A method for purifying mud crab by temporary culture

Abstract

The application discloses a temporary culture treatment method for purifying blue crabs, and specifically comprises the following steps: (1) a pretreatment step, (2) a mixed liquid preparation step, (3) a culture liquid preparation step, and (4) a culture step. The application provides a temporary culture treatment method for purifying blue crabs, which can clean the bodies of the blue crabs, reduce antibiotic and heavy metal residues, and reduce spoilage bacteria, so as to improve the quality and activity of the blue crabs and reduce the loss of the blue crabs.

Description

Technical Field

[0001] This invention relates to the field of biological aquaculture technology, and more specifically, to a method for the temporary holding of purified mud crabs. Background Technology

[0002] Sanmen mud crab is a specialty of Sanmen County, Zhejiang Province, and a Chinese national geographical indication product. Sanmen mud crabs are characterized by their bright blue-green color, thin shells, large claws, rich aroma, and tender meat, making them a favorite among consumers.

[0003] Heavy metals in water bodies typically originate from industrial wastewater and domestic sewage pollution, easily accumulating in aquatic animals. This not only directly harms aquatic products but also poses a potential risk to human health through the food chain. Mud crabs, living in pond mud, are easily contaminated with heavy metals such as phosphorus (Pd) and carbon dioxide (Cd), affecting food safety. Antibiotics are generally used for disease prevention and growth promotion in aquatic products; improper use can lead to antibiotic residues in crabs. Furthermore, mud crabs often carry a lot of dirt and impurities, affecting the quality of the crab meat and potentially carrying bacteria and parasites. During transportation, due to their high protein and free amino acid content, mud crabs are highly susceptible to spoilage bacteria, leading to reduced vitality and muscle quality. Therefore, reducing or even eliminating heavy metal residues in mud crabs, while simultaneously reducing bacterial counts and antibiotic residues, purifying the crabs can not only improve their quality and vitality but also reduce losses during transportation and sales. Summary of the Invention

[0004] This invention overcomes the shortcomings of existing technologies and addresses the risks of antibiotic and heavy metal residues and putrefactive bacteria in mud crabs. It provides a method for cleaning the body of mud crabs, reducing antibiotic and heavy metal residues, and minimizing putrefactive bacteria, thereby improving the quality and vitality of mud crabs and reducing losses.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A method for the temporary holding and treatment of purified mud crabs includes the following steps:

[0007] (1) Pretreatment steps: Set the water level in the temporary holding tank where the crabs to be purified are placed between 28cm and 32cm;

[0008] (2) Preparation steps of the mixture: Dissolve 200mg of tea polyphenols and 40μL of formaldehyde in 40ml of pure water, and then run the mixture at 30℃ and 1200r / min under a magnetic stirrer for 30min to fully dissolve them and obtain a stable tea polyphenol-formaldehyde solution.

[0009] The tea polyphenol-formaldehyde solution was mixed with 40 mg of lysine and kept for 5 minutes after it was fully mixed. The resulting mixture was then centrifuged at high speed, the supernatant was removed, and then the mixture was washed three times with pure water to obtain the tea polyphenol-lysine conjugate.

[0010] (3) Preparation of aquaculture solution: Dissolve the tea polyphenol-lysine conjugate in the water of the temporary holding tank, and make the concentration of the tea polyphenol-lysine conjugate obtained in step (2) between 3.3 mg / L and 6.6 mg / L in the water; and add chlorine dioxide solution at the same time, with the concentration of chlorine dioxide dissolved in the water being 0.5 mg / L;

[0011] Turn on the ozone generator and control the ozone concentration in the water at 0.05 mg / L.

[0012] (4) Breeding steps: Put the mud crabs in the pond and raise them for 72 hours, then take them out.

[0013] Furthermore, the tea polyphenol-lysine conjugate was stored at 4°C.

[0014] Furthermore, the ultracentrifuge operates at a speed of 10,000 r / min.

[0015] Furthermore, the water temperature in the temporary holding tank is controlled between 10℃ and 32℃, and the pH value is between 6 and 8.

[0016] Furthermore, the aerator should be turned on from 10 PM to 11 PM every night until it is turned off from 3 AM to 5 AM the next day, so that the dissolved oxygen content can be controlled between 5 mg / L and 9 mg / L.

[0017] Furthermore, the ratio of the number of mud crabs released to the water quality in the temporary holding pond is controlled between 1:9 and 1:11.

[0018] The advantages of this invention compared to the prior art are:

[0019] The temporary holding method of the present invention not only cleans the body of the mud crab and reduces antibiotic and heavy metal residues, but also reduces the treatment of putrefactive bacteria, thereby improving the quality and vitality of the mud crab and reducing its loss.

[0020] This invention utilizes the fact that tea polyphenol nanoparticles have a diameter of less than 1000 nm, exhibiting good stability, which can compensate for the poor stability of tea polyphenols. Formaldehyde undergoes a Mannish reaction with tea polyphenols, in which formaldehyde provides carbonyl groups, enhancing the entanglement and interaction between derivative molecules. Amino acids, as small molecules, are characterized by simple structures and abundant sources, providing a good foundation for the performance and function of nanoparticles. Lysine, compared to other amino acids, has a smaller average particle size and more effective adsorption capacity. Therefore, by utilizing the rapid Mannish reaction between tea polyphenols, formaldehyde, and lysine, linear tea polyphenol oligomer derivatives are generated, which self-assemble into polyphenol nanoparticles with an average particle size of 293±50 nm. The hydroxyl groups on the benzene rings can bind with heavy metals to form complexes, thereby achieving the effect of heavy metal adsorption.

[0021] This invention utilizes ozone, also known as superoxide, which consists of oxygen molecules carrying an oxygen atom. It can directly destroy bacterial organelles, affecting metabolism and leading to bacterial death. It can also oxidize antibiotics in water, significantly reducing their concentration. Chlorine dioxide has a strong adsorption and penetration ability against microbial cell walls, killing bacteria without them developing resistance. The combination of ozone and chlorine dioxide achieves broad-spectrum bactericidal and antibiotic-degrading effects with high safety. Furthermore, the hydroxyl radicals generated during ozone disinfection readily bind to heavy metals, reducing the harmful effects of heavy metals on crabs. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments.

[0023] Example 1:

[0024] A method for the temporary holding and treatment of purified mud crabs includes the following steps:

[0025] (1) Pretreatment steps: Set the water level in the temporary holding tank for the crabs to be purified between 28cm and 32cm. Generally, it is set at around 30cm.

[0026] (2) Preparation steps of the mixture: Dissolve 200mg of tea polyphenols (EGCG) and 40μL of formaldehyde in 40ml of purified water (i.e., deionized water), and then run the mixture at 30℃ and 1200r / min under a magnetic stirrer for 30min to fully dissolve it and obtain a stable tea polyphenol-formaldehyde solution.

[0027] A tea polyphenol-formaldehyde solution was mixed with 40 mg of lysine (Lys), and the mixture was kept for 5 minutes after thorough mixing. The resulting mixture was then centrifuged at high speed, the supernatant was removed, and the mixture was washed three times with pure water to obtain the tea polyphenol-lysine conjugate (EGCG–lys-NPs).

[0028] Tea polyphenol-lysine conjugates should be stored at 4°C.

[0029] The ultracentrifuge operates at a speed of 10,000 r / min.

[0030] (3) Preparation of the aquaculture solution: Dissolve the tea polyphenol-lysine conjugate in the water of the temporary holding tank, ensuring that the concentration of the tea polyphenol-lysine conjugate obtained in step (2) in the water is between 3.3 mg / L and 6.6 mg / L. Simultaneously add chlorine dioxide solution, with a chlorine dioxide concentration of 0.5 mg / L in the water. The concentration of the tea polyphenol-lysine conjugate should ideally be 6.6 mg / L.

[0031] Turn on the ozone generator and control the ozone concentration in the water at 0.05 mg / L. Connect the air pump and the ozone generator's outlet pipe to the water tank. Turn on the ozone generator and air pump to purge ozone for 1 minute, initially controlling the ozone concentration at approximately 0.05 mg / L. Thereafter, turn on the ozone generator for 1 minute every 6 hours.

[0032] (4) Breeding steps: Place the mud crabs in a holding tank for 72 hours, then remove them. Maintain the water temperature in the holding tank between 10℃ and 32℃, and the pH between 6 and 8 throughout the process. During this period, turn on the aerator from 10 PM to 11 PM daily until 3 AM to 5 AM the following day, keeping the dissolved oxygen content between 5 mg / L and 9 mg / L. Maintain the water temperature between 25℃ and 28℃, and the salinity between 15‰ and 20‰.

[0033] The ratio of the number of mud crabs released to the water in the temporary holding pond should be controlled between 1:9 and 1:11, with 1:10 being the most suitable.

[0034] Example 2:

[0035] Compared with Example 1, Example 2 changed the step of preparing the mixture by dissolving 100 mg of tea polyphenols (EGCG) and 20 mL of formaldehyde in 40 mL of purified water (i.e., deionized water).

[0036] Add 3.3 mg / L of tea polyphenol-lysine compound in the aquaculture solution preparation step.

[0037] To demonstrate that the prepared tea polyphenol-lysine conjugate was free of formaldehyde residue, 20 mg of the conjugate was used, and the formaldehyde residue was determined by HPLC. 20 mg of the conjugate was mixed with 5 mL of acetonitrile, and the mixture was sonicated for 30 min to extract formaldehyde. The extract was then centrifuged at 10000 r / min for 10 min. The supernatant was filtered through 0.22 μm nylon filter paper at 4 °C. An appropriate amount of 2,4-DNPH was dissolved in acetonitrile (containing 1% phosphoric acid solution) to obtain a 2,4-DNPH working solution (3.0 mg mL–1). 2.5 mL of the 2,4-DNPH working solution was added to the supernatant (5 mL), and the mixture was incubated at 40 °C for 60 min to convert formaldehyde to Schiff base. The sample was separated by isocratic elution (methanol / water = 65:35) on an XDB-C18 column (5 μm, 4.6 × 150 mm), detected at 355 nm, with an injection volume of 20 μL and a run time of 6 min. The formaldehyde content of the tea polyphenol-lysine conjugate was below the detection range (0.24 μg mg–1), and no formaldehyde residue was found.

[0038] In summary, the heavy metal content, total bacterial count, vitality index, amino acid content, and polyunsaturated fatty acid content of mud crabs after temporary holding treatment in Examples 1 and 2 were tested. Heavy metal content is a key indicator for assessing the safety and quality of mud crabs. After temporary holding and purification treatment, the lead, cadmium, and chromium content in the mud crabs was significantly lower than that in the control group, as shown in Table 1 below.

[0039]

[0040] Table 1

[0041] Table 1 shows the heavy metal content of mud crabs in different example groups. As can be seen from the table, the hydroxyl groups on polyphenols can bind with heavy metals in mud crabs, effectively reducing their content. Furthermore, ozone promotes the binding of tea polyphenol hydroxyl groups with heavy metals. Therefore, after temporary rearing and purification in Examples 1 and 2, the lead, cadmium, and chromium contents of the mud crabs were reduced by at least 47.1%, 40.0%, and 25.7% respectively compared to the control group.

[0042] The total bacterial count in the muscle of mud crabs is a key indicator of quality. After temporary holding and purification treatment, the total bacterial count in the mud crabs was significantly lower than that in the control group, as shown in Table 2 below:

[0043]

[0044] Table 2

[0045] Ozone and chlorine dioxide can disrupt the structure of putrefactive bacteria, slow their reproduction and growth, and improve the quality of mud crabs. The vitality index of mud crabs is a direct indicator of their vitality; the higher the vitality index, the healthier the crab. In the examples, the vitality index of the mud crabs was significantly higher than that of the control group, indicating that the health level of the mud crabs was significantly improved after the temporary holding and purification treatments in Examples 1 and 2. The specific vitality index statistics are shown in the table below:

[0046] deal with Vitality Index Example 1 2.94±0.21 Example 2 2.56±0.20 Temporary care blank processing 2.05±0.12

[0047] Amino acids and fatty acids are important nutrients in mud crabs. Sixteen amino acids were detected in the muscle of mud crabs, of which 13 had a content greater than 400 mg / 100g. See below for details.

[0048] Table 4:

[0049]

[0050]

[0051]

[0052] Table 4

[0053] The amino acid content in the table above is in mg / 100g. The total amino acid content in Examples 1 and 2 was higher than that in the blank treatment, indicating that the temporary holding and purification treatment can slow down the rate of amino acid production from the degradation of muscle protein in mud crabs. After the temporary holding treatment, the content of saturated and unsaturated fatty acids decreased significantly, but the content of polyunsaturated fatty acids increased significantly. The statistical analysis of fatty acid content is shown in Table 5 below:

[0054]

[0055]

[0056] Table 5

[0057] Table 5 above shows the fatty acid content of blue crabs in different example groups, with the unit being mg / 100g.

[0058] Therefore, the temporary holding method in this plan not only cleans the crab's body and reduces antibiotic and heavy metal residues, but also reduces the treatment of putrefactive bacteria, thereby improving the quality and vitality of the crab and reducing its loss.

[0059] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the concept of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Where the present invention is not described in detail, it can be implemented using conventional technical means.

Claims

1. A method for the temporary holding and treatment of purified mud crabs, characterized in that, Specifically, the steps include the following: (1) Pretreatment steps: Set the water level in the temporary holding tank where the crabs to be purified are placed between 28cm and 32cm; (2) Preparation steps of the mixture: Dissolve 200 mg of tea polyphenols and 40 μL of formaldehyde in 40 ml of purified water, and then run the mixture at 30℃ and 1200 r / min under a magnetic stirrer for 30 min to fully dissolve them and obtain a stable tea polyphenol-formaldehyde solution. The tea polyphenol-formaldehyde solution was mixed with 40 mg of lysine and kept for 5 minutes after thorough mixing. The resulting mixture was then centrifuged at high speed, the supernatant was removed, and the mixture was washed three times with pure water to obtain the tea polyphenol-lysine conjugate. (3) Preparation of aquaculture solution: Dissolve the tea polyphenol-lysine conjugate in the water of the temporary holding tank, and make the concentration of the tea polyphenol-lysine conjugate obtained in step (2) between 3.3 mg / L and 6.6 mg / L in the water; and add chlorine dioxide solution at the same time, with the concentration of chlorine dioxide dissolved in the water being 0.5 mg / L; Turn on the ozone generator to control the ozone concentration in the water at 0.05 mg / L; (4) Breeding steps: Put the mud crabs in the pond and raise them for 72 hours, then take them out.

2. The method for temporary holding of purified mud crabs according to claim 1, characterized in that: Tea polyphenol-lysine conjugates are stored at 4 ℃.

3. The method for temporary holding of purified mud crabs according to claim 1, characterized in that: The ultracentrifuge operates at a speed of 10,000 r / min.

4. The method for temporary holding of purified mud crabs according to claim 1, characterized in that: The water temperature in the temporary holding tank is controlled between 10℃ and 32℃, and the pH value is between 6 and 8.

5. The method for temporary holding of purified mud crabs according to claim 1, characterized in that: Turn on the aerator from 10 PM to 11 PM every night until it is turned off from 3 AM to 5 AM the next day, keeping the dissolved oxygen content between 5 mg / L and 9 mg / L.

6. The method for temporary holding of purified mud crabs according to claim 1, characterized in that: The ratio of the number of mud crabs released to the water in the temporary holding pond should be controlled between 1:9 and 1:11.

Images