A method for shrimp non-aqueous live-keeping transportation

Abstract

The present application belongs to the field of live transportation of aquatic products, and relates to a method for live transportation of prawns without water. The core of the method is to use a staged treatment strategy and the synergistic effect of dormancy induction liquid and moisture supply liquid. First, the prawns are adapted to the environment by fasting and gradient cooling, then they are soaked in pre-cooled dormancy induction liquid to enter a pseudo-death dormancy state and reduce metabolism. Then, the moisture supply liquid soaked sponge is used as a transportation medium, which can form a protective film on the surface of the prawn body to effectively prevent water loss and provide energy. During transportation, the prawns treated with the moisture supply liquid are placed in a sealed box filled with pure oxygen together with the moist sponge, and the prawns are transported without water at low temperature. After the transportation is completed, the prawns are placed in warm water to quickly recover. The prawn live transportation method without water prolongs the survival time of prawns in a water-free environment, improves the transportation efficiency, is simple to operate, and is suitable for large-scale long-distance transportation of prawns.

Description

Technical Field

[0001] This invention belongs to the field of aquatic product preservation and transportation technology, specifically relating to a method for waterless preservation and transportation of shrimp. Background Technology

[0002] Shrimp are delicious and economically valuable, but they are highly susceptible to death after being removed from water. Traditional shrimp transportation methods include both water-based and waterless transport. Water-based transport requires carrying large volumes of water, resulting in high costs, low carrying efficiency, and significant risks due to water quality deterioration during transit. Waterless transport, on the other hand, induces shrimp dormancy through low temperatures, significantly increasing transport density and reducing costs, and has become the mainstream approach in the industry. However, during waterless transport, shrimp still face multiple threats such as severe stress, water loss, and microbial infection, seriously affecting their survival rate and market quality. Therefore, shrimp survival preservatives are often used to improve transport efficiency.

[0003] Early methods of waterless aquaculture transport relied primarily on low-temperature dormancy, oxygenation, and the addition of sodium chloride to regulate osmotic pressure. Currently, survival agents used in aquaculture transport mainly consist of highly effective anesthetics, such as MS-222 and CO2, which reduce metabolic rate by inhibiting central nervous system activity, thus minimizing struggle during transport. Alternatively, low-temperature-induced dormancy and anti-stress agents are used synergistically to reduce mechanical and oxidative stress damage during transport. Although existing survival agents have improved transport survival rates to some extent, they generally suffer from problems such as limited functionality, poor synergy, insufficient long-term protection, and high costs. This leads to unstable shrimp survival rates and poor recovery vitality after long-term transport, limiting their large-scale application. Chinese patent CN107950636A uses a combination of sorbitol, ichthyol, and ascorbic acid as a shrimp survival agent, focusing on anti-stress, but it fails to simultaneously address key issues such as continuous energy supply, antibacterial protection, and maintenance of dormancy effects. For long-term waterless transportation, developing a novel, multifunctional, synergistic, green, safe, and effective survival agent and waterless transportation method for shrimp that combines immediate anesthesia and long-term protection has become a critical technical challenge that needs to be overcome for achieving high survival rates in waterless shrimp transportation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for waterless live transport of shrimp to solve the above-mentioned technical problems.

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

[0006] A method for waterless live transport of shrimp includes the following steps:

[0007] S1. Select healthy shrimp and keep them in seawater without feeding for 24-48 hours, while maintaining oxygenation;

[0008] S2. Cool the seawater where the shrimp are located to 10~12℃ at a rate of 1℃ / h, and add the dormancy induction solution.

[0009] Pre-cool to 10-12℃; immerse the shrimp in the dormancy induction solution for 3-5 minutes to induce all shrimp to enter a state of suspended animation.

[0010] S3. Soak the sponge in the moisturizing and energy-generating solution until it is fully moistened. Dip the shrimp in the moisturizing and energy-generating solution and take them out, then place them on the moistened sponge.

[0011] S4. Place the moistened sponge and shrimp into a transport box, fill it with pure oxygen and seal it. Transport the shrimp in a constant temperature environment of 12~15℃ without water to keep them alive.

[0012] S5. After transportation, remove the shrimp and place them in clean water at 18~22℃ until they are fully recovered.

[0013] As a further improvement, the dormancy induction solution comprises: 0.025-0.05 parts of eugenol, 30-50 parts of peppermint extract, 20-50 parts of sodium chloride, 1-5 parts of zinc sulfate, 5-20 parts of magnesium sulfate, 10-20 parts of betaine, 0.1-0.3 parts of ascorbic acid, and 900 parts of deionized water.

[0014] As a further improvement, the method for preparing the dormancy induction solution is as follows:

[0015] S1. Dry the peppermint leaves, crush them, and pass them through a 20-40 mesh sieve to obtain peppermint powder; mix the peppermint powder with water, perform steam distillation, collect the distillate for oil-water separation, filter, collect the oil phase, and obtain peppermint extract;

[0016] S2. Weigh eugenol, peppermint extract, sodium chloride, zinc sulfate, magnesium sulfate, betaine, ascorbic acid and Tween 80, add them to deionized water, stir well, and sonicate for 3-5 minutes to obtain the dormancy induction solution.

[0017] As a further improvement, the ratio of peppermint powder to water is 10-15 mL of water per gram of peppermint powder; the steam distillation time is 4-6 h; and the amount of Tween 80 added is 10%-30% of the total mass of eugenol and peppermint extract.

[0018] As a further improvement, the moisturizing and energy-providing liquid comprises: 2-5 parts of aloe vera gel extract, 1-3 parts of trehalose, 0.5-1.5 parts of sodium pyruvate, 0.2-0.5 parts of chitosan, and 90-95 parts of deionized water.

[0019] As a further improvement, the preparation method of the moisturizing and energy-supplying liquid is as follows:

[0020] S1. Rinse the aloe vera leaves with sterile water, remove the green epidermis of the leaves, and take out the gel block; crush the gel block with a crusher to obtain the slurry; homogenize the slurry in two stages through a high-pressure homogenizer, centrifuge the homogenized slurry, and filter the supernatant through a 5μm filter membrane to obtain aloe vera gel extract.

[0021] S2. Weigh aloe vera gel extract, trehalose, sodium pyruvate and chitosan, add them to deionized water, stir well, adjust the pH to 6.5, and sonicate for 3-5 minutes to obtain a moisturizing and energy-providing solution.

[0022] As a further improvement, the specific steps of the two-stage homogenization are as follows: high-pressure homogenization is performed using a high-pressure homogenizer, with the first-stage pressure set to 25~35MPa and homogenization performed twice; the second-stage pressure is set to 60~80MPa and homogenization is performed once; the centrifugation step is centrifugation at 10000~12000rpm for 3~5min.

[0023] The present invention also provides a shrimp survival agent, including a dormancy induction solution and a moisturizing and energy-providing solution.

[0024] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0025] The waterless live transport method for shrimp provided by this invention has significant beneficial effects. Its core innovation lies in the staged targeted treatment strategy of using a functionally complementary dormancy induction fluid and a moisturizing and energy-supplying fluid: the dormancy induction fluid induces dormancy and has an anti-stress effect, while the moisturizing and energy-supplying fluid solves the problems of moisturization and energy supply, and the two work synergistically. Eugenol, as a fish anesthetic, has a high effective concentration and a narrow effective concentration window, which hinders its application in shrimp live-keeping agents. This invention uses peppermint extract to synergize with low concentrations of eugenol to exert a highly effective sedative and anesthetizing effect, which can quickly induce deep dormancy in shrimp at low doses of eugenol, reduce the physiological activity level of shrimp during transportation, ensure anti-stress during transportation, and reduce energy consumption and physical damage during transportation. The scientific compounding of sodium chloride, magnesium sulfate, and zinc sulfate can regulate osmotic pressure and ion balance. Betaine can exert anti-inflammatory activity, while ascorbic acid can scavenge free radicals, reduce oxidative stress, and protect important tissues and organs from oxidative damage, so that shrimp have a healthy physiological state after recovery. The use of moisturizing and energy-providing liquid breaks through the limitations of single-formulation soaking treatment. The aloe vera gel extract can form a natural moisturizing layer on the shrimp's body surface, preventing moisture loss from the shrimp's gills and body surface. The combination of trehalose and sodium pyruvate provides energy support. Trehalose, as a stable energy source, is directly absorbed and utilized, while sodium pyruvate can act as a metabolic intermediate to rapidly generate energy through the tricarboxylic acid cycle. The two work synergistically to reduce the shrimp's own glycogen consumption, fundamentally avoiding acidosis caused by lactic acid accumulation. Chitosan provides antibacterial properties, creating a healthier environment for transportation and preventing the invasion of microorganisms in the transportation environment.

[0026] The shrimp waterless survival transport method provided by this invention adopts a phased treatment strategy of pretreatment to induce dormancy and continuous protection during transportation. This enables shrimp to enter a dormant state in a low-stress, mild environment. The shrimp survival agent treatment combined with oxygenation and low-temperature constant-temperature transportation provides continuous protection for shrimp during transportation, solving the problems of low survival rate, large damage, and poor vitality of shrimp after long-distance waterless transportation. Attached Figure Description

[0027] Figure 1 This is a graph showing the results of a two-factor experiment involving eugenol and peppermint extract;

[0028] Figure 2 The graph shows the muscle glycogen detection results of shrimp before simulated waterless transportation, after 12 hours of simulated waterless transportation, and after 24 hours of simulated waterless transportation in Application Example 3 and Comparative Application Examples 1-5.

[0029] Figure 3 The graph shows the results of muscle lactate detection in shrimp before, after 12 hours and 24 hours of simulated waterless transportation, in Application Example 3 and Comparative Application Examples 1-5.

[0030] Figure 4 The results show the detection results of malondialdehyde and superoxide dismutase in the hepatopancreas of shrimp in Application Example 3, Comparative Application Example 2, Comparative Application Example 3, and Comparative Application Example 5. Figure a shows the detection results of malondialdehyde, and Figure b shows the detection results of superoxide dismutase.

[0031] Figure 5 The results show the detection results of acid phosphatase and alkaline phosphatase in the hepatopancreas of shrimp used in Application Example 3 and Comparative Application Example 5. Figure a shows the detection results of acid phosphatase, and Figure b shows the detection results of alkaline phosphatase.

[0032] Figure 6 The results show the detection results of acid phosphatase and alkaline phosphatase in the hemolymph of shrimp used in Application Example 3 and Comparative Application Example 5. Figure a shows the detection results of acid phosphatase, and Figure b shows the detection results of alkaline phosphatase. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0034] Example 1: A method for preparing a shrimp survival agent, the specific steps of which are as follows:

[0035] S1. Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 52℃ for 24 hours. Crush the peppermint leaves and pass them through a 20-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 2L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. Distill for 4 hours and collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0036] S2. Preparation of Aloe Vera Gel Extract: Select fresh leaves of Aloe vera with thick leaves and free from pests and diseases. Rinse the aloe vera leaves three times with sterile water. Use a stainless steel tool to remove the epidermis of the leaves and take out the whole transparent mesophyll gel block, ensuring that the epidermis is completely removed. Use a fruit and vegetable crusher to initially crush the gel block to obtain a slurry. Use a high-pressure homogenizer for two-stage homogenization. The first stage pressure is set to 25 MPa and homogenize twice. The second stage pressure is set to 60 MPa and homogenize once. Centrifuge the homogenized slurry at 12,000 rpm for 3 minutes. Take the supernatant and filter it through a 5 μm filter membrane to obtain the aloe vera gel extract.

[0037] S3. Preparation of dormancy induction solution: Weigh 0.125g of eugenol, 150g of peppermint extract, 100g of sodium chloride, 5g of zinc sulfate, 25g of magnesium sulfate, 50g of betaine, and 0.5g of ascorbic acid, add them to 4.5L of deionized water, add 15g of Tween 80, stir well, and sonicate for 3 minutes to obtain the dormancy induction solution.

[0038] S4. Preparation of moisturizing and energizing solution: Weigh 100g of aloe vera gel extract, 50g of trehalose, 25g of sodium pyruvate, and 10g of chitosan, add them to 4.5L of deionized water, stir well, adjust the pH to 6.5, and sonicate for 3 minutes to obtain the moisturizing and energizing solution.

[0039] Example 2: A method for preparing a shrimp survival agent, the specific steps of which are as follows:

[0040] S1. Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 60℃ for 24 hours. Crush the peppermint leaves and pass them through a 40-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 3L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. Distill for 6 hours and collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0041] S2. Preparation of Aloe Vera Gel Extract: Select fresh leaves of Aloe vera with thick leaves and free from pests and diseases. Rinse the aloe vera leaves three times with sterile water. Use a stainless steel tool to remove the epidermis of the leaves and take out the whole transparent mesophyll gel block, ensuring that the epidermis is completely removed. Use a fruit and vegetable crusher to initially crush the gel block to obtain a slurry. Use a high-pressure homogenizer for two-stage homogenization. The first stage pressure is set to 35 MPa and homogenization is performed twice. The second stage pressure is set to 80 MPa and homogenization is performed once. Centrifuge the homogenized slurry at 10,000 rpm for 5 minutes. Take the supernatant and filter it through a 5 μm filter membrane to obtain the aloe vera gel extract.

[0042] S3. Preparation of dormancy induction solution: Weigh 0.25g of eugenol, 250g of peppermint extract, 250g of sodium chloride, 25g of zinc sulfate, 100g of magnesium sulfate, 100g of betaine, and 1.5g of ascorbic acid, add them to 4.5L of deionized water, add 75g of Tween 80, stir well, and sonicate for 5 minutes to obtain the dormancy induction solution.

[0043] S4. Preparation of moisturizing and energizing solution: Weigh 250g of aloe vera gel extract, 150g of trehalose, 75g of sodium pyruvate, and 25g of chitosan, add them to 4.75L of deionized water, stir well, adjust the pH to 6.5, and sonicate for 5 minutes to obtain the moisturizing and energizing solution.

[0044] Example 3: A method for preparing a shrimp survival agent, the specific steps of which are as follows:

[0045] S1. Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 60℃ for 20 hours. Crush the peppermint leaves and pass them through a 20-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 2L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. Distill for 6 hours and collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0046] S2. Preparation of Aloe Vera Gel Extract: Select fresh leaves of Aloe vera with thick leaves and free from pests and diseases. Rinse the aloe vera leaves three times with sterile water. Use a stainless steel tool to remove the epidermis of the leaves and take out the whole transparent mesophyll gel block, ensuring that the epidermis is completely removed. Use a fruit and vegetable crusher to initially crush the gel block to obtain a slurry. Homogenize the slurry in two stages using a high-pressure homogenizer. The first stage pressure is set to 35 MPa and homogenizes twice. The second stage pressure is set to 80 MPa and homogenizes once. Centrifuge the homogenized slurry at 12,000 rpm for 5 minutes. Take the supernatant and filter it through a 5 μm filter membrane to obtain the aloe vera gel extract.

[0047] S3. Preparation of dormancy induction solution: Weigh 0.25g of eugenol, 250g of peppermint extract, 150g of sodium chloride, 15g of zinc sulfate, 50g of magnesium sulfate, 100g of betaine, and 1.5g of ascorbic acid, add them to 4.5L of deionized water, add 50g of Tween 80, stir well, and sonicate for 5 minutes to obtain the dormancy induction solution.

[0048] S4. Preparation of moisturizing and energizing solution: Weigh 250g of aloe vera gel extract, 100g of trehalose, 50g of sodium pyruvate, and 15g of chitosan, add them to 4.5L of deionized water, stir well, adjust the pH to 6.5, and sonicate for 5 minutes to obtain the moisturizing and energizing solution.

[0049] Comparative Example 1: A method for preparing a shrimp survival agent, differing from Example 3 in that it does not include a moisturizing and energy-providing liquid. The specific steps are as follows:

[0050] S1. Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 60℃ for 20 hours. Crush the peppermint leaves and pass them through a 20-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 2L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. Distill for 6 hours and collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0051] S2. Preparation of dormancy induction solution: Weigh 0.25g of eugenol, 250g of peppermint extract, 150g of sodium chloride, 15g of zinc sulfate, 50g of magnesium sulfate, 100g of betaine, and 1.5g of ascorbic acid, add them to 4.5L of deionized water, add 50g of Tween 80, stir well, and sonicate for 5 minutes to obtain the dormancy induction solution.

[0052] Comparative Example 2: A method for preparing a shrimp survival agent, differing from Example 3 in that no peppermint extract was added. The specific steps are as follows:

[0053] S1. Preparation of Aloe Vera Gel Extract: Select fresh leaves of Aloe Vera with thick leaves and free from pests and diseases. Rinse the aloe vera leaves three times with sterile water. Use a stainless steel tool to remove the epidermis of the leaves and take out the whole transparent pulp gel block, ensuring that the epidermis is completely removed. Use a fruit and vegetable crusher to initially crush the gel block to obtain a slurry. Use a high-pressure homogenizer for two-stage homogenization. The first stage pressure is set to 35 MPa and homogenize twice. The second stage pressure is set to 80 MPa and homogenize once. Centrifuge the homogenized slurry at 12,000 rpm for 5 minutes. Take the supernatant and filter it through a 5 μm filter membrane to obtain the aloe vera gel extract.

[0054] S2. Preparation of dormancy induction solution: Weigh 0.25g of eugenol, 150g of sodium chloride, 15g of zinc sulfate, 50g of magnesium sulfate, 100g of betaine, and 1.5g of ascorbic acid and add them to 4.5L of deionized water. Add 0.05g of Tween 80, stir well, and sonicate for 5 minutes to obtain the dormancy induction solution.

[0055] S3. Preparation of moisturizing and energizing solution: Weigh 250g of aloe vera gel extract, 100g of trehalose, 50g of sodium pyruvate, and 15g of chitosan, add them to 4.5L of deionized water, stir well, adjust the pH to 6.5, and sonicate for 5 minutes to obtain the moisturizing and energizing solution.

[0056] Comparative Example 3: A method for preparing a shrimp survival agent, differing from Example 3 in that aloe vera gel extract was not added. The specific steps are as follows:

[0057] S1. Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 60℃ for 20 hours. Crush the peppermint leaves and pass them through a 20-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 2L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. Distill for 6 hours and collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0058] S2. Preparation of dormancy induction solution: Weigh 0.25g of eugenol, 250g of peppermint extract, 150g of sodium chloride, 15g of zinc sulfate, 50g of magnesium sulfate, 100g of betaine, and 1.5g of ascorbic acid, add them to 4.5L of deionized water, add 50g of Tween 80, stir well, and sonicate for 5 minutes to obtain the dormancy induction solution.

[0059] S3. Preparation of moisturizing and energy-supplying solution: Weigh 100g of trehalose, 50g of sodium pyruvate and 15g of chitosan and add them to 4.5L of deionized water. Stir well, adjust the pH to 6.5, and sonicate for 5 minutes to obtain the moisturizing and energy-supplying solution.

[0060] Comparative Example 4: A method for preparing a shrimp survival agent, differing from Example 3 in that trehalose and sodium pyruvate were not added. The specific steps are as follows:

[0061] S1. Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 60℃ for 20 hours. Crush the peppermint leaves and pass them through a 20-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 2L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. Distill for 6 hours and collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0062] S2. Preparation of Aloe Vera Gel Extract: Select fresh leaves of Aloe vera with thick leaves and free from pests and diseases. Rinse the aloe vera leaves three times with sterile water. Use a stainless steel tool to remove the epidermis of the leaves and take out the whole transparent mesophyll gel block, ensuring that the epidermis is completely removed. Use a fruit and vegetable crusher to initially crush the gel block to obtain a slurry. Homogenize the slurry in two stages using a high-pressure homogenizer. The first stage pressure is set to 35 MPa and homogenizes twice. The second stage pressure is set to 80 MPa and homogenizes once. Centrifuge the homogenized slurry at 12,000 rpm for 5 minutes. Take the supernatant and filter it through a 5 μm filter membrane to obtain the aloe vera gel extract.

[0063] S3. Preparation of dormancy induction solution: Weigh 0.25g of eugenol, 250g of peppermint extract, 150g of sodium chloride, 15g of zinc sulfate, 50g of magnesium sulfate, 100g of betaine, and 1.5g of ascorbic acid, add them to 4.5L of deionized water, add 50g of Tween 80, stir well, and sonicate for 5 minutes to obtain the dormancy induction solution.

[0064] S4. Preparation of moisturizing and energizing solution: Weigh 250g of aloe vera gel extract and 15g of chitosan, add them to 4.5L of deionized water, stir well, adjust the pH to 6.5, and sonicate for 5 minutes to obtain the moisturizing and energizing solution.

[0065] Application Example 1: A method for waterless live transport of shrimp, using the shrimp preservative from Example 1 for treatment, the specific steps are as follows:

[0066] Healthy shrimp were selected and kept in seawater without food for 24 hours while maintaining oxygenation. The seawater containing the shrimp was cooled to 10°C at a rate of 1°C / h, and the dormancy induction solution was pre-cooled to 10°C. The shrimp were immersed in the dormancy induction solution for 3 minutes to induce a state of suspended animation. A sponge was soaked in a moisturizing and energy-providing solution until fully moistened, and the shrimp were dipped in the solution for 5 seconds and then placed on the moist sponge. The sponge and shrimp were placed in a transport box, filled with pure oxygen, and sealed. The shrimp were then transported in a constant temperature environment of 12°C without water. After the transport, the shrimp were removed and placed in clean water at 18°C ​​to allow them to fully recover.

[0067] Application Example 2: A method for waterless live transport of shrimp, using the shrimp preservative from Example 2 for treatment, the specific steps are as follows:

[0068] Healthy shrimp were selected and kept in seawater without food for 48 hours while maintaining oxygenation. The seawater containing the shrimp was cooled to 12°C at a rate of 1°C / h, and the dormancy induction solution was pre-cooled to 12°C. The shrimp were immersed in the dormancy induction solution for 5 minutes to induce a state of suspended animation. A sponge was soaked in a moisturizing and energy-providing solution until fully moistened, and the shrimp were dipped in the solution for 5 seconds and then placed on the moist sponge. The sponge and shrimp were placed in a transport box, filled with pure oxygen, and sealed. The shrimp were then transported in a constant temperature environment of 15°C without water. After the transport, the shrimp were removed and placed in clean water at 22°C to allow them to fully recover.

[0069] Application Example 3: A method for waterless live transport of shrimp, using the shrimp preservative from Example 3 for treatment, the specific steps are as follows:

[0070] Healthy shrimp were selected and kept in seawater without food for 48 hours while maintaining oxygenation. The seawater containing the shrimp was cooled to 10°C at a rate of 1°C / h, and the dormancy induction solution was pre-cooled to 10°C. The shrimp were immersed in the dormancy induction solution for 5 minutes to induce a state of suspended animation. A sponge was soaked in a moisturizing and energy-providing solution until fully moistened, and the shrimp were dipped in the solution for 5 seconds and then placed on the moist sponge. The sponge and shrimp were placed in a transport box, filled with pure oxygen, and sealed. The shrimp were then transported in a constant temperature environment of 12°C without water. After the transport, the shrimp were removed and placed in clean water at 22°C to allow them to fully recover.

[0071] Comparative Application Example 1: A method for waterless live transport of shrimp, using the shrimp preservative from Comparative Example 1 for treatment, the specific steps are as follows:

[0072] Healthy shrimp were selected and kept in seawater without food for 48 hours while maintaining oxygenation. The seawater containing the shrimp was cooled to 10°C at a rate of 1°C / h, and the dormancy induction solution was pre-cooled to 10°C. The shrimp were then immersed in the dormancy induction solution for 5 minutes to induce a state of suspended animation. A sponge was soaked in physiological saline to fully moisten it, and the shrimp were placed on the moistened sponge. The sponge and shrimp were placed in a transport box, filled with pure oxygen, and sealed. The shrimp were then transported in a constant temperature environment of 12°C without water. After the transport, the shrimp were removed and placed in clean water at 22°C to allow them to fully recover.

[0073] Comparative Application Example 2: A method for waterless live transport of shrimp, using the shrimp preservative of Comparative Example 2 for treatment, with specific steps consistent with Application Example 3.

[0074] Comparative Application Example 3: A method for waterless live transport of shrimp, using the shrimp preservative of Comparative Example 3 for treatment, with specific steps consistent with Application Example 3.

[0075] Comparative Application Example 4: A method for waterless live transport of shrimp, using the shrimp preservative of Comparative Example 4 for treatment, with specific steps consistent with Application Example 3.

[0076] Comparative Application Example 5: A method for waterless live transport of shrimp, using physiological saline as a shrimp preservative. The specific steps are as follows: Select healthy shrimp and feed them in seawater for 48 hours while maintaining oxygenation; cool the seawater containing the shrimp to 10°C at a rate of 1°C / h, and pre-cool the physiological saline to 10°C; immerse the shrimp in the physiological saline for 5 minutes to induce a state of suspended animation due to the low temperature; soak a sponge in the physiological saline until fully moistened, and place the shrimp on the moistened sponge; place the sponge and shrimp into a transport box, fill it with pure oxygen, seal it, and conduct waterless live transport in a constant temperature environment of 12°C; after transport, remove the shrimp and place them in clean water at 22°C to allow them to fully recover.

[0077] Study on the synergistic induction of deep dormancy in shrimp by eugenol and peppermint extract

[0078] Eugenol is a commonly used anesthetic for fish with significant anesthetic effects; however, in the application of shrimp transport and preservation, eugenol suffers from a narrow effective dose window and a high effective dose concentration: too low a dose leads to insufficient anesthesia and inadequate dormancy, while too high a dose may cause difficulties in resuscitation or safety issues. Therefore, this experiment uses peppermint extract to compensate for the above problems of eugenol, and determines the optimal ratio of eugenol to peppermint extract through concentration gradient experiments.

[0079] Preparation of peppermint extract: Select clean peppermint leaves that are free from mold and have a strong aroma. Dry the peppermint leaves at 60℃ for 20 hours. Crush the peppermint leaves and pass them through a 20-mesh sieve to obtain peppermint powder. Mix 200g of peppermint powder with 2L of water and place it in a distillation apparatus. Heat and evaporate the mixture, maintaining a steady steam flow. After distilling for 6 hours, collect the distillate. Place the distillate in an oil-water separator for oil-water separation. Filter the mixture and collect the oil phase extract, which is the peppermint extract.

[0080] Peppermint extract and eugenol were compounded and added at mass fractions of 0.02, 0.025, 0.05, and 0.1 parts eugenol, 20, 30, 50, and 80 parts peppermint extract, respectively. A control group with 0 parts peppermint extract was also included. 900 parts deionized water were added uniformly, and Tween 80 was added at 20% of the total mass of eugenol and peppermint extract. The mixture was centrifuged at 12,000 rpm for 5 minutes, and the solution was stirred at high speed to ensure homogeneity. This solution was used as a shrimp anesthetic and was prepared fresh for each use.

[0081] A two-factor orthogonal experiment was conducted, and the experimental group design is shown in Table 1:

[0082] Table 1. Two-factor orthogonal experimental group design

[0083]

[0084] The two-factor experimental reagents were prepared according to Table 1 and used as shrimp anesthetics in the experiment. Healthy shrimp were selected and divided into groups of 20 shrimp per group. The shrimp were first kept in seawater without food for 48 hours with oxygenation. The seawater where the shrimp were kept was cooled to 10°C at a rate of 1°C / h, and the prepared shrimp anesthetic was pre-cooled to 10°C. The shrimp were then immersed in the shrimp anesthetic for 10 minutes. A sponge was soaked in physiological saline to fully moisten it, and the shrimp were placed on the moistened sponge. The sponge and shrimp were placed in a transport box, filled with pure oxygen and sealed. The shrimp were transported in a constant temperature environment of 12°C without water for survival. After the transport, the shrimp were removed and placed in clean water at 22°C. The shrimp were then fed normally for 48 hours, and their condition was observed and the survival rate was calculated. The results are as follows: Figure 1 As shown.

[0085] from Figure 1 The results show that the shrimp survival rates in groups B and C were higher, indicating that adding 30 and 50 parts of peppermint extract resulted in the best waterless transport effect for shrimp. Figure 1The study also showed that the highest shrimp survival rate and best anesthesia effect were observed when the eugenol content was 0.025 and 0.05 parts by weight. Based on the above data, it can be concluded that when the eugenol content is 0.025–0.05 parts by weight, the peppermint extract content is 30–50 parts by weight, and the deionized water content is 900 parts by weight, the shrimp survival rate is highest after anesthesia, achieving the best anesthesia effect.

[0086] Eugenol, an anesthetic for fish, is rarely used in the anhydrous transport of shrimp because its effective concentration window is small. Improper concentration control can lead to shrimp mortality during anhydrous transport. Experiments showed that without peppermint extract, shrimp at 0.02–0.05 parts eugenol experienced awakening during anhydrous transport, resulting in stress and death. This indicates that 0.02–0.05 parts eugenol is insufficient for deep anesthesia. However, this study demonstrates that by combining eugenol with an appropriate amount of peppermint extract, effective anesthesia can be achieved even at low doses, resulting in good post-resuscitation activity and significantly reducing mortality.

[0087] Eugenol regulates the GABA neurotransmitter in the central nervous system, making it difficult for neurons to generate excitatory action potentials, thus hindering the generation and transmission of nerve signals. Eugenol can also affect the generation of action potentials by influencing sodium ion channels, thereby producing an anesthetic effect. The main component of peppermint extract is menthol, which can activate TRPM8 ion channels. Continuous stimulation leads to neuronal desensitization, paralyzing the nervous system and reducing the body's response to external stimuli. Eugenol and menthol affect the shrimp's nervous system at different levels and through different targets. The addition of peppermint extract allows eugenol to achieve deep dormancy in shrimp even at extremely low dosages.

[0088] Shrimp from Application Examples 1-3 and Control Examples 1-5 were subjected to simulated waterless transportation for 12 hours and 24 hours. After recovery, the following tests were performed:

[0089] (1) Survival rate and recovery time

[0090] Shrimp from Application Examples 1-3 and Comparative Application Examples 1-5 were subjected to 24 hours of simulated waterless transportation and given corresponding resuscitation treatments. The survival rate and time to full recovery were recorded. Full recovery was characterized by the shrimp being able to stand upright with their backs facing up and abdomens facing down, and upon stimulation, they could quickly contract their abdominal limbs to jump and swim in a directional manner, with their bodies appearing semi-transparent.

[0091] The test results are shown in Table 2:

[0092] Table 2. Survival rate and recovery time of shrimp after waterless transport

[0093]

[0094] As can be seen from Table 2, the shrimp survival rate of Application Examples 1-3 was all above 95%, indicating that the shrimp survival agent prepared by the method of the present invention can effectively ensure the survival rate of shrimp during 24-hour waterless transportation, and the average recovery time is only 4.5 minutes. In contrast, the shrimp survival rate of Comparative Application Examples 1-5 was lower, and the recovery time was longer.

[0095] Comparative Application Example 1, without the use of a moisturizing and energy-supplying solution, resulted in a survival rate of only 70% and a recovery time as long as 11 minutes. The lack of the moisturizing, nutrient-supplying, and energy-supplying functions of the solution affected the shrimp's basic life activities during transportation. Comparative Application Example 2, without the addition of peppermint extract, which promotes anesthesia and sedation, may have prevented the shrimp from achieving deep anesthesia, affecting the final survival rate. Comparative Application Example 3, without the addition of aloe vera gel extract, lacked long-lasting moisturizing and oxygen supply, affecting the shrimp's basic life activities. Comparative Application Example 4, without the addition of trehalose and sodium pyruvate, lacked exogenous energy substrates, causing the shrimp to tend to deplete their own energy, leading to excessive energy consumption and an inability to maintain basic life activities. Comparative Application Example 5, using physiological saline, resulted in the lowest shrimp survival rate and the longest recovery time after transportation. These results fully demonstrate the advantages of the waterless shrimp preservation and transportation method of this invention.

[0096] (2) Detection of muscle physicochemical indicators

[0097] Shrimp were randomly sampled from three groups: shrimp before simulated waterless transport (0h), shrimp after simulated waterless transport for 12h (12h), and shrimp after simulated waterless transport for 24h (24h). The abdominal segments were shelled, the tail muscle was removed with tweezers, and 100mg of muscle tissue was weighed and flash-frozen in liquid nitrogen for subsequent lactic acid and glycogen detection. Another 100mg of muscle tissue was used to detect pH.

[0098] Muscle pH determination: The weighed muscle sample was added to 9 times its volume of deionized water, homogenized in an ice bath, centrifuged at 4000 rpm for 10 min at 4℃, the supernatant was collected, and the pH value was immediately measured and recorded using a pH meter; the test results are shown in Table 3:

[0099] Table 3. Results of pH test on shrimp muscle

[0100]

[0101] Muscle glycogen assay: Take 100 mg of muscle tissue, add 2 mL of 30% potassium hydroxide solution, boil in a water bath for 20 min to completely dissolve the tissue, cool, add 4 mL of 95% ethanol, mix thoroughly, centrifuge at 3000 rpm for 10 min, discard the supernatant, dissolve the precipitate with 2 mL of distilled water, perform anthrone reagent color development, and measure the absorbance to calculate the glycogen content; the test results are as follows. Figure 2 As shown.

[0102] Muscle lactate assay: Take 100 mg of muscle tissue, add 9 times its weight of physiological saline, homogenize in an ice bath, centrifuge at 3500 rpm for 10 min at 4℃, and collect the supernatant for detection; use a kit to detect lactate and calculate the lactate content; the test results are as follows. Figure 3 As shown.

[0103] The results show that before the simulated waterless transport, the muscle pH of all shrimp groups was within the normal range. After simulating 12h and 24h of waterless transport, the muscle pH of shrimp in Application Examples 1-3 remained above 7, within the normal range, indicating that the dual measures significantly reduced lactic acid production and maintained the normal acid-base balance of shrimp muscle. However, in contrast, after 24h of simulated waterless transport, the pH of shrimp in Application Examples 1-5 all dropped below 7, indicating that lactic acid was produced in the muscle, the acid-base balance was disrupted, and metabolic acidosis occurred, thus affecting the survival rate and recovery ability of shrimp.

[0104] Glycogen in muscles is readily fermented into lactic acid under anaerobic conditions. The continuous decline in muscle glycogen may be due to the shrimp's secretion of large amounts of adrenaline and noradrenaline under low-temperature conditions, which causes more glycogen to be broken down to provide glucose and generate heat to resist the survival environment. This invention uses a moisturizing and energy-supplying liquid to treat the shrimp, playing a dual role of moisturizing and energy supply, reducing stress and energy consumption of the shrimp, and using trehalose and sodium pyruvate as exogenous energy substrates to replace and conserve the consumption of its own glycogen, avoiding energy depletion and reducing anaerobic metabolism and acidosis.

[0105] (3) Detection of liver physicochemical indicators

[0106] Shrimp were randomly sampled from three groups: shrimp before simulated waterless transport (0h), shrimp after simulated waterless transport for 12h (12h), and shrimp after simulated waterless transport for 24h (24h). The complete hepatopancreas was dissected and removed, rinsed thoroughly with pre-cooled physiological saline to remove blood, blotted dry with filter paper, and 50mg of tissue was weighed. Nine times the volume of pre-cooled physiological saline was added, and the mixture was homogenized. The homogenate was centrifuged at 3000rpm for 10min at 4℃, and the supernatant was collected. Malondialdehyde (MDA) and superoxide dismutase (SOD) were detected using a kit. The results are shown below. Figure 4As shown in the figure, Figure a shows the detection results of malondialdehyde; Figure b shows the detection results of superoxide dismutase.

[0107] Malondialdehyde (MDA) and superoxide dismutase (SOD) can assess the level of oxidative stress in the body. MDA reflects the degree of free radical production and lipid oxidation; severe lipid peroxidation of cell membranes can damage liver tissue, thus affecting normal survival. SOD reflects the body's ability to scavenge free radicals; a decrease in SOD levels indicates impaired free radical scavenging ability, which can affect normal metabolism and physiological activities. Figure 4 As can be seen, the malondialdehyde (MDA) content in Application Example 3 remained at a low level, while the MDA content in the Comparative Application Example continued to increase, indicating that free radicals accumulated continuously in the body and lipid oxidation was severe. The superoxide dismutase (SOD) content in Application Example 3 did not show a significant downward trend, indicating that the body's antioxidant capacity remained at a high level, ensuring the normal physiological activities of the shrimp.

[0108] (4) Detection of immune system indicators

[0109] Shrimp were randomly sampled before simulated waterless transport (0h), after 12h simulated waterless transport (12h), and after 24h simulated waterless transport (24h). The hepatopancreas sampling method was the same as the liver physicochemical index detection experiment. Lymphatic blood sampling involved puncturing the sinusoids with a sterile syringe and extracting hemolymph, centrifuging at 3500rpm for 10min at 4℃, and collecting the supernatant for analysis. Alkaline phosphatase and acid phosphatase were detected in the hepatopancreas and hemolymph samples. The results of the hepatopancreas sample analysis are shown below. Figure 5 As shown in the figure, Figure a represents the acid phosphatase test results, and Figure b represents the alkaline phosphatase test results; the test results of the hemolymph sample are as follows. Figure 6 As shown in the figure, Figure a represents the results of acid phosphatase detection, and Figure b represents the results of alkaline phosphatase detection.

[0110] The hepatopancreas is the core immune organ of shrimp, and the hemolymph is the main site of immune response. Immune-related enzymes in the hepatopancreas and hemolymph reflect the shrimp's immune capacity. Acid phosphatase and alkaline phosphatase are key indicators for measuring immune capacity: acid phosphatase is a marker enzyme of lysosomes; higher acid phosphatase levels indicate stronger cellular immunity in shrimp. Alkaline phosphatase represents the shrimp's immune regulation and basal metabolic capacity. As shown in the figure, during anhydrous transport, the levels of acid phosphatase and alkaline phosphatase in Comparative Application Example 5 decreased significantly, indicating significant inhibition of both immune capacity and basal metabolic capacity. However, while the levels of acid phosphatase and alkaline phosphatase in the hepatopancreas of Application Example 3 decreased, they remained at relatively high levels. Notably, the levels of acid phosphatase and alkaline phosphatase in the hemolymph of Application Example 3 remained consistently high, even exceeding initial values, indicating that its immune system was highly active and not subject to immunosuppression during transport, thus maintaining the shrimp's health and contributing to its survival.

[0111] (5) Detection of eugenol residue

[0112] Using shrimp from Application Example 1, after resuscitation, they were immersed in seawater at 22°C for 1 hour, 3 hours, and 6 hours as the experimental group; fresh, physiologically healthy, untreated shrimp were used as the blank control group; the sampling site was shrimp muscle tissue, the shell was removed, and the edible abdominal tail muscle was accurately taken; the muscle tissue was thoroughly homogenized using a homogenizer and stored in a -20°C refrigerator for later use; the detection process was carried out in accordance with GB31656.6-2021 "National Food Safety Standard for Determination of Eugenol Residues in Aquatic Products by Gas Chromatography-Mass Spectrometry".

[0113] Shrimp treated with the preservative agent of this invention showed that after soaking in seawater for 1 hour, the residual amount of eugenol in the muscle was below the quantitative detection limit of the method, and eugenol was not detected. In the anhydrous preservative transportation method for shrimp provided by this invention, the concentration of eugenol in the shrimp is low after transportation, reducing the potential food safety problem of eugenol residue. This result fully demonstrates that the eugenol used in this invention is rapidly metabolized in shrimp and has no accumulation, and the shrimp products treated according to the method of this invention fully meet the requirements of national food safety standards.

[0114] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for waterless live transport of shrimp, characterized in that, Includes the following steps: S1. Select healthy shrimp and keep them in seawater without feeding for 24-48 hours, while maintaining oxygenation; S2. Cool the seawater where the shrimp are located to 10~12℃ at a rate of 1℃ / h, and add the dormancy induction solution. Pre-cool to 10-12℃; immerse the shrimp in the dormancy induction solution for 3-5 minutes to induce all shrimp to enter a state of suspended animation. S3. Soak the sponge in the moisturizing and energy-generating solution until it is fully moistened. Dip the shrimp in the moisturizing and energy-generating solution and take them out, then place them on the moistened sponge. S4. Place the moistened sponge and shrimp into a transport box, fill it with pure oxygen and seal it. Transport the shrimp in a constant temperature environment of 12~15℃ without water to keep them alive. S5. After transportation, remove the shrimp and place them in clean water at 18~22℃ until they are fully recovered. The dormancy induction solution comprises: 0.025-0.05 parts eugenol, 30-50 parts peppermint extract, 20-50 parts sodium chloride, 1-5 parts zinc sulfate, 5-20 parts magnesium sulfate, 10-20 parts betaine, 0.1-0.3 parts ascorbic acid, and 900 parts deionized water; the dormancy induction solution also includes Tween 80, wherein the amount of Tween 80 added is 10%-30% of the total mass of eugenol and peppermint extract. The method for preparing the peppermint extract is as follows: dry peppermint leaves, crush them, and pass them through a 20-40 mesh sieve to obtain peppermint powder; mix the peppermint powder with water, perform steam distillation, collect the distillate for oil-water separation, filter, collect the oil phase, and obtain the peppermint extract. The moisturizing and energy-providing liquid comprises: 2-5 parts aloe vera gel extract, 1-3 parts trehalose, 0.5-1.5 parts sodium pyruvate, 0.2-0.5 parts chitosan, and 90-95 parts deionized water; The preparation method of the aloe vera gel extract is as follows: the aloe vera leaves are rinsed with sterile water, the green epidermis of the leaves is removed, and the gel block is taken out; the gel block is crushed using a crusher to obtain a slurry; the slurry is homogenized in two stages using a high-pressure homogenizer, the homogenized slurry is centrifuged, and the supernatant is filtered through a 5μm filter membrane to obtain the aloe vera gel extract.

2. The method for waterless live transport of shrimp according to claim 1, characterized in that, The method for preparing the dormancy induction solution is as follows: weigh eugenol, peppermint extract, sodium chloride, zinc sulfate, magnesium sulfate, betaine, ascorbic acid and Tween 80, add them to deionized water, stir evenly, and sonicate for 3-5 minutes to obtain the dormancy induction solution.

3. The method for waterless live transport of shrimp according to claim 1, characterized in that, The ratio of peppermint powder to water is 10-15 mL of water per gram of peppermint powder; the steam distillation time is 4-6 hours.

4. The method for waterless live transport of shrimp according to claim 1, characterized in that, The preparation method of the moisturizing and energy-providing liquid is as follows: weigh aloe vera gel extract, trehalose, sodium pyruvate and chitosan, add them to deionized water, stir evenly, adjust the pH to 6.5, and sonicate for 3-5 minutes to obtain the moisturizing and energy-providing liquid.

5. The method for waterless live transport of shrimp according to claim 1, characterized in that, The specific steps of the two-stage homogenization are as follows: high-pressure homogenization is performed using a high-pressure homogenizer. The first-stage pressure is set to 25~35MPa, and homogenization is performed twice. The second-stage pressure is set to 60~80MPa, and homogenization is performed once. The centrifugation step is centrifugation at 10000~12000rpm for 3~5min.

6. A shrimp survival agent, characterized in that, Includes the dormancy induction solution and moisturizing energy supply solution in the method for waterless live transport of shrimp as described in claim 1.

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