Preparation method of algae microcapsule and application of pig feed

Abstract

The invention belongs to the technical field of preparation of active substance preparations, and particularly relates to a preparation method of an algae microcapsule. Haematococcus pluvialis is used as a main raw material, astaxanthin is prepared into algae astaxanthin nano solid particles, firstly, haematococcus pluvialis protein is used as a protective layer, then emulsion is adsorbed to prepare self-emulsified algae astaxanthin solid particles, meanwhile, active wrappage of a net-shaped cross-linked structure is utilized, and the self-emulsified algae astaxanthin nano solid particles are prepared into algae astaxanthin nano solid particles. According to the present invention, the haematococcus pluvialis protein and the active wrappage with the network cross-linked structure are subjected to the positive and negative charge effect with the protein solution so as to obtain the haematococcus pluvialis protein dual-wrapped algae microcapsule, and the astaxanthin in the algae microcapsule can be easily released in the small intestine, and the bioavailability can achieve more than 60%. After the astaxanthin is microencapsulated, the influence of environmental factors such as light, heat, oxygen and the like on the stability of the astaxanthin is avoided, the activity can be kept for a long time, and the microencapsulated astaxanthin is suitable for being used in animal feed additives.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance preparation preparation, and specifically relates to a preparation method of 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 of 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 wall and pectin inside and outside. The gelatinous substance fills the gap between the cell wall and the cytoplasm. The thick and dense cell wall composed of cellulose, hemicellulose, lignin and other substances protects the inside 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, 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 in a vacuum at low temperature and away from light, which seriously limits its wide application. Therefore, how to make astaxanthin able to be stably stored simply and for a long time is a key issue that needs to be solved urgently in order to be able to use it in health foods and medicines.

[0004] The Chinese patent with publication number CN108530939B discloses a foam spray drying method for preparing astaxanthin microcapsules in 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 astaxanthin microencapsulation, the effects of environmental factors such as light, heat, and oxygen on the stability of astaxanthin are avoided, thereby maintaining the functionality of astaxanthin and extending the storage period. Although this method maintains the functionality of astaxanthin and extends the storage period, it does not solve the problem that astaxanthin cannot be directly applied to an aqueous environment, nor can it solve the problem of biological absorption of astaxanthin.

[0005] A Chinese patent with publication number CN118319001B discloses a Haematococcus pluvialis capsule and a preparation method thereof, and discloses that the preparation of Haematococcus pluvialis polymer microspheres includes the following steps: firstly, dehydrating part of the cellulose on the cell wall of Haematococcus pluvialis to produce aldehyde groups; then subjecting the dehydrated cellulose on the cell wall to hydroxyalkylation and aromatic substitution with lignin to cause the Haematococcus pluvialis to coagulate with each other to form coacervates; then, the coacervates of Haematococcus pluvialis are broken, and then corn starch is used for inclusion to form Haematococcus pluvialis polymer microspheres. This method solves the problem of efficiently extracting astaxanthin from Haematococcus pluvialis, but there is a high-temperature extraction link, which has a certain influence on the activity of astaxanthin, and also solves the problem that astaxanthin is not easily destroyed after long-term storage, but does not solve the problem of biological absorption of astaxanthin.

[0006] However, there are still many severe challenges in the application and development of astaxanthin. Astaxanthin is unstable and is prone to oxidative degradation during processing and storage, which is not conducive to commodity production and storage. Astaxanthin has low bioavailability in the body, serious waste of resources, and it is difficult to reach an effective absorption dose. The above challenges have greatly limited the application and promotion of astaxanthin. Therefore, how to change the dosage form of natural astaxanthin and how to improve the water dispersibility, stability and bioavailability of natural astaxanthin have become key technical issues in the application and promotion of astaxanthin. 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 choice or create conditions.

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

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

[0010] S001, drying the Haematococcus pluvialis at a low temperature of 0 to 10° C. for 20 to 40 minutes, then taking out 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 astaxanthin, separating the Haematococcus pluvialis residue and the liquid by centrifugation, taking the upper layer of liquid, adding hexadecyl dimethyl ammonium chloride and mixing evenly, allowing astaxanthin and hexadecyl dimethyl ammonium chloride to react with each other, and generating solid floccules after cooling to 5-10° C.; separating the solid from the liquid, washing and drying the lower layer of solid, and obtaining positively charged astaxanthin solid particles; wherein the mass ratio of hexadecyl dimethyl ammonium chloride to Haematococcus pluvialis powder is 1:10;

[0012] S003, enriching the Haematococcus pluvialis protein with the Haematococcus pluvialis residue separated in S002 by a polyethylene glycol / sodium phosphate two-phase aqueous system, and separating the Haematococcus pluvialis protein in the top phase to obtain a protein solution, the isoelectric point of which is 4.2; adding gelatin to the protein solution, heating and stirring at 50-60° C. for 30-50 minutes until it is completely dispersed, adding citric acid, adjusting the pH to 5.0-5.5, and standing for 30-50 minutes to coagulate, so that the protein in the protein solution is negatively charged, and a mixed solution containing coagulation is obtained; centrifuging for solid-liquid separation, washing and drying the lower layer solid, and obtaining negatively charged protein particles; the mass ratio of the protein solution to the gelatin is 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 solids 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.

[0014] The accumulation of astaxanthin in Haematococcus pluvialis can reach up to 5%, which is the best biological source of natural astaxanthin. Astaxanthin in Haematococcus pluvialis mainly exists in free and esterified forms, with astaxanthin monoester accounting for about 70%, astaxanthin diester 25%, and the rest in free form. In addition to astaxanthin, Haematococcus pluvialis also includes a variety of amino acids, fatty acids, polysaccharides and protein nutrients. Among them, the content of Haematococcus pluvialis protein can reach 30%. Haematococcus pluvialis protein has good oil absorption, emulsification and solubility, and can replace other protein raw materials as microcapsule wall materials, achieving a good embedding effect. At the same time, Haematococcus pluvialis polysaccharide is a small molecule substance, which can be used as a filler for microcapsule wall materials and play a role in supporting wall materials. Therefore, the obtained algae astaxanthin solid particles can effectively protect the activity of astaxanthin after being embedded by Haematococcus pluvialis protein.

[0015] The protein of Haematococcus pluvialis is extracted by flocculation through gelatin isoelectric precipitation. The microstructure of Haematococcus pluvialis protein is rod-shaped, with a rough and porous surface and negative charge. When it is combined with the positively charged astaxanthin solid particles under the action of positive and negative charges, it has a good embedding effect. However, because of its rough and porous surface, it is also conducive to the release of the embedded astaxanthin in the organism, thereby facilitating the absorption of astaxanthin.

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

[0017] When adding algae microcapsules to feed for animals, we found that the algae microcapsules were easily destroyed in the gastric juice and could not reach the small intestine where they were more easily absorbed and utilized. In addition, astaxanthin exists in the form of esters, and even if it can reach the small intestine, it is not easy to disperse in the intestinal juice and be absorbed by animals.

[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 improve the wrapping material, which is an active wrapping material with a network cross-linked structure. The specific preparation process is:

[0019] S101, at a temperature of 50 to 60° C., chitosan is prepared into a chitosan solution with a concentration of 8 to 12% using a 0.01% citric acid aqueous solution; and the chitosan double helix molecular chain structure is hydrolyzed into a single-chain coil structure to obtain a liquid with a single-chain coil structure.

[0020] S102, raising the temperature of 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 peptide chains, and the obtained short-chain gelatin low-molecular peptide chain liquid is added to the liquid of the single-chain coil structure in S101, and interacts with the single-chain coil structure to transform into an intertwined network cross-linked structure, and reacts for 150-200 minutes, the hydroxyl groups of the short-chain gelatin low-molecular peptide chains and the amino groups of chitosan react, and cross-linking points are added to 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, which is recorded as an 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 with emulsion at 10-20° C. so that the surface of the porous algae astaxanthin solid particles absorbs the emulsion, and after soaking for 100-130 minutes, filtering and drying at low temperature to obtain algae astaxanthin solid particles with an emulsion layer on the surface, which are recorded 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, washing the obtained precipitate by centrifugation, and freeze-drying the sample obtained by centrifugation to obtain a network cross-linked structure active inclusion and astaxanthin solid microgels doubly coated with Haematococcus pluvialis protein, 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, while gelatin will release negative charges at pH 6.0 after modification. The short-chain gelatin low-molecular peptide chain interacts with the single-chain coil structure to transform into an intertwined mesh 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 of the mesh cross-linked structure releases hydrogen ions and carries a positive charge. At this time, the negatively charged algae astaxanthin solid particles are used as the adsorption core, and the active substance of the biological macromolecular cross-linked structure is used as the coating film. Through the electrostatic combination of positive and negative charges, the active liquid of the positively charged mesh cross-linked structure is cross-linked and self-assembled and coated on the algae astaxanthin solid particles, forming a mesh cross-linked structure layer by layer. Finally, the algae microcapsules double-wrapped with the active wrapper of the mesh cross-linked structure and the Haematococcus pluvialis protein are obtained, which plays a role in maintaining activity for a long time. 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 is composed of 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. The nano-scale emulsion is prepared by high-pressure homogenization. The emulsion type is oil-in-water type, and the temperature stability and centrifugal stability are good. The emulsification technology can disperse astaxanthin in water to form a self-emulsifying system, which is easier to be absorbed by the human body. Since it is composed of several auxiliary materials, the compatibility between the auxiliary materials needs to be considered, so the auxiliary materials with high solubility and good compatibility are selected through compatibility experiments to prepare the self-emulsifying system. Soybean oil is not compatible with emulsifiers. After comprehensive consideration, tea tree oil with high solubility and good compatibility is selected as the oil phase of the self-emulsifying system. The particle size of the emulsion is relatively small, between 10 and 80 nm, and it is transparent. It can be spontaneously formed under the action of a surfactant and belongs to a thermodynamically stable system. The emulsion has good properties, including excellent stability, low viscosity and strong solubilization ability of lipophilic compounds, and shows good effects in the extraction and stability of astaxanthin. The mixture of small molecule emulsifier, protein and sugar can significantly improve the properties of the prepared emulsion, which can not only protect the activity of astaxanthin in the microcapsule system and reduce the degradation rate of astaxanthin. When dissolved and released in the intestinal fluid of the organism, it can quickly form a self-emulsifying system, enhance the water solubility of astaxanthin, and promote the absorption rate of astaxanthin.

[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 temperature has a significant destructive effect on most bioactive substances. Astaxanthin should be stored at low temperature to slow down its degradation. Many studies have shown that the stability of astaxanthin extract decreases with increasing temperature. For example, the absorbance of astaxanthin extract stored at 4°C remains unchanged, while the residual rate of astaxanthin is only about 30% after storage at 70°C for 6 hours. Although the solubility and antioxidant activity of astaxanthin are significantly improved under acidic conditions, excessive acidity will affect the stability of astaxanthin. Therefore, during the astaxanthin extraction process, we keep it in a low temperature environment, dissolve more astaxanthin under acidic conditions, and then encapsulate it to make algae microcapsules.

[0030] The composition and selection of wall materials are crucial to the performance of microcapsules, and are also the conditions for obtaining microcapsule products with high efficiency and superior performance. Chitosan and colloid compounding, combined with algae protein, work together to form a dense network structure on the molecular surface, and compound protein and hydrophilic colloids. Although algae protein has good emulsifying properties, it has poor mechanical properties. Hydrophilic colloids can improve the surface activity and viscosity of proteins and enhance the stability of wall materials. It was found that the microcapsules prepared by this method have good resistance to strong acid environments, ensuring that they are not destroyed in gastric juice.

[0031] Finally, a method for adding algae microcapsules to pig feed is provided, which can be directly added to the feed, and the amount of algae microcapsules in the feed is 1-3%. Algae microcapsules can promote animal digestion and absorption of feed, and have good safety, can promote animal growth and development, and improve immunity. It can increase the bioavailability of astaxanthin, have good absorption and utilization rate, improve the antioxidant capacity of cells, can greatly improve the colonization performance of astaxanthin in the feed in the intestine, and can give full play to the active ingredients of astaxanthin.

[0032] The beneficial effects of the invention are:

[0033] 1) The present invention uses Haematococcus pluvialis as the main raw material, not only extracts astaxanthin with strong stability and high activity, but also makes full use of the protein of Haematococcus pluvialis, prepares astaxanthin into algae astaxanthin nano solid particles, first uses Haematococcus pluvialis protein as a protective layer, and prepares self-emulsifying algae astaxanthin solid particles through an emulsion, and at the same time utilizes the active wrapping of a network cross-linked structure, and obtains an algae microcapsule doubly wrapped with the active wrapping of a network cross-linked structure and Haematococcus pluvialis protein through the positive and negative charge action with the protein solution, providing a high biological activity, no biological toxicity, and good stability algae astaxanthin microcapsule for the prior art. By adopting a specific material combination and ratio, the obtained algae astaxanthin microcapsule is not destroyed by the strong acid environment in gastric juice, and it is very easy to be absorbed into the lymphatic circulation through the intestinal mucosa, and then enters the blood circulation, so as to efficiently exert its biological efficacy, and shows significant advantages in terms of stability, intestinal absorption efficiency and usage. It not only improves the economic benefits, but also prevents astaxanthin from being oxidized or the structure from being 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 of the network cross-linked structure have good film-forming properties and high strength. The obtained algae microcapsules doubly wrapped by the active inclusions of the network cross-linked structure and the Haematococcus pluvialis protein are observed to be regular spherical under a scanning electron microscope with a smooth surface. The obtained algae microcapsules have high stability 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 form of the astaxanthin raw material is changed, and at the same time, astaxanthin is ensured not to be released in the pig's stomach, thereby preventing it from combining with digestive enzymes in the stomach to reduce 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 the active wrapping material of the mesh cross-linked structure and the double wrapping of the Haematococcus pluvialis protein, giving full play to the good film-forming ability and mucosal adhesion characteristics of the active substance of the coil cross-linked structure, which can be colonized on the surface of the small intestinal mucosa, can reach the intestinal part in the largest amount, and 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) After astaxanthin is microencapsulated, the influence of environmental factors such as light, heat, and oxygen on the stability of astaxanthin is avoided, thereby maintaining the functionality of astaxanthin and extending the storage period; the self-emulsifying algae astaxanthin solid particles and porous structure prepared by the present invention improve the solubility and dispersibility of astaxanthin in water, and have good dispersion stability; the results show that within 4 hours, more than 90% of astaxanthin is released from the solid self-emulsifying system. After digestion, the bioavailability reaches more than 68%. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0039] Example 1

[0040] An algae microcapsule comprises the following steps: firstly, algae astaxanthin nano solid particles are prepared, specifically, 100 parts of Haematococcus pluvialis are subjected to low-temperature drying treatment at 5°C for 30 minutes, and then taken out and crushed to break the wall; Haematococcus pluvialis powder is obtained; 50 parts of Haematococcus pluvialis powder are added to an organic solvent uniformly mixed with dichloromethane and methanol in a volume ratio of 1:1 at 20°C, and mixed with a magnetic stirrer for 150 minutes to fully dissolve the astaxanthin, the Haematococcus pluvialis residue and liquid are separated by centrifugation, the upper layer of liquid is taken, 5 parts of hexadecyl dimethyl ammonium chloride are added and mixed uniformly, astaxanthin and hexadecyl dimethyl ammonium chloride are reacted with each other, and solid flocs are generated after the temperature is lowered to 5°C; the solid and liquid are separated, and the lower layer of solid is washed and dried to obtain positively charged astaxanthin solid particles.

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

[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 to separate the solid and liquid, wash and dry the lower layer of solid, and obtain algae astaxanthin solid particles.

[0043] Furthermore, the active inclusions of the network cross-linked structure are specifically:

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

[0045] 10 parts of 4% gelatin aqueous solution were heated to 90°C, 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 peptide chains, and the obtained short-chain gelatin low-molecular peptide chain liquid was added to the liquid with 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 obtain a nano-scale emulsion.

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

[0048] Take 10 parts of self-emulsified algae astaxanthin solid particles and add them to 30 parts of active liquid with a positively charged network cross-linked structure, adjust the pH to about 4.0, slowly stir to evenly disperse the solid particles, then stop stirring, and let it react for 30 minutes; then slowly stir for 120 minutes, and then let it react for 30 minutes to obtain a uniformly dispersed microsphere mixture, filter the microsphere mixture, centrifuge and wash the obtained precipitate, and freeze-dry the sample obtained by centrifugation to obtain algae microcapsules, the water content of the algae microcapsules is 8%, and the average particle size is about 152 μm.

[0049] Example 2

[0050] An algae microcapsule, firstly preparing algae astaxanthin nano solid particles, specifically: 100 parts of Haematococcus pluvialis are subjected to low-temperature drying treatment at 5°C for 30 minutes, then taken out and crushed to break the wall; Haematococcus pluvialis powder is obtained; 50 parts of Haematococcus pluvialis powder are added to an organic solvent in which dichloromethane and methanol are evenly mixed in a volume ratio of 1:1 at 20°C, and mixed with a magnetic stirrer for 150 minutes to fully dissolve astaxanthin, and Haematococcus pluvialis residue and liquid are separated by centrifugation, and the upper layer of liquid is taken, and 5 parts of hexadecyl dimethyl ammonium chloride are added and mixed evenly to react astaxanthin and hexadecyl dimethyl ammonium chloride, and solid floccules are generated after the temperature is lowered to 5°C; the solid and liquid are separated, and the lower layer of solid is washed and dried to obtain positively charged astaxanthin solid particles;

[0051] 50 parts of Haematococcus pluvialis residue are enriched with Haematococcus pluvialis protein by using a polyethylene glycol / sodium phosphate aqueous two-phase system, and 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, the pH is adjusted to 5.3, and the mixture is allowed to stand for 30 minutes to coagulate, so that the protein in the protein liquid is negatively charged, and a mixed liquid containing coagulates is obtained; the solid-liquid separation is carried out by centrifugation, and the lower layer of solid is washed and dried to obtain negatively charged protein particles.

[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 to separate the solid and liquid, wash and dry the lower layer of solid, and obtain algae astaxanthin solid particles.

[0053] Furthermore, the active inclusions of the network cross-linked structure are specifically:

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

[0055] Heat 10 parts of 4% gelatin aqueous solution to 90°C, adjust the pH to 6.0, and continue to stir rapidly for 6 hours. The gelatin will continue to swell and break into short-chain gelatin low-molecular peptide chains. The obtained short-chain gelatin low-molecular peptide chain liquid is 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 is 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 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 are soaked in 20 parts of emulsion, so that the surface of the porous algae astaxanthin solid particles absorbs the emulsion, and after soaking for 100 minutes, the particles are filtered and dried at low temperature to obtain self-emulsified algae astaxanthin solid particles;

[0058] Take 10 parts of self-emulsified algae astaxanthin solid particles and add them to 40 parts of active liquid with a positively charged network cross-linked structure, adjust the pH to about 4.0, slowly stir to evenly disperse the solid particles, then stop stirring, and let it react for 30 minutes; then slowly stir for 10 minutes, and then let it react for 30 minutes to obtain a uniformly dispersed microsphere mixture, filter the microsphere mixture, centrifuge the obtained precipitate and wash it, freeze-dry the sample obtained by centrifugation to obtain algae microcapsules, the water content of the algae microcapsules is 8%, and the average particle size is about 172μm. The water content of the algae microcapsules is 8-10%, and the particle size is 150-200μm.

[0059] Example 3

[0060] An algae microcapsule, firstly preparing algae astaxanthin nano solid particles, specifically: 100 parts of Haematococcus pluvialis are subjected to low-temperature drying treatment at 5°C for 30 minutes, then taken out and crushed to break the wall; Haematococcus pluvialis powder is obtained; 50 parts of Haematococcus pluvialis powder are added to an organic solvent in which dichloromethane and methanol are evenly mixed in a volume ratio of 1:1 at 20°C, and mixed with a magnetic stirrer for 150 minutes to fully dissolve astaxanthin, and Haematococcus pluvialis residue and liquid are separated by centrifugation, and the upper layer of liquid is taken, and 5 parts of hexadecyl dimethyl ammonium chloride are added and mixed evenly to react astaxanthin and hexadecyl dimethyl ammonium chloride, and solid floccules are generated after the temperature is lowered to 5°C; the solid and liquid are separated, and the lower layer of solid is washed and dried to obtain positively charged astaxanthin solid particles;

[0061] 50 parts of Haematococcus pluvialis residue are enriched with Haematococcus pluvialis protein by using a polyethylene glycol / sodium phosphate aqueous two-phase system, and 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, the pH is adjusted to 5.5, and the mixture is allowed to stand for 30 minutes to coagulate, so that the protein in the protein liquid is negatively charged, and a mixed liquid containing coagulates is obtained; the solid-liquid separation is carried out by centrifugation, and the lower layer of solid is washed and dried to obtain negatively charged protein particles.

[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 to separate the solid and liquid, wash and dry the lower layer of solid, and obtain algae astaxanthin solid particles.

[0063] Furthermore, the active inclusions of the network cross-linked structure are specifically:

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

[0065] 8 parts of 4% gelatin aqueous solution were heated to 90°C, 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 peptide chains, and the obtained short-chain gelatin low-molecular peptide chain liquid was added to the liquid with 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 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 are soaked in 20 parts of emulsion, so that the surface of the porous algae astaxanthin solid particles absorbs the emulsion, and after soaking for 100 minutes, the particles are filtered and dried at low temperature to obtain self-emulsified algae astaxanthin solid particles;

[0068] Take 10 parts of self-emulsified algae astaxanthin solid particles and add them to 30 parts of active liquid with a positively charged network cross-linked structure, adjust the pH to about 4.0, slowly stir to evenly disperse the solid particles, then stop stirring, and let it react for 30 minutes; then slowly stir for 20 minutes, and let it react for another 30 minutes to obtain a uniformly dispersed microsphere mixture, filter the microsphere mixture, centrifuge and wash the obtained precipitate, and freeze-dry the sample obtained by centrifugation to obtain algae microcapsules, the water content of the algae microcapsules is 10%, and the average particle size is about 196 μm.

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

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

[0071] Comparative Example 3, the active wrapping material of the network cross-linked structure is replaced with an equal amount of corn starch wrapping material, and the step of soaking in the emulsion is omitted, and the rest is 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 content of astaxanthin was measured by high performance liquid chromatography, and the chromatographic conditions were as follows: the chromatographic column was a reverse C18 chromatographic column (150 mm*4.6 mm, 5 μm), isocratic elution; the mobile phase A was methanol, the mobile phase B was ethanol, and the mobile phase C was an aqueous solution (80+15+5, V / V / V); the detector was an ultraviolet UV detector: the detection wavelength was 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: Weigh 2.000g of samples of Examples 1 to 3 and Comparative Examples 1 to 4 respectively into dissolution cups, add 250mL of simulated porcine gastric juice to each, place in a dissolution apparatus, set the temperature to 37°C, and the speed to 75r / min. After reacting for 1 hour, 2 hours, and 4 hours, take 10mL of the upper reaction liquid, and measure the astaxanthin content 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 is plotted as follows: Figure 1 .from Figure 1 It can be seen that the stability of Example 1 in gastric juice is significantly improved compared with Comparative Examples 2-3.

[0078] As can be seen from Table 1: Examples 1 to 3 use active wrappers with a network cross-linked structure as the coating protective layer of the self-emulsified algae astaxanthin solid particles. After reacting in simulated pig gastric juice for 4 hours, the astaxanthin content is reduced by no more than 10%, indicating that the active wrappers with a network cross-linked structure are insoluble in strong acid and the shell is almost not destroyed. However, in Comparative Example 1, after being directly added to simulated pig gastric juice for 4 hours, the loss of astaxanthin in gastric juice reached 95%, indicating that astaxanthin is released in the pig stomach, and the protective effect of ordinary chitosan on algae microcapsules in gastric juice is weak, and the coating layer is almost completely destroyed. Comparative Example 4 uses corn starch as a wrapper, and the loss of astaxanthin in gastric juice also reaches 90%, so that astaxanthin is destroyed in large quantities in gastric juice, which affects the absorption effect of astaxanthin 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] Preparation of simulated porcine intestinal fluid: Take 13.6g potassium dihydrogen phosphate in a beaker, add 1000mL deionized water, stir to fully dissolve, adjust the pH value to 6.8 with 0.1mol / L NaOH solution. Take 2g porcine pancreatic lipase, mix well, and filter with a 0.2μm sterile filter to obtain simulated porcine intestinal fluid.

[0083] Experimental method: Weigh 2.000g of samples of Examples 1 to 3, Comparative Examples 1, 3 and 4 respectively into dissolution cups, add 250mL of simulated porcine intestinal fluid to each, place in a dissolution apparatus, set the temperature to 37°C, and the speed to 100r / min. After reacting for 1 hour, 2 hours and 4 hours respectively, take 10mL of the upper reaction liquid and detect the astaxanthin content therein. 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 astaxanthin solid microspheres in Examples 1 to 3 is an active package with a positively charged network cross-linked structure, which remains stable under the acidic conditions of the pig stomach and is destroyed under the neutral conditions of the pig small intestine. After reacting in the simulated pig small intestinal fluid for 4 hours, the release amount of astaxanthin is more than 90%, and almost all of it is released. It shows that the self-emulsified algae astaxanthin solid particles can better control the release of astaxanthin; the protective layer of Comparative Examples 1 and 3 is ordinary chitosan, whether it is under the strong acid conditions in the stomach or under the neutral conditions of the small intestine, astaxanthin is easily released, and it cannot be guaranteed to effectively reach the small intestine after passing through the stomach, thereby affecting the absorption of astaxanthin in the small intestine. The package in Comparative Example 4 is corn starch, whether it is under the strong acid conditions in the stomach or under the neutral conditions of the small intestine, it is very easy to release, so it can neither guarantee that it can effectively reach the small intestine after passing through the stomach, nor can it be effectively absorbed in the small intestine. Therefore, the algae microcapsules with double wrapping of the active wrapping material of the network cross-linked structure and the Haematococcus pluvialis protein prepared in the present invention can simultaneously ensure that the coating layer will not gradually absorb water, swell and disintegrate in the pig stomach, and can remain stable under strong acidic conditions, but be unstable and gradually destroyed under neutral conditions. In this way, the algae microcapsules containing astaxanthin can remain stable in the stomach, and gradually disintegrate and release astaxanthin after reaching the small intestine, thereby exerting the efficacy.

[0087] 2. Bioavailability Test

[0088] The microcapsule preparations of Examples 1-2, Comparative Examples 2 and Comparative Examples 4 were added to feed at 5% concentration, and 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 feed respectively. Blood was collected 0, 4, 8, 12, 15, 18, 24, 48 and 72 hours after feeding, and placed in a centrifuge tube containing EDTA. After standing at room temperature at 25-26°C for 30 minutes and layering, the blood was centrifuged at 4°C and 7500 rpm for 15 minutes, and the upper serum was carefully drawn 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 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-emulsifiable 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] 150 28-day-old weaned piglets from the same batch were selected and randomly divided into 5 groups, 30 in each group; 2% by mass was added to ordinary feed and the piglets were fed for 150 days; the weight before and after feeding, the average feed intake during feeding, the incidence of disease and the mortality rate were measured, and the test results are shown in Table 4 below; it can be obtained that compared with ordinary feed on the market, feeding ordinary piglets with feed added with algae microcapsules prepared by the present invention can promote the growth of raised animals and enhance the disease resistance of animals, significantly reduce breeding costs, and achieve significant economic benefits. Its meat quality is also significantly better than ordinary feed. This is because astaxanthin can improve the antioxidant capacity of the animal body, promote the integrity of the structure and function of jejunal cells, and improve the digestion capacity of the intestine for nutrients, thereby improving growth performance. Prevent unsaturated fatty acids and protein molecules in the muscle from being oxidized into peroxides, prevent lipid decomposition and polypeptide chain breakage, thereby significantly improving the flavor of the meat.

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

[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 rather than to limit it. Any equal modifications and changes made to the technical solution of the present invention by ordinary persons in the art, as long as they do not deviate from the overall concept of the present invention, are still within the scope 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 cross-linked structure and algae astaxanthin solid particles. The preparation of the algae astaxanthin nano solid particles comprises the following steps: S001, drying the Haematococcus pluvialis at a low temperature of 0 to 10° C. for 20 to 40 minutes, then taking out 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 astaxanthin, separating the Haematococcus pluvialis residue and the liquid by centrifugation, taking the upper layer of liquid, adding hexadecyl dimethyl ammonium chloride and mixing evenly, allowing astaxanthin and hexadecyl dimethyl ammonium chloride to react with each other, and generating solid floccules after cooling to 5-10° C.; separating the solid from the liquid, washing and drying the lower layer of solid, and obtaining positively charged astaxanthin solid particles; wherein the mass ratio of hexadecyl dimethyl ammonium chloride to Haematococcus pluvialis powder is 1:10; S003, enriching the Haematococcus pluvialis protein with the Haematococcus pluvialis residue separated in S002 by a polyethylene glycol / sodium phosphate aqueous two-phase system, and separating the Haematococcus pluvialis protein in the top phase to obtain a protein solution, wherein the isoelectric point of the protein solution is 4.2; adding gelatin to the protein solution, heating and stirring at 50-60° C. for 30-50 minutes until the solution is completely dispersed, adding citric acid, adjusting the pH to 5.0-5.5, and standing for 30-50 minutes to cause coagulation, so that the protein in the protein solution is negatively charged, and obtaining a mixed solution containing coagulation bodies; centrifuging for solid-liquid separation, washing and drying the lower layer of solid, and obtaining negatively charged protein particles; the mass ratio of the protein solution to the gelatin is 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 solids 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 positively charged astaxanthin solid particles to negatively charged protein particles is 1:2-4;.

2. The method for preparing algae microcapsules according to claim 1, characterized in that: The wrapping material is an active wrapping material with a network cross-linked structure, and the specific preparation process is as follows: S101, at a temperature of 50 to 60° C., chitosan is prepared into a chitosan solution with a concentration of 8 to 12% using a 0.01% citric acid aqueous solution; and the chitosan double helix molecular chain structure is hydrolyzed into a single-chain coil structure to obtain a liquid with a single-chain coil structure. S102, raising the temperature of 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 peptide chains, and the obtained short-chain gelatin low-molecular peptide chain liquid is added to the liquid of the single-chain coil structure in S101, and interacts with the single-chain coil structure to transform into an intertwined network cross-linked structure, and reacts for 150-200 minutes, the hydroxyl groups of the short-chain gelatin low-molecular peptide chains and the amino groups of chitosan react, and cross-linking points are added to 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, which is recorded as an active liquid with a network cross-linked structure with positive charges; Among them, the mass ratio of gelatin to chitosan is 1-2:3-5; S103, soaking with emulsion at 10-20° C. so that the surface of the porous algae astaxanthin solid particles absorbs the emulsion, and after soaking for 100-130 minutes, filtering and drying at low temperature to obtain algae astaxanthin solid particles with an emulsion layer on the surface, which are recorded 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 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, washing the obtained precipitate by centrifugation, and freeze-drying the sample obtained by centrifugation to obtain a network cross-linked structure active inclusion and astaxanthin solid microgels doubly coated with Haematococcus pluvialis protein, namely, algae microcapsules.

3. 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.

4. The method for preparing algae microcapsules according to claim 3, 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.

5. The method for preparing algae microcapsules according to claim 2, 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.

6. The method for preparing algae microcapsules according to claim 2, characterized in that: The water content of the algae microcapsules is 8-10%, and the particle size is 150-200 μm.

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

Images