East star spot original red preservation culture method

Abstract

The present application provides a kind of east star spot original native red keeps breeding method, comprising the following steps: A1. breeding platform site selection and system layout;A2. fry release;A3. breeding period management;A4. fish management.In the fry release before using morinda citrifolia fermentation liquor and Moringa oleifera leaf water extract as the core active component to assist the disinfectant of sodium alginate film forming protection to disinfect.In the meantime of overcoming the defect of broad-spectrum bactericidal chemical disinfectant damage fish body surface mucus barrier.The embedded astaxanthin component constructs "acid protection-alkali disintegration" intestinal tract targeted delivery system, solves the core problem that astaxanthin absorption rate is extremely low in east star spot digestion process.Through the delivery design of "fixed point release-in situ solubilization-double path absorption", astaxanthin bioavailability is significantly improved.The breeding method of the present application not only improves the external performance of body color, but also enhances the antioxidant defense system of fish body from the internal physiological level, so that the east star spot is comprehensively improved in body color and stress resistance.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for maintaining the original red color of the red grouper in aquaculture. Background Technology

[0002] The red grouper, scientifically known as *Scophagus leopardii*, belongs to the genus *Scophagus* in the family Serranidae, order Perciformes. With its slender and graceful body, the red grouper is covered in numerous blue-white spots, its base a vibrant red to orange-red with extremely high color saturation, earning it the nickname "the ruby ​​of the sea." Not only is it considered a top-grade sashimi and steamed ingredient, but its red color also symbolizes joy and good fortune in traditional Chinese culture, making it a signature species for festive banquets and high-end dining. A single grouper commands a price far exceeding other grouper of the same size, highlighting its significant economic value. In recent years, with the continued decline of wild resources and increasingly stringent fishing regulations, the commercial supply of red grouper has become highly dependent on aquaculture. Currently, the mainstream aquaculture model is recirculating aquaculture systems (RAS). RAS, by controlling environmental parameters such as temperature, light, and water quality, demonstrates significant advantages in achieving stable year-round production, shortening the breeding cycle, and preventing disease transmission, making it an important production method for both fry and fattening of red grouper. However, factory farming also brings a phenotypic defect that seriously affects the commodity value: the body color of farmed grouper gradually darkens and turns black, forming a stark contrast with the bright red appearance of wild individuals. This difference in body color directly leads to significant price grading in the market for farmed grouper, with the price difference between red and black individuals reaching several times. This severely weakens the overall efficiency of the factory farming model and has become a key technological bottleneck restricting the high-quality development of the industry. Summary of the Invention

[0003] In view of this, the present invention proposes a method for maintaining the original red color of the Oriental Star Spot to solve the above problems.

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

[0005] A method for maintaining the original red coloration of the Oriental Star Spotted Spot during cultivation includes the following steps:

[0006] A1. Site selection and system layout of aquaculture platform: Select an open sea area with salinity of 32.3-34.6 psu, and use a gravity-type offshore aquaculture platform for aquaculture. The aquaculture platform is equipped with gravity-type aquaculture cages, and shade nets are erected above the cages.

[0007] A2. Fish fry release: Healthy fry are released into the net cages, and the fry are disinfected before release;

[0008] A3. Management during the breeding period: During the breeding period, feed the fish with special compound feed containing encapsulated astaxanthin components. Different feeds are fed according to the juvenile, medium and adult stages. The daily feeding amount is 1.5-2.5% of the total weight of the fish, and the feed is divided into two feedings in the morning and evening.

[0009] A4. Fish Management: 14-21 days before fish are harvested, adjust the feeding frequency to once a day and adjust the feeding amount to 1.0-1.5% of the total fish weight; stop feeding 1-2 days before fish are harvested; when harvesting, complete the grading, weighing and packing on the operating platform, and maintain a low temperature and dark environment throughout the entire process after the fish are harvested.

[0010] Furthermore, in step A1, the open sea area has a seawater current velocity of 0.3-0.8 m / s, a water depth of ≥15 m, a water temperature of 20-32℃, a transparency of ≥5 m, dissolved oxygen of ≥5 mg / L, and a pH of 7.8-8.4. The net cages have a net depth of 13-14 m and a mesh size of 2.5-4.0 cm. The net cages are made of HDPE material. Shading nets are erected 1.8-2.0 m above the net cages, with a shading rate of 90%.

[0011] Furthermore, in step A2, the fish fry should be 60-80g in size, and the stocking density should be 18-22 fish / m3, released in the early morning or evening.

[0012] Furthermore, in step A2, disinfection is carried out as follows: fish fry are immersed in disinfectant at a mass-to-volume ratio of 1:8-12 for 8-12 minutes. The mass-to-volume ratio is in kg / L. The total weight of fish fry in a single immersion is ≤5kg. Fresh disinfectant is used between each batch.

[0013] Furthermore, the disinfectant is prepared by the following method: Sodium alginate is prepared into a 1.5-2.0% aqueous solution and swelled for 12-24 hours to obtain a sodium alginate aqueous solution for later use; 0.6-0.8 kg of sea salt is dissolved in 2-3 L of purified water and stirred until completely dissolved to obtain a sea salt solution for later use; 6.0-6.5 L of noni fruit fermentation broth, 1.8-2.2 L of moringa leaf water extract, and 0.3-0.5 L of sodium alginate aqueous solution are mixed and stirred at 150-200 rpm / min for 10-15 min; the sea salt solution is added to bring the total volume to 10 L, and the mixture is stirred at 100-150 rpm / min for 5-10 min to obtain the disinfectant.

[0014] Furthermore, the noni fruit fermentation broth is prepared by the following method: Ripe noni fruits are washed, drained, and chopped. The chopped noni fruits are then placed into a fermentation container, filling it to 75-80% of its total volume. 0.5-1.0% of the total mass of the chopped noni fruits is inoculated with *Lactobacillus plantarum* activation solution. The mixture is then subjected to anaerobic fermentation at 25-35℃ under dark conditions for 30-45 days. After fermentation, the fruit residue is removed by coarse filtration through multiple layers of gauze. The resulting filtrate is centrifuged at 5000-8000 rpm for 15-20 min, and the supernatant is collected. The supernatant is then filtered through a 0.22 μm microfiltration membrane for sterilization to obtain the noni fruit fermentation broth.

[0015] Furthermore, the activated Lactobacillus plantarum solution was prepared by the following method: Lactobacillus plantarum was inoculated into MRS liquid medium and incubated statically at 35-37℃ for 12-16 hours to obtain a viable count ≥10. 8 CFU / mL of Lactobacillus activating solution.

[0016] Furthermore, the water extract of Moringa leaves is prepared by the following method: Moringa leaves are washed, drained, and dried at 40-50℃ until the moisture content is ≤10%. The leaves are then pulverized and passed through a 40-60 mesh sieve to obtain Moringa powder. The Moringa powder is added to purified water at a mass-to-volume ratio of 1:15-25 (kg / L) at 50-70℃ and extracted in a constant temperature water bath for 1.5-2.5 hours, stirring every 20-30 minutes. After extraction, the solution is filtered through a 200-mesh filter cloth, and the filtrate is collected. The residue is added to purified water at a mass-to-volume ratio of 1:8-12 and extracted a second time under the same conditions for 1.0-1.5 hours. The mixture is filtered again, and the filtrate is collected. The two filtrates are combined and concentrated under reduced pressure at 50-60℃ and a vacuum degree of -0.08 to -0.10 MPa to 20-25% of the original volume to obtain a concentrated solution. The concentrate was filtered through a 0.45 μm microfiltration membrane and then through a 0.22 μm microfiltration membrane to obtain the water extract of Moringa leaves.

[0017] Furthermore, in step A3, the encapsulated astaxanthin component comprises the following raw materials in parts by weight: 10-15 parts astaxanthin, 30-40 parts amidated pectin, 5-10 parts calcium chloride or calcium lactate, 3-8 parts sodium taurocholate, 5-10 parts krill oil phospholipids, 2-5 parts chitosan oligosaccharide with a molecular weight of 1-3 kDa, 3-6 parts zein, and 10-20 parts maltodextrin.

[0018] Furthermore, the astaxanthin-encapsulated component was prepared by the following method:

[0019] B1. Preparation of Astaxanthin-Zezyme Composite Nanoparticles: Astaxanthin and zezyme were dissolved in an aqueous ethanol solution with a concentration of 65-75% at a total mass of 10-20 times to obtain an organic phase; the organic phase was added to an aqueous phase composed of deionized water at a volume ratio of 1:5-8 at a rate of 5-10 mL / min under a stirring speed of 6000-8000 rpm, and stirring was continued at 500-1000 rpm for 20-30 min to obtain an astaxanthin-zezyme composite nanoparticle suspension; the ethanol was removed by vacuum distillation under conditions of 40-50℃ and a vacuum degree of -0.08 to -0.10 MPa to obtain a nanoparticle suspension for later use;

[0020] B2. Preparation of pre-assembled micelles: Sodium taurocholate and krill oil phospholipids were dissolved in 15-25 times their total weight of phosphate buffer solution with a pH of 7.2-7.4. The solution was then sonicated at 200-400W for 10-15 min under ice bath conditions, with a work / interval ratio of 3 s / 3 s, to obtain an empty pre-assembled nanomicelle solution for later use.

[0021] B3. Assembly of the co-encapsulation system: Dissolve amidated pectin in 10-15 times its weight of deionized water and stir at 500-800 rpm until completely dissolved to obtain a pectin solution. Add nano-core suspension, empty pre-assembled nano micelle solution and chitosan oligosaccharide sequentially while stirring at 500-800 rpm. After the addition is complete, stir at 500-800 rpm for 15-20 min to obtain a mixed aqueous phase.

[0022] B4. Ion crosslinking molding: Dissolve calcium chloride or calcium lactate in 40-60 times its weight of deionized water to obtain a calcium ion solution; under magnetic stirring at 300-500 rpm, add the mixed aqueous phase dropwise to the calcium ion solution at a dropping rate of 1-3 mL / min. After the addition is complete, stir at 300-500 rpm to solidify for 30-45 min to form a suspension of nanogel particles.

[0023] B5. Separation and Drying: Centrifuge the nanogel particle suspension at 8000-10000 rpm for 10-15 min, discard the supernatant, and collect the nanogel particle precipitate; dissolve maltodextrin in 1-2 times its weight of purified water to obtain a maltodextrin solution; add the nanogel particle precipitate to the maltodextrin solution, stir at 50-100 rpm for 5-10 min to uniformly wet and disperse it, spread it on a freeze-drying tray, and freeze-dry at -45~-50℃ and a vacuum degree <20 Pa for 24-48 h to obtain the encapsulated astaxanthin component.

[0024] Furthermore, in step A3, the juvenile fish are fed juvenile feed at a daily rate of 1.5-2.5% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of juvenile feed includes the following ingredients by weight: 280-320g white fish meal, 180-220g steamed fish meal, 50-80g Antarctic krill meal, 80-120g soybean protein concentrate, 40-60g fish oil, 3-5g astaxanthin-encapsulated components, 20-30g yeast hydrolysate, 4-6g mineral premix, 0.01-0.02g vitamin A, 0.005-0.01g vitamin D3, 1-3g tartrate, 3-5g galacto-oligosaccharides, 40-60g flour, and the remainder is a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 3.0-4.0mm.

[0025] Furthermore, in step A3, during the mid-stage of the fish, feed them mid-stage feed at a daily rate of 1.5-2.5% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of mid-stage feed includes the following ingredients by weight: 250-280g white fish meal, 200-250g steamed fish meal, 80-120g Antarctic krill meal, 100-140g soybean protein concentrate, 50-70g fish oil, 4-6g astaxanthin-encapsulated components, 25-35g yeast hydrolysate, 5-10g mineral premix, 0.01-0.02g vitamin A, 0.005-0.01g vitamin D3, 1.5-2.5g tribocylate, 4-6g galactooligosaccharides, 40-60g flour, with the remainder being a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 5.0-6.0mm.

[0026] Furthermore, in step A3, adult fish are fed adult fish feed at a daily rate of 1.5-2.5% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of adult fish feed includes the following ingredients by weight: 220-260g white fish meal, 220-260g steamed fish meal, 60-90g Antarctic krill meal, 80-100g soybean protein concentrate, 60-80g fish oil, 5-8g astaxanthin-encapsulated components, 20-30g yeast hydrolysate, 5-10g mineral premix, 0.015-0.025g vitamin A, 0.008-0.012g vitamin D3, 1-2g tribocylate, 3-5g galactooligosaccharides, with the remainder being a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 7.0-8.0mm.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention solves the core problem of extremely low absorption rate of astaxanthin during the digestion of grouper by constructing an "acid protection-alkali disintegration" intestinal targeted delivery system. On the one hand, a nanogel carrier formed by the cross-linking of amidated high-methoxyl pectin and calcium ions is used. In the strongly acidic environment of the stomach, this carrier exists in a dense state, physically confining astaxanthin within the carrier and effectively resisting gastric acid damage, allowing it to pass through the stomach intact and reach the intestinal absorption site. On the other hand, the carrier responsively disintegrates in the weakly alkaline environment of the intestine, simultaneously releasing sodium taurocholate / krill oil phospholipid pre-assembled nanomicelles that capture astaxanthin molecules in situ within the intestinal lumen, forming ultra-small mixed micelles that can be directly taken up by the intestinal epithelium. Simultaneously, chitosan oligosaccharides open the tight junctions of the intestinal epithelium, creating an alternative absorption pathway. Through this "point-to-point release-in-situ solubilization-dual-pathway absorption" delivery design, the bioavailability of astaxanthin is significantly improved. Feed containing this encapsulated astaxanthin component resulted in redder grouper body color and better antioxidant activity.

[0029] 2. The fish fry disinfectant of this invention uses noni fruit fermentation broth and moringa leaf water extract as core active components, supplemented by sodium alginate for film-forming protection. The organic acids in the noni fruit fermentation broth provide a pH barrier and broad-spectrum bactericidal efficacy, while scopolamine specifically interferes with the Vibrio quorum sensing system; moringin and isothiocyanates in the moringa leaf water extract synergistically fight bacteria, and flavonoids provide antioxidant protection; sodium alginate forms a protective gel film on the fish's surface. This overcomes the defect of chemical disinfectants damaging the fish's mucus barrier while providing broad-spectrum bactericidal activity.

[0030] 3. This invention organically integrates shading and light control on offshore aquaculture platforms, nutritional regulation, precise feeding with encapsulated astaxanthin, and green disinfection of seedlings, forming a comprehensive color maintenance technology system covering the entire lifecycle of grouper farming. Differentiated feed formulations for the juvenile, mid-adult, and adult stages, combined with the dual protection of intestinal health provided by tributyric acid glycerides and galactooligosaccharides, achieve full-process color management from seedling placement to market launch. After adopting this solution, the activity of liver antioxidant enzymes (SOD, GSH-Px) and total antioxidant capacity (T-AOC) in grouper significantly increased, while the content of lipid peroxidation product MDA significantly decreased. This demonstrates that this solution not only improves the external appearance of color but also enhances the fish's antioxidant defense system from an internal physiological perspective, resulting in a comprehensive improvement in the color and stress resistance of farmed grouper. Detailed Implementation

[0031] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0034] Example 1

[0035] A method for maintaining the original red coloration of the Oriental Star Spotted Spot during cultivation includes the following steps:

[0036] A1. Site selection and system layout of aquaculture platform: Select an open sea area with salinity of 32.3-34.6 psu, seawater velocity of 0.3-0.8 m / s, water depth ≥15 m, water temperature of 20-32℃, transparency ≥5 m, dissolved oxygen ≥5 mg / L, and pH of 7.8-8.4. Gravity-type offshore aquaculture platform will be used for aquaculture. The aquaculture platform is equipped with gravity-type aquaculture cages with a net depth of 13 m and a mesh size of 2.5 cm. The cages are made of HDPE material. A shade net is erected above the cages, with a shade rate of 90% and an installation height of 1.8 m above the cages.

[0037] A2. Fish Fry Stocking: Healthy fry, weighing 80g, are stocked into the net cages at a density of 18 fry / m². 3 Release the fry in the early morning or evening. Disinfect the fry before release by means of the following method: Soak the fry in a disinfectant solution at a mass-to-volume ratio of 1:8 for 12 minutes. The mass-to-volume ratio is in kg / L. The total weight of the fry soaked at one time is 5 kg. Replace the disinfectant solution between each batch.

[0038] A3. Management during the breeding period: During the breeding period, feed the fish with special compound feed containing encapsulated astaxanthin components. Different feeds are given for the juvenile, medium and adult stages. The daily feeding amount is 2.0% of the total weight of the fish, and the feed is given twice a day, in the morning and evening.

[0039] A4. Fish Management: 14 days before the fish are harvested, the feeding frequency is adjusted to once a day, and the feeding amount is adjusted to 1.2% of the total weight of the fish; feeding is stopped 1 day before the fish are harvested; when harvesting, the fish are graded, weighed and packed on the operating platform, and the fish are kept in a low temperature and dark environment throughout the entire process after harvesting.

[0040] The above-mentioned disinfectant was prepared by the following method: sodium alginate was prepared into a 1.5% aqueous solution and swollen for 12 hours to obtain a sodium alginate aqueous solution for later use; 0.6 kg of sea salt was dissolved in 2 L of purified water and stirred until completely dissolved to obtain a sea salt solution for later use; 6.0 L of noni fruit fermentation broth, 1.8 L of moringa leaf water extract, and 0.3 L of sodium alginate aqueous solution were mixed and stirred at 150 rpm / min for 15 min; the sea salt solution was added to make up to 10 L, and the mixture was stirred at 100 rpm / min for 10 min to obtain the disinfectant.

[0041] The noni fruit fermentation broth was prepared by the following method: Ripe noni fruits were washed, drained, and chopped. The chopped noni fruits were then placed into a fermentation container, filling it to 75% of its total volume. 0.5% (by weight of the chopped noni fruits) of *Lactobacillus plantarum* activated solution was inoculated into the container. The mixture was then subjected to anaerobic fermentation at 25°C in the dark for 45 days. After fermentation, the fruit residue was removed by coarse filtration through multiple layers of gauze. The resulting filtrate was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm microfiltration membrane for sterilization to obtain the noni fruit fermentation broth. The *Lactobacillus plantarum* activated solution was prepared by the following method: *Lactobacillus plantarum* was inoculated into MRS liquid medium and cultured at 35°C for 16 h to obtain a viable count ≥10⁻⁶. 8 CFU / mL of Lactobacillus activating solution.

[0042] The water extract of Moringa leaves was prepared by the following method: Moringa leaves were washed, drained, dried at 40℃ to a moisture content of 10%, and pulverized through a 40-mesh sieve to obtain Moringa powder. The Moringa powder was added to purified water at 50℃ at a mass-to-volume ratio of 1:15 (kg / L), and extracted in a constant-temperature water bath for 2.5 hours, stirring every 30 minutes. After extraction, the solution was filtered through a 200-mesh filter cloth, and the filtrate was collected. The residue was added to purified water at a mass-to-volume ratio of 1:8 and extracted a second time for 1.5 hours under the same conditions, followed by filtration and collection of the filtrate. The two filtrates were combined and concentrated under reduced pressure at 50℃ and a vacuum of -0.08 MPa to 25% of the original volume to obtain a concentrated solution. The concentrated solution was filtered through a 0.45 μm microfiltration membrane and then through a 0.22 μm microfiltration membrane to obtain the water extract of Moringa leaves.

[0043] The above-mentioned astaxanthin encapsulation components include the following raw materials in parts by weight: 10 parts astaxanthin, 30 parts amidated pectin, 5 parts calcium chloride or calcium lactate, 3 parts sodium taurocholate, 5 parts krill oil phospholipids, 2 parts chitosan oligosaccharide with a molecular weight of 1 kDa, 3 parts zein, and 10 parts maltodextrin.

[0044] The above-mentioned encapsulated astaxanthin components were prepared according to the following method:

[0045] B1. Preparation of Astaxanthin-Zezyme Composite Nanonuclei: Astaxanthin and zezyme were dissolved in an aqueous solution of 75% ethanol at a total mass of 10 times to obtain an organic phase; the organic phase was added to an aqueous phase composed of deionized water at a volume ratio of 1:5 at a rate of 5 mL / min under a stirring speed of 6000 rpm, and stirring was continued at 500 rpm for 30 min to obtain an astaxanthin-zezyme composite nanoparticle suspension; the ethanol was removed by vacuum distillation at 40℃ and a vacuum degree of -0.08 MPa to obtain a nanonuclei suspension for later use;

[0046] B2. Preparation of pre-assembled micelles: Sodium taurocholate and krill oil phospholipids were dissolved in 15 times their total weight of phosphate buffer at pH 7.2. The solution was then sonicated at 200W for 15 min under ice bath conditions, with a work / interval ratio of 3 s / 3 s, to obtain an empty pre-assembled nanomicelle solution for later use.

[0047] B3. Assembly of the co-embedding system: The amidated pectin was dissolved in 10 times its weight of deionized water and stirred at 500 rpm until completely dissolved to obtain a pectin solution. The nano-core suspension, empty pre-assembled nano micelle solution, and chitosan oligosaccharide were added sequentially while stirring at 500 rpm. After the addition was completed, the mixture was stirred at 500 rpm for 20 min to obtain a mixed aqueous phase.

[0048] B4. Ionic crosslinking molding: Dissolve calcium chloride or calcium lactate in 40 times its weight of deionized water to obtain a calcium ion solution; add the mixed aqueous phase dropwise to the calcium ion solution at a rate of 1 mL / min under magnetic stirring at 300 rpm; after the addition is complete, stir at 300 rpm to solidify for 45 min to form a suspension of nanogel particles.

[0049] B5. Separation and drying: The nanogel particle suspension was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the nanogel particle precipitate was collected; maltodextrin was dissolved in 1 times its weight of purified water to obtain a maltodextrin solution; the nanogel particle precipitate was added to the maltodextrin solution and stirred at 50 rpm for 10 min to make it uniformly wetted and dispersed, spread on a freeze-drying tray, and freeze-dried at -45℃ and 15 Pa vacuum for 48 h to obtain the encapsulated astaxanthin component.

[0050] In step A3 above, the juvenile fish are fed juvenile feed at a daily rate of 2.0% of the total fish weight, divided into two feedings, one in the morning and one in the evening. Each kg of juvenile feed includes the following ingredients by weight: 280g white fish meal, 180g steamed fish meal, 50g Antarctic krill meal, 80g soybean protein concentrate, 40g fish oil, 3g astaxanthin-encapsulated component, 20g yeast hydrolysate, 4g mineral premix, 0.01g vitamin A, 0.005g vitamin D3, 1g tartrate, 3g galactooligosaccharides, 40g flour, and the remainder is a filler, which is zeolite powder or diatomaceous earth. The feed particle size is 3.0mm.

[0051] In step A3 above, during the mid-stage of the fish, feed should be given at a rate of 2.0% of the total fish weight per day, divided into two feedings, one in the morning and one in the evening. Each kg of mid-stage feed should include the following ingredients by weight: 250g white fish meal, 200g steamed fish meal, 80g Antarctic krill meal, 100g soybean protein concentrate, 50g fish oil, 4g astaxanthin-encapsulated component, 25g yeast hydrolysate, 5g mineral premix, 0.01g vitamin A, 0.005g vitamin D3, 1.5g tartrate, 4g galactooligosaccharides, 40g flour, and the remainder should be a filler, which should be zeolite powder or diatomaceous earth. The feed particle size should be 5.0mm.

[0052] In step A3 above, adult fish are fed adult fish feed at a daily rate of 2.0% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of adult fish feed includes the following ingredients by weight: 220g white fish meal, 220g steamed fish meal, 60g Antarctic krill meal, 80g soybean protein concentrate, 60g fish oil, 5g astaxanthin-encapsulated component, 20g yeast hydrolysate, 5g mineral premix, 0.015g vitamin A, 0.008g vitamin D3, 1g tartrate, 3g galactooligosaccharides, and the remainder is a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 7.0mm.

[0053] Example 2

[0054] A method for maintaining the original red coloration of the Oriental Star Spotted Spot during cultivation includes the following steps:

[0055] A1. Site Selection and System Layout of Aquaculture Platform: An open sea area with a salinity of 32.3-34.6 psu, seawater current velocity of 0.3-0.8 m / s, water depth ≥15 m, water temperature of 20-32℃, transparency ≥5 m, dissolved oxygen ≥5 mg / L, and pH of 7.8-8.4 will be selected. Gravity-type offshore aquaculture platforms will be used for aquaculture. The platforms will be equipped with gravity-type aquaculture cages with a net depth of 14 m and a mesh size of 3.0 cm. The cages will be made of HDPE material, and a shade net will be erected above the cages at a height of 1.9 m, with a 90% shading rate. A2. Fish Fry Stocking: Healthy fish fry (80g each) will be stocked into the cages at a stocking density of 20 fry / m². 3 Release the fry in the early morning or evening. Disinfect the fry before release by means of the following method: Soak the fry in a disinfectant solution at a mass-to-volume ratio of 1:10 for 10 minutes (mass-to-volume ratio unit: kg / L), with a total weight of 5 kg of fry soaked at a time. Replace the disinfectant solution between batches.

[0056] A3. Management during the breeding period: During the breeding period, feed the fish with special compound feed containing encapsulated astaxanthin components. Different feeds are given for the juvenile, medium and adult stages. The daily feeding amount is 2.0% of the total weight of the fish, and the feed is given twice a day, in the morning and evening.

[0057] A4. Fish Management: 14 days before the fish are harvested, the feeding frequency is adjusted to once a day, and the feeding amount is adjusted to 1.2% of the total weight of the fish; feeding is stopped 1 day before the fish are harvested; when harvesting, the fish are graded, weighed and packed on the operating platform, and the fish are kept in a low temperature and dark environment throughout the entire process after harvesting.

[0058] The above-mentioned disinfectant was prepared by the following method: sodium alginate was prepared into a 1.8% aqueous solution and swollen for 18 hours to obtain a sodium alginate aqueous solution for later use; 0.7 kg of sea salt was dissolved in 2.5 L of purified water and stirred until completely dissolved to obtain a sea salt solution for later use; 6.3 L of noni fruit fermentation broth, 2.0 L of moringa leaf water extract, and 0.4 L of sodium alginate aqueous solution were mixed and stirred at 180 rpm / min for 12 min, and the sea salt solution was added to make up to 10 L, and stirred at 120 rpm / min for 8 min to obtain the disinfectant.

[0059] The noni fruit fermentation broth was prepared by the following method: Ripe noni fruits were washed, drained, and chopped. The chopped noni fruits were then placed into a fermentation container, filling it to 78% of its total volume. 0.8% (by weight of the chopped noni fruits) of *Lactobacillus plantarum* activated solution was inoculated into the container. The mixture was then subjected to anaerobic fermentation at 30°C in the dark for 37 days. After fermentation, the fruit residue was removed by coarse filtration through multiple layers of gauze. The resulting filtrate was centrifuged at 6500 rpm for 18 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm microfiltration membrane for sterilization to obtain the noni fruit fermentation broth. The *Lactobacillus plantarum* activated solution was prepared by the following method: *Lactobacillus plantarum* was inoculated into MRS liquid medium and cultured at 36°C for 14 h to obtain a viable count ≥10⁻⁶. 8 CFU / mL of Lactobacillus activating solution.

[0060] The water extract of Moringa leaves was prepared by the following method: Moringa leaves were washed, drained, dried at 45℃ to a moisture content of 10%, and pulverized through a 50-mesh sieve to obtain Moringa powder. The Moringa powder was added to purified water at 60℃ at a mass-to-volume ratio of 1:20 (kg / L), and extracted in a constant-temperature water bath for 2.0 h, stirring every 25 min. After extraction, the solution was filtered through a 200-mesh filter cloth, and the filtrate was collected. The residue was added to purified water at a mass-to-volume ratio of 1:10 and extracted a second time under the same conditions for 1.2 h, followed by filtration, and the filtrate was collected again. The two filtrates were combined and concentrated under reduced pressure at 55℃ and a vacuum of -0.09 MPa to 22% of the original volume to obtain a concentrated solution. The concentrated solution was filtered through a 0.45 μm microfiltration membrane and then through a 0.22 μm microfiltration membrane to obtain the water extract of Moringa leaves.

[0061] The above-mentioned astaxanthin encapsulation components include the following raw materials in parts by weight: 12 parts astaxanthin, 35 parts amidated pectin, 8 parts calcium chloride or calcium lactate, 6 parts sodium taurocholate, 8 parts krill oil phospholipids, 4 parts chitosan oligosaccharide with a molecular weight of 2 kDa, 5 parts zein, and 15 parts maltodextrin.

[0062] The above-mentioned encapsulated astaxanthin components were prepared according to the following method:

[0063] B1. Preparation of Astaxanthin-Zezyme Composite Nanonuclei: Astaxanthin and zezyme were dissolved in an aqueous solution of 70% ethanol at a total mass of 15 times to obtain an organic phase; the organic phase was added to an aqueous phase composed of deionized water at a volume ratio of 1:6.5 at a rate of 8 mL / min under a stirring speed of 7000 rpm, and stirring was continued at 800 rpm for 25 min to obtain an astaxanthin-zezyme composite nanoparticle suspension; the ethanol was removed by vacuum distillation at 45℃ and a vacuum degree of -0.09 MPa to obtain a nanonuclei suspension for later use;

[0064] B2. Preparation of pre-assembled micelles: Sodium taurocholate and krill oil phospholipids were dissolved in 20 times their total weight of phosphate buffer at pH 7.3. The solution was then sonicated at 300W for 12 min under ice bath conditions, with a work / interval ratio of 3 s / 3 s, to obtain an empty pre-assembled nanomicelle solution for later use.

[0065] B3. Assembly of the co-encapsulation system: The amidated pectin was dissolved in 12 times its weight of deionized water and stirred at 600 rpm until completely dissolved to obtain a pectin solution. The nano-core suspension, empty pre-assembled nano micelle solution, and chitosan oligosaccharide were added sequentially while stirring at 600 rpm. After the addition was completed, the mixture was stirred at 600 rpm for 18 min to obtain a mixed aqueous phase.

[0066] B4. Ionic crosslinking molding: Dissolve calcium chloride or calcium lactate in 50 times its weight of deionized water to obtain a calcium ion solution; add the mixed aqueous phase dropwise to the calcium ion solution at a dropping rate of 2 mL / min under magnetic stirring at 400 rpm; after the addition is complete, stir at 400 rpm to solidify for 38 min to form a suspension of nanogel particles.

[0067] B5. Separation and drying: The nanogel particle suspension was centrifuged at 9000 rpm for 12 min, the supernatant was discarded, and the nanogel particle precipitate was collected; maltodextrin was dissolved in 1.5 times its weight of purified water to obtain a maltodextrin solution; the nanogel particle precipitate was added to the maltodextrin solution and stirred at 80 rpm for 8 min to make it uniformly wetted and dispersed, spread on a freeze-drying tray, and freeze-dried at -48℃ and 15 Pa vacuum for 36 h to obtain the encapsulated astaxanthin component.

[0068] In step A3 above, the juvenile fish are fed juvenile feed at a daily rate of 2.0% of the total fish weight, divided into two feedings, one in the morning and one in the evening. Each kg of juvenile feed includes the following ingredients by weight: 300g white fish meal, 200g steamed fish meal, 65g Antarctic krill meal, 100g soybean protein concentrate, 50g fish oil, 4g astaxanthin-encapsulated component, 25g yeast hydrolysate, 5g mineral premix, 0.02g vitamin A, 0.01g vitamin D3, 2g tartrate, 4g galactooligosaccharides, 50g flour, and the remainder is a filler, which is zeolite powder or diatomaceous earth. The feed particle size is 3.5mm.

[0069] In step A3 above, during the mid-stage of the fish, feed should be given at a rate of 2.0% of the total fish weight per day, divided into two feedings, one in the morning and one in the evening. Each kg of mid-stage feed should include the following ingredients by weight: 265g white fish meal, 225g steamed fish meal, 100g Antarctic krill meal, 120g soybean protein concentrate, 60g fish oil, 5g astaxanthin-encapsulated component, 30g yeast hydrolysate, 8g mineral premix, 0.02g vitamin A, 0.01g vitamin D3, 2.0g tartrate, 5g galactooligosaccharides, 50g flour, and the remainder should be a filler, which should be zeolite powder or diatomaceous earth. The feed particle size should be 5.5mm.

[0070] In step A3 above, adult fish are fed adult fish feed at a daily rate of 2.0% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of adult fish feed includes the following ingredients by weight: 240g white fish meal, 240g steamed fish meal, 75g Antarctic krill meal, 90g soybean protein concentrate, 70g fish oil, 6.5g astaxanthin-encapsulated component, 25g yeast hydrolysate, 8g mineral premix, 0.025g vitamin A, 0.012g vitamin D3, 1.5g tartrate, 4g galactooligosaccharides, and the remainder is a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 7.5mm.

[0071] Example 3

[0072] A method for maintaining the original red coloration of the Oriental Star Spotted Spot during cultivation includes the following steps:

[0073] A1. Site selection and system layout of aquaculture platform: Select an open sea area with salinity of 32.3-34.6 psu, seawater velocity of 0.3-0.8 m / s, water depth ≥15 m, water temperature of 20-32℃, transparency ≥5 m, dissolved oxygen ≥5 mg / L, and pH of 7.8-8.4. Gravity-type offshore aquaculture platform will be used for aquaculture. The aquaculture platform is equipped with gravity-type aquaculture cages with a net depth of 14 m and a mesh size of 4.0 cm. The cages are made of HDPE material. A shade net is erected above the cages, with a shade rate of 90% and an installation height of 2.0 m above the cages.

[0074] A2. Fish Fry Stocking: Healthy fry, weighing 80g, are stocked into the net cages at a density of 22 fry / m². 3 Release the fry in the early morning or evening. Disinfect the fry before release by means of the following method: Soak the fry in disinfectant at a mass-to-volume ratio of 1:12 for 8 minutes (mass-to-volume ratio unit: kg / L), with a total weight of 5 kg of fry soaked at a time. Replace the disinfectant solution between batches.

[0075] A3. Management during the breeding period: During the breeding period, feed the fish with special compound feed containing encapsulated astaxanthin components. Different feeds are given for the juvenile, medium and adult stages. The daily feeding amount is 2.0% of the total weight of the fish, and the feed is given twice a day, in the morning and evening.

[0076] A4. Fish Management: 21 days before fish are harvested, the feeding frequency is adjusted to once a day, and the feeding amount is adjusted to 1.2% of the total weight of the fish; feeding is stopped 1 day before fish are harvested; when harvesting, the fish are graded, weighed and packed on the operating platform, and a low-temperature and dark environment is maintained throughout the entire process after the fish are harvested.

[0077] The above-mentioned disinfectant was prepared by the following method: sodium alginate was prepared into a 2.0% aqueous solution and allowed to swell for 24 hours to obtain a sodium alginate aqueous solution for later use; 0.8 kg of sea salt was dissolved in 3 L of purified water and stirred until completely dissolved to obtain a sea salt solution for later use; 6.5 L of noni fruit fermentation broth, 2.2 L of moringa leaf water extract, and 0.5 L of sodium alginate aqueous solution were mixed and stirred at 200 rpm / min for 10 min; the sea salt solution was added to make up to 10 L, and the mixture was stirred at 150 rpm / min for 5 min to obtain the disinfectant.

[0078] The noni fruit fermentation broth was prepared by the following method: Ripe noni fruits were washed, drained, and chopped. The chopped noni fruits were then placed into a fermentation container, filling it to 80% of its total volume. 1.0% (by weight of the chopped noni fruits) of *Lactobacillus plantarum* activated solution was inoculated into the container. The mixture was then anaerobically fermented at 35°C in the dark for 30 days. After fermentation, the fruit residue was removed by coarse filtration through multiple layers of gauze. The resulting filtrate was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm microfiltration membrane for sterilization to obtain the noni fruit fermentation broth. The *Lactobacillus plantarum* activated solution was prepared by the following method: *Lactobacillus plantarum* was inoculated into MRS liquid medium and incubated at 37°C for 12 hours to obtain a viable count ≥10⁻⁶. 8 CFU / mL of Lactobacillus activating solution.

[0079] The water extract of Moringa leaves was prepared by the following method: Moringa leaves were washed, drained, dried at 50℃ to a moisture content of 10%, and pulverized through a 60-mesh sieve to obtain Moringa powder. The Moringa powder was added to purified water at 70℃ at a mass-to-volume ratio of 1:25 (kg / L), and extracted in a constant-temperature water bath for 1.5 hours, stirring every 20 minutes. After extraction, the solution was filtered through a 200-mesh filter cloth, and the filtrate was collected. The residue was added to purified water at a mass-to-volume ratio of 1:12 and extracted a second time for 1.0 hour under the same conditions, followed by filtration, and the filtrate was collected. The two filtrates were combined and concentrated under reduced pressure at 60℃ and a vacuum of -0.10 MPa to 20% of the original volume to obtain a concentrated solution. The concentrated solution was filtered through a 0.45 μm microfiltration membrane and then through a 0.22 μm microfiltration membrane to obtain the water extract of Moringa leaves.

[0080] The above-mentioned astaxanthin encapsulation components include the following raw materials in parts by weight: 15 parts astaxanthin, 40 parts amidated pectin, 10 parts calcium chloride or calcium lactate, 8 parts sodium taurocholate, 10 parts krill oil phospholipids, 5 parts chitosan oligosaccharide with a molecular weight of 3 kDa, 6 parts zein, and 20 parts maltodextrin.

[0081] The above-mentioned encapsulated astaxanthin components were prepared according to the following method:

[0082] B1. Preparation of Astaxanthin-Zezyme Composite Nanonuclei: Astaxanthin and zezyme were dissolved in an aqueous solution of 65% ethanol at a total mass of 20 times to obtain an organic phase; the organic phase was added to an aqueous phase composed of deionized water at a volume ratio of 1:8 at a rate of 10 mL / min under a stirring speed of 8000 rpm, and stirring was continued at 1000 rpm for 20 min to obtain an astaxanthin-zezyme composite nanoparticle suspension; the ethanol was removed by vacuum distillation at 50℃ and a vacuum degree of -0.10 MPa to obtain a nanonuclei suspension for later use;

[0083] B2. Preparation of pre-assembled micelles: Sodium taurocholate and krill oil phospholipids were dissolved in 25 times their total weight of phosphate buffer at pH 7.4. The solution was then sonicated at 400W for 10 min under ice bath conditions, with a work / interval ratio of 3 s / 3 s, to obtain an empty pre-assembled nanomicelle solution for later use.

[0084] B3. Assembly of the co-embedding system: The amidated pectin was dissolved in 15 times its weight of deionized water and stirred at 800 rpm until completely dissolved to obtain a pectin solution. The nano-core suspension, empty pre-assembled nano micelle solution, and chitosan oligosaccharide were added sequentially while stirring at 800 rpm. After the addition was completed, the mixture was stirred at 800 rpm for 15 min to obtain a mixed aqueous phase.

[0085] B4. Ionic crosslinking molding: Dissolve calcium chloride or calcium lactate in 60 times its weight of deionized water to obtain a calcium ion solution; add the mixed aqueous phase dropwise to the calcium ion solution at a rate of 3 mL / min under magnetic stirring at 500 rpm; after the addition is complete, stir at 500 rpm for 30 min to solidify and form a suspension of nanogel particles.

[0086] B5. Separation and drying: The nanogel particle suspension was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the nanogel particle precipitate was collected; maltodextrin was dissolved in twice the amount of purified water to obtain a maltodextrin solution; the nanogel particle precipitate was added to the maltodextrin solution and stirred at 100 rpm for 5 min to make it uniformly wetted and dispersed, spread on a freeze-drying tray, and freeze-dried at -50℃ and 25 Pa for 24 h to obtain the encapsulated astaxanthin component.

[0087] In step A3 above, the juvenile fish are fed juvenile feed at a daily rate of 2.0% of the total fish weight, divided into two feedings, one in the morning and one in the evening. Each kg of juvenile feed includes the following ingredients: 320g white fish meal, 220g steamed fish meal, 80g Antarctic krill meal, 120g soybean protein concentrate, 60g fish oil, 5g astaxanthin-encapsulated component, 30g yeast hydrolysate, 6g mineral premix, 0.02g vitamin A, 0.01g vitamin D3, 3g tartrate, 5g galactooligosaccharides, 60g flour, and the remainder is a filler, which is zeolite powder or diatomaceous earth. The feed particle size is 4.0mm.

[0088] In step A3 above, during the mid-stage of the fish, feed should be given at a rate of 2.0% of the total fish weight per day, divided into two feedings, one in the morning and one in the evening. Each kg of mid-stage feed should include the following ingredients by weight: 280g white fish meal, 250g steamed fish meal, 120g Antarctic krill meal, 140g soybean protein concentrate, 70g fish oil, 6g astaxanthin-encapsulated component, 35g yeast hydrolysate, 10g mineral premix, 0.02g vitamin A, 0.01g vitamin D3, 2.5g tartrate, 6g galactooligosaccharides, 60g flour, and the remainder should be a filler, which should be zeolite powder or diatomaceous earth. The feed particle size should be 5.5mm.

[0089] In step A3 above, adult fish are fed adult fish feed at a daily rate of 2.0% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of adult fish feed includes the following ingredients by weight: 260g white fish meal, 260g steamed fish meal, 90g Antarctic krill meal, 100g soybean protein concentrate, 80g fish oil, 8g astaxanthin-encapsulated components, 30g yeast hydrolysate, 10g mineral premix, 0.025g vitamin A, 0.012g vitamin D3, 2g tartrate, 5g galactooligosaccharides, and the remainder is a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 8.0mm.

[0090] Comparative Example 1

[0091] The difference between this comparative example and Example 2 is that commercially available astaxanthin was used instead of the encapsulated astaxanthin component.

[0092] Comparative Example 2

[0093] The difference between this comparative example and Example 2 is that the raw material for encapsulating astaxanthin does not contain chitosan oligosaccharides.

[0094] Comparative Example 3

[0095] Compared with Example 2, this comparative example differs in that the raw material for encapsulating astaxanthin does not contain sodium taurocholate or krill oil phospholipids. The encapsulated astaxanthin component was prepared according to the following method:

[0096] B1. Preparation of Astaxanthin-Zezyme Composite Nanonuclei: Astaxanthin and zezyme were dissolved in an aqueous solution of 70% ethanol at a total mass of 15 times to obtain an organic phase; the organic phase was added to an aqueous phase composed of deionized water at a volume ratio of 1:6.5 at a rate of 8 mL / min under a stirring speed of 7000 rpm, and stirring was continued at 800 rpm for 25 min to obtain an astaxanthin-zezyme composite nanoparticle suspension; the ethanol was removed by vacuum distillation at 45℃ and a vacuum degree of -0.09 MPa to obtain a nanonuclei suspension for later use;

[0097] B2. Assembly of the co-encapsulation system: The amidated pectin was dissolved in 12 times its weight of deionized water and stirred at 600 rpm until completely dissolved to obtain a pectin solution. The nano-core suspension and chitosan oligosaccharide were added sequentially while stirring at 600 rpm. After the addition was completed, the mixture was stirred at 600 rpm for 18 min to obtain a mixed aqueous phase.

[0098] B3. Ionic crosslinking molding: Dissolve calcium chloride or calcium lactate in 50 times its weight of deionized water to obtain a calcium ion solution; add the mixed aqueous phase dropwise to the calcium ion solution at a dropping rate of 2 mL / min under magnetic stirring at 400 rpm; after the addition is complete, stir at 400 rpm to solidify for 38 min to form a suspension of nanogel particles.

[0099] B4. Separation and drying: The nanogel particle suspension was centrifuged at 9000 rpm for 12 min, the supernatant was discarded, and the nanogel particle precipitate was collected; maltodextrin was dissolved in 1.5 times its weight of purified water to obtain a maltodextrin solution; the nanogel particle precipitate was added to the maltodextrin solution and stirred at 80 rpm for 8 min to make it uniformly wetted and dispersed, spread on a freeze-drying tray, and freeze-dried at -48℃ and 15 Pa vacuum for 36 h to obtain the encapsulated astaxanthin component.

[0100] Comparative Example 4

[0101] The difference between this comparative example and Example 2 is that, in step A2, the sample was disinfected by soaking in a 20 mg / L potassium permanganate solution for 10 minutes.

[0102] Comparative Example 5

[0103] The difference between this comparative example and Example 2 is that the disinfectant does not contain noni fruit fermentation liquid.

[0104] Comparative Example 6

[0105] The difference between this comparative example and Example 2 is that the disinfectant does not contain Moringa leaf water extract.

[0106] I. Body color and antioxidant properties testing

[0107] In a sea area of ​​Dongfang City, Hainan Province, fish were cultured according to the methods described in Examples 1-3 and Comparative Examples 1-3, with three replicates for each group. Fry were released on the same day in March, and the culture period was 8 months. Body color and antioxidant properties were tested on the same day after the culture period ended.

[0108] Body color was tested using the following method: a CR-400 colorimeter was used to measure body color, and the a* value of the abdomen was measured uniformly. Three bodies were randomly selected from each cage for testing. The results are expressed as mean ± standard deviation. The test results are shown in Table 1.

[0109] Antioxidant properties were tested as follows: Three animals were randomly selected from each cage, their bodies were dried with filter paper, and their liver tissue was quickly dissected on an ice tray using a scalpel sterilized with alcohol. The tissue mass was accurately weighed, rinsed three times with 0.86% cold saline solution to remove blood, and then dried with filter paper. The tissues were then placed in numbered test tubes and stored in a -20°C freezer. Remove the tissue to be tested from the refrigerator and accurately weigh it. Place it in a clean centrifuge tube and add 9 times its weight of 0.86% physiological saline. Under ice bath conditions, cut the tissue into small pieces with small scissors, then homogenize it using a homogenizer at 12000 r / min for 10 seconds each time, with 30-second intervals, for 3-4 consecutive times to prepare a 10% tissue homogenate. Then, place the tissue homogenate in an ultra-low temperature centrifuge at 4℃ and 2500 r / min for 12 min. Collect the supernatant and store it at 4℃. Within 24 hours, use a superoxide dismutase (SOD) test kit to detect SOD activity, a malondialdehyde (MDA) test kit to detect MDA activity, a glutathione peroxidase (GSH-Px) test kit to detect GSH-Px activity, and a total antioxidant capacity (T-AOC) test kit to detect T-AOC activity. The results are expressed as mean ± standard deviation. The test results are shown in Table 1.

[0110] Table 1

[0111]

[0112] As can be seen from Table 1, the grouper cultured in Examples 1-3 has a redder color and higher antioxidant activity.

[0113] Comparing Example 2 with Comparative Example 1, Comparative Example 1 used commercially available, unencapsulated astaxanthin, which was directly exposed to gastric acid in the highly acidic environment of the anchovy's stomach. Most astaxanthin molecules were destroyed by acid hydrolysis or isomerized and inactivated, significantly reducing the amount of intact astaxanthin actually reaching the intestinal absorption site. Simultaneously, the small amount of astaxanthin reaching the intestine also had low absorption efficiency due to the lack of absorption-promoting channels, resulting in insufficient astaxanthin deposition in the skin. In the liver, due to the limited availability of astaxanthin, the antioxidant enzyme system was not fully activated, leading to weak free radical scavenging ability and MDA accumulation. In contrast, Example 2, through the "acid protection-alkali disintegration" delivery mechanism of pectin-calcium gel, protected astaxanthin from complete passage through the stomach and precise release into the intestine. Simultaneously, the pre-assembled micelles and chitosan oligosaccharides synergistically solubilized and promoted absorption, significantly improving the bioavailability of astaxanthin. This resulted in sufficient skin pigment deposition, presenting a bright red body color, and comprehensive activation of liver antioxidant enzyme activity.

[0114] Comparing Example 2 with Comparative Example 2, the astaxanthin encapsulated component in Comparative Example 2 lacked chitosan oligosaccharides. Although the pectin-calcium carrier could still protect astaxanthin and pre-assembled micelles through the stomach and release them into the intestine, two key functions were missing: First, the lack of additional ionic cross-linking reinforcement from chitosan oligosaccharides in the stomach resulted in slightly lower carrier density compared to Example 2, potentially leading to premature leakage and destruction of a small amount of astaxanthin by gastric acid. Second, after release into the intestine, the absence of chitosan oligosaccharides to open the tight junctions of the intestinal epithelium meant that astaxanthin was absorbed solely through the transcellular pathway of the pre-assembled micelles, with the bypass absorption pathway closed. The absorption efficiency was lower than in Example 2, resulting in significantly lower body color vibrancy and antioxidant indicators. This demonstrates that chitosan oligosaccharides make an irreplaceable contribution to improving the bioavailability of astaxanthin.

[0115] Comparing Example 2 with Comparative Example 3, the astaxanthin-encapsulated component in Comparative Example 3 lacked sodium taurocholate and krill oil phospholipids, meaning it lacked pre-assembled nanomicelles. When the carrier disintegrated in the intestine, astaxanthin and chitosan oligosaccharides were released simultaneously. However, astaxanthin molecules are hydrophobic and have extremely low solubility in the aqueous phase of the intestinal lumen, failing to form mixed micelles that can be recognized and taken up by intestinal epithelial cells. Instead, they relied on the fish's endogenous bile acid secretion for gradual emulsification and solubilization. However, under formulated feed conditions, the amount and rhythm of bile secretion in grouper often cannot meet the immediate solubilization requirements of high-dose astaxanthin. A large amount of astaxanthin was not effectively micellized and was excreted with the feces, resulting in a low actual absorption rate. Therefore, although chitosan oligosaccharides opened some alternative absorption pathways, the amount of absorbable micellized astaxanthin was far less than in Example 2, resulting in moderate body color and antioxidant indicators, superior to Comparative Example 1 and Comparative Example 2, but significantly lower than Example 2. It is evident that pre-assembled micelles play a crucial role in relieving fish's dependence on endogenous bile acids and ensuring efficient solubilization and absorption of astaxanthin.

[0116] II. Disinfection Effectiveness Testing

[0117] Healthy grouper fry with an initial weight of 80g and no external damage were selected. Disinfection was performed according to the methods in Example 2 and Comparative Examples 4-6, with three replicates per group and ten fry per replicate. Immediately after disinfection, the fry were transferred to clean seawater for 30 minutes of temporary rearing. After temporary rearing, bacterial samples were collected from the fry's body surface for testing. Sampling was performed before disinfection and 30 minutes after disinfection. Before disinfection, three fry were randomly selected from each group for sampling. After disinfection and 30 minutes of temporary rearing, another three fry were randomly selected for sampling. A 2cm × 2cm area on the left side of the fry's body was wiped with a sterile cotton swab. The swab tip was cut into a test tube containing sterile physiological saline, and the tube was vortexed for 2 minutes to elute the stock solution. The stock solution was then serially diluted 10-fold with sterile physiological saline. 10... -1 10 -2 10 -3 Three dilutions were prepared, with 100 μL of each dilution spread onto TSA agar plates (total bacterial count) and TCBS agar plates (Vibrio count), with two replicates for each dilution. Plates with colony counts between 30 and 300 CFU were selected for counting, and results were expressed as CFU / cm³. 2 Surface area is used as the unit of measurement. The overall bactericidal rate and Vibrio clearance rate are calculated using the following formulas:

[0118] Total sterilization rate (%) = (Total colony count before disinfection - Total colony count after disinfection) / Total colony count before disinfection × 100%

[0119] Vibrio removal rate (%) = (Number of Vibrio colonies before disinfection - Number of Vibrio colonies after disinfection) / Number of Vibrio colonies before disinfection × 100%

[0120] The results are expressed as mean ± standard deviation, and are shown in Table 2.

[0121] 100 μL of each dilution was spread on seawater nutrient agar plates (total bacterial count) and TCBS agar plates (Vibrio count), with two parallel plates for each dilution.

[0122] Table 2

[0123]

[0124] As shown in Table 2, the total bacterial kill rate of Example 2 was not significantly different from that of the potassium permanganate group (Comparative Example 4), indicating that the broad-spectrum bactericidal efficacy of the disinfectant of this invention is comparable to that of chemical disinfectants. The kill rate dropped sharply to 72.54% after removing the noni fruit fermentation broth (Comparative Example 5), and to 81.36% after removing the Moringa leaf water extract (Comparative Example 6), proving that the two plant components are the core sources of the broad-spectrum bactericidal efficacy, and that they have a synergistic effect. The Vibrio scavenging rate of Example 2 was numerically higher than its total bacterial kill rate, suggesting that this disinfectant has a certain selective suppression trend against Vibrio. This is mainly because scopolamine in the noni fruit fermentation broth can specifically interfere with the Vibrio quorum sensing system, hindering biofilm formation and suppressing the expression of virulence factors, making it easier for Vibrio to be killed by organic acids or removed by the protective membrane. Although potassium permanganate also has a high kill rate against Vibrio, it may cause damage to the mucus on the surface of fish. The disinfectant of this invention can effectively kill bacteria without damaging the mucus barrier on the surface of fish. It maintains the secretory function of mucus cells through the protective film of sodium alginate and the antioxidant components of moringa, making it more green and environmentally friendly.

[0125] 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 method for maintaining the original red coloration of the Oriental Star Spotted Spot during cultivation, characterized in that, Includes the following steps: A1. Site selection and system layout of aquaculture platform: Select an open sea area with salinity of 32.3-34.6 psu, and use a gravity-type offshore aquaculture platform for aquaculture. The aquaculture platform is equipped with gravity-type aquaculture cages, and shade nets are erected above the cages. A2. Fish fry release: Healthy fry are released into the net cages, and the fry are disinfected before release; A3. Management during the breeding period: During the breeding period, feed the fish with special compound feed containing encapsulated astaxanthin components. Different feeds are fed according to the juvenile, medium and adult stages. The daily feeding amount is 1.5-2.5% of the total weight of the fish, and the feed is divided into two feedings in the morning and evening. A4. Fish Management: 14-21 days before fish are harvested, adjust the feeding frequency to once a day and adjust the feeding amount to 1.0-1.5% of the total fish weight; stop feeding 1-2 days before fish are harvested; when harvesting, complete the grading, weighing and packing on the operating platform, and maintain a low temperature and dark environment throughout the entire process after the fish are harvested.

2. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A1, the open sea area has a seawater flow velocity of 0.3-0.8 m / s, a water depth of ≥15 m, a water temperature of 20-32℃, a transparency of ≥5 m, dissolved oxygen of ≥5 mg / L, and a pH of 7.8-8.

4. The net cage has a net depth of 13-14 m and a mesh size of 2.5-4.0 cm. The net cage is made of HDPE material. The shade net is erected 1.8-2.0 m above the net cage and has a shading rate of 90%.

3. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A2, the fish fry weigh 60-80g and are stocked at a density of 18-22 fish / m³. 3 It should be released in the early morning or evening.

4. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A2, disinfection is carried out as follows: fish fry are immersed in disinfectant at a mass-to-volume ratio of 1:8-12 for 8-12 minutes. The mass-to-volume ratio is in kg / L. The total weight of fish fry in a single immersion is ≤5kg. Fresh disinfectant is used between each batch.

5. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 4, characterized in that, The disinfectant is prepared by the following method: sodium alginate is prepared into an aqueous solution with a concentration of 1.5-2.0%, and swelled for 12-24 hours to obtain an aqueous solution of sodium alginate for later use; 0.6-0.8 kg of sea salt is dissolved in 2-3 L of purified water and stirred until completely dissolved to obtain a sea salt solution for later use; 6.0-6.5 L of noni fruit fermentation broth, 1.8-2.2 L of moringa leaf water extract, and 0.3-0.5 L of sodium alginate aqueous solution are mixed and stirred at 150-200 rpm / min for 10-15 min; the sea salt solution is added to make up to 10 L, and stirred at 100-150 rpm / min for 5-10 min to obtain the disinfectant.

6. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A3, the encapsulated astaxanthin component comprises the following raw materials in parts by weight: 10-15 parts astaxanthin, 30-40 parts amidated pectin, 5-10 parts calcium chloride or calcium lactate, 3-8 parts sodium taurocholate, 5-10 parts krill oil phospholipids, 2-5 parts chitosan oligosaccharide with a molecular weight of 1-3 kDa, 3-6 parts zein, and 10-20 parts maltodextrin.

7. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 6, characterized in that, The encapsulated astaxanthin component was prepared according to the following method: B1. Preparation of Astaxanthin-Zezyme Composite Nanoparticles: Astaxanthin and zezyme were dissolved in an aqueous ethanol solution with a concentration of 65-75% at a total mass of 10-20 times to obtain an organic phase; the organic phase was added to an aqueous phase composed of deionized water at a volume ratio of 1:5-8 at a rate of 5-10 mL / min under a stirring speed of 6000-8000 rpm, and stirring was continued at 500-1000 rpm for 20-30 min to obtain an astaxanthin-zezyme composite nanoparticle suspension; the ethanol was removed by vacuum distillation under conditions of 40-50℃ and a vacuum degree of -0.08 to -0.10 MPa to obtain a nanoparticle suspension for later use; B2. Preparation of pre-assembled micelles: Sodium taurocholate and krill oil phospholipids were dissolved in 15-25 times their total weight of phosphate buffer solution with a pH of 7.2-7.

4. The solution was then sonicated at 200-400W for 10-15 min under ice bath conditions, with a work / interval ratio of 3 s / 3 s, to obtain an empty pre-assembled nanomicelle solution for later use. B3. Assembly of the co-embedding system: Dissolve amidated pectin in 10-15 times its weight of deionized water and stir at 500-800 rpm until completely dissolved to obtain a pectin solution. Add nano-core suspension, empty pre-assembled nano micelle solution and chitosan oligosaccharide sequentially while stirring at 500-800 rpm. After the addition is complete, stir at 500-800 rpm for 15-20 min to obtain a mixed aqueous phase. B4. Ion crosslinking molding: Dissolve calcium chloride or calcium lactate in 40-60 times its weight of deionized water to obtain a calcium ion solution; under magnetic stirring at 300-500 rpm, add the mixed aqueous phase dropwise to the calcium ion solution at a dropping rate of 1-3 mL / min. After the addition is complete, stir at 300-500 rpm to solidify for 30-45 min to form a suspension of nanogel particles. B5. Separation and Drying: Centrifuge the nanogel particle suspension at 8000-10000 rpm for 10-15 min, discard the supernatant, and collect the nanogel particle precipitate; dissolve maltodextrin in 1-2 times its weight of purified water to obtain a maltodextrin solution; add the nanogel particle precipitate to the maltodextrin solution, stir at 50-100 rpm for 5-10 min to uniformly wet and disperse it, spread it on a freeze-drying tray, and freeze-dry at -45~-50℃ and a vacuum degree <20 Pa for 24-48 h to obtain the encapsulated astaxanthin component.

8. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A3, the juvenile fish are fed juvenile feed at a daily rate of 1.5-2.5% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of juvenile feed includes the following ingredients by weight: 280-320g white fish meal, 180-220g steamed fish meal, 50-80g Antarctic krill meal, 80-120g soybean protein concentrate, 40-60g fish oil, 3-5g astaxanthin-encapsulated components, 20-30g yeast hydrolysate, 4-6g mineral premix, 0.01-0.02g vitamin A, 0.005-0.01g vitamin D3, 1-3g tartrate, 3-5g galacto-oligosaccharides, 40-60g flour, and the remainder is a filler, which is zeolite powder or diatomaceous earth. The feed particle size is 3.0-4.0mm.

9. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A3, the fish are fed a mid-stage feed at a daily rate of 1.5-2.5% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of mid-stage feed includes the following ingredients by weight: 250-280g white fish meal, 200-250g steamed fish meal, 80-120g Antarctic krill meal, 100-140g soybean protein concentrate, 50-70g fish oil, 4-6g astaxanthin-encapsulated components, 25-35g yeast hydrolysate, 5-10g mineral premix, 0.01-0.02g vitamin A, 0.005-0.01g vitamin D3, 1.5-2.5g tribocylate, 4-6g galacto-oligosaccharides, 40-60g flour, and the remainder is a filler, which is zeolite powder or diatomaceous earth. The feed particle size is 5.0-6.0mm.

10. The method for maintaining the original red color of the Oriental Star Spotted Spot as described in claim 1, characterized in that, In step A3, adult fish are fed juvenile fish feed at a daily rate of 1.5-2.5% of their total weight, divided into two feedings, one in the morning and one in the evening. Each kg of adult fish feed includes the following ingredients by weight: 220-260g white fish meal, 220-260g steamed fish meal, 60-90g Antarctic krill meal, 80-100g soybean protein concentrate, 60-80g fish oil, 5-8g astaxanthin-encapsulated components, 20-30g yeast hydrolysate, 5-10g mineral premix, 0.015-0.025g vitamin A, 0.008-0.012g vitamin D3, 1-2g tribocylate, 3-5g galactooligosaccharides, with the remainder being a filler carrier, which is zeolite powder or diatomaceous earth. The feed particle size is 7.0-8.0mm.

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