[0130] Similar to Example 1, except that inositol hexaphosphate is replaced with sodium tripolyphosphate (BASF (China) Co., Ltd.).

[0131] Comparative Example 15

[0132] Similar to Example 1, except that EGCG is replaced with vitamin C (ascorbic acid, BASF (China) Co., Ltd.).

[0133] Comparative Example 16

[0134] Similar to Example 1, except that EGCG is replaced with vitamin E (α-tocopherol, BASF (China) Co., Ltd.).

[0135] Comparative Example 17

[0136] Similar to Example 1, except that EGCG was replaced with tea polyphenols (a mixture other than EGCG). This material was customized by Xi'an Zhongyan Kangze Biotechnology Co., Ltd., and EGCG was efficiently separated from other catechin monomers by high-speed countercurrent chromatography (HSCCC) to obtain a mixture without EGCG.

[0137] Comparative Example 18

[0138] Similar to Example 1, except that EGCG is replaced with resveratrol (BASF (China) Co., Ltd.).

[0139] Example 1: Effects of different treatments on delaying hardening in soft-shell crabs

[0140] 1. Experimental Grouping

[0141] 1) Low-temperature precooling + film-forming blocking (film-forming blocking agent prepared in Example 1)

[0142] 2) Low-temperature precooling + film-forming blocking (film-forming blocking agent prepared in Example 2)

[0143] 3) Low-temperature precooling + film-forming blocking (film-forming blocking agent prepared in Example 3)

[0144] 4) Separate pre-cooling treatment;

[0145] 5) Standalone film-forming blocking;

[0146] 6) Blank control.

[0147] 2. Experimental Methods

[0148] The procedures for experimental groups 1-3 are as follows:

[0149] Pre-cooling procedure: 10 soft-shell crabs per group, 3 replicates per group. Immediately place them on a tray lined with damp sterile gauze (to prevent friction damage to the crabs) and put them in a refrigerator at 0-4℃ for 4 minutes. After processing, measure the surface temperature of the crabs with an electronic thermometer, and let it drop to 5-10℃.

[0150] Film-forming blocking: After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming blocking agent (the sprayer was 15 cm away from the crab body, and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crab body.

[0151] Temporary holding treatment: Transfer the crabs to a temporary holding tank. 12 hours in advance, adjust water parameters: water temperature 20±0.5℃, aerator turned on to maintain dissolved oxygen ≥6.5mg / L. Monitor water quality every 2 hours: adjust pH to 8.0±0.1 with sodium bicarbonate, and detect ammonia nitrogen using Nessler's reagent spectrophotometry. If it exceeds 0.1mg / L, replace 1 / 3 of the water. Continue temporary holding for 96 hours without feeding (to avoid uneaten feed contaminating the water).

[0152] Experimental group 4) is the same as experimental groups 1-3, except that film formation blocking is not performed.

[0153] Experimental group 5) is the same as experimental groups 1-3, except that it does not undergo pre-cooling treatment.

[0154] Experimental group 6) is the same as experimental groups 1-3, except that: no pre-cooling and film-forming blocking treatment is performed, and it is directly temporarily incubated.

[0155] 3. Testing Standards

[0156] Hardening time: Starting from the end of the treatment, the hardness of the central area of ​​the crab carapace was measured every hour using an LD-A digital display Shore C hardness tester (3 points were measured each time and the average value was taken). When the hardness reached 20HA (the stable hardness after natural hardening is about 35HA, and 20HA is the critical value for edible soft shell), it was recorded as "hardening time".

[0157] Survival rate: The number of surviving individuals in each group was counted at 24h, 48h, 72h and 96h after treatment (survival criteria: normal crab activity and voluntary extension and retraction of appendages), and the survival rate was calculated (survival rate = number of surviving individuals / initial sample size × 100%).

[0158] 4. Experimental Results

[0159] The experimental results are shown in Table 1.

[0160] Table 1

[0161]

[0162] Note: Different superscript letters in the same column indicate significant differences between groups. P <0.05), the same applies below.

[0163] According to the experimental results in Table 1, different treatment methods showed significant differences in their effects on delaying hardening and survival rate of soft-shell crabs.

[0164] In terms of delaying hardening, the combined treatment of pre-cooling and film-forming inhibition was significantly better than either treatment alone. Groups 1) to 3) used different film-forming inhibitors in combination with pre-cooling, and the average hardening time exceeded 75 hours. Group 1) had the longest hardening time, reaching 88.6 ± 3.2 hours, significantly higher than pre-cooling alone (22.5 ± 1.8 hours) and film-forming inhibition alone (55.8 ± 2.1 hours), and far exceeding the blank control (7.2 ± 0.6 hours). This indicates that the synergistic effect of pre-cooling and film-forming inhibition can effectively delay the hardening of soft-shell crabs, and there are differences in effectiveness among different film-forming inhibitors, with the film-forming inhibitor in group 1) showing the best effect.

[0165] Regarding survival rates, all treatment groups achieved 100% survival at 24 hours, but differences gradually emerged over time. The combined treatment groups (groups 1-3) consistently had higher survival rates than the single treatment groups and the control group. At 96 hours, group 1) maintained a survival rate of 90±10%, significantly higher than pre-cooling alone (73.3±5.7%), film-forming inhibition alone (80±5.7%), and the control group (66.7±5.7%). This indicates that the combined treatment of pre-cooling and film-forming inhibition not only delays hardening but also improves the survival rate of soft-shell crabs during temporary rearing, with the effect becoming more pronounced over time.

[0166] In summary, the combined treatment of precooling and film-forming inhibition is an effective method to delay hardening of soft-shell crabs and improve their survival rate, with the film-forming inhibitor in group 1) showing the best effect. This result provides a scientific basis for the preservation and temporary holding of soft-shell crabs, and helps to improve their economic value and market supply stability.

[0167] Example 2: Effect of natural plant colloids of different viscosities on delaying hardening in soft-shell crabs

[0168] 1. Pre-cooling treatment

[0169] Take 90 soft-shell crabs, divide them into 3 groups of 10 crabs each, with three replicates per group, and immediately place them on a tray lined with damp sterile gauze (to prevent friction damage to the crabs), and put them in a refrigerator at 0-4℃ for 4 minutes. After the treatment, measure the surface temperature of the crabs with an electronic thermometer, and they all dropped to 5-8℃.

[0170] 2. Film-forming blockade

[0171] After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming inhibitor (the sprayer was 15 cm away from the crab and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crabs.

[0172] Three groups of soft-shell crabs were sprayed with the film-forming blocking agents prepared in Example 1, Comparative Examples 1 and 2, respectively.

[0173] 3. Temporary care

[0174] The treated soft-shell crabs were transferred to a polypropylene holding tank (4 m × 5 m × 1.2 m, effective water depth 1.0 m). The tank water was treated by sand filtration, ultraviolet sterilization, and a protein skimmer. Operating parameters: water temperature 20 ℃, dissolved oxygen 6.8 mg / L. Water quality was sampled every 2 hours: pH 8.0 (fine-tuned with NaHCO3), ammonia nitrogen 0.06 mg / L. Each group of crabs was marked to avoid confusion.

[0175] 4. Effect Measurement

[0176] Hardening time was determined using an LD-A digital Shore C hardness tester. Hardness of the central region of the crab carapace was measured every hour (3 points were measured each time, and the average value was taken). The hardening time was recorded when the hardness reached 20 HA. For 96-hour survival rate statistics, the crabs were transferred to a standard breeding environment after the experiment, and the number of dead individuals was recorded and the survival rate was calculated (survival rate = number of surviving crabs / total number of crabs × 100%). Film-forming performance indicators included viscosity measurement (using a Brookfield DV2T rotational viscometer to measure the viscosity of a 1 wt% flaxseed gum solution at 25℃, unit: mPa·s) and water vapor transmission rate (WVTR, calculated using an ASTM E96 standard tester based on the mass difference between high / low humidity chambers, unit: g / m²). 2 •day) and film thickness measurement (the film thickness was measured at 5 random locations on the carapace of the crab using a laser confocal microscope or digital thickness gauge, and the average value was taken, in μm).

[0177] 5. Experimental Results

[0178] The results are as follows Figure 1 As shown.

[0179] Based on the provided bar chart, the effects of natural plant colloids of different viscosities on delaying hardening in soft-shell crabs were analyzed.

[0180] Regarding water vapor transmission rate (WVTR), Example 1 exhibited a relatively low transmittance, showing a significant difference compared to Comparative Examples 1 and 2. This indicates that the colloidal membrane of Example 1 has good water vapor barrier properties, effectively reducing moisture exchange and potentially helping to delay the hardening process of soft-shell crabs.

[0181] The membrane thickness measurements showed that the membrane thickness in Example 1 was the greatest. A thicker membrane likely provides better protection and helps maintain the soft-shelled crab's soft-shell state.

[0182] The results of the hardening time measurement showed that the colloid of Example 1 could significantly prolong the hardening time of soft-shell crabs. This may be because the film formed by the colloid of Example 1 is denser and thicker, better blocking the exchange of substances between the crab body and the external environment, thereby slowing down the hardening process.

[0183] Regarding the 96-hour survival rate, Example 1 showed the highest survival rate. This may be attributed to its excellent film-forming properties, which provide a more stable microenvironment for the crabs and reduce the adverse effects of the external environment on them.

[0184] In summary, natural plant colloids of different viscosities exhibited significant differences in film-forming properties and the ability to delay the hardening of soft-shell crabs. The colloid of Example 1 demonstrated superior performance in terms of WVTR, film thickness, hardening time, and survival rate. This is likely because the colloid of Example 1 possesses a suitable viscosity, enabling it to form a uniform and dense film that effectively blocks the exchange of substances between the crab and its external environment, thereby delaying hardening and improving survival rate. These results provide valuable reference for selecting suitable natural plant colloids for the preservation and transportation of soft-shell crabs.

[0185] Example 3: Effect of natural plant colloids of different viscosities on delaying hardening in soft-shell crabs

[0186] 1. Pre-cooling treatment

[0187] Take 150 soft-shell crabs, divide them into 5 groups of 10 crabs each, with 3 replicates per group. Immediately place them on a tray lined with damp, sterile gauze (to prevent friction damage to the crabs) and put them in a refrigerator at 0-4℃ for 4 minutes. After processing, measure the surface temperature of the crabs with an electronic thermometer; all crabs have cooled to 5-8℃.

[0188] 2. Film-forming blockade

[0189] After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming inhibitor (the sprayer was 15 cm away from the crab and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crabs.

[0190] Five groups of soft-shell crabs were sprayed with the film-forming blocking agents prepared in Example 1 and Comparative Examples 3-6, respectively.

[0191] 3. Temporary care

[0192] The treated soft-shell crabs were transferred to a polypropylene holding tank (4 m × 5 m × 1.2 m, effective water depth 1.0 m). The tank water was treated by sand filtration, ultraviolet sterilization, and a protein skimmer. Operating parameters: water temperature 20 ℃, dissolved oxygen 6.8 mg / L. Water quality was sampled every 2 hours: pH 8.0 (fine-tuned with NaHCO3), ammonia nitrogen 0.06 mg / L. Each group of crabs was marked to avoid confusion.

[0193] 4. Detection indicators

[0194] Hardening time was determined using an LD-A digital Shore C hardness tester. Hardness of the central region of the crab carapace was measured every hour (3 points were measured each time, and the average value was taken). The hardening time was recorded when the hardness reached 20 HA. For 96-hour survival rate statistics, the crabs were transferred to a standard breeding environment after the experiment, and the number of dead individuals was recorded and the survival rate was calculated (survival rate = number of surviving crabs / total number of crabs × 100%). Film-forming performance indicators included viscosity measurement (using a Brookfield DV2T rotational viscometer to measure the viscosity of a 1 wt% flaxseed gum solution at 25℃, unit: mPa•s) and water vapor transmission rate (WVTR, calculated using an ASTM E96 standard tester based on the mass difference between high / low humidity chambers, unit: g / m²). 2 •day) and film thickness measurement (the film thickness was measured at 5 random locations on the carapace of the crab using a laser confocal microscope or digital thickness gauge, and the average value was taken in μm).

[0195] 5. Experimental Results

[0196] The results are as follows Figure 2 As shown. According to Figure 2 The data were used to re-analyze the effects of different natural plant colloids on delaying hardening in soft-shell crabs.

[0197] In terms of water vapor transmission rate (WVTR), flaxseed gum performed the best, with the lowest value. This means it has excellent water vapor barrier properties, effectively reducing the exchange of water between the crab's internal body and the external environment, thus creating favorable conditions for delaying hardening. Other colloids, such as guar gum and xanthan gum, had relatively higher values, indicating slightly inferior barrier properties.

[0198] Membrane thickness measurements indicate that the membrane formed by flaxseed gum has a certain advantage in thickness. A thicker membrane helps to better protect the crab's body and reduce the impact of external factors.

[0199] In terms of hardening time, flaxseed gum significantly prolonged the hardening time of soft-shell crabs, which fully demonstrates its excellent effect in blocking the hardening process of crabs. This may be due to its good film-forming properties, which effectively inhibit the exchange of hardening-related substances.

[0200] In terms of 96-hour survival rate, the flaxseed gum group also performed best, with the highest survival rate, indicating that its film formation provided a more suitable microenvironment for the crab body, which is conducive to the survival of the crab.

[0201] Overall, among the various natural plant colloids tested in this experiment, flaxseed gum showed significant advantages in all indicators. The film it formed exhibited excellent performance, effectively delaying the hardening of soft-shell crabs and improving their survival rate. This may be related to its unique physicochemical properties, providing a better option for natural plant colloids in soft-shell crab-related applications.

[0202] Example 3: Effects of different antibacterial and film-setting substances on delaying hardening in soft-shell crabs

[0203] 1. Pre-cooling treatment

[0204] Take 150 soft-shell crabs and divide them into 5 groups of 10 crabs each, with 3 replicates per group. Immediately place them on a tray lined with damp, sterile gauze (to prevent friction damage to the crabs) and put them in a refrigerator at 0-4℃ for 4 minutes. After treatment, measure the surface temperature of the crabs with an electronic thermometer; all crabs have cooled to 5-8℃.

[0205] 2. Film-forming blockade

[0206] After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming inhibitor (the sprayer was 15 cm away from the crab and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crabs.

[0207] Five groups of soft-shell crabs were sprayed with the film-forming blocking agents prepared in Example 1 and Comparative Examples 7-10, respectively.

[0208] 3. Temporary care

[0209] The treated soft-shell crabs were transferred to a polypropylene holding tank (4 m × 5 m × 1.2 m, effective water depth 1.0 m). The tank water was treated by sand filtration, ultraviolet sterilization, and a protein skimmer. Operating parameters: water temperature 20 ℃, dissolved oxygen 6.8 mg / L. Water quality was sampled every 2 hours: pH 8.0 (fine-tuned with NaHCO3), ammonia nitrogen 0.06 mg / L. Each group of crabs was marked to avoid confusion.

[0210] 4. Detection indicators

[0211] Antibacterial indicators were determined by plate culture method, which showed the total viable bacteria count (CFU / cm²) on the surface of the crab. 2 The inhibitory effect on Vibrio parahaemolyticus was evaluated using the inhibition zone test (paper disc diffusion method), and the minimum inhibitory concentration (MIC) was determined using the broth dilution method. The hardening time was measured every hour using an LD-A digital display Shore C hardness tester to detect the hardness of the central area of ​​the crab carapace (average of 3 points). The hardening endpoint was recorded when the hardness reached 20 HA. The 96-hour survival rate was calculated by continuously observing the survival of the crabs after the experiment, counting the number of dead individuals and calculating the survival rate (survival rate = number of surviving crabs / total number of crabs × 100%). This comprehensively quantified the overall effect of the antibacterial film-forming substance on delaying hardening and protecting the survival of soft-shell crabs.

[0212] 5. Experimental Results

[0213] The experimental results are shown in Table 2.

[0214] Table 2

[0215]

[0216] Based on the experimental results in Table 2, the effects of different antibacterial film-forming substances on delaying hardening in soft-shell crabs are analyzed as follows:

[0217] In terms of antibacterial properties, the dipotassium glycyrrhizate used in Example 1 showed a significant advantage. It had the lowest total viable bacterial count on the crab surface, at 1200±54 CFU / cm³. 2 The value was much lower than that of the other pairs; the diameter of the inhibition zone was the largest, reaching 18.5±1.6mm, indicating the best inhibitory effect on Vibrio parahaemolyticus; the minimum inhibitory concentration (MIC) was also the lowest, at 0.8±0.1μg / mL, indicating its strong antibacterial efficacy.

[0218] Regarding the hardening time, the soft-shell crab in Example 1 had the longest hardening time, reaching 89.2±3.4h. This indicates that the antibacterial film formed by dipotassium glycyrrhizate can effectively delay the hardening of the crab body. This may be because its good antibacterial properties maintain a stable microenvironment on the surface of the crab body, reducing the promotion of hardening by external factors.

[0219] Regarding the 96-hour survival rate, Example 1 showed the highest survival rate at 86.6 ± 5.7%, further demonstrating that the antibacterial film of dipotassium glycyrrhizate provides good protection for the crab body and is beneficial to the survival of the crab.

[0220] In comparison, the antibacterial film-setting substances in each comparative example were inferior to those in Example 1 in terms of antibacterial properties, delayed hardening, and improved survival rate. For example, sodium benzoate and methylparaben had high total viable bacteria counts, poor antibacterial effects, short hardening times, and low survival rates.

[0221] In summary, dipotassium glycyrrhizate, as an antibacterial and film-setting substance, exhibits excellent performance in inhibiting bacterial growth, delaying hardening of soft-shell crabs, and improving survival rate, demonstrating significant advantages and providing a more effective method for the preservation and storage of soft-shell crabs.

[0222] Example 4: Effect of different calcium ion chelating substances on delaying hardening in soft-shell crabs

[0223] 1. Pre-cooling treatment

[0224] Take 150 soft-shell crabs, divide them into 5 groups of 10 crabs each, with 3 replicates per group. Immediately place them on a tray lined with damp, sterile gauze (to prevent friction damage to the crabs) and put them in a refrigerator at 0-4℃ for 4 minutes. After processing, measure the surface temperature of the crabs with an electronic thermometer; all crabs have cooled to 5-8℃.

[0225] 2. Film-forming blockade

[0226] After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming inhibitor (the sprayer was 15 cm away from the crab and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crabs.

[0227] Five groups of soft-shell crabs were sprayed with the film-forming blocking agents prepared in Example 1 and Comparative Examples 11-14, respectively.

[0228] 3. Temporary care

[0229] The treated soft-shell crabs were transferred to a polypropylene holding tank (4 m × 5 m × 1.2 m, effective water depth 1.0 m). The tank water was treated by sand filtration, ultraviolet sterilization, and a protein skimmer. Operating parameters: water temperature 20 ℃, dissolved oxygen 6.8 mg / L. Water quality was sampled every 2 hours: pH 8.0 (fine-tuned with NaHCO3), ammonia nitrogen 0.06 mg / L. Each group of crabs was marked to avoid confusion.

[0230] 4. Detection indicators

[0231] The testing indicators cover three aspects: calcium ion chelating ability, hardening process, and survival rate. Calcium ion chelating ability was determined by complexometric titration, specifically using a 0.05 mol / L EDTA standard solution. The endpoint was determined by the color change of the calcium indicator, and the chelation value (mg CaCO3 / g) was calculated. Hardening time was measured every hour using an LD-A digital display Shore C hardness tester, with the hardness of the central area of ​​the crab carapace being recorded as the hardening endpoint when the hardness value reached 20 HA. The 96-hour survival rate was determined by continuously observing the survival of the crabs after the experiment, counting the number of dead individuals, and calculating the survival rate (survival rate = number of surviving crabs / total number of crabs × 100%). These three indicators synergistically quantify the comprehensive effect of calcium ion chelating substances on delaying hardening in soft-shell crabs.

[0232] 5. Experimental Results

[0233] The results are as follows Figure 3 As shown. According to Figure 3 The experimental results and their effects on different calcium ion chelating substances in delaying hardening in soft-shell crabs are analyzed as follows:

[0234] In terms of calcium ion chelating ability, Example 1 showed significant superiority, with a chelation value of approximately 850 mg CaCO3 / g, far exceeding that of the comparative examples. This indicates that the calcium ion chelating substance used in Example 1 has a stronger chelating ability and can more effectively bind calcium ions, thereby potentially delaying the hardening process of the crab body.

[0235] The hardening time results confirmed this. Example 1 had the longest hardening time, indicating that it effectively slowed down the hardening rate of the crab body by chelating calcium ions. In contrast, the hardening times of Comparative Examples 11 to 14 were all shorter than those of Example 1, and the differences between the groups were not significant, indicating that the hardening-retarding effect of these calcium ion chelating substances was not as good as that of Example 1.

[0236] Regarding the 96-hour survival rate, Example 1 also showed the best results, with the highest survival rate. This may be because the calcium ion chelating substance in Example 1 not only delayed hardening but may also have a protective effect on the crab body, reducing the adverse effects of the external environment on the crab body.

[0237] In summary, the calcium ion chelating substance in Example 1 demonstrated significant advantages in chelating ability, delaying hardening, and improving survival rate. This is likely due to the substance's unique chemical structure or properties, which enable it to bind more effectively to calcium ions, thereby effectively delaying the hardening of soft-shell crabs. The results of this experiment provide valuable reference for the preservation and storage of soft-shell crabs.

[0238] Example 5: Effects of different antioxidants on delaying hardening in soft-shell crabs

[0239] 1. Pre-cooling treatment

[0240] Take 150 soft-shell crabs, divide them into 5 groups of 10 crabs each, with 3 replicates per group. Immediately place them on a tray lined with damp, sterile gauze (to prevent friction damage to the crabs) and put them in a refrigerator at 0-4℃ for 4 minutes. After processing, measure the surface temperature of the crabs with an electronic thermometer; all crabs have cooled to 5-8℃.

[0241] 2. Film-forming blockade

[0242] After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming inhibitor (the sprayer was 15 cm away from the crab and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crabs.

[0243] Five groups of soft-shell crabs were sprayed with the film-forming blocking agents prepared in Example 1 and Comparative Examples 15-18, respectively.

[0244] 3. Temporary care

[0245] The treated soft-shell crabs were transferred to a polypropylene holding tank (4 m × 5 m × 1.2 m, effective water depth 1.0 m). The tank water was treated by sand filtration, ultraviolet sterilization, and a protein skimmer. Operating parameters: water temperature 20℃, dissolved oxygen 6.8 mg / L. Water quality was sampled every 2 hours: pH 8.0 (fine-tuned with NaHCO3), ammonia nitrogen 0.06 mg / L. Each group of crabs was marked to avoid confusion.

[0246] 4. Detection indicators

[0247] The testing indicators include three quantitative dimensions: Total Antioxidant Capacity (TEAC), which is determined by the FRAP method to measure the iron ion reduction capacity in crab tissues, reflecting the enhancement effect of film-forming inhibitors on the antioxidant defense system of soft-shell crabs; Hardening Time, which is monitored every hour by an LD-A digital display Shore C hardness tester, with a hardness of 20HA as the hardening endpoint, to accurately quantify the delay in the hardening process; and 96-hour Survival Rate, which is calculated by continuously observing the survival status of crabs after the experiment, counting the number of dead individuals and calculating the survival rate (survival rate = number of surviving crabs / total number of crabs × 100%), to comprehensively evaluate the efficacy of antioxidants in protecting the survival of soft-shell crabs.

[0248] 5. Experimental Results

[0249] The results are as follows Figure 4 As shown.

[0250] according to Figure 4 The experimental results and their effects on the delay of hardening in soft-shell crabs are analyzed below:

[0251] In terms of total antioxidant capacity (TEAC), Example 1 showed a significant advantage, with a TEAC value much higher than that of Comparative Examples 15-18. This indicates that the antioxidants used in Example 1 can significantly enhance the crab's antioxidant defense system, effectively scavenge free radicals, reduce oxidative damage, and provide a good foundation for delaying crab hardening.

[0252] Regarding hardening time, the soft-shell crab in Example 1 hardened for a significantly longer time than the comparative examples. This indicates that the antioxidant effectively delayed the hardening process by enhancing the crab's antioxidant capacity and slowing down the internal physiological and biochemical reactions. In contrast, the relatively short hardening times of Comparative Examples 15-18 suggest that their antioxidants were less effective at delaying hardening.

[0253] In terms of 96-hour survival rate, Example 1 also performed best, with the highest survival rate. This may be attributed to its strong antioxidant capacity, which maintained the normal function and structure of crab cells, reduced cell damage and death caused by oxidative stress, and improved the survival ability of crabs in temporary holding environments.

[0254] In summary, the antioxidants selected in Example 1 showed significant effects in enhancing the antioxidant capacity of crabs, delaying hardening, and improving survival rates. This may be related to their antioxidant mechanisms, stability, and compatibility with crab tissues. These results provide a clear direction for the selection of antioxidants in the preservation and storage of soft-shell crabs, contributing to improved quality and economic benefits.

[0255] Example 6: Effect of different emulsifier selection on delaying hardening in soft-shell crabs

[0256] This effect example tested the effects of Examples 1, 4, 5, and 6. The results showed that different emulsifiers had little effect on the hardening time of soft-shell crab. The role of the emulsifier is to help the components mix evenly and make the film more uniform. Conventional non-toxic emulsifiers in the art can be used as alternatives to the present invention.

[0257] 1. Pre-cooling treatment

[0258] Take 50 soft-shell crabs, divide them into 5 groups of 10 each, and immediately place them on a tray lined with damp, sterile gauze (to prevent friction damage to the crabs). Place them in a refrigerator at 0-4℃ for 4 minutes. After processing, measure the surface temperature of the crabs with an electronic thermometer; all crabs should have cooled to 5-8℃.

[0259] 2. Film-forming blockade

[0260] After pre-cooling, the soft-shell crabs were transferred to a clean bench surface covered with sterile non-woven fabric (65% moisture content). Each crab was evenly sprayed with 5 mL of film-forming inhibitor (the sprayer was 15 cm away from the crab and sprayed in 3 times to avoid local liquid accumulation). After that, the crabs were left to stand and dry for 6 minutes. A uniform and transparent film was observed to form on the surface of the crabs.

[0261] Four groups of soft-shell crabs were sprayed with the film-forming blocking agents prepared in Examples 1, 4, 5, and 6, respectively.

[0262] 3. Temporary care

[0263] The treated soft-shell crabs were transferred to a polypropylene holding tank (4 m × 5 m × 1.2 m, effective water depth 1.0 m). The tank water was treated by sand filtration, ultraviolet sterilization, and a protein skimmer. Operating parameters: water temperature 20 ℃, dissolved oxygen 6.8 mg / L. Water quality was sampled every 2 hours: pH 8.0 (fine-tuned with NaHCO3), ammonia nitrogen 0.06 mg / L. Each group of crabs was marked to avoid confusion.

[0264] 4. Detection indicators

[0265] The testing indicators cover the physical stability assessment of the film-forming inhibitor emulsified system and the quantification of physiological indicators of soft-shell crabs: the emulsification indicators are directly tested through three dimensions: centrifugation stability test (layer volume ratio after 3000 rpm for 30 min), average particle size (nm) and Zeta potential (mV) measured by laser particle size analyzer, to directly detect the colloidal system (emulsion formed by emulsifier, flaxseed gum and other components) after film-forming inhibitor emulsification, to quantify its anti-layering ability, particle size uniformity and charge repulsion to evaluate the effect of emulsifier; the hardening time is monitored every hour by an LD-A digital display Shore C hardness tester to monitor the hardness of the central area of ​​the crab carapace, with a hardness of 20HA as the hardening endpoint; the 96-hour survival rate is calculated by continuously observing the survival status of the crabs after the experiment, counting the number of dead individuals and calculating the survival rate (survival rate = number of surviving crabs / total number of crabs × 100%). The three indicators together constitute a complete quantitative chain of "emulsification stability - film protection effect - delayed hardening - survival protection", and the emulsification indicators only apply to the film-forming inhibitor system itself and do not involve the direct properties of the crab or water.

[0266] 5. Experimental Results

[0267] Table 3

[0268]

[0269] Based on the experimental data in Table 3, the effects of different emulsifiers on delaying hardening in soft-shell crabs are analyzed as follows:

[0270] Regarding emulsification parameters, different emulsifiers exhibited significant differences. Example 1 (polyglycerol fatty acid ester) showed the highest centrifugal stability, reaching 98.4 ± 0.3%, indicating that its emulsion system had the strongest anti-stratification ability; it also had the smallest average particle size, at 120 ± 1.0 nm, indicating that the emulsion formed had a more uniform particle size; and its Zeta potential was -35.2 ± 0.2 mV, indicating moderate charge repulsion, which contributes to the stability of the emulsion. In contrast, the emulsification parameters of other examples were not as good as those of Example 1. For example, Example 6 (PG10L) had the lowest centrifugal stability and the largest average particle size.

[0271] Regarding the hardening time, the soft-shell crab in Example 1 had the longest hardening time, which may be attributed to its excellent emulsification properties, resulting in a more uniform film formation and thus more effectively delaying the hardening of the crab body. In contrast, the hardening times of Examples 4-6 were relatively short, indicating that their emulsification effect had limited impact on delaying hardening.

[0272] Regarding the 96-hour survival rate, Example 1 also performed best, with the highest survival rate. This may be because its excellent emulsifying properties provide a more stable protective film for the crab body, reducing the adverse effects of the external environment on the crab body.

[0273] Overall, polyglycerol fatty acid esters, as emulsifiers, demonstrate excellent performance in emulsification stability, delaying hardening, and improving survival rates. They effectively enhance the performance of film-forming inhibitors, providing better protection for the freshness and preservation of soft-shell crabs. While other emulsifiers also have some effect, their overall performance is not as good as that of polyglycerol fatty acid esters.

[0274] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A composition for retarding the hardening of the carapace of a soft-shell crab, characterized by: The composition comprises, by weight, the following: Flaxseed gum 6-8 parts, dipotassium glycyrrhizate 1-1.5 parts, inositol hexaphosphate 3-4 parts, epigallocatechin gallate 0.8-1.2 parts, polyglycerol fatty acid ester 2.5-3 parts; The viscosity of the flaxseed gum is 10,000 to 14,000 mPa·s.

2. A film-forming inhibitor for soft-shell crabs, characterized in that: Includes the composition of claim 1 and PBS buffer solution.

3. The soft-shell crab film-forming inhibitor according to claim 2, characterized in that: The film-forming inhibitor, by mass fraction, comprises: Flaxseed gum 0.6-0.8 wt%, dipotassium glycyrrhizate 0.1-0.15 wt%, inositol hexaphosphate 0.3-0.4 wt%, epigallocatechin gallate 0.08-0.12 wt%, polyglycerol fatty acid ester 0.25-0.3 wt%, PBS buffer solution balance.

4. A method for preparing the film-forming inhibitor according to claim 2 or 3, comprising the following steps: 1) Add flaxseed gum to PBS buffer solution and stir to obtain a colloidal solution; 2) Add inositol hexaphosphate, epigallocatechin gallate, dipotassium glycyrrhizate and polyglycerol fatty acid ester to the colloidal solution, mix, and form an emulsion; 3) Add PBS buffer solution to bring the total to 100 wt%.

5. The preparation method according to claim 4, characterized in that: The stirring temperature in step 1) is 48~52℃; Step 2) The mixing temperature is 38~40℃.

6. The use of the composition of claim 1, or the film-forming inhibitor of claim 2 or 3, in delaying the hardening of the shell of mud crab.

7. A method for delaying the hardening of the shell of soft-shell crabs, comprising the following steps: 1) Pre-cooling treatment of soft-shell crabs; 2) Blocking of film formation on the body surface: Treat soft-shell crabs with the film-forming blocking agent as described in claim 2 or 3.

8. The method according to claim 7, characterized in that: The steps also include: 3) Temporary holding management: The soft-shell crabs treated in step (2) are transferred to a temporary holding tank with dissolved oxygen ≥6 mg / L, and the pH of the water is corrected to 7.8–8.2 and ammonia nitrogen ≤0.1 mg / L every 2 hours by online monitoring.

9. The method according to claim 8, characterized in that: Step 1) The soft-shell crabs that have undergone pre-cooling treatment are those that have molted within 1 hour. The temperature of the crab body after pre-cooling is 5~8℃.

10. The method according to claim 9, characterized in that: The film-forming barrier on the body surface is treated on a wet substrate; The moisture content of the wet substrate is 60-70%; The substrate includes non-woven fabric and sponge.

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