Preparation method of algae microcapsules and use of algae microcapsules in pig feed

Abstract

The present invention belongs to the technical field of active substance preparation, specifically relating to a method for preparing algae microcapsules. Using Haematococcus pluvialis as the primary raw material, the method prepares astaxanthin into algae astaxanthin nanoparticles. Haematococcus pluvialis protein is first used as a protective layer, followed by adsorption of an emulsion to form self-emulsifying algae astaxanthin solid particles. Furthermore, an active coating with a cross-linked network structure is utilized. Through positive and negative charge interactions with a protein solution, algae microcapsules are formed, each doubly coated with the cross-linked network structure and Haematococcus pluvialis protein. The algae microcapsules of the present invention allow for easy release of astaxanthin in the small intestine, with a bioavailability exceeding 60%. After microencapsulation, the astaxanthin is protected from the effects of environmental factors such as light, heat, and oxygen on its stability, maintaining its activity for extended periods, making it suitable for use in animal feed additives.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance preparation, and particularly relates to a method for preparing algae microcapsules. Background Art

[0002] The astaxanthin molecular structure contains 13 conjugated double bonds, hydroxyl and unsaturated ketone groups on the terminal ring structure, and α-hydroxyketone composed of hydroxyl and ketone groups. The multiple unsaturated double bond structure of astaxanthin determines that it is easily degraded and oxidized by light, oxygen, and heat; Haematococcus pluvialis is the species with the highest accumulation of all known astaxanthin-synthesizing organisms. The astaxanthin in Haematococcus pluvialis exists in the form of esters with a content of up to 5%. The cells of Haematococcus pluvialis are wrapped by two layers of cell walls and pectin. The gelatinous substance fills the gap between the cell wall and the cytoplasm. The thick and dense cell wall composed of substances such as cellulose, hemicellulose and lignin protects the interior of the cell, making it difficult for the astaxanthin in the cytoplasm to be released and absorbed. Therefore, the Haematococcus pluvialis needs to be broken into pieces, but the extracted astaxanthin is insoluble in water and cannot be directly applied to aqueous environments.

[0003] Moreover, both the broken Haematococcus pluvialis powder and its extracts need to be stored at low temperature, away from light and in a vacuum, which seriously limits its wide application. Therefore, how to make astaxanthin able to be stored stably for a simple and long time is a key issue that needs to be solved urgently to ensure its application in health foods and medicines.

[0004] Chinese patent publication number CN108530939B discloses a foam spray drying method for preparing astaxanthin microcapsules from Haematococcus pluvialis and a preparation method thereof. The microcapsules are prepared from the following raw materials in parts by weight: 4-5g of gum arabic, 1-2g of variable starch, 3-6g of soy protein, 1-2g of lactitol, 8-12g of astaxanthin, and an emulsifier. After microencapsulation, the astaxanthin is protected from the effects of environmental factors such as light, heat, and oxygen on its stability, thereby maintaining its functionality and extending its storage life. Although this method maintains the functionality of astaxanthin and extends its storage life, it does not address the problem that astaxanthin cannot be directly applied in aqueous environments or that it is not readily absorbed by organisms.

[0005] Chinese patent publication number CN118319001B discloses a Haematococcus pluvialis capsule and a preparation method thereof, disclosing that the preparation of Haematococcus pluvialis polymer microspheres includes the following steps: first, dehydrating a portion of the cellulose on the Haematococcus pluvialis cell wall to produce aldehyde groups; then, subjecting the dehydrated cellulose on the cell wall to aromatic substitution with lignin, causing the Haematococcus pluvialis to coagulate with each other to form aggregates; then, breaking the Haematococcus pluvialis aggregates, and then encapsulating them with corn starch to form Haematococcus pluvialis polymer microspheres. This method solves the problem of efficiently extracting astaxanthin from Haematococcus pluvialis, but involves a high-temperature extraction step, which has a certain impact on the activity of astaxanthin. It also solves the problem of astaxanthin being difficult to be destroyed after long-term storage, but does not solve the problem of biological absorption of astaxanthin.

[0006] However, astaxanthin faces numerous challenges in its application and development. Its unstable nature makes it susceptible to oxidative degradation during processing and storage, hindering its commercial production and storage. Furthermore, astaxanthin suffers from low bioavailability in vivo, leading to significant resource waste and difficulty achieving an effective absorption dose. These challenges significantly limit its application and promotion. Therefore, key technical challenges in the application and promotion of astaxanthin include modifying the dosage form of natural astaxanthin and improving its water dispersibility, stability, and bioavailability. Summary of the Invention

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

[0008] The present invention mainly provides a method for preparing algae microcapsules that are not easily destroyed by gastric acid, are easily absorbed by the small intestine, and can improve the water dispersibility, stability, and bioavailability of natural astaxanthin. The technical solution is as follows:

[0009] An algae microcapsule comprises an active encapsulation with a network-like cross-linked structure and algae astaxanthin solid particles. The preparation of the algae astaxanthin nanoparticles comprises the following steps:

[0010] S001, drying Haematococcus pluvialis at a low temperature of 0 to 10° C. for 20 to 40 minutes, then removing and crushing the algae to obtain Haematococcus pluvialis powder;

[0011] S002, adding Haematococcus pluvialis powder to an organic solvent uniformly mixed with dichloromethane and methanol in a volume ratio of 1:1 at 20-30° C., mixing with a magnetic stirrer for 120-150 minutes to fully dissolve the astaxanthin, separating the Haematococcus pluvialis residue and the liquid by centrifugation, taking the upper liquid, adding cetyldimethylammonium chloride and mixing uniformly to allow the astaxanthin and cetyldimethylammonium chloride to react with each other, and cooling to 5-10° C. to form solid flocs; separating the solid and the liquid, washing and drying the lower solid layer to obtain positively charged astaxanthin solid microparticles; wherein the mass ratio of cetyldimethylammonium chloride to Haematococcus pluvialis powder is 1:10;

[0012] S003, enriching the Haematococcus pluvialis protein from the Haematococcus pluvialis residue separated in S002 using a polyethylene glycol / sodium phosphate aqueous two-phase system, separating the Haematococcus pluvialis protein in the top phase to obtain a protein solution having an isoelectric point of 4.2; adding gelatin to the protein solution and heating and stirring at 50-60° C. for 30-50 minutes until completely dispersed; adding citric acid to adjust the pH to 5.0-5.5; and standing for 30-50 minutes to allow coagulation, so that the protein in the protein solution has a negative charge, thereby obtaining a mixed solution containing coagulation; centrifuging for solid-liquid separation, washing and drying the lower solid layer to obtain negatively charged protein particles; the mass ratio of the protein solution to gelatin being 1:1;

[0013] S004, adding positively charged astaxanthin solid particles and negatively charged protein particles to deionized water, heating and stirring at 50-60° C. for 30-50 minutes, adding citric acid, adjusting the pH to 4.5, so that the protein particles and astaxanthin solid are fully coagulated, centrifuging for solid-liquid separation, washing and drying the lower layer of solid to obtain algae astaxanthin solid particles; the mass ratio of the positively charged astaxanthin solid particles to the negatively charged protein particles is 1:2-4.

[0014] Haematococcus pluvialis can accumulate up to 5% astaxanthin, making it an excellent biological source of natural astaxanthin. Astaxanthin in Haematococcus pluvialis exists primarily in free and esterified forms, with astaxanthin monoesters accounting for approximately 70%, astaxanthin diesters 25%, and the remainder in free form. In addition to astaxanthin, Haematococcus pluvialis also contains a variety of amino acids, fatty acids, polysaccharides, and protein nutrients. Haematococcus pluvialis protein accounts for up to 30% of the total. Its excellent oil absorption, emulsification, and solubility make it a viable alternative to other protein-based materials for microcapsule wall materials, achieving excellent encapsulation results. Furthermore, Haematococcus pluvialis polysaccharides are small molecules that can serve as fillers and support the microcapsule wall. Consequently, the resulting algal astaxanthin solid microparticles, encapsulated by Haematococcus pluvialis protein, effectively preserve the activity of the astaxanthin.

[0015] The protein from Haematococcus pluvialis is extracted by flocculating it using gelatin isoelectric precipitation. The rod-shaped microstructure of Haematococcus pluvialis protein, with a rough, porous surface and a negative charge, creates a strong encapsulation effect when combined with the positively charged astaxanthin solid particles. This rough, porous surface also facilitates the release of the encapsulated astaxanthin within the organism, thus facilitating its absorption.

[0016] Then, corn starch was dissolved in water to prepare a 0.5 g / mL aqueous solution. The algae astaxanthin solid microspheres were dispersed by shaking and then slowly and evenly added to the corn starch aqueous solution. The mixture was stirred at 200-300 r / min at 65-75°C for 3-5 hours. The mixture was cooled to 5-10°C and allowed to stand for 5-10 hours. The mixture was washed with water, dried, and ground to obtain algae microcapsules.

[0017] When algae microcapsules are added to animal feed, we found that they are easily broken down in gastric fluid, preventing them from reaching the small intestine where they are more readily absorbed. Furthermore, astaxanthin exists in an ester form, so even if it does reach the small intestine, it is not easily dispersed in the intestinal fluid and absorbed by the animal.

[0018] Therefore, while maintaining the stability of astaxanthin, it is also very important to improve the bioavailability of astaxanthin, that is, choosing a suitable delivery system is crucial. Therefore, we have improved the wrapping material, which is an active wrapping material with a network cross-linked structure. The specific preparation process is as follows:

[0019] S101, preparing chitosan into a chitosan solution with a concentration of 8-12% using a 0.01% citric acid aqueous solution at a temperature of 50-60° C.; stirring for 50-80 minutes to hydrolyze the chitosan double helix molecular chain structure into a single-chain coil structure, thereby obtaining a liquid with a single-chain coil structure;

[0020] S102, heating the gelatin aqueous solution to 80-90°C, adjusting the pH to 6.0, and continuing to stir rapidly for 6-8 hours. The gelatin continuously swells and breaks into short-chain gelatin low-molecular-weight peptide chains. The obtained short-chain gelatin low-molecular-weight peptide chain liquid is added to the liquid with the single-chain coil structure in S101, and interacts with the single-chain coil structure to transform into an intertwined network cross-linked structure. The reaction takes 150-200 minutes. The hydroxyl groups of the short-chain gelatin low-molecular-weight peptide chains and the amino groups of the chitosan react, increasing cross-linking points on the chitosan, forming a network structure of cross-linked short-chain peptide chains of chitosan, and a large amount of positive charges are enriched on the surface. This is recorded as the active liquid with a network cross-linked structure with positive charges.

[0021] Among them, the mass ratio of gelatin to chitosan is 1-2:3-5;

[0022] S103, soaking the porous algae astaxanthin solid particles with an emulsion at 10-20° C. to allow the emulsion to be absorbed by the surface of the particles. After soaking for 100-130 minutes, filtering the particles and drying at low temperature to obtain algae astaxanthin solid particles having an emulsion layer on the surface, which are referred to as self-emulsified algae astaxanthin solid particles.

[0023] S104, adding the self-emulsified algae astaxanthin solid particles to the active liquid with a positively charged network cross-linked structure, adjusting the pH to about 4.0, slowly stirring to evenly disperse the solid particles, then stopping the stirring, and allowing the reaction to stand for 20 to 30 minutes; then slowly stirring for 10 to 20 minutes, and then allowing the reaction to stand for 20 to 30 minutes to obtain a uniformly dispersed microsphere mixture, wherein the microspheres are coated microspheres formed by the active liquid with a positively charged network cross-linked structure and the self-emulsified algae astaxanthin solid particles, filtering the microsphere mixture, and washing the obtained precipitate by centrifugation. The sample obtained by centrifugation is freeze-dried to obtain a network cross-linked structure active encapsulation and Haematococcus pluvialis protein doubly coated astaxanthin solid microgels, namely algae microcapsules.

[0024] The mass ratio of the algae astaxanthin solid particles to the active liquid containing a positively charged network cross-linked structure is 1:3-4;

[0025] The water content of the algae microcapsules is 8-10%, and the particle size is 150-200 μm.

[0026] Chitosan, as a natural cationic polysaccharide, has good biosafety and film-forming properties. Gelatin, after modification, releases negative charges at a pH of 6.0. This allows the short-chain gelatin low-molecular peptide chains to interact with the single-chain coil structure, transforming into an intertwined network cross-linked structure. This structure, as a wrapper, can firmly lock the algae astaxanthin solid particles. When the pH is 4.0, the active liquid in the network cross-linked structure releases hydrogen ions and becomes positively charged. At this time, the negatively charged algae astaxanthin solid particles serve as the adsorption core, and the active substance of the biomacromolecule cross-linked structure serves as the coating. Through the electrostatic bonding of positive and negative charges, the active liquid with a positively charged network cross-linked structure self-assembles and coats the algae astaxanthin solid particles, forming a network cross-linked structure that wraps the layers. Finally, the algae microcapsules are double-wrapped with the network cross-linked structure active wrapper and Haematococcus pluvialis protein, which plays a role in maintaining long-term activity. When the algae microcapsules reach the small intestine, astaxanthin in the form of esters and the emulsifying substances on its surface are dispersed in the intestinal fluid to form a self-emulsifying system. The self-emulsifying system emulsifies the astaxanthin, dispersing it in the intestinal fluid and turning it into an aqueous state that is easily absorbed by organisms.

[0027] The emulsion comprises 5 parts soybean lecithin, 2 parts glucose, 3 parts polyethylene glycol 400, 20 parts tea tree oil, and 10 parts deionized water. The nano-scale emulsion is produced using a high-pressure homogenization method. The emulsion is a water-in-oil type with excellent temperature and centrifugal stability. This emulsification technology disperses astaxanthin in water, forming a self-emulsifying system that is more easily absorbed by the human body. Because the emulsion is composed of several excipients, the compatibility between the excipients must be considered. Therefore, compatibility experiments were conducted to select excipients with high solubility and good compatibility to prepare the self-emulsifying system. Soybean oil is not compatible with emulsifiers, so after comprehensive consideration, tea tree oil, which has high solubility and good compatibility, was selected as the oil phase of the self-emulsifying system. The emulsion has a relatively small particle size of between 10 and 80 nm and is transparent. It forms spontaneously under the action of a surfactant and is a thermodynamically stable system. This emulsion exhibits favorable properties, including excellent stability, low viscosity, and strong solubilization of lipophilic compounds, demonstrating promising results in the extraction and stability of astaxanthin. The mixture of a small molecule emulsifier, protein, and sugar significantly enhances the properties of the prepared emulsion, protecting the activity of astaxanthin within the microencapsulated system and reducing its degradation rate. Furthermore, upon dissolution and release in the intestinal fluid of an organism, it rapidly forms a self-emulsifying system, enhancing the water solubility of astaxanthin and promoting its absorption.

[0028] The high-pressure homogenization method comprises the following steps: the homogenization pressure is 20-30 MPa, the temperature is 40-50° C., and the processing time is 10-20 minutes.

[0029] High temperatures have a significant destructive effect on most bioactive substances, and astaxanthin should be stored at low temperatures to slow its degradation. Multiple studies have shown that the stability of astaxanthin extracts decreases with increasing temperature. For example, the absorbance of astaxanthin extracts stored at 4°C remains unchanged, while after 6 hours of storage at 70°C, the residual astaxanthin is only around 30%. Although the solubility and antioxidant activity of astaxanthin are significantly improved under acidic conditions, excessive acidity can affect astaxanthin stability. Therefore, we maintain low temperatures during the astaxanthin extraction process, allowing more astaxanthin to dissolve under acidic conditions before encapsulating it into algae microcapsules.

[0030] The composition and selection of the wall material are crucial to the performance of microcapsules and are essential for achieving high efficiency and superior performance. Chitosan and colloids are combined with algae proteins to create a synergistic network on the molecular surface. The combination of protein and hydrophilic colloids, while possessing excellent emulsifying properties, also has poor mechanical properties. Hydrophilic colloids improve the surface activity and viscosity of the protein, enhancing the stability of the wall material. Microcapsules produced using this method have been shown to exhibit excellent resistance to strong acid environments, ensuring their survival in gastric fluid.

[0031] Finally, a method for adding algae microcapsules to pig feed is provided. The algae microcapsules can be added directly to the feed at a dosage of 1-3%. These algae microcapsules promote animal digestion and absorption of feed, are safe, and can promote animal growth and development, enhancing immunity. They can also increase the bioavailability of astaxanthin, improve absorption and utilization, enhance cellular antioxidant capacity, and significantly improve the colonization of astaxanthin in the intestine, allowing the active ingredients of astaxanthin to be fully utilized.

[0032] The beneficial effects of the invention are:

[0033] 1) The present invention uses Haematococcus pluvialis as the main raw material, not only extracting astaxanthin with strong stability and high activity, but also making full use of the protein of Haematococcus pluvialis to prepare astaxanthin into algae astaxanthin nanoparticles. The Haematococcus pluvialis protein is first used as a protective layer, and then prepared into self-emulsifying algae astaxanthin solid particles through an emulsion. At the same time, an active wrapper with a network cross-linked structure is utilized. Through the interaction of positive and negative charges with the protein solution, an algae microcapsule with a double wrapper of the active wrapper with a network cross-linked structure and Haematococcus pluvialis protein is obtained, providing a high biological activity, non-biotoxic, and good stability algae astaxanthin microcapsule for the prior art. By using a specific material combination and ratio, the obtained algae astaxanthin microcapsules are not destroyed by the strong acid environment in gastric juice, and are easily absorbed through the intestinal mucosa into the lymphatic circulation and then into the blood circulation, thereby efficiently exerting their biological efficacy, showing significant advantages in terms of stability, intestinal absorption efficiency, and usage. It not only improves economic benefits, but also prevents astaxanthin from being oxidized or structurally destroyed, and can maintain activity for a long time.

[0034] 2) The algae microcapsules prepared by the present invention have good effects; the active inclusions with a network cross-linked structure have good film-forming properties and high strength. The obtained algae microcapsules, which are doubly coated with the active inclusions with a network cross-linked structure and Haematococcus pluvialis protein, are regular spherical in shape and have a smooth surface when observed under a scanning electron microscope. The obtained algae microcapsules are highly stable and still have high physical stability under strong acid, strong alkali and high temperature conditions.

[0035] 3) The present invention also provides the use of the algae microcapsules as feed additives. The morphology of the astaxanthin raw material is changed, while ensuring that astaxanthin is not released in the pig's stomach, thereby preventing it from combining with digestive enzymes in the stomach and reducing the digestion and absorption of nutrients, so that it is released after reaching the small intestine to exert antioxidant, antibacterial, antiviral and other effects. The present invention utilizes a double wrapping of an active wrapping material with a network cross-linked structure and Haematococcus pluvialis protein, giving full play to the good film-forming ability and mucosal adhesion properties of the active substance of the coil cross-linked structure, capable of colonizing on the surface of the small intestinal mucosa, and can reach the intestinal area in the largest amount. Under neutral or weakly alkaline conditions, the self-emulsified algae astaxanthin solid particles are effectively released, and a self-emulsifying system is formed to promote the bioavailability of astaxanthin.

[0036] 4) Microencapsulation of astaxanthin protects it from environmental factors such as light, heat, and oxygen, thereby maintaining its functionality and extending its storage life. The self-emulsifying algae astaxanthin solid microparticles and porous structure prepared by the present invention enhance its solubility and dispersibility in water, resulting in excellent dispersion stability. Results show that within four hours, over 90% of the astaxanthin is released from the solid self-emulsifying system. After digestion, the bioavailability reaches over 68%. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the release concentration graph of algae microcapsules in simulated gastric fluid over time. DETAILED DESCRIPTION

[0038] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0039] Example 1

[0040] An algae microcapsule comprises the following steps: first, preparing algae astaxanthin nanoparticles, specifically: subjecting 100 parts of Haematococcus pluvialis to low-temperature drying at 5°C for 30 minutes, then taking out and crushing the algae to obtain Haematococcus pluvialis powder; taking 50 parts of Haematococcus pluvialis powder and adding them to an organic solvent uniformly mixed with dichloromethane and methanol in a volume ratio of 1:1 at 20°C, mixing them with a magnetic stirrer for 150 minutes to fully dissolve the astaxanthin; separating the Haematococcus pluvialis residue and the liquid by centrifugation; taking the upper liquid, adding 5 parts of hexadecyldimethylammonium chloride and mixing them uniformly to allow the astaxanthin and the hexadecyldimethylammonium chloride to react with each other; and cooling the liquid to 5°C to generate solid flocs; separating the solid and the liquid, and washing and drying the lower solid layer to obtain positively charged astaxanthin solid particles.

[0041] 50 parts of Haematococcus pluvialis residue are enriched with Haematococcus pluvialis protein using a polyethylene glycol / sodium phosphate aqueous two-phase system. The Haematococcus pluvialis protein is separated in the top phase to obtain a protein liquid. 10 parts of gelatin are added to 10 parts of the protein liquid, heated and stirred at 50°C for 30 minutes until completely dispersed. Citric acid is added to adjust the pH to 5.2, and the mixture is allowed to stand for 30 minutes to cause coagulation, so that the protein in the protein liquid is negatively charged, thereby obtaining a mixed liquid containing coagulates. The solid-liquid separation is carried out by centrifugation, and the lower solid layer is washed and dried to obtain negatively charged protein microparticles.

[0042] Take 10 parts of positively charged astaxanthin solid particles and 20 parts of negatively charged protein particles, add them to deionized water, heat and stir at 50°C for 50 minutes, add citric acid, adjust the pH to 4.5, so that the protein particles and astaxanthin solids are fully condensed, centrifuge the solid-liquid separation, wash and dry the lower layer of solid to obtain algae astaxanthin solid particles.

[0043] Furthermore, the active packaging material of the network cross-linked structure is specifically:

[0044] At a temperature of 50°C, chitosan was prepared into 10 parts of an 8% chitosan solution with a 0.01% citric acid aqueous solution; the chitosan double helix molecular chain structure was hydrolyzed into a single chain coil structure by high-speed stirring for 80 minutes, thereby obtaining a liquid with a single chain coil structure;

[0045] Ten parts of a 4% gelatin aqueous solution were heated to 90°C, the pH was adjusted to 6.0, and rapid stirring was continued for 8 hours. The gelatin continued to swell and break into short-chain gelatin low-molecular-weight peptide chains. The obtained short-chain gelatin low-molecular-weight peptide chain liquid was added to the liquid with a single-chain coil structure and reacted for 200 minutes to obtain a cross-linked network structure of chitosan cross-linked short-chain peptide chains, which was recorded as an active liquid with a positively charged network cross-linked structure.

[0046] 5 parts of soybean lecithin, 2 parts of glucose, 3 parts of polyethylene glycol 400, 20 parts of tea tree oil and 10 parts of deionized water were homogenized by high pressure homogenization at a pressure of 20 MPa, a temperature of 40° C. and a treatment time of 20 minutes to prepare a nano-emulsion.

[0047] At 10°C, 10 parts of porous algae astaxanthin solid particles were soaked in 20 parts of emulsion, so that the surface of the porous algae astaxanthin solid particles absorbed the emulsion. After soaking for 100 minutes, the particles were filtered and dried at low temperature to obtain self-emulsified algae astaxanthin solid particles.

[0048] 10 parts of self-emulsified algae astaxanthin solid particles are added to 30 parts of an active liquid with a positively charged network cross-linked structure, the pH is adjusted to about 4.0, and stirring is continued slowly to uniformly disperse the solid particles. Stirring is then stopped and the reaction is allowed to stand for 30 minutes. Stirring is continued slowly for 120 minutes, and the reaction is allowed to stand for another 30 minutes to obtain a uniformly dispersed microsphere mixture. The microsphere mixture is filtered, and the resulting precipitate is centrifuged and washed. The sample obtained by centrifugation is freeze-dried to obtain algae microcapsules. The algae microcapsules have a moisture content of 8% and an average particle size of about 152 μm.

[0049] Example 2

[0050] An algae microcapsule comprises: first, preparing algae astaxanthin nanoparticles, specifically: drying 100 parts of Haematococcus pluvialis at 5°C for 30 minutes, then removing and crushing the algae to obtain Haematococcus pluvialis powder; adding 50 parts of Haematococcus pluvialis powder to an organic solvent uniformly mixed with dichloromethane and methanol in a volume ratio of 1:1 at 20°C, mixing with a magnetic stirrer for 150 minutes to fully dissolve the astaxanthin; separating the Haematococcus pluvialis residue and the liquid by centrifugation; taking the upper liquid, adding 5 parts of hexadecyldimethylammonium chloride, and mixing uniformly to allow the astaxanthin and the hexadecyldimethylammonium chloride to react with each other; cooling to 5°C to generate solid flocs; separating the solid and the liquid, washing and drying the lower solid layer, and obtaining positively charged astaxanthin solid particles;

[0051] 50 parts of Haematococcus pluvialis residue are enriched with Haematococcus pluvialis protein using a polyethylene glycol / sodium phosphate aqueous two-phase system. The Haematococcus pluvialis protein is separated in the top phase to obtain a protein liquid. 10 parts of gelatin are added to 10 parts of the protein liquid, heated and stirred at 50°C for 30 minutes until completely dispersed. Citric acid is added to adjust the pH to 5.3, and the mixture is allowed to stand for 30 minutes to cause coagulation, so that the protein in the protein liquid is negatively charged, thereby obtaining a mixed liquid containing coagulates. The solid-liquid separation is carried out by centrifugation, and the lower solid layer is washed and dried to obtain negatively charged protein microparticles.

[0052] Take 10 parts of positively charged astaxanthin solid particles and 40 parts of negatively charged protein particles, add them to deionized water, heat and stir at 50°C for 50 minutes, add citric acid, adjust the pH to 4.5, so that the protein particles and astaxanthin solids are fully condensed, centrifuge the solid-liquid separation, wash and dry the lower layer of solid to obtain algae astaxanthin solid particles.

[0053] Furthermore, the active packaging material of the network cross-linked structure is specifically:

[0054] At a temperature of 50°C, chitosan was prepared into 10 parts of a 12% chitosan solution with a 0.01% citric acid aqueous solution; the chitosan double helix molecular chain structure was hydrolyzed into a single chain coil structure by high-speed stirring for 80 minutes, thereby obtaining a liquid with a single chain coil structure;

[0055] Ten parts of a 4% gelatin aqueous solution were heated to 90°C, the pH was adjusted to 6.0, and rapid stirring was continued for 6 hours. The gelatin continued to swell and break into short-chain gelatin low-molecular-weight peptide chains. The obtained short-chain gelatin low-molecular-weight peptide chain liquid was added to the liquid with a single-chain coil structure and reacted for 200 minutes to obtain a cross-linked network structure of chitosan cross-linked short-chain peptide chains, which was recorded as an active liquid with a positively charged network cross-linked structure.

[0056] 5 parts of soybean lecithin, 2 parts of glucose, 3 parts of polyethylene glycol 400, 20 parts of tea tree oil and 10 parts of deionized water were homogenized by high pressure homogenization at a pressure of 25 MPa, a temperature of 50° C. and a treatment time of 10 minutes to prepare a nano-emulsion;

[0057] At 10°C, 10 parts of porous algae astaxanthin solid particles were soaked in 20 parts of emulsion, so that the surface of the porous algae astaxanthin solid particles absorbed the emulsion. After soaking for 100 minutes, the particles were filtered and dried at low temperature to obtain self-emulsified algae astaxanthin solid particles.

[0058] Ten parts of self-emulsified algae astaxanthin solid particles are added to 40 parts of a positively charged network cross-linked active liquid. The pH is adjusted to approximately 4.0, and the mixture is slowly stirred until the solid particles are evenly dispersed. Stirring is then stopped and the mixture is allowed to react for 30 minutes. After another 10 minutes of slow stirring and another 30 minutes of reaction, a uniformly dispersed microsphere mixture is obtained. The microsphere mixture is filtered, the resulting precipitate is centrifuged and washed, and the centrifuged sample is freeze-dried to obtain algae microcapsules. The algae microcapsules have a moisture content of 8% and an average particle size of approximately 172 μm. The algae microcapsules have a moisture content of 8-10% and a particle size of 150-200 μm.

[0059] Example 3

[0060] An algae microcapsule comprises: first, preparing algae astaxanthin nanoparticles, specifically: drying 100 parts of Haematococcus pluvialis at 5°C for 30 minutes, then removing and crushing the algae to obtain Haematococcus pluvialis powder; adding 50 parts of Haematococcus pluvialis powder to an organic solvent uniformly mixed with dichloromethane and methanol in a volume ratio of 1:1 at 20°C, mixing with a magnetic stirrer for 150 minutes to fully dissolve the astaxanthin; separating the Haematococcus pluvialis residue and the liquid by centrifugation; taking the upper liquid, adding 5 parts of hexadecyldimethylammonium chloride, and mixing uniformly to allow the astaxanthin and the hexadecyldimethylammonium chloride to react with each other; cooling to 5°C to generate solid flocs; separating the solid and the liquid, washing and drying the lower solid layer, and obtaining positively charged astaxanthin solid particles;

[0061] 50 parts of Haematococcus pluvialis residue are enriched with Haematococcus pluvialis protein using a polyethylene glycol / sodium phosphate aqueous two-phase system. The Haematococcus pluvialis protein is separated in the top phase to obtain a protein liquid. 10 parts of gelatin are added to 10 parts of the protein liquid, heated and stirred at 50°C for 30 minutes until completely dispersed. Citric acid is added to adjust the pH to 5.5, and the mixture is allowed to stand for 30 minutes to cause coagulation, so that the protein in the protein liquid is negatively charged, thereby obtaining a mixed liquid containing coagulates. The solid-liquid separation is carried out by centrifugation, and the lower solid layer is washed and dried to obtain negatively charged protein microparticles.

[0062] Take 10 parts of positively charged astaxanthin solid particles and 30 parts of negatively charged protein particles, add them to deionized water, heat and stir at 50°C for 50 minutes, add citric acid, adjust the pH to 4.5, so that the protein particles and astaxanthin solids are fully condensed, centrifuge the solid-liquid separation, wash and dry the lower layer of solid to obtain algae astaxanthin solid particles.

[0063] Furthermore, the active packaging material of the network cross-linked structure is specifically:

[0064] At a temperature of 50°C, chitosan was prepared into 10 parts of a 10% chitosan solution with a 0.01% citric acid aqueous solution; the chitosan double helix molecular chain structure was hydrolyzed into a single chain coil structure by high-speed stirring for 80 minutes, thereby obtaining a liquid with a single chain coil structure;

[0065] 8 parts of a 4% gelatin aqueous solution were heated to 90°C, the pH was adjusted to 6.0, and rapid stirring was continued for 7 hours. The gelatin continued to swell and break into short-chain gelatin low-molecular-weight peptide chains. The obtained short-chain gelatin low-molecular-weight peptide chain liquid was added to the liquid with a single-chain coil structure and reacted for 200 minutes to obtain a cross-linked network structure of chitosan cross-linked short-chain peptide chains, which was recorded as an active liquid with a positively charged network cross-linked structure.

[0066] 5 parts of soybean lecithin, 2 parts of glucose, 3 parts of polyethylene glycol 400, 20 parts of tea tree oil and 10 parts of deionized water were homogenized by high pressure homogenization at a pressure of 30 MPa, a temperature of 40° C. and a treatment time of 20 minutes to prepare a nano-emulsion;

[0067] At 10°C, 10 parts of porous algae astaxanthin solid particles were soaked in 20 parts of emulsion, so that the surface of the porous algae astaxanthin solid particles absorbed the emulsion. After soaking for 100 minutes, the particles were filtered and dried at low temperature to obtain self-emulsified algae astaxanthin solid particles.

[0068] 10 parts of self-emulsified algae astaxanthin solid particles are added to 30 parts of an active liquid with a positively charged network cross-linked structure, the pH is adjusted to about 4.0, and stirring is continued slowly to uniformly disperse the solid particles. Stirring is then stopped and the mixture is allowed to react for 30 minutes. Stirring is continued slowly for another 20 minutes, and the mixture is allowed to react for another 30 minutes to obtain a uniformly dispersed microsphere mixture. The microsphere mixture is filtered, and the resulting precipitate is washed by centrifugation. The sample obtained by centrifugation is freeze-dried to obtain algae microcapsules. The algae microcapsules have a moisture content of 10% and an average particle size of about 196 μm.

[0069] Comparative Example 1: The active wrapping material with a cross-linked network structure was replaced with an equal amount of ordinary chitosan wrapping material, and the rest was the same as in Example 1;

[0070] Comparative Example 2, the step of soaking in the emulsion was omitted, and the active wrapping material with a network cross-linked structure was directly used for wrapping, and the rest was the same as in Example 1;

[0071] Comparative Example 3: The active coating of the network cross-linked structure was replaced with corn starch coating in equal amounts, and the emulsion soaking step was omitted. Other aspects were the same as in Example 1.

[0072] Comparative Example 4 is a Haematococcus pluvialis capsule product involved in Example 4 of the Chinese patent publication number CN118319001B.

[0073] 1. Gastrointestinal dissolution test

[0074] The astaxanthin content was measured by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: reverse-phase 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 an aqueous solution (80+15+5, V / V / V); the detector was an ultraviolet (UV) detector with a detection wavelength of 478 nm; the injection volume was 10 μL, and the column temperature was room temperature. 1. Pig stomach solubility determination:

[0075] Preparation of simulated porcine gastric juice: Take 7 mL of 1 mol / L dilute hydrochloric acid and add it to 1000 mL of deionized water, adjust the pH to 1.5, add 2 g of porcine pepsin and stir to mix evenly, and filter with a 0.2 μm sterile filter to obtain simulated porcine gastric juice.

[0076] Experimental Method: 2.000 g of each sample from Examples 1-3 and Comparative Examples 1-4 was weighed into a dissolution cup, and 250 mL of simulated porcine gastric fluid was added to each. The dissolution apparatus was then placed at a temperature of 37°C and a speed of 75 rpm. After reacting for 1 hour, 2 hours, and 4 hours, 10 mL of the upper reaction liquid was collected and the astaxanthin content was measured by high-performance liquid chromatography. The results are shown in Table 1.

[0077] The amount of astaxanthin released from the microcapsules prepared in Example 1, Comparative Example 1 and Comparative Example 4 in gastric juice over time was plotted as follows: Figure 1 .from Figure 1 It can be seen that Example 1 has significantly improved stability in gastric juice compared to Comparative Examples 2-3.

[0078] As can be seen from Table 1, Examples 1 to 3 used a network-crosslinked active coating as the protective coating for the self-emulsifying algae astaxanthin solid particles. After a 4-hour reaction in simulated porcine gastric fluid, the astaxanthin content decreased by no more than 10%, indicating that the network-crosslinked active coating was insoluble in strong acid and the shell was barely damaged. In contrast, Comparative Example 1, which used ordinary chitosan as the coating and was directly added to simulated porcine gastric fluid for a 4-hour reaction, lost 95% of the astaxanthin in the gastric fluid, indicating that astaxanthin was released in the pig stomach and that ordinary chitosan had a weak protective effect on the algae microcapsules in gastric fluid, with the coating almost completely destroyed. Comparative Example 4, which used corn starch as the coating, also lost 90% of the astaxanthin in gastric fluid, resulting in a significant destruction of the astaxanthin in the gastric fluid, thus affecting its absorption in the small intestine.

[0079] Table 1 Astaxanthin content after simulated pig stomach dissolution (%)

[0080] Experimental group 1 hour 2 hours 4 hours Example 1 4.18 5.32 8.01 Example 2 5.12 6.77 9.57 Example 3 4.55 5.89 8.63 Comparative Example 1 48.59 75.14 95.05 Comparative Example 3 44.82 84.53 92.24 Comparative Example 4 65.93 82.38 90.06

[0081] 2. Pig small intestine dissolution test:

[0082] To prepare simulated porcine intestinal fluid: Place 13.6g of potassium dihydrogen phosphate in a beaker, add 1000mL of deionized water, and stir to fully dissolve. Adjust the pH to 6.8 with 0.1mol / L NaOH solution. Add 2g of porcine pancreatic lipase, mix thoroughly, and filter through a 0.2μm sterile filter to obtain simulated porcine intestinal fluid.

[0083] Experimental Method: 2.000 g of each sample from Examples 1-3 and Comparative Examples 1, 3, and 4 was weighed into a dissolution cup. 250 mL of simulated porcine intestinal fluid was added to each sample. The dissolution apparatus was then placed at a temperature of 37°C and a speed of 100 rpm. After reaction for 1 hour, 2 hours, and 4 hours, 10 mL of the upper reaction liquid was collected and assayed for astaxanthin content. The results are shown in Table 2.

[0084] Table 2 Astaxanthin content after simulated pig small intestine dissolution (%)

[0085] Experimental group 1 hour 2 hours 4 hours Example 1 30.16 72.18 93.65 Example 2 31.72 75.55 93.89 Example 3 32.92 73.27 92.43 Comparative Example 1 42.73 62.38 85.12 Comparative Example 3 53.87 74.35 82.54 Comparative Example 4 64.61 80.92 86.37

[0086] The protective layer of the astaxanthin solid microspheres in Examples 1 to 3 is an active coating with a positively charged network cross-linked structure. It remains stable under the acidic conditions of the pig stomach and is destroyed under the neutral conditions of the pig small intestine. After 4 hours of reaction in simulated pig small intestinal fluid, the astaxanthin release rate is over 90%, and almost all astaxanthin is released. This shows that self-emulsifying algae astaxanthin solid particles can better control the release of astaxanthin. The protective layer of Comparative Examples 1 and 3 is ordinary chitosan. Whether under the strong acid conditions in the stomach or under the neutral conditions in the small intestine, astaxanthin is easily released. It cannot be guaranteed to effectively reach the small intestine after passing through the stomach, thus affecting the absorption of astaxanthin in the small intestine. The coating in Comparative Example 4 is corn starch. Whether under the strong acid conditions in the stomach or under the neutral conditions in the small intestine, it is very easy to release. Therefore, it cannot be guaranteed to effectively reach the small intestine after passing through the stomach, nor can it be effectively absorbed in the small intestine. Therefore, the algae microcapsules produced in this invention, which are dually coated with a cross-linked active coating and Haematococcus pluvialis protein, simultaneously ensure that the coating layer does not gradually absorb water, swell, and disintegrate in the pig's stomach. They remain stable under strongly acidic conditions, but are unstable and gradually degrade under neutral conditions. This ensures that the algae microcapsules containing astaxanthin remain stable in the stomach and gradually disintegrate upon reaching the small intestine, releasing the astaxanthin and thus exerting its therapeutic effect.

[0087] 2. Bioavailability test

[0088] The microcapsule preparations of Examples 1-2, Comparative Examples 2 and 4 were added to feed at 5% concentration. 150 white-striped pigs in good condition, similar in weight and at the same stage were randomly divided into 5 groups, 30 in each group, and fed 1 kg of the feed. Blood was collected at 0, 4, 8, 12, 15, 18, 24, 48 and 72 hours after feeding, placed in a centrifuge tube containing EDTA, and allowed to stand at room temperature at 25-26°C for 30 minutes. After separation, the blood was centrifuged at 4°C and 7500 rpm for 15 minutes, and the upper serum was carefully collected to determine the shrimp The content of astaxanthin was measured, and the metabolic kinetic curve of astaxanthin concentration-time in root serum was drawn. The area under the curve was calculated by the trapezoidal method. The bioavailability of astaxanthin was measured by the size of the area under the curve (AUC0-t). As shown in Table 3, the results showed that the area under the curve AUC values ​​in the pig serum of the sample groups of Examples 1-2 were about 6 times higher than those of the comparative example. The bioavailability of astaxanthin in the dispersible self-emulsifying algae astaxanthin solid particles prepared by the present invention was significantly improved. After digestion, the bioavailability reached more than 68%.

[0089] project Example 1 Example 2 Comparative Example 2 Comparative Example 4 AUC value 24.5 23.8 3.1 3.9

[0090] 3. Piglet growth status

[0091] A total of 150 28-day-old weaned piglets were randomly divided into five groups of 30 each. Astaxanthin was added to standard feed at a 2% mass fraction and fed for 150 days. Body weight before and after feeding, average feed intake during the feeding period, disease incidence, and mortality were measured. The test results are shown in Table 4. Compared with standard commercial feed, feeding standard piglets with feed supplemented with the algae microcapsules prepared in this invention promoted growth and enhanced disease resistance, significantly reducing breeding costs and achieving significant economic benefits. The meat quality was also significantly better than that of standard feed. This is because astaxanthin can enhance the animal's antioxidant capacity, promote the structural and functional integrity of jejunal cells, and improve the intestinal digestion capacity, thereby enhancing growth performance. It also prevents the oxidation of unsaturated fatty acids and protein molecules in muscle into peroxides, preventing lipid breakdown and polypeptide chain scission, significantly improving the flavor of the meat.

[0092] Table 4 Data record table of white striped pig breeding

[0093] project Ordinary feed Example 1 Example 2 Example 3 Comparative Example 4 Average daily weight gain (kg / day) 0.65 1.30 1.32 1.31 0.85 Disease incidence (%) 8.0 2.6 2.0 2.0 3.3 mortality rate(%) 2.6 1.3 1.3 0.66 0.66

[0094] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any modifications and changes made to the technical solution of the present invention by ordinary persons in the art shall still fall within the scope of the present invention as long as they do not depart from the overall concept of the present invention.

Claims

1. A method for preparing algae microcapsules, characterized in that: An algae microcapsule comprises an active encapsulation with a network-like cross-linked structure and algae astaxanthin nanoparticles. The preparation of the algae astaxanthin nanoparticles comprises the following steps: S001, drying Haematococcus pluvialis at a low temperature of 0 to 10° C. for 20 to 40 minutes, then removing and crushing the algae to obtain Haematococcus pluvialis powder; S002, adding Haematococcus pluvialis powder to an organic solvent uniformly mixed with dichloromethane and methanol in a volume ratio of 1:1 at 20-30° C., mixing with a magnetic stirrer for 120-150 minutes to fully dissolve the astaxanthin, separating the Haematococcus pluvialis residue and the liquid by centrifugation, taking the upper liquid, adding cetyldimethylammonium chloride and mixing uniformly to allow the astaxanthin and cetyldimethylammonium chloride to react with each other, and cooling to 5-10° C. to form solid flocs; separating the solid and the liquid, washing and drying the lower solid layer to obtain positively charged astaxanthin solid microparticles; wherein the mass ratio of cetyldimethylammonium chloride to Haematococcus pluvialis powder is 1:10; S003, enriching the Haematococcus pluvialis protein from the Haematococcus pluvialis residue separated in S002 using a polyethylene glycol / sodium phosphate aqueous two-phase system, separating the Haematococcus pluvialis protein in the top phase to obtain a protein solution having an isoelectric point of 4.2; adding gelatin to the protein solution and heating and stirring at 50-60° C. for 30-50 minutes until completely dispersed; adding citric acid to adjust the pH to 5.0-5.5; and standing for 30-50 minutes to allow coagulation, so that the protein in the protein solution has a negative charge, to obtain a mixed solution containing coagulates; centrifuging for solid-liquid separation, washing and drying the lower solid layer to obtain negatively charged protein particles; the mass ratio of the protein solution to gelatin being 1:1; S004, adding positively charged astaxanthin solid particles and negatively charged protein particles to deionized water, heating and stirring at 50-60° C. for 30-50 minutes, adding citric acid, adjusting the pH to 4.5, so that the protein particles and astaxanthin solid are fully condensed, centrifuging for solid-liquid separation, washing and drying the lower layer of solid to obtain algae astaxanthin solid particles; the mass ratio of the positively charged astaxanthin solid particles to the negatively charged protein particles is 1:2-4; the active coating with a network cross-linked structure is formed by the entanglement of short-chain gelatin low-molecular peptide chains and chitosan single-chain coils, and the algae astaxanthin nanoparticles are prepared into self-emulsifying algae astaxanthin solid particles by an emulsion before forming microcapsules with the coating.

2. The method for preparing algae microcapsules according to claim 1, characterized in that: The preparation process of the active packaging material with a network cross-linked structure is as follows: S101, preparing chitosan into a chitosan solution with a concentration of 8-12% using a 0.01% citric acid aqueous solution at a temperature of 50-60° C.; stirring for 50-80 minutes to hydrolyze the chitosan double helix molecular chain structure into a single-chain coil structure, thereby obtaining a liquid with a single-chain coil structure; S102, heat the gelatin aqueous solution to 80-90°C, adjust the pH to 6.0, and continue to stir rapidly for 6-8 hours. The gelatin continues to swell and break into short-chain gelatin low-molecular-weight peptide chains. The obtained short-chain gelatin low-molecular-weight peptide chain liquid is added to the liquid with the single-chain coil structure in S101, and interacts with the single-chain coil structure to transform into an intertwined network cross-linked structure. The reaction takes 150-200 minutes. The hydroxyl groups of the short-chain gelatin low-molecular-weight peptide chains and the amino groups of the chitosan react, and cross-linking points are added to the chitosan, forming a cross-linked network structure of the chitosan cross-linked short-chain peptide chains, and a large amount of positive charges are enriched on the surface, which is recorded as the active liquid with a positively charged network cross-linked structure.

3. The method for preparing algae microcapsules according to claim 2, characterized in that: S103, soaking the porous algae astaxanthin solid particles with an emulsion at 10-20° C. to allow the emulsion to be absorbed by the surface of the particles. After soaking for 100-130 minutes, filtering the particles and drying at low temperature to obtain algae astaxanthin solid particles having an emulsion layer on the surface, which are referred to as self-emulsified algae astaxanthin solid particles. S104, adding the self-emulsified algae astaxanthin solid particles to the active liquid with a positively charged network cross-linked structure, adjusting the pH to 4.0, slowly stirring to evenly disperse the solid particles, then stopping the stirring, and allowing the reaction to stand for 20 to 30 minutes; then slowly stirring for 10 to 20 minutes, and then allowing the reaction to stand for 20 to 30 minutes to obtain a uniformly dispersed microsphere mixture, wherein the microspheres are coated microspheres formed by the active liquid with a positively charged network cross-linked structure and the self-emulsified algae astaxanthin solid particles, filtering the microsphere mixture, and washing the obtained precipitate by centrifugation. The sample obtained by centrifugation is freeze-dried to obtain a network cross-linked structure active encapsulation and Haematococcus pluvialis protein doubly coated astaxanthin solid microgel, which is the algae microcapsule; the mass ratio of gelatin to chitosan is 1 to 2:3 to 5.

4. The method for preparing algae microcapsules according to claim 1, characterized in that: 5 parts of soybean lecithin, 2 parts of glucose, 3 parts of polyethylene glycol 400, 20 parts of tea tree oil and 10 parts of deionized water are used to prepare a nano-scale emulsion by high-pressure homogenization. The emulsion type is water-in-oil type.

5. The method for preparing algae microcapsules according to claim 4, characterized in that: The high-pressure homogenization method comprises the following steps: the homogenization pressure is 20-30 MPa, the temperature is 40-50° C., and the processing time is 10-20 minutes.

6. The method for preparing algae microcapsules according to claim 3, characterized in that: The mass ratio of the algae astaxanthin solid particles to the active liquid containing a positively charged network cross-linked structure is 1:3-4.

7. The method for preparing algae microcapsules according to claim 3, characterized in that: The water content of the algae microcapsules is 8-10%, and the particle size is 150-200 μm.

8. The method for preparing algae microcapsules according to claim 3, characterized in that: The algae microcapsules are added in an amount of 1 to 2% in pig feed.

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