A kind of shrimp fry micro-particle immune enhanced feed and its preparation method

Abstract

The application discloses a kind of to shrimp fry microgranule immunity enhancement feed and preparation method thereof, the feed includes the following raw materials: high gluten flour, pre-gelatinized starch, enzymatic fish solution, krill powder, refined fish oil, earthworm powder, schizochytrium sp. Powder, soybean lecithin, marine red yeast, sodium alginate, brown algae powder, coffee residue fermentation, saltmarsh rush extract, cat's claw extract, mineral premix, vitamin premix, molting hardener, natural mixed tocopherol, rosemary extract, citric acid, cinnamon essential oil, wherein coffee residue fermentation, saltmarsh rush extract, cat's claw extract and the like are compounded into immunity enhancement component, and molting hardener is compounded by horsetail extract and alfalfa extract.The application is synergized by the triple immune barrier of "bacteriostasis-anti-stress-antiviral" and the molting hardening mechanism of "silicon-sterol" double path, combined with slow-release coating process, which significantly improves the survival rate and growth uniformity of shrimp fry.

Description

Technical Field

[0001] This invention relates to the field of feed and its preparation technology, specifically to a microparticle immune-enhancing feed for shrimp larvae and its preparation method. Background Technology

[0002] Shrimp farming is a vital pillar of my country's aquaculture economy, and the health management of the larval stage (typically PL5-PL20) directly determines the survival rate and economic benefits during the rearing period. This stage is a critical period for shrimp larvae, transitioning from endogenous nutrition to exogenous feeding. They exhibit physiological characteristics such as short intestines, low digestive enzyme activity, an immature immune system, and frequent molting. Simultaneously, the high-density environment of the larval ponds easily leads to water quality deterioration, resulting in infections by pathogens such as Vibrio, white spot syndrome virus, and Enterocytozoa, causing shrimp mortality. However, most existing micro-particle feeds only focus on basic nutrition and lack targeted immune-enhancing components, failing to meet the multiple needs of shrimp larvae for disease resistance, stress resistance, and molting hardening, thus resulting in low larval survival rates and poor growth uniformity. Summary of the Invention

[0003] In view of this, the present invention proposes a microparticle immune-enhancing feed for shrimp larvae and its preparation method to solve the above problems.

[0004] The technical solution of this invention is implemented as follows:

[0005] A microparticle-based immune-enhancing feed for shrimp larvae comprises the following ingredients in parts by weight: 35.0-65.0 parts high-gluten flour, 10.0-15.0 parts pregelatinized starch, 15.0-20.0 parts enzymatically hydrolyzed fish lysate, 5.0-10.0 parts krill meal, 4.0-6.0 parts refined fish oil, 3.0-5.0 parts earthworm meal, 2.0-4.0 parts Schizochyptella powder, 2.5-3.5 parts soybean lecithin, 1.0-3.0 parts marine red yeast, 1.5-3.0 parts sodium alginate, and 1.0-2... 0.0 parts brown algae powder, 1.0-2.0 parts coffee grounds ferment, 0.2-0.5 parts glycerin, 0.1-0.3 parts Salicornia glutinosa extract, 0.1-0.3 parts Cat's Claw vine extract, 1.0-2.0 parts mineral premix, 0.5-1.0 parts vitamin premix, 0.2-0.5 parts molting and hardening agent, 0.05-0.10 parts natural mixed tocopherols, 0.04-0.06 parts rosemary extract, 0.05-0.10 parts citric acid, 0.05-0.10 parts cinnamon essential oil.

[0006] Furthermore, the coffee grounds ferment is prepared by the following method: dried coffee grounds are soaked in water for 1.5-2.5 hours, then the coffee grounds are removed and steam-sterilized at 115-125℃ for 15-20 minutes. A suspension of Aspergillus oryzae spores (3.042) is inoculated at 5-10% of the coffee grounds mass, with a spore suspension concentration of 10. 6-10 7 Add spores / mL, then add 3-5% glucose and 0.3-0.6% sodium nitrate (by weight of coffee grounds), mix well, and adjust the pH to 4.5-5.0 with lactic acid to obtain the fermentation material. Place the fermentation material in a fermentation tank, cover the top of the fermentation tank with gauze, and then ferment at 30-35℃ for 2-3 days, stirring once every 12 hours for 5-10 minutes each time. Then ferment at 30-35℃ for 3-5 days, stirring once every 24 hours for 5-10 minutes each time. Then ferment at 28-30℃ for 2-4 days, stirring once every 48 hours for 5-10 minutes each time. After fermentation, dry the material at 60-70℃ for 6-10 hours, and then pulverize it through an 80-120 mesh sieve to obtain the coffee grounds fermentation product.

[0007] Furthermore, the *Salvia splendens* extract is prepared by the following method: Fresh *Salvia splendens* is rinsed 3-4 times with clean water, dried at 60-70℃ until the moisture content is ≤10%, and then pulverized through a 30-50 mesh sieve to obtain *Salvia splendens* powder; the *Salvia splendens* powder is added to purified water at a material-to-liquid ratio of 1:10-15 (material-to-liquid ratio unit: g / mL), citric acid is added to adjust the pH to 5.5-6.0, and extraction is carried out in a water bath at 80-90℃ for 2-3 hours. After extraction, the mixture is filtered through an 80-120 mesh filter cloth, the filtrate is collected, the residue is extracted once more, the two filtrates are combined, and the combined filtrate is... Concentrate the *Salvia splendens* extract under reduced pressure at 60-70℃ to 20-25% of its original volume to obtain a concentrated *Salvia splendens* solution. Add 95% ethanol to the concentrated *Salvia splendens* solution at a volume ratio of 1:3-5, let it stand at 3-5℃ for 10-15 hours, then centrifuge at 3500-4500 rpm for 12-18 minutes, collect the supernatant, then concentrate the supernatant under reduced pressure at 50-60℃ until there is no ethanol odor, then vacuum dry at 55-65℃ and -0.08 to -0.10 until the water content is ≤8%, then pulverize and pass through an 80-120 mesh sieve to obtain the *Salvia splendens* extract.

[0008] Furthermore, the extract of *Cat's Claw Vine* is prepared by the following method: Dried *Cat's Claw Vine* stems are cut into 2-3 cm segments, pulverized, and passed through a 30-50 mesh sieve to obtain *Cat's Claw Vine* powder; the *Cat's Claw Vine* powder is added to purified water at a material-to-liquid ratio of 1:4-6 (g / mL), citric acid is added to adjust the pH to 4.5-5.0, 0.1% (by weight of *Cat's Claw Vine*) of cellulase is added, and enzymatic hydrolysis is carried out at 48-52℃ for 1.5-2.0 h, followed by enzyme inactivation at 85-90℃ for 9-11 min, and then cooled to ≤60℃ to obtain *Cat's Claw Vine* enzymatic hydrolysate; the *Cat's Claw Vine* enzymatic hydrolysate is added to 65% ethanol at a volume ratio of 1:9-11, and heated in a water bath at 60-65℃. Heat extraction for 2-2.5 hours. After extraction, filter through an 80-120 mesh filter cloth and collect the filtrate. Concentrate the filtrate under reduced pressure at 50-60℃ until no ethanol odor remains, obtaining a concentrated cat's claw vine extract. Add 1.5-2.5 times the volume of purified water to the cat's claw vine extract, stir well, and let stand at 3-5℃ for 10-15 hours. Then centrifuge at 3500-4500 rpm for 12-18 minutes and collect the supernatant. Concentrate the supernatant at 50-60℃ to 15-25% of its original volume, and then vacuum dry at 50-60℃ and -0.08 to -0.10 MPa until the water content is ≤8%. Finally, pulverize and pass through an 80-120 mesh sieve to obtain the cat's claw vine extract.

[0009] Furthermore, the molting and hardening agent is composed of horsetail extract and alfalfa extract in a mass ratio of 0.5-1:1.

[0010] Furthermore, the horsetail extract was prepared by the following method: The whole horsetail herb was pulverized and passed through a 30-50 mesh sieve to obtain horsetail powder; the horsetail powder was added to a 0.1-0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:10-15 (material-to-liquid ratio in g / mL), and extracted in a water bath at 80-90℃ for 2-3 hours. After extraction, the solution was filtered through an 80-120 mesh filter cloth, and the filtrate was collected; the residue was added to a 0.1-0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:7-9, and extracted again at the same temperature for 1.5-2.0 hours, and the two filtrates were combined; food-grade calcium carbonate was added to the combined filtrate, stirred evenly, and the pH was adjusted to 6.5-7.0. The solution was allowed to stand at 25-30℃ for 1.0-1.5 hours, then filtered through a 150-200 mesh filter cloth, and the filtrate was collected. The filtrate was concentrated under reduced pressure at 60-70℃ to 20-25% of its original volume to obtain a concentrated solution. 95% ethanol was added to the concentrated solution at a volume ratio of 1:3-5, stirred evenly, and allowed to stand at 3-5℃ for 10-15 hours. The solution was then centrifuged at 3500-4500 rpm for 12-18 minutes, and the supernatant was collected. The supernatant was concentrated at 50-60℃ to 15-25% of its original volume, and then vacuum dried at 50-60℃ and -0.08 to -0.10 MPa until the water content was ≤8%. The solution was then pulverized and passed through an 80-120 mesh sieve to obtain the horsetail extract.

[0011] Furthermore, the alfalfa extract was prepared by the following method: dried alfalfa whole herb was pulverized and passed through a 30-50 mesh sieve to obtain alfalfa powder; the alfalfa powder was added to 65% ethanol at a material-to-liquid ratio of 1:8-12, and extracted in a water bath at 60-70℃ for 2.0-2.5 hours. After extraction, the mixture was filtered through an 80-120 mesh filter cloth, and the filtrate was collected; the residue was added to 65% ethanol at a material-to-liquid ratio of 1:7-9, and extracted again at the same temperature for 1.5-2.0 hours. The two filtrates were combined; the combined filtrate was then subjected to a 50°C water bath. Concentrate under reduced pressure at -60℃ until no ethanol odor remains, obtaining a concentrated solution. Add 2.5-3.5 times the volume of purified water to the concentrated solution, stir well, and let stand at 3-5℃ for 10-15 hours. Then centrifuge at 3500-4500 rpm for 12-18 minutes and collect the supernatant. Concentrate the supernatant under reduced pressure at 50-60℃ to 15-25% of the original volume, and then vacuum dry at 55-65℃ and -0.08 to -0.10 MPa until the water content is ≤8%. Finally, pulverize the shavings through an 80-120 mesh sieve to obtain the alfalfa extract.

[0012] Furthermore, the above-mentioned method for preparing a microparticle-based immune-enhancing feed for shrimp larvae includes the following steps:

[0013] Step 1: Pass the high-gluten flour, pregelatinized starch, krill powder, earthworm powder, Schizochytrium powder, marine red yeast, brown algae powder, coffee grounds fermentation product, Salicornia glutinosa extract, Cat's Claw vine extract, mineral premix, vitamin premix, molting and hardening agent, and sodium alginate through an 80-100 mesh sieve. Weigh them according to their weight parts. Take two-thirds of the weight of sodium alginate and the remaining components and put them into a three-dimensional mixer and mix them at 10-20 rpm for 20-30 minutes to obtain mixture I.

[0014] Step 2: Weigh the enzymatically hydrolyzed fish slurry, refined fish oil, soybean lecithin, and citric acid according to their respective weight parts. Add the citric acid to warm water at 40-50℃ at a mass-to-volume ratio of 1:1-2 and stir to dissolve. The mass-to-volume ratio is in kg / L. Then, mix the refined fish oil, four-fifths of the weight of soybean lecithin, and the citric acid aqueous solution, and stir at 800-1200 rpm for 5-10 minutes to obtain mixture II.

[0015] Step 3: Add mixture I, enzymatically hydrolyzed fish slurry, and mixture II into the conditioning unit of a twin-screw extruder granulator. Add water to adjust the material moisture content to 28-32%. Condition at 45-50℃ for 1-2 minutes. Then, extrude the mixture into strips under conditions of 0.2-0.5mm orifice diameter, 150-200rpm screw speed, and 55-60℃ die temperature. Cut the strips into 1.0-1.5mm lengths to obtain wet granules.

[0016] Step 4: Let the wet granules stand in a curing machine at 40-45℃ for 30-40 minutes, and then dry them at 45-50℃ and -0.08 to -0.10 MPa pressure until the moisture content is ≤8% to obtain dried granules;

[0017] Step 5: Cool the dried granules to 25-35℃, put them into a vacuum spraying machine, evacuate to -0.08~-0.10MPa, spray the coating liquid into the vacuum, maintain vacuum rolling for 3-5 minutes, release the vacuum and take out the granules, and blow them in a fluidized bed at 35-40℃ for 12-20 minutes; then use a vibrating screen to screen out 100-300μm and 300-500μm granules, vacuum pack them in aluminum foil composite bags or nitrogen-filled packaging, add deoxidizer, and store them in the dark at ≤10℃.

[0018] Furthermore, the coating solution is prepared by the following method: Weigh glycerol, natural mixed tocopherols, rosemary extract, and cinnamon essential oil according to the weight parts. Dissolve the remaining one-third sodium alginate in warm water at 50-60℃ at a mass-volume ratio of 1:50-100 (kg / L). Add the remaining soybean lecithin and glycerol, and shear at 8000-12000 rpm for 5-8 min. Add the natural mixed tocopherols, rosemary extract, and cinnamon essential oil, and shear at 8000-12000 rpm for 3-5 min to obtain the coating solution.

[0019] The feed described in this application is specifically designed for shrimp larvae in the PL5-PL20 stage.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. This invention adds coffee grounds ferment, Salicornia glutinosa extract and Cat's Claw Vine extract, which respectively form a triple immune barrier of "antibacterial-antistress-antiviral" from three dimensions: anti-vibrio, anti-stress and antiviral, thus making a complementary contribution to improving the survival rate of shrimp larvae.

[0022] 2. This invention constructs a synergistic molting mechanism through a combination of horsetail and alfalfa extracts, utilizing a dual-pathway "silicon-sterol" pathway to significantly improve the growth performance and uniformity of shrimp larvae. Horsetail extract is rich in bioavailable hydrated silica, which directly participates in shell mineralization and hardening; alfalfa extract provides phytosterols (such as β-sitosterol), serving as precursors for the synthesis of endogenous molting hormones in shrimp larvae. The combination of the two creates a synergistic effect of "silicon-provided hardening + sterol-promoted molting".

[0023] 3. This invention employs low-temperature conditioning and low-temperature die extrusion granulation to protect the heat-sensitive active substances in earthworm powder, Schizochyptella powder, marine red yeast, and plant extracts. Simultaneously, it combines natural mixed tocopherols, rosemary extract, cinnamon essential oil, and other fat-soluble components with sodium alginate, soybean lecithin, and glycerin to form a coating emulsion. This emulsion is then vacuum-sprayed into the micropores on the surface of the particles to form a sodium alginate slow-release gel film. This allows the functional components to be continuously released in water for 2-4 hours, significantly reducing washout loss and ensuring that the active ingredients are effectively ingested by the shrimp larvae. Detailed Implementation

[0024] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0027] Example 1

[0028] A microparticle-based immune-enhancing feed for shrimp larvae comprises the following ingredients in parts by weight: 35.0 parts high-gluten flour, 10.0 parts pregelatinized starch, 15.0 parts enzymatically hydrolyzed fish lysate, 5.0 parts krill powder, 4.0 parts refined fish oil, 3.0 parts earthworm powder, 2.0-part Schizochytrium powder, 2.5-part soybean lecithin, 1.0-part marine red yeast, 1.5 parts sodium alginate, 1.0-part brown algae powder, 1.0 part coffee grounds ferment, 0.2 parts glycerin, 0.1 parts Salicornia glutinosa extract, 0.1 parts Cat's Claw vine extract, 1.0-part mineral premix, 0.5 parts vitamin premix, 0.2 parts molting and hardening agent, 0.05 parts natural mixed tocopherols, 0.04 parts rosemary extract, 0.05 parts citric acid, and 0.05 parts cinnamon essential oil. The molting and hardening agent consists of horsetail extract and alfalfa extract in a mass ratio of 0.5:1.

[0029] The coffee grounds fermentation product was prepared by the following method: dried coffee grounds were soaked in water for 1.5 hours, then the coffee grounds were removed and steam-sterilized at 115°C for 20 minutes. Aspergillus oryzae spore suspension was inoculated at 5% of the coffee grounds mass, resulting in a spore suspension concentration of 10. 7The spores were added at a concentration of 1 / mL, along with 3% glucose and 0.3% sodium nitrate (by weight of coffee grounds). The mixture was thoroughly mixed, and the pH was adjusted to 4.5 with lactic acid to obtain the fermentation material. The fermentation material was placed in a fermentation tank, and the top of the tank was covered with gauze. Fermentation was then carried out at 30°C for 3 days, with stirring every 12 hours for 5 minutes each time. Fermentation was then carried out at 30°C for another 5 days, with stirring every 24 hours for 5 minutes each time. Fermentation was then carried out at 28°C for 2 days, with stirring every 48 hours for 5 minutes each time. After fermentation, the material was dried at 60°C for 10 hours, and then ultra-finely pulverized through an 80-mesh sieve to obtain the coffee grounds fermentation product.

[0030] The *Salvia splendens* extract was prepared by the following method: Fresh *Salvia splendens* was rinsed three times with water, dried at 60℃ to a moisture content of 10%, and then pulverized through a 30-mesh sieve to obtain *Salvia splendens* powder; the *Salvia splendens* powder was added to purified water at a ratio of 1:10 (g / mL), citric acid was added to adjust the pH to 5.5, and the mixture was extracted in an 80℃ water bath for 2 hours. After extraction, the mixture was filtered through an 80-mesh filter cloth, and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The filtrate was concentrated under reduced pressure at 60°C to 25% of its original volume to obtain a concentrated Salicornia glutinosa extract. 95% ethanol was added to the Salicornia glutinosa extract at a volume ratio of 1:3, and the mixture was allowed to stand at 3°C ​​for 10 hours. Then, it was centrifuged at 3500 rpm for 18 minutes, and the supernatant was collected. The supernatant was then concentrated under reduced pressure at 50°C until there was no ethanol odor, and then vacuum dried at 55°C and -0.08 MPa to a water content of 8%. Finally, it was pulverized and passed through an 80-mesh sieve to obtain the Salicornia glutinosa extract.

[0031] The cat's claw vine extract was prepared by the following method: dried cat's claw vine stems were cut into 2cm segments, pulverized, and passed through a 30-mesh sieve to obtain cat's claw vine powder; the cat's claw vine powder was added to purified water at a material-to-liquid ratio of 1:4 (material-to-liquid ratio unit: g / mL), citric acid was added to adjust the pH to 4.5, and 0.1% (by weight of cat's claw vine) of cellulase was added. Enzymatic hydrolysis was carried out at 48℃ for 2.0h, followed by warming to 85℃ for 11min to inactivate the enzyme, and then cooling to 60℃ to obtain cat's claw vine enzymatic hydrolysate; the cat's claw vine enzymatic hydrolysate was added to 65% ethanol at a volume ratio of 1:9. Extraction was carried out in a water bath at 60℃ for 2.5 h. After extraction, the solution was filtered through an 80-mesh filter cloth, and the filtrate was collected. The filtrate was concentrated under reduced pressure at 50℃ until no ethanol odor was detected, yielding a concentrated cat's claw vine extract. 1.5 times the volume of purified water was added to the cat's claw vine extract, and the mixture was stirred evenly. The solution was allowed to stand at 3℃ for 10 h, and then centrifuged at 3500 rpm for 18 min. The supernatant was collected. The supernatant was concentrated to 25% of its original volume at 50℃, and then vacuum dried at 50℃ and -0.08 MPa to a water content of 8%. The extract was then pulverized and passed through an 80-mesh sieve to obtain the cat's claw vine extract.

[0032] The horsetail extract was prepared by the following method: The whole horsetail herb was pulverized and passed through a 30-mesh sieve to obtain horsetail powder; the horsetail powder was added to a 0.1% citric acid aqueous solution at a material-to-liquid ratio of 1:10 (material-to-liquid ratio in g / mL), and extracted in a water bath at 80℃ for 3 hours. After extraction, the solution was filtered through an 80-mesh filter cloth, and the filtrate was collected; the residue was added to a 0.1% citric acid aqueous solution at a material-to-liquid ratio of 1:7, and extracted again at the same temperature for 2.0 hours. The two filtrates were combined; food-grade calcium carbonate was added to the combined filtrate and stirred until homogeneous. Adjust the pH to 6.5, let stand at 25℃ for 1.0 h, then filter with a 150-mesh filter cloth and collect the filtrate; concentrate the filtrate under reduced pressure at 60℃ to 25% of the original volume to obtain a concentrated solution; add 95% ethanol to the concentrated solution at a volume ratio of 1:3, stir well, let stand at 3℃ for 10 h, centrifuge at 3500 rpm for 18 min, and collect the supernatant; concentrate the supernatant at 50℃ to 25% of the original volume, then vacuum dry at 50℃ and -0.08 MPa to a water content of 8%, and then pulverize through an 80-mesh sieve to obtain the horsetail extract.

[0033] The alfalfa extract was prepared by the following method: dried alfalfa whole herb was pulverized and passed through a 30-mesh sieve to obtain alfalfa powder; the alfalfa powder was added to 65% ethanol at a material-to-liquid ratio of 1:8, and extracted in a water bath at 60℃ for 2.5h. After extraction, the mixture was filtered through an 80-mesh filter cloth, and the filtrate was collected; the residue was added to 65% ethanol at a material-to-liquid ratio of 1:7, and extracted again at the same temperature for 2.0h, and the two filtrates were combined; the combined filtrate was concentrated under reduced pressure at 50℃ until no ethanol odor was detected, and the concentrate was obtained; 2.5 times the volume of purified water was added to the concentrate, stirred evenly, and allowed to stand at 3℃ for 10h, followed by centrifugation at 3500rpm for 18min, and the supernatant was collected; the supernatant was concentrated under reduced pressure at 50℃ to 25% of the original volume, and then vacuum dried at 55℃ and -0.08MPa to a moisture content of 8%, and then pulverized and passed through an 80-mesh sieve to obtain the alfalfa extract.

[0034] Example 2

[0035] A microparticle-based immune-enhancing feed for shrimp larvae comprises the following ingredients in parts by weight: 65.0 parts high-gluten flour, 15.0 parts pregelatinized starch, 20.0 parts enzymatically hydrolyzed fish lysate, 10.0 parts krill meal, 6.0 parts refined fish oil, 5.0 parts earthworm meal, 4.0 parts Schizochytrium meal, 3.5 parts soybean lecithin, 3.0 parts marine red yeast, 3.0 parts sodium alginate, 2.0 parts brown algae meal, 2.0 parts coffee grounds ferment, 0.5 parts glycerin, 0.3 parts Salicornia caryophylla extract, 0.3 parts Uncaria rhynchophylla extract, 2.0 parts mineral premix, 1.0 part vitamin premix, 0.5 parts molting and hardening agent, 0.10 parts natural mixed tocopherols, 0.06 parts rosemary extract, 0.10 parts citric acid, and 0.10 parts cinnamon essential oil. The molting and hardening agent is composed of Equisetum hyemale extract and Alfalfa extract in a 1:1 mass ratio.

[0036] The coffee grounds fermentation product was prepared by the following method: dried coffee grounds were soaked in water for 2.5 hours, then the coffee grounds were removed and steam-sterilized at 125°C for 15 minutes. Aspergillus oryzae spore suspension was inoculated at 10% of the coffee grounds mass, resulting in a spore suspension concentration of 10%. 6 The spores were added at a concentration of 5% glucose and 0.6% sodium nitrate (by weight of coffee grounds) per mL. The mixture was thoroughly mixed, and the pH was adjusted to 5.0 with lactic acid to obtain the fermentation material. The fermentation material was placed in a fermentation tank, and the top of the fermentation tank was covered with gauze. Fermentation was carried out at 35°C for 2 days, with stirring every 12 hours for 10 minutes each time. Fermentation was then carried out at 35°C for 3 days, with stirring every 24 hours for 10 minutes each time. Fermentation was then carried out at 30°C for 2 days, with stirring every 48 hours for 10 minutes each time. After fermentation, the material was dried at 70°C for 6 hours, and then ultra-finely pulverized through an 80-mesh sieve to obtain the coffee grounds fermentation product.

[0037] The *Salvia splendens* extract was prepared by the following method: Fresh *Salvia splendens* was rinsed four times with water, dried at 70℃ to a moisture content of 10%, and then pulverized through a 50-mesh sieve to obtain *Salvia splendens* powder; the *Salvia splendens* powder was added to purified water at a ratio of 1:15 (g / mL), citric acid was added to adjust the pH to 6.0, and the mixture was extracted in a water bath at 80-90℃ for 2 hours. After extraction, the mixture was filtered through a 120-mesh filter cloth, and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The combined filtrates were concentrated under reduced pressure at 70°C to 20% of their original volume to obtain a concentrated Salicornia glutinosa extract. 95% ethanol was added to the Salicornia glutinosa extract at a volume ratio of 1:5, and the mixture was allowed to stand at 5°C for 15 hours. Then, it was centrifuged at 4500 rpm for 12 minutes, and the supernatant was collected. The supernatant was then concentrated under reduced pressure at 60°C until there was no ethanol odor, and then vacuum dried at 65°C and -0.10 MPa to a water content of 8%. Finally, it was pulverized and passed through a 120-mesh sieve to obtain the Salicornia glutinosa extract.

[0038] The cat's claw vine extract was prepared by the following method: dried cat's claw vine stems were cut into 3cm segments, pulverized, and passed through a 50-mesh sieve to obtain cat's claw vine powder; the cat's claw vine powder was added to purified water at a material-to-liquid ratio of 1:6 (material-to-liquid ratio unit: g / mL), citric acid was added to adjust the pH to 5.0, and 0.1% (by weight of cat's claw vine) of cellulase was added. Enzymatic hydrolysis was carried out at 52℃ for 1.5h, followed by heating to 90℃ to inactivate the enzyme for 9min, and then cooling to 55℃ to obtain cat's claw vine enzymatic hydrolysate; the cat's claw vine enzymatic hydrolysate was added to 65% ethanol at a volume ratio of 1:11. Extraction was carried out by heating in a water bath at 65℃ for 2 hours. After extraction, the solution was filtered through a 120-mesh filter cloth, and the filtrate was collected. The filtrate was concentrated under reduced pressure at 60℃ until no ethanol odor was detected, yielding a concentrated cat's claw vine extract. 2.5 times the volume of purified water was added to the cat's claw vine extract, and the mixture was stirred evenly. The solution was allowed to stand at 5℃ for 15 hours, followed by centrifugation at 4500 rpm for 12 minutes, and the supernatant was collected. The supernatant was concentrated to 15% of its original volume at 60℃, and then vacuum dried at 60℃ and -0.10 MPa to a water content of 8%. The extract was then pulverized and passed through a 120-mesh sieve to obtain the cat's claw vine extract.

[0039] The horsetail extract was prepared by the following method: The whole horsetail herb was pulverized and passed through a 50-mesh sieve to obtain horsetail powder; the horsetail powder was added to a 0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:15 (material-to-liquid ratio in g / mL), and extracted in a 90℃ water bath for 2 hours. After extraction, the solution was filtered through a 120-mesh filter cloth, and the filtrate was collected; the residue was added to a 0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:9, and extracted again at the same temperature for 1.5 hours. The two filtrates were combined; food-grade calcium carbonate was added to the combined filtrate and stirred until homogeneous. The pH was adjusted to 7.0, and the mixture was allowed to stand at 30°C for 1.5 hours. Then, it was filtered through a 200-mesh filter cloth, and the filtrate was collected. The filtrate was concentrated under reduced pressure at 70°C to 20% of its original volume to obtain a concentrated solution. 95% ethanol was added to the concentrated solution at a volume ratio of 1:5, stirred evenly, and allowed to stand at 5°C for 15 hours. The solution was then centrifuged at 4500 rpm for 12 minutes, and the supernatant was collected. The supernatant was concentrated at 60°C to 15% of its original volume, and then vacuum dried at 60°C and -0.10 MPa to a water content of 8%. The solution was then pulverized and passed through a 120-mesh sieve to obtain the horsetail extract.

[0040] The alfalfa extract was prepared by the following method: dried whole alfalfa herb was pulverized and passed through a 50-mesh sieve to obtain alfalfa powder; the alfalfa powder was added to 65% ethanol at a material-to-liquid ratio of 1:12, and extracted in a water bath at 70℃ for 2.0 h; after extraction, the mixture was filtered through a 120-mesh filter cloth, and the filtrate was collected; the residue was added to 65% ethanol at a material-to-liquid ratio of 1:9, and extracted again at the same temperature for 1.5 h, and the two filtrates were combined; the combined filtrate was concentrated under reduced pressure at 60℃ until no ethanol odor was detected, and the concentrate was obtained; 3.5 times the volume of purified water was added to the concentrate, stirred evenly, and allowed to stand at 5℃ for 15 h, followed by centrifugation at 4500 rpm for 12 min, and the supernatant was collected; the supernatant was concentrated under reduced pressure at 60℃ to 15% of the original volume, and then vacuum dried at 65℃ and -0.10 MPa to a moisture content of 8%, and then pulverized and passed through a 120-mesh sieve to obtain the alfalfa extract.

[0041] Example 3

[0042] A microparticle-based immune-enhancing feed for shrimp larvae comprises the following ingredients in parts by weight: 50.0 parts high-gluten flour, 12.5 parts pregelatinized starch, 17.5 parts enzymatically hydrolyzed fish lysate, 7.5 parts krill meal, 5.0 parts refined fish oil, 4.0 parts earthworm meal, 3.0 parts Schizochyptella meal, 3.0 parts soybean lecithin, 2.0 parts marine red yeast, 2.2 parts sodium alginate, 1.5 parts brown algae meal, 1.5 parts coffee grounds ferment, 0.4 parts glycerin, 0.2 parts Salicyla extract, 0.2 parts Cat's Claw vine extract, 1.5 parts mineral premix, 0.8 parts vitamin premix, 0.4 parts molting and hardening agent, 0.08 parts natural mixed tocopherols, 0.05 parts rosemary extract, 0.08 parts citric acid, and 0.08 parts cinnamon essential oil. The molting and hardening agent is composed of Equisetum hyemale extract and Alfalfa extract in a mass ratio of 0.8:1.

[0043] The coffee grounds fermentation product was prepared by the following method: dried coffee grounds were soaked in water for 2.0 hours, then the coffee grounds were removed and steam-sterilized at 120°C for 18 minutes. Aspergillus oryzae spore suspension was inoculated at 8% of the coffee grounds mass, resulting in a spore suspension concentration of 10. 7 The spores were added at a concentration of 4% glucose and 0.5% sodium nitrate (by weight of coffee grounds) per mL. The mixture was then thoroughly mixed and the pH was adjusted to 4.8 with lactic acid to obtain the fermentation material. The fermentation material was placed in a fermentation tank, and the top of the tank was covered with gauze. Fermentation was then carried out at 33°C for 3 days, with stirring every 12 hours for 8 minutes each time. Fermentation was then carried out at 32°C for 4 days, with stirring every 24 hours for 8 minutes each time. Fermentation was then carried out at 29°C for 3 days, with stirring every 48 hours for 8 minutes each time. After fermentation, the material was dried at 65°C for 8 hours, and then ultra-finely pulverized through a 100-mesh sieve to obtain the coffee grounds fermentation product.

[0044] The *Salvia splendens* extract was prepared by the following method: Fresh *Salvia splendens* was rinsed four times with water, dried at 65℃ to a moisture content of 10%, and then pulverized through a 40-mesh sieve to obtain *Salvia splendens* powder; the *Salvia splendens* powder was added to purified water at a material-to-liquid ratio of 1:12 (g / mL), citric acid was added to adjust the pH to 5.8, and the mixture was extracted in a water bath at 85℃ for 2.5 hours. After extraction, the mixture was filtered through a 100-mesh filter cloth, and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The filtrate was concentrated under reduced pressure at 65°C to 22% of its original volume to obtain a concentrated Salicornia glutinosa extract. 95% ethanol was added to the Salicornia glutinosa extract at a volume ratio of 1:4, and the mixture was allowed to stand at 4°C for 12 hours. Then, it was centrifuged at 4000 rpm for 16 minutes, and the supernatant was collected. The supernatant was then concentrated under reduced pressure at 55°C until there was no ethanol odor, and then vacuum dried at 60°C and -0.09 MPa to a water content of 8%. Finally, it was pulverized and passed through a 100-mesh sieve to obtain the Salicornia glutinosa extract.

[0045] The cat's claw vine extract was prepared by the following method: dried cat's claw vine stems were cut into 3cm segments, pulverized, and passed through a 40-mesh sieve to obtain cat's claw vine powder; the cat's claw vine powder was added to purified water at a material-to-liquid ratio of 1:5 (material-to-liquid ratio unit: g / mL), citric acid was added to adjust the pH to 4.8, and 0.1% (by weight of cat's claw vine) of cellulase was added. Enzymatic hydrolysis was carried out at 50℃ for 1.8h, followed by warming to 88℃ to inactivate the enzyme for 10min, and then cooling to 58℃ to obtain cat's claw vine enzymatic hydrolysate; the cat's claw vine enzymatic hydrolysate was added to 65% ethanol at a volume ratio of 1:10, and... Extraction was performed by heating in a water bath at 63℃ for 2.2 hours. After extraction, the solution was filtered through a 100-mesh filter cloth, and the filtrate was collected. The filtrate was concentrated under reduced pressure at 55℃ until no ethanol odor was detected, yielding a concentrated cat's claw vine extract. Two times the volume of purified water was added to the cat's claw vine extract, and the mixture was stirred evenly. The extract was allowed to stand at 4℃ for 12.5 hours, followed by centrifugation at 4000 rpm for 15 minutes, and the supernatant was collected. The supernatant was concentrated to 20% of its original volume at 55℃, and then vacuum dried at 55℃ and -0.09 MPa until the water content was 8%. The extract was then pulverized and passed through a 100-mesh sieve to obtain the cat's claw vine extract.

[0046] The horsetail extract was prepared by the following method: The whole horsetail herb was pulverized and passed through a 40-mesh sieve to obtain horsetail powder; the horsetail powder was added to a 0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:12.5 (material-to-liquid ratio in g / mL), and extracted in a water bath at 85℃ for 2.5 h. After extraction, the solution was filtered through a 100-mesh filter cloth, and the filtrate was collected; the residue was added to a 0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:8, and extracted again at the same temperature for 1.8 h, and the two filtrates were combined; food-grade calcium carbonate was added to the combined filtrate and stirred thoroughly. The solution was homogenized, the pH was adjusted to 6.8, and the mixture was allowed to stand at 28℃ for 1.2 h. Then, it was filtered through a 180-mesh filter cloth, and the filtrate was collected. The filtrate was concentrated under reduced pressure at 65℃ to 22% of its original volume to obtain a concentrated solution. 95% ethanol was added to the concentrated solution at a volume ratio of 1:4, stirred evenly, and allowed to stand at 4℃ for 12 h. The solution was then centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The supernatant was concentrated at 55℃ to 20% of its original volume, and then vacuum dried at 55℃ and -0.09 MPa to a water content of 8%. The solution was then pulverized and passed through a 100-mesh sieve to obtain the horsetail extract.

[0047] The alfalfa extract was prepared by the following method: dried alfalfa whole herb was pulverized and passed through a 40-mesh sieve to obtain alfalfa powder; the alfalfa powder was added to 65% ethanol at a material-to-liquid ratio of 1:10, and extracted by heating in a water bath at 65℃ for 2.2h. After extraction, the extract was filtered through a 100-mesh filter cloth and the filtrate was collected; the residue was added to 65% ethanol at a material-to-liquid ratio of 1:8 and extracted again at the same temperature for 1.8h, and the two filtrates were combined; the combined filtrate was concentrated under reduced pressure at 55℃ until there was no ethanol odor to obtain a concentrate; 3.0 times the volume of purified water was added to the concentrate, stirred evenly, and allowed to stand at 4℃ for 12.5h, followed by centrifugation at 4000rpm for 15min, and the supernatant was collected; the supernatant was concentrated under reduced pressure at 55℃ to 20% of the original volume, and then vacuum dried at 60℃ and -0.09MPa to a moisture content of 8%, and then pulverized and passed through a 100-mesh sieve to obtain the alfalfa extract.

[0048] The method for preparing a microparticle-based immune-enhancing feed for shrimp larvae as described in Examples 1-3 includes the following steps:

[0049] Step 1: Pass high-gluten flour, pregelatinized starch, krill powder, earthworm powder, Schizochytrium powder, marine red yeast, brown algae powder, coffee grounds fermentation product, Salicornia glutinosa extract, Cat's Claw vine extract, mineral premix, vitamin premix, molting and hardening agent, and sodium alginate through a 90-mesh sieve. Weigh them according to their weight parts. Take two-thirds of the weight of sodium alginate and the remaining components and put them into a three-dimensional mixer and mix them at 15 rpm for 25 minutes to obtain mixture I.

[0050] Step 2: Weigh the enzymatically hydrolyzed fish slurry, refined fish oil, soybean lecithin, and citric acid according to their weight parts. Add the citric acid to 45℃ warm water at a mass-to-volume ratio of 1:1.5 and stir to dissolve. The mass-to-volume ratio is in kg / L. Then mix the refined fish oil, four-fifths of the weight of soybean lecithin, and the citric acid aqueous solution and stir at 1000 rpm for 8 minutes to obtain mixture II.

[0051] Step 3: Add mixture I, enzymatically hydrolyzed fish slurry, and mixture II into the conditioning unit of a twin-screw extruder granulator, add water to adjust the material moisture content to 30%, condition at 48℃ for 1.5 min, then extrude into strips under conditions of 0.4 mm orifice, 180 rpm screw speed, and 58℃ die temperature, and cut into 1.5 mm lengths with a cutter to obtain wet granules;

[0052] Step 4: Let the wet granules stand in a curing machine at 42℃ for 35 minutes, and then dry them at 48℃ and -0.09MPa pressure until the moisture content is 8% to obtain dried granules;

[0053] Step 5: Cool the dried granules to 30°C, put them into a vacuum spraying machine, evacuate to -0.09MPa, spray the coating liquid into the vacuum, maintain vacuum rolling for 4 minutes, release the vacuum and take out the granules, and blow them in a fluidized bed at 38°C for 15 minutes; then use a vibrating screen to screen out 100-300μm and 300-500μm granules, vacuum pack them in aluminum foil composite bags or nitrogen-filled packaging, add deoxidizer, and store them in the dark at 10°C.

[0054] The coating solution was prepared by the following method: glycerol, natural mixed tocopherols, rosemary extract, and cinnamon essential oil were weighed according to their weight parts. The remaining one-third sodium alginate was added to 55°C warm water at a mass-volume ratio of 1:80 and stirred to dissolve it. The remaining soybean lecithin and glycerol were added, and the mixture was sheared at 10,000 rpm for 6 minutes. Natural mixed tocopherols, rosemary extract, and cinnamon essential oil were added, and the mixture was sheared at 10,000 rpm for 4 minutes to obtain the coating solution.

[0055] Example 4

[0056] Compared with Example 3, Example 4 differs in that the method for preparing a microparticle immune-enhancing feed for shrimp larvae includes the following steps:

[0057] Step 1: Pass the high-gluten flour, pregelatinized starch, krill powder, earthworm powder, Schizochytrium powder, marine red yeast, brown algae powder, coffee grounds fermentation product, Salicornia glutinosa extract, Cat's Claw vine extract, mineral premix, vitamin premix, molting and hardening agent, and sodium alginate through an 80-mesh sieve. Weigh them according to their weight parts. Take two-thirds of the weight of sodium alginate and the remaining components and put them into a three-dimensional mixer and mix them at 10 rpm for 30 minutes to obtain mixture I.

[0058] Step 2: Weigh the enzymatically hydrolyzed fish slurry, refined fish oil, soybean lecithin, and citric acid according to their respective weight parts. Add the citric acid to 40°C warm water at a mass-to-volume ratio of 1:1 and stir to dissolve. The mass-to-volume ratio is in kg / L. Then, mix the refined fish oil, four-fifths of the weight of soybean lecithin, and the citric acid aqueous solution, and stir at 800 rpm for 10 minutes to obtain mixture II.

[0059] Step 3: Add mixture I, enzymatically hydrolyzed fish slurry, and mixture II to the conditioning unit of a twin-screw extruder granulator, add water to adjust the material moisture content to 28%, condition at 45℃ for 2 minutes, and then extrude into strips under conditions of 0.2mm orifice diameter, 150rpm screw speed, and 55℃ die temperature. Cut into 1.0mm lengths with a cutter to obtain wet granules.

[0060] Step 4: Let the wet granules stand in a curing machine at 40℃ for 40 minutes, and then dry them at 45℃ and -0.08MPa pressure until the moisture content is 8% to obtain dried granules;

[0061] Step 5: Cool the dried granules to 25°C, put them into a vacuum spraying machine, evacuate to -0.08MPa, spray the coating liquid into the vacuum, maintain vacuum rolling for 5 minutes, release the vacuum and take out the granules, and blow them in a fluidized bed at 35°C for 20 minutes; then use a vibrating screen to screen out 100-300μm and 300-500μm granules, vacuum pack them in aluminum foil composite bags or nitrogen-filled packaging, add deoxidizer, and store them in the dark at 10°C.

[0062] The coating solution was prepared by the following method: glycerol, natural mixed tocopherols, rosemary extract, and cinnamon essential oil were weighed according to their weight parts. The remaining one-third sodium alginate was dissolved in warm water at 50°C by stirring at a mass-volume ratio of 1:50 (kg / L). The remaining soybean lecithin and glycerol were added, and the mixture was sheared at 8000 rpm for 8 min. The natural mixed tocopherols, rosemary extract, and cinnamon essential oil were added, and the mixture was sheared at 8000 rpm for 5 min to obtain the coating solution.

[0063] Example 5

[0064] Compared with Example 3, Example 4 differs in that the method for preparing a microparticle immune-enhancing feed for shrimp larvae includes the following steps:

[0065] Step 1: Pass the high-gluten flour, pregelatinized starch, krill powder, earthworm powder, Schizochytrium powder, marine red yeast, brown algae powder, coffee grounds fermentation product, Salicornia glutinosa extract, Cat's Claw vine extract, mineral premix, vitamin premix, molting and hardening agent, and sodium alginate through a 100-mesh sieve. Weigh them according to their weight parts. Take two-thirds of the weight of sodium alginate and the remaining components and put them into a three-dimensional mixer. Mix at 20 rpm for 20 minutes to obtain mixture I.

[0066] Step 2: Weigh the enzymatically hydrolyzed fish slurry, refined fish oil, soybean lecithin, and citric acid according to their weight parts. Add the citric acid to 50℃ warm water at a mass-to-volume ratio of 1:2 and stir to dissolve. The mass-to-volume ratio is in kg / L. Then mix the refined fish oil, four-fifths of the weight of soybean lecithin, and the citric acid aqueous solution and stir at 1200 rpm for 5 minutes to obtain mixture II.

[0067] Step 3: Add mixture I, enzymatically hydrolyzed fish slurry, and mixture II into the conditioning unit of a twin-screw extruder granulator, add water to adjust the material moisture content to 32%, condition at 50℃ for 1 min, and then extrude into strips under conditions of 0.5 mm orifice diameter, 200 rpm screw speed, and 60℃ die temperature. Cut into 1.5 mm lengths with a cutter to obtain wet granules.

[0068] Step 4: Let the wet granules stand in a curing machine at 45℃ for 30 minutes, and then dry them at 50℃ and -0.08MPa pressure until the moisture content is 8% to obtain dried granules;

[0069] Step 5: Cool the dried granules to 35°C, put them into a vacuum spraying machine, evacuate to -0.10MPa, spray the coating liquid into the vacuum, maintain vacuum rolling for 5 minutes, release the vacuum and take out the granules, and blow them in a fluidized bed at 40°C for 12 minutes; then use a vibrating screen to screen out 100-300μm and 300-500μm granules, vacuum pack them in aluminum foil composite bags or nitrogen-filled packaging, add deoxidizer, and store them in the dark at 10°C.

[0070] The coating solution was prepared by the following method: glycerol, natural mixed tocopherols, rosemary extract, and cinnamon essential oil were weighed according to their weight parts. The remaining one-third sodium alginate was dissolved in 60°C warm water at a mass-volume ratio of 1:100 (kg / L). The remaining soybean lecithin and glycerol were added, and the mixture was sheared at 12000 rpm for 5 min. The natural mixed tocopherols, rosemary extract, and cinnamon essential oil were added, and the mixture was sheared at 12000 rpm for 3 min to obtain the coating solution.

[0071] The feed in this application is specifically designed for shrimp larvae in the PL5-PL20 stage, with the 100-300μm size feed suitable for the PL5-PL10 stage and the 300-500μm size feed suitable for the PL11-PL20 stage.

[0072] Comparative Example 1

[0073] The difference between this comparative example and Example 3 is that the raw materials do not contain coffee grounds fermentation products.

[0074] Comparative Example 2

[0075] The difference between this comparative example and Example 3 is that the raw materials do not contain coffee grounds fermentation products.

[0076] Comparative Example 3

[0077] The difference between this comparative example and Example 3 is that the raw materials do not contain cat's claw vine extract.

[0078] Comparative Example 4

[0079] The difference between this comparative example and Example 3 is that the raw materials do not contain a molting and hardening agent.

[0080] Comparative Example 5

[0081] The difference between this comparative example and Example 3 is that the molting and hardening agent consists only of horsetail extract.

[0082] Comparative Example 6

[0083] The difference between this comparative example and Example 3 is that the molting and hardening agent consists only of alfalfa extract.

[0084] Healthy, uniformly sized PL5 whiteleg shrimp larvae from the same batch, with an average body length of 0.8 cm, were selected and randomly divided into Example 1-5 and Comparative Examples 1-6, with three replicates per group. Indoor rearing ponds were used, with each rearing unit measuring 1.0 m². 3The water depth is 0.5m, with 2000 shrimp larvae stocked per pond. The water salinity is 26‰, temperature is 28℃, pH is 8.0, dissolved oxygen is ≥5mg / L, ammonia nitrogen is <0.2mg / L, and nitrite is <0.05mg / L. 30% of the water is changed daily, and the photoperiod is 12L:12D. From day 1 to day 6, shrimp are fed 100-300μm pellets six times a day at 6:00, 9:00, 12:00, 15:00, 18:00, and 21:00. From day 1 to day 3, 4g of feed is given each time; from day 4 to day 6, 6g of feed is given each time. From day 7 to day 15, shrimp were fed 300-500μm pellets four times a day at 6:00, 11:00, 16:00, and 21:00. From day 7 to day 10, each feeding was 8 g, and from day 11 to day 15, each feeding was 10 g. On the morning of day 16, the survival rate was recorded in each pond. Thirty shrimp were randomly selected from each pond, and the weight of each individual shrimp was measured to calculate the average weight and uniformity of growth per shrimp. Uniformity of growth was calculated as (standard deviation of body weight / average body weight) × 100%. The results are shown in Table 1.

[0085] Table 1

[0086]

[0087] As can be seen from Table 1, the feeds prepared in Examples 1-5 of the present invention are more suitable for the shrimp larvae stage and have a better effect on the growth performance of shrimp larvae.

[0088] Comparing Example 3 with Comparative Example 1, Example 3, by adding coffee grounds fermentation product, produced active metabolites such as monacolin K and red yeast rice pigment after solid-state fermentation of coffee grounds with Aspergillus oryzae 3.042. Monacolin K can inhibit the proliferation of pathogenic bacteria such as Vibrio parahaemolyticus by inhibiting Vibrio reductase activity and interfering with its cell membrane synthesis. Furthermore, the organic acids and antimicrobial peptides produced during fermentation can synergistically reduce the Vibrio load in the shrimp larvae's intestines. Therefore, the addition of coffee grounds fermentation product inhibits Vibrio and enhances the shrimp larvae's resistance to infection, thereby improving survival rate.

[0089] Comparing Example 3 with Comparative Example 2, Example 3, by adding Salicornia glutinosa extract, which is rich in triterpenoid saponins and flavonoids, demonstrates that the triterpenoid saponins can enhance the adaptability of shrimp larvae to salinity fluctuations by regulating the osmotic pressure balance within the larvae. Simultaneously, the flavonoids possess free radical scavenging activity, which can alleviate oxidative stress induced by stress factors such as ammonia nitrogen and nitrite in high-density aquaculture environments. Therefore, Salicornia glutinosa extract plays an important role in maintaining the physiological stability of shrimp larvae during frequent water changes in the larval stage and reducing stress-induced mortality.

[0090] Comparing Example 3 with Comparative Example 3, Example 3, by adding *Cephalotaxus fortunei* extract, shows that its triterpenoid saponins and tetracyclooxygenated indole alkaloids can inhibit viral invasion by blocking the binding of white spot syndrome virus envelope proteins to shrimp cell membrane receptors. Simultaneously, its active ingredients can upregulate the expression of prophenoloxidase system and antimicrobial peptide genes in shrimp larvae hemocytes, enhancing non-specific immune responses. Therefore, *Cephalotaxus fortunei* extract has an irreplaceable targeting role in antiviral immune defense in shrimp larvae.

[0091] Comparing Example 3 with Comparative Examples 4-6, Example 3, by adding a molting and hardening agent, works by synergistically promoting molting and shell hardening in shrimp larvae. The horsetail extract is rich in bioavailable hydrated silica, which can directly participate in the mineralization process of the shell, accelerating the cross-linking and deposition of chitin with silicon and calcium in the new shell, thus shortening the soft-shell period. The phytosterols (such as β-sitosterol) in the alfalfa extract are important precursors for the synthesis of endogenous molting hormones in shrimp larvae, promoting the normal initiation of molting signals. The combination of these two agents forms a dual pathway of "silicon + sterol," balancing molting frequency and hardening speed. Compared to Comparative Example 4, which did not contain the molting and hardening agent, Example 3 outperformed Comparative Example 4 in three dimensions: survival rate, average body weight, and uniform growth. Furthermore, Table 1 shows that the survival rates of Comparative Examples 5 and 6 (which only added horsetail or alfalfa as single components) were between those of Comparative Example 4 and Example 3, but both were lower than that of Example 3. This indicates that the effect of a single component is weaker than that of a compound, further verifying that horsetail and alfalfa extracts have a synergistic and complementary effect in promoting molting and hardening, and that neither can be omitted.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microparticle-based immune-enhancing feed for shrimp larvae, characterized in that, The product comprises the following ingredients by weight: 35.0-65.0 parts high-gluten flour, 10.0-15.0 parts pregelatinized starch, 15.0-20.0 parts enzymatically hydrolyzed fish paste, 5.0-10.0 parts krill powder, 4.0-6.0 parts refined fish oil, 3.0-5.0 parts earthworm powder, 2.0-4.0 parts Schizochyptella powder, 2.5-3.5 parts soybean lecithin, 1.0-3.0 parts marine red yeast, 1.5-3.0 parts sodium alginate, 1.0-2.0 parts brown algae powder, 1 0.0-2.0 parts coffee grounds ferment, 0.2-0.5 parts glycerin, 0.1-0.3 parts Salicornia zedoaria extract, 0.1-0.3 parts Cat's Claw vine extract, 1.0-2.0 parts mineral premix, 0.5-1.0 parts vitamin premix, 0.2-0.5 parts molting and hardening agent, 0.05-0.10 parts natural mixed tocopherols, 0.04-0.06 parts rosemary extract, 0.05-0.10 parts citric acid, 0.05-0.10 parts cinnamon essential oil.

2. The shrimp larvae microparticle immune-enhancing feed as described in claim 1, characterized in that, The coffee grounds fermentation product is prepared by the following method: dried coffee grounds are soaked in water for 1.5-2.5 hours, then the coffee grounds are removed and steam-sterilized at 115-125℃ for 15-20 minutes. Aspergillus oryzae spore suspension is inoculated at 5-10% of the coffee grounds mass, with a spore suspension concentration of 10. 6 -10 7 Add spores / mL, then add 3-5% glucose and 0.3-0.6% sodium nitrate (by weight of coffee grounds), mix well, and adjust the pH to 4.5-5.0 with lactic acid to obtain the fermentation material. Place the fermentation material in a fermentation tank, cover the top of the fermentation tank with gauze, and then ferment at 30-35℃ for 2-3 days, stirring once every 12 hours for 5-10 minutes each time. Then ferment at 30-35℃ for 3-5 days, stirring once every 24 hours for 5-10 minutes each time. Then ferment at 28-30℃ for 2-4 days, stirring once every 48 hours for 5-10 minutes each time. After fermentation, dry the material at 60-70℃ for 6-10 hours, and then pulverize it through an 80-120 mesh sieve to obtain the coffee grounds fermentation product.

3. The shrimp larvae microparticle immune-enhancing feed as described in claim 1, characterized in that, The *Salvia splendens* extract is prepared by the following method: Fresh *Salvia splendens* is rinsed 3-4 times with clean water, dried at 60-70℃ until the moisture content is ≤10%, and then pulverized through a 30-50 mesh sieve to obtain *Salvia splendens* powder; the *Salvia splendens* powder is added to purified water at a material-to-liquid ratio of 1:10-15 (material-to-liquid ratio unit: g / mL), citric acid is added to adjust the pH to 5.5-6.0, and the mixture is extracted in a water bath at 80-90℃ for 2-3 hours. After extraction, the mixture is filtered through an 80-120 mesh filter cloth, the filtrate is collected, the residue is extracted once more, the two filtrates are combined, and the combined filtrate is... Concentrate the *Salvia splendens* extract under reduced pressure at 60-70℃ to 20-25% of its original volume to obtain a concentrated *Salvia splendens* solution. Add 95% ethanol to the concentrated *Salvia splendens* solution at a volume ratio of 1:3-5, let it stand at 3-5℃ for 10-15 hours, then centrifuge at 3500-4500 rpm for 12-18 minutes, collect the supernatant, then concentrate the supernatant under reduced pressure at 50-60℃ until there is no ethanol odor, then vacuum dry at 55-65℃ and -0.08 to -0.10 until the water content is ≤8%, then pulverize and pass through an 80-120 mesh sieve to obtain the *Salvia splendens* extract.

4. The shrimp larvae microparticle immune-enhancing feed as described in claim 1, characterized in that, The cat's claw vine extract is prepared by the following method: dried cat's claw vine stems are cut into 2-3 cm segments, pulverized, and passed through a 30-50 mesh sieve to obtain cat's claw vine powder; the cat's claw vine powder is added to purified water at a material-to-liquid ratio of 1:4-6 (g / mL), citric acid is added to adjust the pH to 4.5-5.0, 0.1% (by weight of cat's claw vine) of cellulase is added, and enzymatic hydrolysis is carried out at 48-52℃ for 1.5-2.0 h, followed by inactivation of the enzyme at 85-90℃ for 9-11 min, and then cooled to ≤60℃ to obtain cat's claw vine enzymatic hydrolysate; the cat's claw vine enzymatic hydrolysate is added to 65% ethanol at a volume ratio of 1:9-11 and heated in a water bath at 60-65℃. Extract for 2-2.5 hours. After extraction, filter through an 80-120 mesh filter cloth and collect the filtrate. Concentrate the filtrate under reduced pressure at 50-60℃ until no ethanol odor remains, obtaining a concentrated cat's claw vine extract. Add 1.5-2.5 times the volume of purified water to the cat's claw vine extract, stir well, and let stand at 3-5℃ for 10-15 hours. Then centrifuge at 3500-4500 rpm for 12-18 minutes and collect the supernatant. Concentrate the supernatant at 50-60℃ to 15-25% of its original volume, and then vacuum dry at 50-60℃ and -0.08 to -0.10 MPa until the water content is ≤8%. Finally, pulverize and pass through an 80-120 mesh sieve to obtain the cat's claw vine extract.

5. The shrimp larvae microparticle immune-enhancing feed as described in claim 1, characterized in that, The molting and hardening agent is composed of horsetail extract and alfalfa extract in a mass ratio of 0.5-1:

1.

6. The shrimp larvae microparticle immune-enhancing feed as described in claim 1, characterized in that, The horsetail extract is prepared by the following method: The whole horsetail herb is pulverized and passed through a 30-50 mesh sieve to obtain horsetail powder; the horsetail powder is added to a 0.1-0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:10-15 (material-to-liquid ratio in g / mL), and extracted in a water bath at 80-90℃ for 2-3 hours. After extraction, the solution is filtered through an 80-120 mesh filter cloth, and the filtrate is collected; the filter residue is added to a 0.1-0.2% citric acid aqueous solution at a material-to-liquid ratio of 1:7-9, and extracted again at the same temperature for 1.5-2.0 hours, and the two filtrates are combined; food-grade calcium carbonate is added to the combined filtrate, stirred evenly, and the pH is adjusted to 6.5-7.

0. The solution is allowed to stand at 25-30℃ for 1.0-1.5 hours, then filtered through a 150-200 mesh filter cloth, and the filtrate is collected. The filtrate is concentrated under reduced pressure at 60-70℃ to 20-25% of its original volume to obtain a concentrated solution. 95% ethanol is added to the concentrated solution at a volume ratio of 1:3-5, stirred evenly, and allowed to stand at 3-5℃ for 10-15 hours. The solution is then centrifuged at 3500-4500 rpm for 12-18 minutes, and the supernatant is collected. The supernatant is concentrated at 50-60℃ to 15-25% of its original volume, and then vacuum dried at 50-60℃ and -0.08 to -0.10 MPa until the water content is ≤8%. The solution is then pulverized and passed through an 80-120 mesh sieve to obtain the horsetail extract.

7. The shrimp larvae microparticle immune-enhancing feed as described in claim 1, characterized in that, The alfalfa extract is prepared by the following method: dried whole alfalfa herb is pulverized and passed through a 30-50 mesh sieve to obtain alfalfa powder; the alfalfa powder is added to 65% ethanol at a material-to-liquid ratio of 1:8-12, and extracted in a water bath at 60-70℃ for 2.0-2.5 hours. After extraction, the mixture is filtered through an 80-120 mesh filter cloth, and the filtrate is collected; the residue is added to 65% ethanol at a material-to-liquid ratio of 1:7-9, and extracted again at the same temperature for 1.5-2.0 hours. The two filtrates are combined; the combined filtrate is then heated at 50-60℃. Concentrate under reduced pressure at 0℃ until no ethanol odor remains to obtain a concentrated solution; add 2.5-3.5 times the volume of purified water to the concentrated solution, stir well, and let stand at 3-5℃ for 10-15 hours, then centrifuge at 3500-4500 rpm for 12-18 minutes and collect the supernatant; concentrate the supernatant under reduced pressure at 50-60℃ to 15-25% of the original volume, then vacuum dry at 55-65℃ and -0.08 to -0.10 MPa until the water content is ≤8%, then pulverize and pass through an 80-120 mesh sieve to obtain the alfalfa extract.

8. A method for preparing a shrimp larvae microparticle immune-enhancing feed according to any one of claims 1-7, characterized in that, Includes the following steps: Step 1: Pass the high-gluten flour, pregelatinized starch, krill powder, earthworm powder, Schizochytrium powder, marine red yeast, brown algae powder, coffee grounds fermentation product, Salicornia glutinosa extract, Cat's Claw vine extract, mineral premix, vitamin premix, molting and hardening agent, and sodium alginate through an 80-100 mesh sieve. Weigh them according to their weight parts. Take two-thirds of the weight of sodium alginate and the remaining components and put them into a three-dimensional mixer and mix them at 10-20 rpm for 20-30 minutes to obtain mixture I. Step 2: Weigh the enzymatically hydrolyzed fish slurry, refined fish oil, soybean lecithin, and citric acid according to their respective weight parts. Add the citric acid to warm water at 40-50℃ at a mass-to-volume ratio of 1:1-2 and stir to dissolve. The mass-to-volume ratio is in kg / L. Then, mix the refined fish oil, four-fifths of the weight of soybean lecithin, and the citric acid aqueous solution, and stir at 800-1200 rpm for 5-10 minutes to obtain mixture II. Step 3: Add mixture I, enzymatically hydrolyzed fish slurry, and mixture II into the conditioning unit of a twin-screw extruder granulator. Add water to adjust the material moisture content to 28-32%. Condition at 45-50℃ for 1-2 minutes. Then, extrude the mixture into strips under conditions of 0.2-0.5mm orifice diameter, 150-200rpm screw speed, and 55-60℃ die temperature. Cut the strips into 1.0-1.5mm lengths to obtain wet granules. Step 4: Let the wet granules stand in a curing machine at 40-45℃ for 30-40 minutes, and then dry them at 45-50℃ and -0.08 to -0.10MPa pressure until the moisture content is ≤8% to obtain dried granules; Step 5: Cool the dried granules to 25-35℃, put them into a vacuum spraying machine, evacuate to -0.08~-0.10MPa, spray the coating liquid into the vacuum, maintain vacuum rolling for 3-5 minutes, release the vacuum and take out the granules, and blow them in a fluidized bed at 35-40℃ for 12-20 minutes. The particles of 100-300μm and 300-500μm were then screened out using a vibrating screen, vacuum-packed in aluminum foil composite bags or nitrogen-filled packaging, with an internal deoxidizer, and stored away from light at ≤10℃.

9. The method for preparing a shrimp larvae microparticle immune-enhancing feed as described in claim 8, characterized in that, The coating solution in step five is prepared by the following method: Weigh glycerin, natural mixed tocopherols, rosemary extract, and cinnamon essential oil according to the weight parts. Dissolve the remaining one-third sodium alginate in warm water at 50-60℃ at a mass-volume ratio of 1:50-100 (kg / L). Add the remaining soybean lecithin and glycerin, and shear at 8000-12000 rpm for 5-8 minutes. Add natural mixed tocopherols, rosemary extract, and cinnamon essential oil, and shear at 8000-12000 rpm for 3-5 minutes to obtain the coating solution.

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