Preparation method of composite algae microcapsule and application of composite algae microcapsule in tilapia mossambica feed

Abstract

The invention belongs to the technical field of preparation of active substance preparations, and discloses a preparation method of a composite algae microcapsule. The amphoteric starch containing hydrophilic and oleophylic groups is used as a main inclusion, is suitable for the stomachless physiological characteristic of tilapia, and can significantly improve the bioavailability; the wrapped active multi-nutrient particles contain various active nutrient substances such as algae astaxanthin solid particles, emulsified liquid of nanoscale emulsified particles, fish soluble, yeast cell wall powder and hylothrix powder. When the composite algae microcapsule is added into a tilapia compound feed, the brood quantity, the egg laying quality and the fertilization rate of parent tilapia can be remarkably increased, the hatching emergence rate is increased, the anti-stress capability, the immunocompetence and the growth performance of fry are enhanced, the breeding efficiency and the fry quality of tilapia culture are optimized, and the survival rate of tilapia is increased. The technical problems that in existing tilapia mossambica breeding, parent fish reproductive performance is low, fry are weak in physique, and the breeding survival rate is low are solved.

Description

Technical Field

[0001] This invention belongs to the field of active substance preparation technology, specifically relating to a method for preparing a composite algae microcapsule and its application in tilapia feed. Background Technology In the artificial breeding of tilapia, the reproductive performance of the broodstock directly determines the quantity and quality of fry supply, while the fry's physical condition directly affects the survival rate, growth rate, and ultimate breeding efficiency. Currently, tilapia farming in my country generally suffers from problems such as low broodstock egg production, scattered spawning, high egg deformity rate, and low fertilization rate. Furthermore, fertilized eggs are susceptible to environmental stress and pathogen infection during hatching, resulting in a hatching rate of less than 60%. Moreover, hatched fry generally exhibit weak stress resistance, low immune function, slow growth, and low survival rate, which not only increases breeding costs but also seriously hinders the large-scale expansion and standardized production of the tilapia farming industry.

[0002] The core reason for the above problems lies in the unreasonable nutritional ratio of existing tilapia formulated feeds, which lack functional components that can specifically improve the reproductive performance of broodstock and enhance the physical condition of fry. Current tilapia feeds mainly consist of grains, soybean meal, and fishmeal, which can only meet the basic growth needs of tilapia and cannot provide sufficient functional nutrients for broodstock reproduction, nor can they effectively enhance the immune activity and stress resistance of fry.

[0003] Excessive protein content in feed leads to low protein conversion rates, resource waste, and environmental pollution problems such as high ammonia nitrogen levels in the aquaculture water. Meanwhile, current methods often aim to reduce the burden on the liver by decreasing the fat content in feed, or to supplement feed with herbal extracts to improve intestinal digestion and absorption, thus enhancing the digestive capacity of tilapia. However, the former often fails to meet the nutritional needs of intensive tilapia farming, while the latter can significantly reduce the palatability of tilapia feed, resulting in poor feed stability, low feed intake, and reduced feed utilization, leading to slow growth. For a long time, the tilapia farming industry has pursued maximizing yield while neglecting the full utilization of nutrients and the pollution of the aquatic environment, increasing the disease rate of tilapia, which is extremely detrimental to their growth. Therefore, preparing a palatable and stable tilapia feed additive can not only improve the utilization rate of protein and fat by tilapia and enhance the digestion and absorption of feed, but also effectively ensure a stable feeding rate and improve the stability of feed feeding, making it more suitable for the growth and development of tilapia in large-scale farming.

[0004] Chinese patent CN114271412B discloses a mixed feed and its preparation method for improving the resistance of tilapia to saprolegniasis. The method involves drying and pulverizing tea residue, extracting and recovering the protein from the tea residue to obtain low-fiber tea residue protein containing highly stable polyphenols. This tea residue protein is then compounded with soybean oil, vitamins and minerals, fishmeal, flour, and gramineous crop by-products to form a mixed feed. This method is said to improve the non-specific immunity of tilapia and enhance their resistance to saprolegniasis. However, this patent does not address providing sufficient functional nutrients to improve feed utilization, nor does it effectively enhance the immune activity and stress resistance of fish fry.

[0005] Chinese patent CN116218679B discloses a functional feed additive for improving tilapia quality. This patent provides a strain of *Hygrophytes maculatus*, its cultivation method, and the prepared aquatic functional feed. The *Hygrophytes maculatus* provided in this patent is rich in palmitoleic acid, EPA, and *Laminaria japonica* polysaccharides, among other nutrients and active ingredients. It can be used as an immune enhancer to improve tilapia growth performance and accelerate growth rate; enhance tilapia's antioxidant capacity, improve its resistance to external environmental stress during aquaculture, reduce lipid deposition in the liver and susceptibility to pathogens, and improve liver health and disease resistance. It can also replace some antibiotics, disinfectants, and antibacterial agents. However, this patent does not address the utilization rate of protein and fat in tilapia, nor does it improve the conversion rate of feed digestibility and absorption. It cannot provide sufficient functional nutrients for broodstock reproduction, nor can it effectively enhance the immune activity and stress resistance of fry.

[0006] Therefore, how to improve the feed by providing sufficient functional nutrients that are suitable for the digestive mode of tilapia, a fish without a stomach, effectively increase the feed intake of tilapia, improve the utilization rate of active ingredients in the feed, provide sufficient functional nutrients for the breeding of parent fish, and effectively enhance the immune activity and stress resistance of fry are currently the difficult and hot research topics. Summary of the Invention

[0007] In order to solve the above problems, the purpose of this invention is to provide a method for preparing algae microcapsules, so as to solve one or more technical problems existing in the prior art, and at least provide a beneficial option or create conditions.

[0008] This invention primarily provides a method for preparing composite algae microcapsules that provide sufficient functional nutrients for broodstock reproduction, effectively enhance the immune activity and stress resistance of fry, are easily absorbed by the tilapia digestive system, and improve the water dispersibility, stability, and bioavailability of active ingredients such as astaxanthin. The technical solution is as follows: A composite algae microcapsule includes an encapsulating material and active multi-nutrient particles, wherein the active multi-nutrient particles are composed of the following raw materials in parts by weight: 30-40 parts of algal astaxanthin solid microparticles, 15-18 parts of emulsion of nano-sized emulsified microparticles, 15-20 parts of fish lysate, 3-5 parts of yeast cell wall powder, and 8-12 parts of filamentous algae powder.

[0009] The preparation method of the algal astaxanthin solid particles is as follows: S1. After drying Haematococcus pluvialis at a low temperature of 0-10℃ for 20-40 minutes, take it out and pulverize it with low temperature ultrasonic cell disruption at 0-5℃ to obtain Haematococcus pluvialis powder. S2, add Haematococcus pluvialis powder to an organic solvent containing a 3:1 volume ratio of ethanol to ethyl acetate at 20-30℃, with a material-to-liquid ratio of 1:30; mix with a magnetic stirrer for 120-150 minutes to fully dissolve the astaxanthin; separate the Haematococcus pluvialis residue and liquid by centrifugation at 8000-10000 r / min, and take the upper liquid; S3, the upper liquid is distilled under reduced pressure to obtain concentrated astaxanthin oil, vitamin E is added and stirred evenly; then silica is added and mixed evenly to load astaxanthin in silica, thus obtaining algal astaxanthin solid particles; wherein, the mass ratio of vitamin E, silica and astaxanthin oil is 0.2:2:3.

[0010] The silica is feed-grade oleophilic mesoporous silica with an oil absorption value of ≥2.2g / g and a pore size of 30-50nm; In aquatic compound feeds, fish oil is commonly used because it is rich in high-quality unsaturated fatty acids. Fish oil can improve the palatability and digestibility of feed, increase feed intake in aquatic animals, improve feed utilization, and promote growth and development, increasing weight gain rate and yield. However, compared to vegetable oils, fish oil is more prone to oxidation and rancidity, and may also contain persistent organic pollutants. Oxidized fish oil in feed can lead to reduced feed utilization, affect fish body composition, and inhibit growth performance in farmed animals. Studies have found that adding appropriate amounts of vegetable oil to fish feed affects fish muscle by increasing muscle fat content, improving muscle texture, adjusting the fatty acid composition of muscle, and improving the nutritional value of the feed.

[0011] Therefore, to better promote the growth performance of tilapia, improve the reproductive capacity of broodstock and the growth rate and disease resistance of juveniles, we also provide an emulsion containing positively charged nano-sized emulsified microparticles with plant-based fat sources and active disease-resistant factors, specifically: Eight parts of soybean lecithin, ten parts of soybean oil, three parts of glyceryl tartrate, three parts of glyceryl monostearate, five parts of tea tree oil, twelve parts of traditional Chinese medicine extract, and twelve parts of 1.5% chitosan aqueous solution were mixed using a high-pressure homogenization method to prepare a nano-sized emulsion. The pH value was adjusted to 5.5-6.0, a flavoring agent was added, and the mixture was stirred thoroughly to obtain an emulsion with uniformly dispersed positively charged nano-sized emulsion particles. The flavor compound is a mixture of lysine and arginine, with a mass ratio of lysine to arginine of 1:3 and a ratio of flavor compound to water of 1:6.

[0012] The aqueous solution of the herbal extracts, by weight, comprises 10-20 parts of Astragalus membranaceus extract, 8-15 parts of Angelica sinensis extract, 5-10 parts of Leonurus japonicus extract, 4-8 parts of Eucommia ulmoides extract, 1-2 parts of β-cyclodextrin, and 20-30 parts of deionized water. The raw materials are stirred for 20-30 minutes, allowed to stand for 5 minutes, and the supernatant is filtered.

[0013] The homogenization process is carried out 2 to 3 times using a high-pressure homogenization method, with a homogenization pressure of 30 to 60 MPa, a temperature of 40 to 50°C, and a processing time of 10 to 20 minutes.

[0014] Astragalus has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, and enhancing immunity. Its astragalus polysaccharides can significantly enhance the immune activity of tilapia. Angelica has the effects of nourishing blood and promoting blood circulation, regulating menstruation and relieving pain, and can promote the development of reproductive organs in tilapia broodstock. Eucommia has the effects of tonifying the liver and kidneys and strengthening tendons and bones, and can enhance the constitution of tilapia. Motherwort has the effect of promoting ovarian development and increasing the number and quality of eggs laid. Aqueous solutions of these herbal extracts have the effects of disease resistance, enhancing immune activity, and promoting reproduction and spawning.

[0015] Tributyric acid esters, as a precursor of butyric acid, can slowly release butyric acid in the small intestine. They possess various physiological benefits, including repairing intestinal villi, maintaining intestinal flora balance, enhancing the body's antioxidant capacity, and improving weight gain and feed utilization efficiency in tilapia. They can also replace antibiotic growth promoters. However, tributyric acid esters currently suffer from drawbacks such as poor water solubility, poor flowability, and a bitter taste. By combining them with vegetable oil to form emulsion microparticles and adding flavoring agents, the performance and stability of tributyric acid esters can be improved, enabling sustained release in animals and increasing bioavailability. The flavoring agents also enhance the palatability of the microcapsules, and the overall system's charge makes the emulsion highly stable and less prone to aggregation.

[0016] Preparation of filamentous algae powder: Select fresh, bright green, and odorless filamentous algae, rinse them 3-5 times with deionized water to remove surface impurities, attached bacteria, and salt; blanch the rinsed filamentous algae in boiling water for 2-3 minutes to remove the fishy smell and anti-nutritional factors such as phytic acid and tannins, then quickly cool them in cold water and drain; place the cooled filamentous algae in an oven and dry them at 55-65℃ for 5-7 hours until the moisture content is ≤5%; pulverize the dried filamentous algae into powder using a high-speed shearing machine to obtain filamentous algae powder, and seal and store it in a cool, dry place for later use.

[0017] The fish lysate mentioned is an enzymatically hydrolyzed fish lysate, specifically: S01, the collected fish scales, fish skin and fish bones are washed clean with water, soaked in 1% citric acid solution for 1 hour, rinsed clean with deionized water, and ground in a colloid mill to obtain a paste. Add S02 to the extract, vortex for 10 minutes at a material-to-liquid ratio of 1:1 to 3, centrifuge at 15000 r / min for 15 minutes, and take the supernatant; place it in a vacuum freeze dryer, rapidly freeze at -60 to -40℃ for 3 hours, and dry at low temperature for 10 hours to obtain freeze-dried powder. S03, take the freeze-dried powder, add the compound enzyme solution, and vortex for 5 minutes at a material-to-liquid ratio of 40:1; S04 is subjected to ultrasonic enzymatic hydrolysis at 50℃ for 3-6 hours, and the hydrolysate is purified by nanofiltration for 2-5 hours to obtain a high-concentration enzymatically hydrolyzed fish slurry.

[0018] The extract consists of betaine, citric acid, vitamin C, and deionized water in a mass ratio of 3:5:2:50. The complex enzyme solution consists of flavor protease, subtilisin, and deionized water in a mass ratio of 2–3:1–2:10.

[0019] The fish slurry is derived from the scales, skin, bones, and viscera of one or more of the following: tilapia, grass carp, silver carp, or crucian carp.

[0020] The betaine-citric acid-vitamin C system allows for the gentle and efficient extraction of active substances, providing antioxidant and structural protection immediately upon extraction, ultimately improving the yield, stability, and flavor of fish paste. Betaine, a natural zwitterionic compound, possesses extremely strong buffering capacity, creating an environment that efficiently dissolves active substances while minimizing protein acid denaturation. During extraction, betaine balances intracellular and extracellular osmotic pressure, thereby increasing the yield of active ingredients.

[0021] The compound enzyme solution is first deeply cleaved by Bacillus subtilis protease, and then the ends are refined by flavor protease, achieving the best balance between flavor and degree of hydrolysis. This results in rapid degradation of large protein molecules and a high proportion of small peptides with a molecular weight of less than 3000D in the enzymatic hydrolysate. The final enzymatically hydrolyzed fish lysate is rich in small peptides, contains a complete range of amino acids, and is free of bitterness. It is very suitable for the characteristics of tilapia, such as short intestines and rapid absorption, and is especially suitable for juvenile fish feed, reducing the digestive burden.

[0022] The aforementioned coating is a modified corn starch, prepared by combining it with chitosan and an emulsifier, specifically: S101, add corn starch to deionized water, stir evenly, slowly heat to 80-90℃, stir for 20-40 minutes, corn starch goes through the first and second stages and begins to gelatinize, continue stirring and reacting, add glycerol and stir for 20-40 minutes, dry, crush, grind to obtain modified small molecule starch granules. The mass ratio of glycerol, corn starch and deionized water is 1:20:80; S102: Modified small molecule starch granules are dispersed in deionized water, sodium bicarbonate is added, the pH value is adjusted to 8.5-9, arginine is added, and the temperature is controlled at 35-45℃ to introduce amino groups into the small molecule starch granules, generating cationic small molecule starch granules, which are then dried for later use. S103: Sodium tripolyphosphate is added to deionized water, sodium bicarbonate is added, and the pH is adjusted to 8.5-9. Then, it is added dropwise to cationic small molecule starch granules at room temperature while stirring rapidly. The mixture is then heated in a constant temperature water bath at 85-90°C for 40-60 minutes, and then cooled to 30-40°C with continued stirring. Octenyl succinic anhydride diluted with glycerol is then added, and the mixture is stirred at 30-40°C for 30-40 minutes. After that, the mixture is dried at low temperature to remove the glycerol, and then ground to obtain amphoteric starch granules.

[0023] The mass ratio of sodium tripolyphosphate, arginine, octenyl succinic anhydride and modified small molecule starch granules is 0.8–1: 0.8–1.2: 0.5–0.6: 15–20. Sodium tripolyphosphate can form ester bonds with small starch molecules containing hydroxyl groups, and simultaneously promote cross-linking between starch molecules. Under alkaline conditions, the hydroxyl groups of the cationic small starch molecule particles dissociate into oxygen anions, which undergo a nucleophilic substitution reaction with some of the positive charge on octenyl succinic anhydride to form octenyl succinic anhydride starch ester. The anhydride ring is opened, with one end binding to the starch molecule through the ester group and the other end producing a carboxylic acid. The treated starch granules contain both hydrophilic and hydrophobic groups, making them suitable as coatings. They possess some solubility and can remain in water for a period of time, preventing rapid nutrient loss upon contact with water.

[0024] S104. Chitosan is added to a 0.3% citric acid aqueous solution at 50-60℃ and stirred rapidly to dissolve it. Then, amphoteric starch granules and glyceryl stearate are added sequentially. The mixture is placed in a constant temperature water bath and heated at 60-70℃ with a stirring speed of 300-500 r / min for 40-60 min until it is evenly dispersed. The pH is then adjusted to 6.5-7 to obtain a uniform encapsulated mixture. The mixture is then cooled to 40-50℃ for later use.

[0025] The mass ratio of amphoteric starch granules, chitosan, glyceryl stearate, and 0.3% citric acid aqueous solution is 15–20:3–5:2–3:30–40. Finally, a method for preparing a composite algae microcapsule is as follows: S201: Mix half of the fish slurry and filamentous algae powder evenly, then add yeast cell wall powder and mix evenly. Finally, add the remaining half of the fish slurry, add deionized water and stir evenly to obtain a mixed fluid. Process the mixed fluid through a high-pressure homogenizer 1-2 times, and then spray dry and granulate to obtain mixed particles of 40-80μm. S202 involves adding algal astaxanthin solid microparticles to an emulsion of nano-sized emulsified microparticles, adjusting the pH to 7.0–7.5, stirring for 15–20 minutes, and letting it stand for 5–10 minutes; then adding mixed particles, stirring for 15–20 minutes, and letting it stand for 5–10 minutes; through the interaction of positive and negative charges, the particles are layered and coated, and then granulated at low temperature to obtain active multi-nutrient particles. The core layer is algal astaxanthin solid microparticles, the middle layer is nano-sized emulsified microparticles, and the outer layer is a mixture of fish lysate, yeast cell wall powder, and filamentous algae powder.

[0026] In a weakly acidic or neutral environment, the silanol groups on the silica carrier of algal astaxanthin solid particles form silicate groups and exhibit negative charge, with a large number of negative charges on the surface. Meanwhile, the nano-sized emulsion particles carry positive charges. Through emulsification dispersion-electrostatic recombination, the core layer is first formed by algal astaxanthin solid particles, the middle layer is by nano-sized emulsion particles, and the outer layer is a mixture of fish lysate, yeast cell wall powder, and filamentous algae powder. In a neutral environment, the surface of the fish lysate, yeast cell wall powder, and filamentous algae powder all carry negative charges. Therefore, through the electrostatic interaction of positive and negative charges, layers of active multi-nutrient particles are formed.

[0027] The core layer consists of solid algal astaxanthin particles, the middle layer is composed of nano-sized emulsified particles, and the outer layer is a mixture of fish lysate, yeast cell wall powder, and filamentous algae powder. These three layers are dispersed through emulsification and electrostatic compounding to ultimately produce active, multi-nutrient particles. This design ensures that the active substances astaxanthin and herbal active ingredients are not easily lost or oxidized, maintaining their activity for a long time. Simultaneously, the nano-sized emulsified particles in the middle layer provide a large source of plant-based fats, and their flavor compounds mask the taste of the herbal ingredients and astaxanthin, greatly enhancing palatability and increasing tilapia's feed intake. The outer layers of fish lysate and filamentous algae powder are also specifically formulated for tilapia, increasing nutrition while making the nutrients abundant and easily absorbed. The yeast cell wall powder is rich in β-glucan and mannan oligosaccharides, which can activate immune cells such as macrophages in fish, clear harmful pathogens from the intestines, prevent their colonization, improve the intestinal morphology of aquatic animals, and enhance the resistance and stress resistance of tilapia.

[0028] S203, then add the active multi-nutrient particles to the uniform encapsulation mixture, maintain the pH at 7.0-7.5, slowly stir to disperse the active multi-nutrient particles evenly, then stop stirring and let it stand for 20-30 minutes; then stir slowly at 100-120 r / min for 10-20 minutes, and let it stand for 20-30 minutes to obtain a uniformly dispersed microsphere mixture. Filter the microsphere mixture, centrifuge the resulting precipitate at 3000-4000 r / min for 5-8 minutes, wash 2-3 times, and freeze-dry the centrifuged sample at -50 to -40℃ for 4-6 hours to obtain composite algae microcapsules; The composite algae microcapsules have a particle size of 250–350 μm. The mass ratio of the active multinutrient particles to the encapsulated liquid is 3–4:1.5–1.8. Using active multi-nutrient granules as the adsorption core, the outer layer carries a negative charge and cross-links with cationic starch macromolecules in the coating. Through electrostatic bonding of positive and negative charges, a composite algae microcapsule is obtained. The dual barrier of hydrophobic groups and phosphate ester cross-linking prevents rapid dissolution in water, maintaining the particle shape in water, reducing nutrient loss, and ensuring long-term activity. The gelatinized starch granules, cross-linked with sodium tripolyphosphate, acquire a granular framework and anti-swelling properties. Their hydrophobic octenyl long chains and charge ensure uniform dispersion in chitosan aqueous solution, improving the hydrophilicity of the coating and enhancing its stability. The resulting coating is soft and thin, slowing down the swelling rate in water. This coating can adsorb active substances in feed, increasing fish's feeding willingness, and leaving fish without any burden after consumption.

[0029] Because tilapia lack gastric glands and gastric acid secretion, they cannot perform preliminary digestion of feed through gastric acid, nor can they store food in their stomachs. Feed remains in the digestive tract for approximately 2-4 hours, resulting in traditionally unencapsulated active ingredients (such as algal astaxanthin and herbal flavonoids) passing through the intestines quickly with extremely low bioavailability. Furthermore, the intestinal pH of tilapia is neutral to slightly alkaline (pH 7.0-8.2), lacking a strongly acidic environment. Therefore, the cross-linking and solidification process of this patented microcapsule does not need to consider gastric acid degradation; the focus is on optimizing the "slow release in the intestine" performance—ensuring that after the microcapsules enter the tilapia's intestines, they gradually break down in a neutral / slightly alkaline environment, releasing the core active ingredient and prolonging the duration of action. By balancing "improving the solubility of the fat-soluble active ingredient (astaxanthin)" and "adapting to the tilapia's intestinal digestive enzyme system," the design avoids excessively thick encapsulation and overly water-soluble active substances dissolving too quickly, which could negatively impact the tilapia's intestinal digestive function and the absorption of active ingredients.

[0030] Given the physiological characteristics of tilapia—namely, the absence of a stomach, a short intestine, and a short digestive retention time—this invention specifically optimizes the thickness of the microcapsule encapsulation material and the cell wall disruption rate. By adjusting the mass ratio of the encapsulation material to the active multinutrient particles, the thickness of the prepared microcapsule wall material is controlled at 10–20 μm, ensuring slow cell wall disruption within the tilapia intestine (pH 7.0–8.2). The disruption time is controlled at 1.5–2.5 hours, precisely matching the retention time of the tilapia feed in the intestine—avoiding both excessively rapid cell wall disruption leading to loss of active ingredients and excessively slow disruption resulting in insufficient release of active ingredients, thus maximizing bioavailability.

[0031] The compound algae microcapsules are added at a concentration of 1-5% in tilapia feed; in broodstock feed, the addition amount is 2.0-5.0% to meet the nutritional needs for gonadal development and improved reproductive performance; and in fry feed, the addition amount is 1-2.0% to suit the digestive capacity of juvenile fish and enhance their physical condition. The basic feed formula ensures a balanced supply of protein, energy, minerals, and vitamins, working synergistically with the microcapsules to comprehensively guarantee the growth and reproductive needs of tilapia.

[0032] Compared with existing technologies, the beneficial effects of this invention are as follows: 1) This invention uses Haematococcus pluvialis as the main raw material to extract astaxanthin, which has strong stability and high activity, and prepares algal astaxanthin solid microparticles. It also provides an emulsion of positively charged nano-sized emulsified microparticles containing plant-based fat sources and active substances from traditional Chinese medicine. Finally, using specific raw materials in synergy, an active multi-nutrient particle is produced through emulsification, dispersion, and electrostatic compounding. This active multi-nutrient particle has a core layer of algal astaxanthin solid microparticles, a middle layer of nano-sized emulsified microparticles, and an outer layer of a mixture of fish lysate, yeast cell wall powder, and filamentous algae powder. This design ensures that the active substances astaxanthin and traditional Chinese medicine active substances are not easily lost or oxidized, maintaining their activity for a long time. Simultaneously, the middle layer of nano-sized emulsified microparticles provides a large amount of plant-based fat source, and its flavor compounds mask the taste of the traditional Chinese medicine and astaxanthin, greatly enhancing palatability and increasing tilapia feeding. The outer layer of fish lysate and filamentous algae powder is also specifically formulated for tilapia, increasing nutrition while making the nutrients rich and easily absorbed. Yeast cell wall powder is rich in β-glucan and mannan oligosaccharide, which can activate immune cells such as macrophages in fish, clear harmful pathogens in the intestines, prevent their colonization, improve the intestinal morphology of aquatic animals, and enhance the resistance and stress resistance of tilapia.

[0033] 2) This invention also provides a coating material. Sodium tripolyphosphate can form ester bonds with small starch molecules containing hydroxyl groups, while simultaneously promoting cross-linking between starch molecules. Under alkaline conditions, the hydroxyl groups of the cationic small starch molecules dissociate into oxygen anions. These anions then undergo a nucleophilic substitution reaction with some of the positive charges on octenyl succinic anhydride, generating a starch ester. The anhydride ring is opened, with one end binding to the starch molecule through the ester group and the other end producing a carboxylic acid. The treated starch particles contain both hydrophilic and lipophilic-hydrophobic groups. Using this material as a coating material provides a certain degree of solubility while maintaining its position in water for a period of time, preventing rapid nutrient loss upon contact with water. This method is well-suited to the physiological characteristics of tilapia, which have no stomach, short intestines, and short digestive retention time. The thickness of the prepared microcapsule wall material is controlled at 10-20 μm, ensuring that it slowly breaks down in the tilapia's intestines. This avoids both the loss of active ingredients due to excessively rapid wall breaking and the inability to fully release active ingredients due to excessively slow wall breaking, thus maximizing bioavailability.

[0034] 3) The present invention also provides a fish lysate that achieves the best balance between flavor and degree of hydrolysis. The enzymatically hydrolyzed fish lysate is rich in small molecule peptides, contains a complete range of amino acids and is free of bitterness. It is very suitable for the characteristics of tilapia with short intestines and fast absorption, and is especially suitable for juvenile fish feed, reducing the digestive burden.

[0035] 4) This invention also provides the application of the aforementioned composite algae microcapsules as a feed additive. It alters the form of astaxanthin raw materials while ensuring that active ingredients such as astaxanthin are released after reaching the intestines, thereby exerting antioxidant, antibacterial, and antiviral effects. The excellent film-forming ability and mucosal adhesion properties of the active substances enable them to colonize the surface of the small intestinal mucosa, maximizing their reach into the intestinal tract. Under neutral or weakly alkaline conditions, the algae astaxanthin solid particles are effectively released, promoting the bioavailability of astaxanthin and traditional Chinese medicine extracts. Furthermore, the fat source in the microcapsules is mainly plant oil, utilizing tricresyl esters to slowly release butyric acid in the small intestine, which helps repair intestinal villi, maintain intestinal flora balance, enhance the body's antioxidant capacity, and increase the growth rate of tilapia. By combining β-glucan and mannan oligosaccharide-rich yeast cell wall powder with broodstock to enhance broodstock reproductive capacity, juvenile growth rate, and disease resistance, and through the synergistic effect of fish lysate and flavor compounding agents, the palatability of the compound algae microcapsules is improved, increasing tilapia feed intake and improving the absorption rate of feed nutrients. Furthermore, the various nutrients in the compound algae microcapsules enhance broodstock reproductive capacity and effectively strengthen the immune activity and stress resistance of fry.

[0036] 5) The addition of the compound algae microcapsules prepared in this invention, with their active multi-nutrient granules, not only helps strengthen the immune system of tilapia and reduce morbidity and mortality, but also helps improve the antioxidant capacity and heavy metal resistance of tilapia, thus enhancing its nutritional value. The active ingredients of the active multi-nutrient granules can improve the intestinal microbiota, enhance nutrient absorption, maintain intestinal health, and reduce the need for antibiotics. They can significantly enhance the antibacterial activity of tilapia, playing a beneficial role in the prevention and treatment of intestinal infections in tilapia, and also helping to improve the growth rate of tilapia and reduce the feed conversion ratio and farming cycle. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Example 1 A composite algae microcapsule is prepared by first preparing algae astaxanthin solid microparticles, specifically: After drying Haematococcus pluvialis at 5℃ for 30 minutes, it was removed, pulverized, and subjected to low-temperature ultrasonic cell disruption at 5℃ to obtain Haematococcus pluvialis powder. 50 parts of Haematococcus pluvialis powder were added to 150 parts of an organic solvent containing ethanol and ethyl acetate in a volume ratio of 3:1 at 30℃ and mixed with a magnetic stirrer for 120 minutes to fully dissolve astaxanthin. The Haematococcus pluvialis residue and liquid were separated by centrifugation at 10000 r / min, and the supernatant liquid was collected. The supernatant liquid was distilled under reduced pressure to obtain concentrated astaxanthin oil. 30 parts of astaxanthin oil were taken, 0.2 parts of vitamin E were added, and the mixture was stirred for 10 minutes to mix evenly. Then, 20 parts of silica were added and stirred for 30 minutes to mix evenly. After standing for 20 minutes, the mixture was stirred for another 30 minutes to load the astaxanthin onto the silica, resulting in algal astaxanthin solid microparticles.

[0038] Weigh out 20 parts of Astragalus membranaceus extract, 8 parts of Angelica sinensis extract, 10 parts of Leonurus japonicus extract, 8 parts of Eucommia ulmoides extract, and 25 parts of deionized water. Stir each ingredient at 40°C for 30 minutes to dissolve it completely. Then add 2 parts of β-cyclodextrin and stir evenly. Let stand for 5 minutes, filter and take the upper liquid to obtain the herbal extract.

[0039] Furthermore, an emulsion of positively charged nanoscale emulsified particles is prepared, specifically as follows: Take 8 parts of soybean lecithin, 10 parts of soybean oil, 3 parts of glyceryl tartrate, 3 parts of glyceryl monostearate, and 5 parts of tea tree oil, and heat at 45°C until completely dissolved to obtain the lipid phase.

[0040] Take 1.5 parts of chitosan and add it to 98.5 parts of 0.05% citric acid aqueous solution. Stir at 70℃ for 30 minutes to dissolve it completely to obtain a 1.5% chitosan aqueous solution. Then take 12 parts of the chitosan aqueous solution and add 12 parts of traditional Chinese medicine extract. Adjust the pH value to 5.6 and stir evenly to obtain the aqueous phase.

[0041] The lipid phase was slowly added to the aqueous phase and stirred at 10,000 r / min for 10 minutes to form a crude emulsion. After homogenization under high pressure three times at 45°C, 4 parts of flavor compound were added and stirred thoroughly to obtain a stable emulsion of positively charged nano-sized emulsion particles. Furthermore, the preparation of fish slurry specifically involves: The collected fish scales, skin and bones were washed clean with water, soaked in a 1% citric acid solution for 1 hour, rinsed with deionized water, and ground in a colloid mill to obtain a paste. An extract was prepared by combining 6 parts betaine, 10 parts citric acid, 4 parts vitamin C and 100 parts deionized water; a compound enzyme solution was prepared by combining 3 parts flavor protease, 2 parts subtilis protease and 10 parts deionized water. Add 100 parts of the paste to 100 parts of the extract, vortex for 10 minutes, centrifuge at 15000 r / min for 15 minutes, and collect the supernatant; place it in a vacuum freeze dryer, rapidly freeze at -50℃ for 3 hours, and dry at low temperature for 10 hours to obtain freeze-dried powder; take 40 parts of the freeze-dried powder and add 1 part of the compound enzyme solution, vortex for 5 minutes at a material-to-liquid ratio of 40:1; sonicate and enzymatically hydrolyze at 50℃ for 5 hours, and purify the enzymatic hydrolysate by nanofiltration for 3 hours to obtain a high concentration of enzymatically hydrolyzed fish lysate.

[0042] Furthermore, a mixture of modified corn starch, chitosan, and emulsifier is prepared to encapsulate the contents, specifically as follows: Add 100 parts of corn starch to 400 parts of deionized water, stir evenly, slowly heat to 80-90℃, stir for 20-40 minutes. After the first and second stages, the corn starch begins to gelatinize. Continue stirring and reacting, add 5 parts of glycerol for 30 minutes, dry, crush, and grind to obtain modified small molecule starch granules. 100 parts of modified small molecule starch granules were dispersed in 400 parts of deionized water, sodium bicarbonate was added, the pH value was adjusted to 8.6, and then 6 parts of arginine were added. The temperature was controlled at 35℃ to generate cationic small molecule starch granules, which were then dried for later use. Add 5 parts sodium tripolyphosphate to 95 parts deionized water, add sodium bicarbonate to adjust the pH to 8.7, and then add it dropwise to the cationic small molecule starch granules at room temperature while stirring rapidly. Then heat in a constant temperature water bath at 85°C for 50 minutes, continue stirring and cool to 30°C, then add octenyl succinic anhydride diluted with 5 parts glycerol (3 parts octenyl succinic anhydride diluted with 2 parts glycerol), stir at 40°C for 30 minutes, dry at low temperature to remove glycerol, grind to obtain amphoteric starch granules.

[0043] Six parts of chitosan were added to 80 parts of 0.3% citric acid aqueous solution at 60℃ and stirred rapidly to dissolve. Then, 40 parts of amphoteric starch granules and 6 parts of glyceryl stearate were added sequentially. The mixture was placed in a constant temperature water bath and heated at 70℃ with a stirring speed of 300 r / min for 60 minutes until it was evenly dispersed. The pH was then adjusted to 6.5 to obtain a uniform encapsulated mixture. The mixture was then cooled to 40℃ and set aside for later use. Finally, a method for preparing a composite algae microcapsule is as follows: Mix 10 parts fish soluble paste and 10 parts filamentous algae powder evenly, then add 5 parts yeast cell wall powder and mix evenly. Finally, add 10 parts fish soluble paste and 1 part deionized water to obtain a mixed fluid. Process the mixed fluid twice through a high-pressure homogenizer, and then spray dry and granulate to obtain mixed particles of 40-80μm. Forty parts of algal astaxanthin solid microparticles were added to an emulsion of 18 parts of nano-sized emulsified microparticles. The pH was adjusted to 7.2, and the mixture was stirred for 15 minutes and allowed to stand for 10 minutes. Then, mixed particles were added, stirred for 18 minutes, and allowed to stand for 10 minutes. The particles were coated layer by layer by positive and negative charges and then granulated by low-temperature drying to obtain active multi-nutrient particles. Add 40 parts of active multinutrient granules to 18 parts of uniform encapsulation mixture, maintain pH at 7.2, slowly stir to disperse the active multinutrient granules evenly, then stop stirring and let it stand for 30 minutes; then stir slowly at 120 r / min for 12 minutes, and let it stand for 25 minutes to obtain a uniformly dispersed microsphere mixture. Filter the microsphere mixture, centrifuge the resulting precipitate at 3000 r / min for 8 minutes, wash 2-3 times, and freeze-dry the centrifuged sample at -40℃ for 6 hours to obtain composite algae microcapsules. Example 2 A composite algae microcapsule is prepared by first preparing algae astaxanthin solid microparticles, specifically: After drying Haematococcus pluvialis at 5℃ for 30 minutes, it was removed, pulverized, and subjected to low-temperature ultrasonic cell disruption at 5℃ to obtain Haematococcus pluvialis powder. 50 parts of Haematococcus pluvialis powder were added to 150 parts of an organic solvent containing ethanol and ethyl acetate in a volume ratio of 3:1 at 25℃ and mixed with a magnetic stirrer for 150 minutes to fully dissolve astaxanthin. The Haematococcus pluvialis residue and liquid were separated by centrifugation at 10000 r / min, and the supernatant liquid was collected. The supernatant liquid was distilled under reduced pressure to obtain concentrated astaxanthin oil. 30 parts of astaxanthin oil were taken, 0.2 parts of vitamin E were added, and the mixture was stirred for 10 minutes to mix evenly. Then, 20 parts of silica were added and stirred for 30 minutes to mix evenly. After standing for 20 minutes, the mixture was stirred for another 30 minutes to load the astaxanthin in the silica, resulting in algal astaxanthin solid microparticles. Weigh out 10 parts of Astragalus membranaceus extract, 15 parts of Angelica sinensis extract, 8 parts of Leonurus japonicus extract, 8 parts of Eucommia ulmoides extract and 20 parts of deionized water. Stir each raw material at 40℃ for 30 minutes to dissolve it completely. Then add 2 parts of β-cyclodextrin and stir evenly. Let stand for 5 minutes, filter and take the upper liquid to obtain the herbal extract.

[0044] Furthermore, an emulsion of positively charged nanoscale emulsified particles is prepared, specifically as follows: Take 8 parts of soybean lecithin, 10 parts of soybean oil, 3 parts of glyceryl tartrate, 3 parts of glyceryl monostearate, and 5 parts of tea tree oil, and heat at 45°C until completely dissolved to obtain the lipid phase.

[0045] Take 1.5 parts of chitosan and add it to 98.5 parts of 0.05% citric acid aqueous solution. Stir at 70℃ for 30 minutes to dissolve it completely to obtain a 1.5% chitosan aqueous solution. Then take 12 parts of the chitosan aqueous solution, add 12 parts of traditional Chinese medicine extract, adjust the pH value to 6.0, and stir evenly to obtain the aqueous phase.

[0046] The lipid phase was slowly added to the aqueous phase and stirred at 10,000 r / min for 10 minutes to form a crude emulsion. After homogenization under high pressure three times at 45°C, 4 parts of flavor compound were added and stirred thoroughly to obtain a stable emulsion of positively charged nano-sized emulsion particles. Further, the fish sol is prepared, specifically as follows: The collected fish scales, skin and bones were washed clean with water, soaked in a 1% citric acid solution for 1 hour, rinsed with deionized water, and ground in a colloid mill to obtain a paste. A compound enzyme solution was prepared by mixing 2 parts flavor protease, 2 parts Bacillus subtilis protease and 10 parts deionized water. Add 100 parts of the paste to 100 parts of the extract, vortex for 10 minutes, centrifuge at 15000 r / min for 15 minutes, and collect the supernatant; place it in a vacuum freeze dryer, rapidly freeze at -50℃ for 3 hours, and dry at low temperature for 10 hours to obtain freeze-dried powder; take 40 parts of the freeze-dried powder and add 1 part of the compound enzyme solution, vortex for 5 minutes at a material-to-liquid ratio of 40:1; sonicate and enzymatically hydrolyze at 50℃ for 4 hours, and purify the enzymatic hydrolysate by nanofiltration for 4 hours to obtain a high concentration of enzymatically hydrolyzed fish lysate.

[0047] Furthermore, a mixture of modified corn starch, chitosan, and emulsifier is prepared to encapsulate the contents, specifically as follows: Add 100 parts of corn starch to 400 parts of deionized water, stir evenly, slowly heat to 80℃, stir for 40 minutes. After the first and second stages, the corn starch begins to gelatinize. Continue stirring and reacting, add 5 parts of glycerol and stir and react for 40 minutes, dry, crush, grind to obtain modified small molecule starch granules. 100 parts of modified small molecule starch granules were dispersed in 400 parts of deionized water, sodium bicarbonate was added, the pH value was adjusted to 9, and 4 parts of arginine were added. The temperature was controlled at 35-45℃ to introduce amino groups into the small molecule starch granules, generating cationic small molecule starch granules, which were then dried for later use. Add 4 parts sodium tripolyphosphate to 96 parts deionized water, add sodium bicarbonate to adjust the pH to 9, and then add it dropwise to the cationic small molecule starch granules at room temperature while stirring rapidly. Then heat in a 90°C constant temperature water bath for 40 minutes, continue stirring and cool to 40°C, then add octenyl succinic anhydride diluted with 4.5 parts glycerol (2.5 parts octenyl succinic anhydride diluted with 2 parts glycerol), stir at 40°C for 30 minutes, dry at low temperature to remove glycerol, grind to obtain amphoteric starch granules.

[0048] Four parts of chitosan were added to 70 parts of 0.3% citric acid aqueous solution at 60℃ and stirred rapidly to dissolve. Then, 30 parts of amphoteric starch granules and 5 parts of glyceryl stearate were added sequentially. The mixture was placed in a constant temperature water bath and heated at 70℃ with a stirring speed of 300 r / min for 60 min until it was evenly dispersed. The pH was then adjusted to 6.8 to obtain a uniform encapsulated mixture. The mixture was then cooled to 40℃ and set aside for later use. Finally, a method for preparing a composite algae microcapsule is as follows: Mix 10 parts fish soluble paste and 12 parts filamentous algae powder evenly, then add 4 parts yeast cell wall powder and mix evenly, finally add 5 parts fish soluble paste and 2 parts deionized water to obtain a mixed fluid. Process the mixed fluid twice through a high-pressure homogenizer, and then spray dry and granulate to obtain mixed particles of 40-80μm. Thirty parts of algal astaxanthin solid microparticles were added to an emulsion of 15 parts of nano-sized emulsified microparticles. The pH was adjusted to 7.0, and the mixture was stirred for 20 minutes and allowed to stand for 8 minutes. Then, mixed particles were added, stirred for 15 minutes, and allowed to stand for 5 minutes. The particles were coated layer by layer by positive and negative charges and then granulated by low-temperature drying to obtain active multi-nutrient particles. Add 35 parts of active multinutrient granules to 16 parts of uniform encapsulation mixture, maintain pH at 7.5, slowly stir to disperse the active multinutrient granules evenly, then stop stirring and let it stand for 30 minutes; then stir slowly at 120 r / min for 15 minutes, and let it stand for 20 minutes to obtain a uniformly dispersed microsphere mixture. Filter the microsphere mixture, centrifuge the resulting precipitate at 4000 r / min for 8 minutes, wash 2-3 times, and freeze-dry the centrifuged sample at -50℃ for 5 hours to obtain composite algae microcapsules.

[0049] Example 3 A composite algae microcapsule is prepared by first preparing algae astaxanthin solid microparticles, specifically: After drying Haematococcus pluvialis at 5℃ for 30 minutes, it was removed, pulverized, and subjected to low-temperature ultrasonic cell disruption at 5℃ to obtain Haematococcus pluvialis powder. 50 parts of Haematococcus pluvialis powder were added to 150 parts of an organic solvent containing ethanol and ethyl acetate in a volume ratio of 3:1 at 25℃ and mixed with a magnetic stirrer for 130 minutes to fully dissolve astaxanthin. The Haematococcus pluvialis residue and liquid were separated by centrifugation at 10000 r / min, and the supernatant liquid was collected. The supernatant liquid was distilled under reduced pressure to obtain concentrated astaxanthin oil. 30 parts of astaxanthin oil were taken, 0.2 parts of vitamin E were added, and the mixture was stirred for 10 minutes to mix evenly. Then, 20 parts of silica were added and stirred for 30 minutes to mix evenly. After standing for 20 minutes, the mixture was stirred for another 30 minutes to load the astaxanthin onto the silica, resulting in algal astaxanthin solid microparticles.

[0050] Weigh out 15 parts of Astragalus membranaceus extract, 12 parts of Angelica sinensis extract, 8 parts of Leonurus japonicus extract, 6 parts of Eucommia ulmoides extract and 28 parts of deionized water. Stir each raw material at 40℃ for 30 minutes to dissolve it completely. Then add 1 part of β-cyclodextrin and stir evenly. Let it stand for 5 minutes, filter and take the upper liquid to obtain the herbal extract.

[0051] Furthermore, an emulsion of positively charged nanoscale emulsified particles is prepared, specifically as follows: Take 8 parts of soybean lecithin, 10 parts of soybean oil, 3 parts of glyceryl tartrate, 3 parts of glyceryl monostearate, and 5 parts of tea tree oil, and heat at 45°C until completely dissolved to obtain the lipid phase.

[0052] Take 1.5 parts of chitosan and add it to 98.5 parts of 0.05% citric acid aqueous solution. Stir at 70℃ for 30 minutes to dissolve it completely to obtain a 1.5% chitosan aqueous solution. Then take 12 parts of the chitosan aqueous solution, add 12 parts of traditional Chinese medicine extract, adjust the pH value to 6.0, and stir evenly to obtain the aqueous phase.

[0053] The lipid phase was slowly added to the aqueous phase and stirred at 10,000 r / min for 10 minutes to form a crude emulsion. After homogenization under high pressure three times at 45°C, 4 parts of flavor compound were added and stirred thoroughly to obtain a stable emulsion of positively charged nano-sized emulsion particles. Further, the fish sol is prepared, specifically as follows: The collected fish scales, skin and bones were washed clean with water, soaked in a 1% citric acid solution for 1 hour, rinsed with deionized water, and ground in a colloid mill to obtain a paste. A compound enzyme solution was prepared by mixing 3 parts flavor protease, 1 part Bacillus subtilis protease and 10 parts deionized water. Add 100 parts of the paste to 100 parts of the extract, vortex for 10 minutes, centrifuge at 15000 r / min for 15 minutes, and collect the supernatant; place it in a vacuum freeze dryer, rapidly freeze at -50℃ for 3 hours, and dry at low temperature for 10 hours to obtain freeze-dried powder; take 40 parts of the freeze-dried powder and add 1 part of the compound enzyme solution, vortex for 5 minutes at a material-to-liquid ratio of 40:1; sonicate and enzymatically hydrolyze at 50℃ for 3 hours, and purify the enzymatic hydrolysate by nanofiltration for 3 hours to obtain a high concentration of enzymatically hydrolyzed fish lysate.

[0054] Furthermore, a mixture of modified corn starch, chitosan, and emulsifier is prepared to encapsulate the contents, specifically as follows: Add 100 parts of corn starch to 400 parts of deionized water, stir evenly, slowly heat to 85℃, stir for 30 minutes. After the first and second stages, the corn starch begins to gelatinize. Continue stirring and reacting, add 5 parts of glycerol and stir and react for 30 minutes, dry, crush, grind to obtain modified small molecule starch granules. 100 parts of modified small molecule starch granules were dispersed in 400 parts of deionized water, sodium bicarbonate was added, the pH value was adjusted to 8.5, and 5 parts of arginine were added. The temperature was controlled at 35-45℃ to introduce amino groups into the small molecule starch granules, generating cationic small molecule starch granules, which were then dried for later use. Add 5 parts sodium tripolyphosphate to 95 parts deionized water, add sodium bicarbonate, adjust the pH to 8.7, and then add it dropwise to cationic small molecule starch granules at room temperature while stirring rapidly. Then heat in a constant temperature water bath at 85°C for 50 minutes, continue stirring and cool to 40°C, then add octenyl succinic anhydride diluted with 5 parts glycerol (3 parts octenyl succinic anhydride diluted with 2 parts glycerol), stir at 40°C for 35 minutes, dry at low temperature to remove glycerol, grind to obtain amphoteric starch granules.

[0055] Four parts of chitosan were added to 60 parts of 0.3% citric acid aqueous solution at 60℃ and stirred rapidly to dissolve. Then, 35 parts of amphoteric starch granules and 4 parts of glyceryl stearate were added sequentially. The mixture was placed in a constant temperature water bath and heated at 70℃ with a stirring speed of 350 r / min for 60 min until it was evenly dispersed. The pH was then adjusted to 7 to obtain a uniform encapsulated mixture. The mixture was cooled to 40℃ and set aside for later use. Finally, a method for preparing a composite algae microcapsule is as follows: Mix 10 parts fish soluble paste and 8 parts filamentous algae powder evenly, then add 3 parts yeast cell wall powder and mix evenly. Finally, add 8 parts fish soluble paste and 1 part deionized water to obtain a mixed fluid. Process the mixed fluid through a high-pressure homogenizer 1-2 times, and then spray dry and granulate to obtain mixed particles of 40-80μm. 35 parts of algal astaxanthin solid microparticles were added to an emulsion of 17 parts of nano-sized emulsified microparticles, the pH was adjusted to 7.0, the mixture was stirred for 20 minutes and allowed to stand for 10 minutes; then the mixed particles were added, stirred for 15 minutes and allowed to stand for 8 minutes; the particles were coated layer by layer by positive and negative charge interaction, and then granulated by low-temperature drying to obtain active multi-nutrient particles. Add 30 parts of active multinutrient granules to 15 parts of uniform encapsulation mixture, maintain pH at 7.0, slowly stir to disperse the active multinutrient granules evenly, then stop stirring and let it stand for 30 minutes; then stir slowly at 100 r / min for 12 minutes, and let it stand for 25 minutes to obtain a uniformly dispersed microsphere mixture. Filter the microsphere mixture, centrifuge the resulting precipitate at 4000 r / min for 6 minutes, wash 2-3 times, and freeze-dry the centrifuged sample at -45℃ for 4 hours to obtain composite algae microcapsules.

[0056] Comparative Example 1: Astaxanthin solid microparticles, emulsions of nano-sized emulsified microparticles, fish slurry, yeast cell wall powder, and filamentous algae powder were mixed, dried, and granulated directly without being made into microcapsules to produce tilapia feed additives. Comparative Example 2: The coating material was a mixture of unmodified corn starch, chitosan and emulsifier, and the rest was the same as in Example 1; Comparative Example 3: The encapsulation material was a mixture of chitosan, sodium alginate and emulsifier, and the rest was the same as in Example 1; Comparative Example 4: The emulsion of nanoemulsion microparticles was directly replaced by an equal amount of fish oil, and the fish slurry was replaced by ordinary non-enzymatically hydrolyzed fish slurry. Other aspects were the same as in Example 1. Comparative Example 5: Commercially available tilapia multi-nutrient feed additive.

[0057] Experimental data and results analysis 1. Enteric dissolution test The astaxanthin content was measured using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: reverse-flow C18 column (150 mm * 4.6 mm, 5 μm), isocratic elution; mobile phase A was methanol, mobile phase B was ethanol, and mobile phase C was aqueous solution (80 + 15 + 5, V / V / V); the detector was a UV detector with a detection wavelength of 478 nm; the injection volume was 10 μL, and the column temperature was room temperature. Preparation of simulated tilapia intestinal fluid: Take 13.6g potassium dihydrogen phosphate, 0.5g sodium chloride and 0.1g potassium chloride in a beaker, add 1000mL deionized water, stir to dissolve completely, and adjust the pH to 7.6 with 0.1mol / L NaOH solution; add 1g amylase (≥2000U / g) + 1g trypsin (≥10000U / g) + 0.2g lipase (≥500U / g) + 0.05g BSA, and mix gently (avoid vigorous stirring to prevent inactivation).

[0058] Aseptic treatment: Filter with a 0.2μm sterile filter head, prepare and use immediately (enzyme solution is stable at room temperature for ≤4h, and refrigerated at 4℃ for ≤24h).

[0059] Experimental Method: 2.000g of samples from Examples 1-3 and Comparative Examples 1-3 were weighed into dissolution vessels, and 250mL of simulated tilapia intestinal fluid was added to each. The vessels were placed in a dissolution apparatus, and the temperature was set to 37℃ and the rotation speed to 100r / min. After reacting for 1 hour, 2 hours, and 3 hours, 10mL of the upper reaction solution was taken, and the astaxanthin content was measured. The results are shown in Table 1.

[0060] Table 1. Astaxanthin content (%) after dissolution from simulated intestinal fluid

[0061] The encapsulation of the active multinutrient particles in Examples 1-3 has a positively charged network cross-linked structure, which can be destroyed under the weakly alkaline conditions of the intestine. After reacting for 3 hours in simulated tilapia intestinal fluid, the release of astaxanthin was over 90%, almost all of it was released. This is because the modified corn starch surface contains a double barrier of hydrophobic groups and phosphate ester cross-linking. The gelatinized starch particles, under the cross-linking of sodium tripolyphosphate, are given a particle skeleton and anti-swelling properties. Its hydrophobic octenyl long chain improves the hydrophilicity of the encapsulation and also enhances the stability of the encapsulation. The resulting encapsulation film is soft and thin, which can slow down the swelling rate in water and better control the release of active substances such as astaxanthin. Since the fish need to be fed into the water, this hydrophilic and lipophilic amphiphilic encapsulation maintains the particle shape in the water, reduces nutrient loss, and plays a role in maintaining activity for a long time. Comparative Example 1 had no encapsulation material, while Comparative Example 2 used unmodified corn starch as the encapsulation material. Both were easily released in water and under the weakly alkaline conditions of the intestine, thus failing to guarantee effective arrival in the small intestine after immersion in water, nor sufficient time for effective absorption by intestinal fluid. Comparative Example 3 used ordinary chitosan and sodium alginate as the protective layer. Under the weakly alkaline conditions of the small intestine, the water solubility of chitosan decreased sharply, existing in a capsule state. It required the amylase in the intestinal fluid to degrade its molecular chains before release. However, the enzymes in the tilapia's intestinal fluid could not effectively degrade chitosan in a short time, thus affecting the absorption of astaxanthin in the small intestine. Therefore, using modified amphoteric starch granules as the encapsulation material in Examples 1-3 precisely matched the residence time in the intestine of tilapia without a stomach—avoiding both excessively rapid microcapsule rupture leading to loss of active ingredients and excessively slow rupture resulting in insufficient release of active ingredients, maximizing bioavailability and thus maximizing efficacy.

[0062] 2. Reproductive performance of broodstock Two hundred healthy, uniformly sized red tilapia broodstock (females weighing 0.8-1.2 kg and males weighing 1.0-1.5 kg) were randomly divided into a control group and an experimental group, with 100 fish in each group (female-to-male ratio 3:1). Both groups were housed in 50 m² ponds under identical conditions (water temperature 24-28℃, pH 7.0-8.5, dissolved oxygen ≥5 mg / L, ammonia nitrogen ≤0.2 mg / L). The control group was fed a basal diet, while the experimental group was fed a specialized diet supplemented with 1.5% compound algae microcapsules. The experiment lasted 90 days, with feeding twice daily (9:00 AM and 4:00 PM), at a rate of 3-5% of the fish's body weight, adjusted according to feeding behavior.

[0063] Reproductive performance of broodstock: After the experiment, the number of eggs carried by each group of broodstock (dissection method), uniformity of egg diameter (100 eggs were randomly selected, the diameter was measured with calipers, and the coefficient of variation was calculated), fertilization rate (number of fertilized eggs / total number of eggs × 100%), and hatching rate (number of hatched fry / number of fertilized eggs × 100%) were counted. The experimental results showed that, compared with the control group, the experimental group of tilapia parent fish had a 22.5% higher number of eggs, a 31.2% lower coefficient of variation in egg diameter (increased uniformity), a 18.3% higher fertilization rate, and a 27.6% higher hatching rate.

[0064] 3. Fish fry growth status Fish fry were raised in 100-liter glass aquariums, with initial weights of approximately 1.3 grams randomly distributed into six tanks. The water used was tap water exposed to sunlight to remove chlorine, and 35% of the water in the tanks was changed daily during the feeding period. Feeding was conducted three times daily (8:30 AM, 12:00 PM, and 6:00 PM), with adjustments made every two weeks. (Water temperature 24-28℃, pH 7.0-8.5, dissolved oxygen ≥5 mg / L, ammonia nitrogen ≤0.2 mg / L), with a light-dark ratio of 14 h:10 h. A five-week feeding experiment was conducted.

[0065] The control group was fed a basal diet. Under the same natural conditions, the same basal diet was mixed with feed additives from Examples 1-2, Comparative Examples 2, 3, and 5, with an addition amount of 2%, resulting in a total of 6 experimental groups. Each group contained 100 tilapia. The tilapia species used in the experiments was red tilapia. The body weights of the tilapia before and after feeding were recorded and the average values ​​were calculated. The number of tilapia that died was also recorded, as shown in Table 2.

[0066] Survival rate (%) = (Number of terminal tails / Number of initial tails) × 100 Feed conversion ratio = Total feed intake / Fish weight gain; Table 2. Growth Performance Record of Red Tilapia Fry

[0067] As shown in Table 2, the growth rate, final weight, feed utilization rate and survival rate of the red tilapia fry with added compound algae microcapsules in Examples 1-2 were significantly improved compared with other tilapia farming conditions. Compared with the control group, the survival rate of the fry in Example 1 was increased by 22% and the weight growth rate was increased by 100%.

[0068] The stress resistance of tilapia cultured for 5 weeks in the control group and in Example 1 was tested (sudden temperature change stress test: water temperature dropped suddenly from 27℃ to 15℃, and the survival rate was observed for 48 hours). The survival rate of tilapia cultured for 48 hours increased by 21.7%.

[0069] Table 2 shows that the fish fry fed with compound algae microcapsules grow faster, absorb nutrients better, and have stronger stress resistance.

[0070] 4. Growth of tilapia fry Nine hundred robust, uniformly sized juvenile red tilapia weighing 100±10g were selected and randomly distributed into nine 50m² ponds, with 100 juveniles per pond and a water depth of 2 meters. Dissolved oxygen levels were maintained above 5mg / L. The experiment lasted for three months. During this period, a basal diet supplemented with 3% of the feed additives prepared in Examples 1-2 and Comparative Examples 1-5 was used, along with a control group fed only the basal diet. The fish were fed twice daily, at 9:00 AM and 6:00 PM, after a period of full feeding. The following growth indicators and the activity of tilapia hepatopancreatic lipase were measured, and the results are shown in Table 3.

[0071] Hepatopancreatic samples were homogenized with approximately 10% (v / v) 0.1M PBS (pH=7.4) and centrifuged at 4°C for 5 minutes (3000 rpm / min). The supernatant was collected and frozen at -80°C. Hepatopancreatic lipase activity was determined according to a kit from Nanjing Jiancheng Biotechnology Co., Ltd.

[0072] Feed intake rate (%) = Feed intake / [(Initial fish weight + Final fish weight) / 2] / day × 100 Feed conversion ratio = total feed intake / fish weight gain; liver-to-body ratio (HSI, %) = liver weight / fish weight × 100%; Visceral weight ratio (VSI, %) = (Weight of viscera in fish / Weight of fish) × 100% Conditionness (CF, g / cm³) 3 = Final body mass of fish / L 3 ×100%, where L is the body length of the fish; Table 3. Growth Record of Tilapia Juveniles

[0073] Table 3 shows that the tilapia in Examples 1-3 had high feed intake rates and feed conversion ratios <1. Furthermore, their hepatobiliary ratio was significantly improved, and their conditionability and hepatopancreatic lipase levels were significantly increased. The livers were purplish-red, and the hepatobiliary ratio increased, remaining between 1-2%, which is not indicative of hepatomegaly. In contrast, Comparative Examples 2-5, compared to Example 3, showed decreased feed intake and conditionability, and significantly reduced hepatopancreatic lipase levels. In Comparative Examples 4 and 5, the hepatobiliary ratio increased excessively, while feed intake and conditionability decreased significantly, resulting in reduced fish growth quality. This indicates that the present invention, through the scientific combination of various nutrients, not only improves the palatability of the tilapia compound feed and promotes feeding, but also effectively reduces the burden on the liver and promotes tilapia growth. It increases the fat utilization rate of tilapia and improves the feed utilization rate.

[0074] 5. Effects of adding compound algae microcapsules to feed on the activity of digestive enzymes in tilapia. Adult tilapia raised for 3 months in Examples 1, Comparative Examples 3 and 5, and the control group were all fasted for 24 hours. Then, liver and intestinal tissues of the tilapia were taken, and the digestive enzyme activities in the intestine and liver were measured using a kit from Nanjing Jiancheng Biotechnology Co., Ltd., as shown in Table 4.

[0075] Table 4. Effects of adding compound algae microcapsules on the activity of digestive enzymes in tilapia.

[0076] As shown in Table 4, the feed containing compound algae microcapsules in Example 1 did not significantly increase the activity of amylase and trypsin in the intestines and liver, but it could significantly improve... The activity of lipases in the liver and intestines. Complex algae microcapsules can increase lipase activity, improve fat transport capacity, and reduce fat deposition, thereby improving liver health in tilapia. Therefore, it can enhance the antioxidant capacity of tilapia, improve its ability to resist external environmental stress during the breeding process, help reduce its susceptibility to pathogens, and improve disease resistance.

[0077] 6. Astaxanthin Bioavailability Test Adult tilapia cultured for 3 months in Examples 1, 1, 3, and 4 were randomly selected from each group at 0, 4, 8, 12, 15, 18, 24, 48, and 72 hours after feeding. Blood was collected from each group and placed in centrifuge tubes containing EDTA. After separation at room temperature (25-26°C) for 30 minutes, the cells were centrifuged at 4°C and 7500 rpm for 15 minutes. The upper serum layer was carefully aspirated, and the astaxanthin content was measured. A metabolic kinetic curve of astaxanthin concentration versus time in serum was plotted. The area under the curve (AUC0-t) was used to measure the bioavailability of astaxanthin, as shown in Table 5. The results showed that the AUC values ​​in the serum of the Example 1 sample group were about 3 times higher than those of the comparative examples. The bioavailability of astaxanthin in the composite algal astaxanthin solid microparticles prepared in this invention was significantly improved, and the bioavailability reached more than 71% after digestion. This is because in Comparative Example 1, the lack of an encapsulation layer resulted in the loss of most nutrients during feeding and after consumption, leading to low bioavailability. In Comparative Example 3, the encapsulation layer made it difficult for tilapia to digest the food, reducing their appetite and preventing the effective release and absorption of astaxanthin from the encapsulation layer. In Comparative Example 4, the composite algae microcapsules lacked an emulsifying layer, resulting in a sharp decrease in the dispersion and water solubility of astaxanthin, which also significantly reduced bioavailability.

[0078] Table 5 Bioavailability of Astaxanthin

[0079] 7. Evaluation of the deliciousness of tilapia flesh Adult tilapia raised for 3 months were taken from Examples 1, 1, 4, and the control. The standard for evaluating the deliciousness of tilapia meat was the determination of the amino acid content in its meat. The standard GB 5009.124-2016 National Food Safety Standard for the Determination of Amino Acids in Food was referenced. The results are shown in Table 6. As can be seen from Table 6, the amino acid composition of the tilapia fed with the composite microcapsules of Examples 1-2 as an additive was relatively balanced and rich in content, which was higher than that of the comparative example, the control example, and commercially available tilapia.

[0080] Table 6. Evaluation Table of Amino Acid Index Tests (mg / 100g)

[0081] As shown in Tables 2-6, the composite algae microcapsules provided in the examples also contain astaxanthin, an antioxidant active ingredient, a plant oil-based fat source, traditional Chinese medicine ingredients, and small molecule peptides from fish lysate. This ensures that astaxanthin and other active ingredients are released after reaching the intestines, thereby exerting antioxidant, antibacterial, and antiviral effects. The excellent film-forming ability and mucosal adhesion properties of the active substances allow them to colonize the surface of the small intestinal mucosa, maximizing their reach into the intestinal tract. Under neutral or weakly alkaline conditions, the algae astaxanthin solid particles are effectively released, promoting the bioavailability of astaxanthin and the traditional Chinese medicine extract. Furthermore, the use of tributyric acid esters allows for the slow release of butyric acid in the small intestine, which helps repair intestinal villi, maintain intestinal flora balance, enhance the body's antioxidant capacity, and increase the growth rate of tilapia. By combining the β-glucan and mannan oligosaccharides rich in yeast cell wall powder to enhance the reproductive capacity of broodstock, the growth rate of juveniles, and their disease resistance, and through the synergistic effect of fish lysate and flavor compounding agents, the palatability of the compound algae microcapsules is improved, increasing tilapia feed intake and improving the absorption rate of feed nutrients. Furthermore, the various nutrients in the compound algae microcapsules enhance the reproductive capacity of broodstock and effectively strengthen the immune activity and stress resistance of fry. Therefore, when using the multi-nutrient functional feed additive of this invention for tilapia farming, the disease incidence rate of tilapia is reduced, the fish population is vigorous, the fish meat has a high amino acid content, and the taste is excellent.

[0082] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Equal modifications and variations made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the overall concept of the present invention, shall still fall within the scope of the present invention.

Claims

1. A method for preparing composite algae microcapsules, characterized in that, The composite algae microcapsule includes an encapsulating material and active multi-nutrient particles. The active multi-nutrient particles are composed of the following raw materials in parts by weight: 30-40 parts of algal astaxanthin solid microparticles, 15-18 parts of emulsion of nano-sized emulsified microparticles, 15-20 parts of fish lysate, 3-5 parts of yeast cell wall powder, and 8-12 parts of filamentous algae powder. The composite algae microcapsules are prepared by the following method: S201, mix half of the fish slurry and filamentous algae powder, then add yeast cell wall powder and mix evenly, finally add the remaining half of the fish slurry, add deionized water and stir evenly to obtain a mixed fluid, process the mixed fluid through a high-pressure homogenizer 1-2 times, and then spray dry and granulate to obtain mixed particles of 40-80μm. S202 involves adding algal astaxanthin solid microparticles to an emulsion of nano-sized emulsified microparticles, adjusting the pH to 7.0–7.5, stirring for 15–20 minutes, and letting it stand for 5–10 minutes; then adding mixed particles, stirring for 15–20 minutes, and letting it stand for 5–10 minutes; through the interaction of positive and negative charges, the particles are layered and coated, and then granulated by low-temperature drying to obtain active multi-nutrient particles. The core layer is algal astaxanthin solid microparticles, the middle layer is nano-sized emulsified microparticles, and the outer layer is a mixture of fish lysate, yeast cell wall powder, and filamentous algae powder. S203, then add the active multi-nutrient particles to the uniform encapsulation mixture, maintain the pH at 7.0-7.5, slowly stir to disperse the active multi-nutrient particles evenly, then stop stirring and let it stand for 20-30 minutes; then stir slowly at 100-120 r / min for 10-20 minutes, and let it stand for 20-30 minutes to obtain a uniformly dispersed microsphere mixture. Filter the microsphere mixture, centrifuge the resulting precipitate at 3000-4000 r / min for 5-8 minutes, wash 2-3 times, and freeze-dry the centrifuged sample at -50 to -40℃ for 4-6 hours to obtain composite algae microcapsules; The mass ratio of active multinutrient granules to the encapsulated liquid is 3-4:1.5-1.

8.

2. The method for preparing the composite algae microcapsules according to claim 1, characterized in that, The preparation method of the algal astaxanthin solid particles is as follows: S1. After drying Haematococcus pluvialis at a low temperature of 0-10℃ for 20-40 minutes, take it out and pulverize it with low temperature ultrasonic cell disruption at 0-5℃ to obtain Haematococcus pluvialis powder. S2, add Haematococcus pluvialis powder to an organic solvent containing a 3:1 volume ratio of ethanol to ethyl acetate at 20-30℃, with a material-to-liquid ratio of 1:30; mix with a magnetic stirrer for 120-150 minutes to fully dissolve the astaxanthin; separate the Haematococcus pluvialis residue and liquid by centrifugation at 8000-10000 r / min, and take the upper liquid; S3, the upper liquid is distilled under reduced pressure to obtain concentrated astaxanthin oil, vitamin E is added and stirred evenly; then silica is added and mixed evenly to load astaxanthin in silica, thus obtaining algal astaxanthin solid particles; wherein, the mass ratio of vitamin E, silica and astaxanthin oil is 0.2:2:3; The silica is feed-grade oleophilic mesoporous silica with an oil absorption value of ≥2.2g / g and a pore size of 30-50nm.

3. The method for preparing the composite algae microcapsules according to claim 1, characterized in that, An emulsion of positively charged nanoscale emulsified microparticles prepared from the following parts by weight of raw materials, specifically: Eight parts of soybean lecithin, ten parts of soybean oil, three parts of glyceryl tartrate, three parts of glyceryl monostearate, five parts of tea tree oil, twelve parts of traditional Chinese medicine extract, and twelve parts of 1.5% chitosan aqueous solution were mixed using a high-pressure homogenization method to prepare a nano-sized emulsion. The pH value was adjusted to 5.5-6.0, a flavoring agent was added, and the mixture was stirred thoroughly to obtain an emulsion with uniformly dispersed positively charged nano-sized emulsion particles. The flavoring agent is a mixture of lysine and arginine, with a mass ratio of lysine to arginine of 1:

3.

4. The method for preparing the composite algae microcapsules according to claim 3, characterized in that, The herbal extract comprises, by weight, 10-20 parts of Astragalus membranaceus extract, 8-15 parts of Angelica sinensis extract, 5-10 parts of Leonurus japonicus extract, 4-8 parts of Eucommia ulmoides extract, 1-2 parts of β-cyclodextrin, and 20-30 parts of deionized water. The raw materials are stirred for 20-30 minutes, allowed to stand for 5 minutes, and the supernatant is filtered.

5. The method for preparing composite algae microcapsules according to claim 1, characterized in that, The fish lysate mentioned is an enzymatically hydrolyzed fish lysate, specifically: S01, the collected fish scales, fish skin and fish bones are washed clean with water, soaked in 1% citric acid solution for 1 hour, rinsed clean with deionized water, and ground in a colloid mill to obtain a paste. Add S02, extract, vortex for 10 minutes at a material-to-liquid ratio of 1:1 to 3, centrifuge at 15000 r / min for 15 minutes, and collect the supernatant; The product was placed in a vacuum freeze dryer and rapidly frozen at -60 to -40°C for 3 hours, then dried at a low temperature for 10 hours to obtain freeze-dried powder. S03, take the freeze-dried powder, add the compound enzyme solution, and vortex for 5 minutes at a material-to-liquid ratio of 40:1; S04 is subjected to ultrasonic enzymatic hydrolysis at 50℃ for 3-6 hours, and the hydrolysate is purified by nanofiltration for 2-5 hours to obtain a high-concentration enzymatically hydrolyzed fish slurry.

6. The method for preparing the composite algae microcapsules according to claim 1, characterized in that, The aforementioned coating is a mixture of modified corn starch, chitosan, and emulsifier, specifically: S101, add corn starch to deionized water, stir evenly, slowly heat to 80-90℃, stir for 20-40 minutes, corn starch goes through the first and second stages and begins to gelatinize, continue stirring and reacting, add glycerol and stir for 20-40 minutes, dry, crush, grind to obtain modified small molecule starch granules. The mass ratio of glycerol, corn starch and deionized water is 1:20:80; S102: Disperse the gelatinized starch granules in deionized water, add sodium bicarbonate, adjust the pH to 8.5-9, then add arginine, and control the temperature at 35-45℃ to introduce amino groups into the small molecule starch granules, generating cationic small molecule starch granules, which are then dried for later use. S103: Sodium tripolyphosphate is added to deionized water, sodium bicarbonate is added, and the pH value is adjusted to 8.5-9. Then, it is added dropwise to small molecule starch granules with cationicity at room temperature while stirring rapidly. Then, it is heated in a constant temperature water bath at 85-90℃ for 40-60 minutes, and then cooled to 30-40℃ with continued stirring. Then, octenyl succinic anhydride diluted with glycerol is added, and it is stirred at 30-40℃ for 30-40 minutes. After that, it is dried at low temperature to remove glycerol, and then ground to obtain amphoteric starch granules. The mass ratio of sodium tripolyphosphate, arginine, octenyl succinic anhydride and modified small molecule starch granules is 0.8–1: 0.8–1.2: 0.5–0.6: 15–20. S104. Chitosan is added to a 0.3% citric acid aqueous solution at 50-60℃ and stirred rapidly to dissolve it. Then, amphoteric starch granules and glyceryl stearate are added sequentially. The mixture is placed in a constant temperature water bath and heated at 60-70℃ with a stirring speed of 300-500 r / min for 40-60 min until it is evenly dispersed. The pH is then adjusted to 6.5-7 to obtain a uniform encapsulated mixture. The mixture is then cooled to 40-50℃ for later use. The mass ratio of amphoteric starch granules, chitosan, glyceryl stearate, and 0.3% citric acid aqueous solution is 15–20:3–5:2–3:30–40.

7. The method for preparing the composite algae microcapsules according to claim 1, characterized in that, The homogenization process is carried out 2 to 3 times using a high-pressure homogenization method, with a homogenization pressure of 30 to 60 MPa, a temperature of 40 to 50°C, and a processing time of 10 to 20 minutes.

8. The method for preparing the composite algae microcapsules according to claim 5, characterized in that, The extract consists of betaine, citric acid, vitamin C, and deionized water in a mass ratio of 3:5:2:50; the complex enzyme solution consists of flavor protease, subtilis protease, and deionized water in a mass ratio of 2-3:1-2:

10. The fish slurry is derived from one or more of tilapia, grass carp, silver carp, or crucian carp.

9. A tilapia feed additive, characterized in that: The method for preparing the composite algae microcapsules according to any one of claims 1-8 results in a composite algae microcapsule that is a tilapia feed additive. The amount of composite algae microcapsules added to broodstock feed is 1.0-5.0% to meet the nutritional requirements for gonadal development and improved reproductive performance. The amount of composite algae microcapsules added to fry feed is 1.0-2.0% to suit the digestive capacity of juvenile fish.

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