Method for producing astaxanthin emulsion by phaffia rhodozyma

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for producing astaxanthin emulsion from phaffia rhodozyma, which specifically comprises the following steps of: performing superfine grinding on dried phaffia rhodozyma, extracting astaxanthin by using a solvent, dissolving the astaxanthin in soybean oil, taking whey protein as a water phase, preparing O / W emulsion, adding tannic acid to protect the astaxanthin from being influenced by enzymatic crosslinking, and preparing the astaxanthin emulsion by using the tannic acid. And a'multilayer 'interfacial film is formed through electrostatic assembly and enzymatic crosslinking. The chitosan-sodium alginate'multi-layer 'interfacial film improves the mechanical strength of the interfacial film, and limits the oxidizing agent from permeating into the internal oil phase to be in contact with the astaxanthin. Flocculation of the astaxanthin emulsion is inhibited through the charge neutralization effect of sodium alginate grafted polyethylene glycol sheltered salt ions, enzymatic crosslinking is conducted on chitosan grafted peptide fragments, and targeted release of the astaxanthin in the intestinal tract is achieved. The use amount of tyrosinase and the enzymatic reaction time are controlled, the residue of tyrosinase with oxidizing property is prevented, and tannic acid prevents the oxidizing substance generated by the reaction of tyrosinase from oxidizing astaxanthin.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically a method for producing astaxanthin emulsion using Rhodotorula rubra. Background Technology

[0002] Astaxanthin possesses excellent antioxidant properties and is widely used as a functional food ingredient, as well as in cosmetics, pharmaceuticals, and health supplements. However, the unsaturated conjugated double bond structure in the astaxanthin molecule leads to poor stability and limited solubility, severely restricting its wider application. To improve the solubility, stability, and bioavailability of astaxanthin, various delivery systems have been explored and applied, such as emulsions, liposomes, and nanoparticles. Among these, emulsion-based delivery systems offer an effective approach to improving the water solubility and bioavailability of hydrophobic bioactive compounds.

[0003] Emulsion delivery systems utilize emulsifiers to disperse the astaxanthin oil phase in the aqueous phase, forming oil droplets (such as O / W emulsions). This process is simple, low-cost, and easily scalable. However, such delivery systems have relatively poor physical stability, and long-term storage may lead to stratification and aggregation. Furthermore, the interfacial film formed by the emulsifier cannot completely block the migration of pro-oxidative factors such as oxygen and metal ions into the oil droplets, thus offering limited protection against astaxanthin oxidation. Therefore, it is necessary to construct "multi-layered" interfacial films to effectively block oxygen and free radicals. However, these "multi-layered" interfacial films are typically assembled through electrostatic adsorption. When such astaxanthin emulsions are added to functional beverages, the presence of salt ions (such as Na+) in these beverages can cause problems. + Cl - This will greatly weaken the electrostatic attraction between the protective layers protecting astaxanthin, ultimately leading to delamination, aggregation, or leakage of the protective structure, thus losing its protective and targeted properties. Summary of the Invention

[0004] (1) Technical problems to be solved The purpose of this invention is to provide a method for producing astaxanthin emulsions using Rhodotorula rubra, in order to solve the problem that astaxanthin emulsions with "multi-layer" interfacial membranes lose their protective and targeting properties against astaxanthin in the salt ion environment of functional beverages due to the electrostatically assembled "multi-layer" interfacial membranes.

[0005] (2) Technical solution To achieve the above objectives, the present invention provides a method for producing astaxanthin emulsion using Rhodotorula rubra, comprising the following steps: S1. Solvent extraction: The dried Rhodotorula rubra yeast was ultra-finely pulverized to 10~20μm, and the obtained powder was extracted with solvent. The supernatant was collected by centrifugation and rotary evaporation to obtain astaxanthin. Vitamin E and tert-butylhydroquinone were dissolved in soybean oil, and astaxanthin was added and stirred to dissolve to obtain astaxanthin soybean oil solution. S2. Primary emulsification: Dissolve whey protein and L-ascorbic acid in deionized water, slowly add astaxanthin soybean oil solution under high-speed shearing, continue shearing pre-homogenization, transfer to high-pressure homogenizer, and cycle homogenize to obtain O / W emulsion; S3. Oxidative protection: Dissolve tannic acid in deionized water to obtain a tannic acid solution. Slowly add the tannic acid solution dropwise to the O / W emulsion and continue stirring to obtain a tannic acid-O / W emulsion. S4. Electrostatic assembly: Tannic acid-O / W emulsion was added to peptide-chitosan solution, stirred continuously, fully adsorbed, centrifuged to collect the precipitate, and redispersed in deionized water to obtain chitosan-tannic acid-O / W emulsion. Slowly added polyethylene glycol-sodium alginate solution, stirred continuously, centrifuged to collect the precipitate, and redispersed in phosphate buffer to obtain multilayer polymer-coated O / W emulsion. S5. Enzymatic cross-linking: Tyrosinase is added to the O / W emulsion coated with a multilayer polymer, and the reaction is continuously stirred. The resulting product is heat-treated, and the precipitate is collected by centrifugation to obtain astaxanthin emulsion.

[0006] Furthermore, the solvent in S1 is one or more of dichloromethane, chloroform, methanol, ethyl acetate, and acetone; preferably acetone.

[0007] Furthermore, the whey protein solution is a neutral solution with a negative charge and a concentration of 1-1.5% (w / v); the peptide-chitosan solution is an acidic acetic acid solution with a positive charge and a concentration of 0.2-0.25% (w / v); and the polyethylene glycol-sodium alginate solution is a neutral solution with a negative charge and a concentration of 0.1-0.2% (w / v).

[0008] Furthermore, the volume ratio of astaxanthin soybean oil solution to whey protein solution in S2 is 1:9.

[0009] Furthermore, the mass ratio of tannic acid solution to O / W emulsion in S3 is 1~3:100.

[0010] Furthermore, the preparation method of the peptide-chitosan solution includes the following steps: S11. Add chitosan to MES buffer, add glacial acetic acid, stir to dissolve, and obtain chitosan solution; S12. Dissolve the peptide with the glycine-glycine-arginine-tyrosine sequence in MES buffer, add EDC·HCl and NHS, and activate at room temperature to obtain a peptide solution. S13. Add the peptide solution dropwise to the chitosan solution, stir the reaction, dialyze the resulting reaction solution for purification, freeze-dry to obtain peptide-chitosan; S14. Add the peptide-chitosan to a 0.5% (w / v) acetic acid solution, stir continuously to dissolve, and filter through an aqueous microporous membrane to obtain a peptide-chitosan solution.

[0011] Furthermore, the preparation method of the polyethylene glycol-sodium alginate solution includes the following steps: S21. Dissolve sodium alginate in MES buffer, add EDC·HCl and NHS, stir to activate, and obtain sodium alginate solution; S22. Dissolve mPEG-NH2 in MES buffer, add it to sodium alginate solution, stir to react, dialyze to purify the reaction solution, freeze dry to obtain polyethylene glycol-sodium alginate; S23. Dissolve polyethylene glycol-sodium alginate in deionized water and stir magnetically to obtain a polyethylene glycol-sodium alginate solution.

[0012] Furthermore, the amount of tyrosinase added in S5 is 50~75 U / mL.

[0013] By using 20μm ultrafine pulverization of Rhodopsinia rubra cell walls, and considering toxicity and isomerization rate, acetone was selected as the preferred solvent for astaxanthin extraction. Astaxanthin was dissolved in soybean oil as the oil phase, and a whey protein solution under neutral conditions was used as the aqueous phase. The hydrophobic segments of whey protein were anchored in the oil phase, while the hydrophilic segments extended into the aqueous phase, forming an interfacial protein film. Due to the steric hindrance of the protein chains and the electrostatic repulsion generated by the negative charge of the whey protein film under neutral conditions, the flocculation of the O / W emulsion could be effectively inhibited.

[0014] However, whey protein has poor physical stability, and the single-layer interfacial film it forms cannot completely block the migration of pro-oxidative factors such as oxygen and metal ions into the oil droplets, thus offering limited protection against astaxanthin oxidation. Therefore, electrostatic assembly is used to construct a "multi-layer" interfacial film to effectively block oxygen and free radicals. This is because chitosan, in acetic acid solution, undergoes protonation from -NH2 to -NH3. +The chitosan, becoming positively charged, is added to the negatively charged O / W emulsion. Through electrostatic attraction, the chitosan is uniformly coated on the surface of the O / W emulsion. Furthermore, the sodium alginate solution, under neutral conditions, exhibits strong negative charge and is uniformly coated on the outside of the chitosan layer through electrostatic attraction. This results in uniformly sized droplets formed through electrostatic layer-by-layer assembly, ensuring that each O / W emulsion is completely encapsulated. The chitosan-sodium alginate combination not only enhances the mechanical strength of the interfacial film, effectively resisting droplet aggregation and gravitational separation, thus significantly extending the product's shelf life, but also constitutes a strong physical barrier, greatly limiting the penetration of oxygen, free radicals, and pro-oxidants (such as metal ions) into the internal oil phase and their contact with astaxanthin.

[0015] Functional beverages commonly contain added salt ions (such as sodium). + Cl - ), Na + Cl - These substances will aggregate around the negatively charged and positively charged sodium alginate and chitosan, respectively, forming an "ionic atmosphere" that neutralizes their net charge. This significantly weakens the electrostatic attraction between chitosan and alginate, making the interfacial film loose, porous, uneven in thickness, and with poor mechanical strength, potentially even leading to structural disintegration and astaxanthin leakage and oxidation. Therefore, grafting polyethylene glycol onto sodium alginate is beneficial. Polyethylene glycol, a strongly hydrophilic chain segment, can form a thick hydration layer on the emulsion surface and enhance steric hindrance. This not only effectively shields against the charge neutralization effect of salt ions but also inhibits the flocculation of astaxanthin emulsions in beverages. Chitosan, modified with a glycine-glycine-arginine-tyrosine peptide sequence, is then cross-linked with tyrosinase using tyrosine as a substrate. The enzyme specifically "bridges" between the phenolic groups of the polymer chain, forming a covalently cross-linked network around the original electrostatic layer. This network strongly resists salt ion interference and has better mechanical strength, effectively preventing droplet aggregation. Meanwhile, in the gastric environment, sodium alginate is protonated, weakening the outer barrier, but the cross-linked network remains basically stable. In the intestine, trypsin specifically cleaves peptides, the cross-linked network disintegrates, sodium alginate is completely dissociated, electrostatic repulsion is enhanced, chitosan is deprotonated, the network relaxes, the chitosan-sodium alginate protective layer fails, bile salts and lipases emulsify the oil phase, and finally astaxanthin is released and absorbed in the intestine, significantly improving the bioavailability of astaxanthin.

[0016] Because tyrosinase is a redox enzyme, it generates excess reactive oxygen species and highly reactive intermediates during the reaction, which may oxidize and degrade astaxanthin, leading to a decrease in its content and loss of activity. Therefore, during the enzymatic cross-linking stage, it is necessary to control the amount of tyrosinase and the reaction time, and after the reaction, heat treatment is required to inactivate the enzyme, thereby avoiding tyrosinase residue in the astaxanthin emulsion. Simultaneously, to further prevent tyrosinase from affecting astaxanthin, tannic acid is added dropwise at the oil / water phase interface after the O / W emulsion is prepared to form a dense protective shell as an additional physical barrier. Due to its large molecular weight, tyrosinase cannot penetrate the protective shell, and the phenolic hydroxyl groups abundant in tannic acid molecules allow highly reactive intermediates generated by the enzymatic reaction to preferentially attack tannic acid, thus protecting astaxanthin from attack. However, the amount of tannic acid added needs to be controlled. If too much tannic acid is added, because tannic acid molecules contain a large number of phenolic hydroxyl groups, it can be strongly adsorbed on the interface of whey protein-coated oil droplets, occupying the adsorption sites originally reserved for subsequent positively charged chitosan. Furthermore, tannic acid itself is negatively charged. If excessive adsorption occurs, it will further enhance the negative charge of the O / W emulsion interface, or form a spatial and charge barrier, hindering the approach and adsorption of chitosan, resulting in the failure of the first layer assembly or poor results.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. By constructing a "multilayer" interfacial membrane of chitosan-sodium alginate through electrostatic assembly, not only is the mechanical strength of the interfacial membrane improved, which can effectively resist the aggregation and gravitational separation between droplets and greatly extend the shelf life of the product, but it also constitutes a strong physical barrier that can greatly limit the penetration of oxygen, free radicals and pro-oxidants (such as metal ions) into the internal oil phase and their contact with astaxanthin.

[0018] 2. To prevent salt ions in functional beverages from weakening the electrostatic attraction between chitosan and sodium alginate, sodium alginate grafted with polyethylene glycol effectively shields the charge neutralization effect of salt ions and inhibits the flocculation of astaxanthin emulsion in beverages. At the same time, chitosan grafted with peptides undergoes enzymatic cross-linking, strongly resisting salt ion interference. The cross-linked network has better mechanical strength, effectively preventing droplet aggregation. Furthermore, the cross-linked network remains stable in the acidic environment of the stomach. In the intestines, the peptides are degraded by trypsin and disintegrated, achieving targeted release of astaxanthin in the intestines, thereby improving its bioavailability.

[0019] 3. By controlling the amount of tyrosinase and the enzyme-catalyzed reaction time, and by performing heat treatment after the reaction is completed to inactivate the enzyme, the residual oxidative tyrosinase is prevented. In addition, tannic acid is added to the O / W emulsion to prevent the oxidative substances produced by the tyrosinase reaction from oxidizing astaxanthin. Attached Figure Description

[0020] Figure 1 This is a flowchart of the preparation process of astaxanthin emulsion in Example 1 of the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: This example discloses a method for producing astaxanthin emulsion using Rhodotorula rubra, comprising the following steps: S1. Solvent extraction: The dried Rhodopseudomonas erythropoiesis was ultra-finely pulverized to 10-20 μm. The powder was added to acetone at a material-to-liquid ratio of 1:10 (w / v) for extraction. The supernatant was collected by centrifugation. The extraction was repeated 3 times. The supernatants were combined and evaporated by rotary evaporation to obtain astaxanthin. 0.05 g of vitamin E and 0.01 g of tert-butylhydroquinone were dissolved in 10 g of soybean oil. 10 mg of astaxanthin was added and stirred to dissolve to obtain an astaxanthin soybean oil solution. S2. Primary emulsification: Dissolve 1.35g whey protein and 0.02g L-ascorbic acid in deionized water, bring the volume to 90g, and slowly add astaxanthin soybean oil solution under high-speed shearing. Continue shearing for 2 minutes for pre-homogenization, then transfer to a high-pressure homogenizer and cycle homogenize 3 times at 100MPa to obtain O / W emulsion. S3. Oxidative protection: Dissolve 0.01g tannic acid in 10mL deionized water to obtain a tannic acid solution. Slowly add 1mL of the tannic acid solution to 100g O / W emulsion and continue stirring for 30min to obtain a tannic acid-O / W emulsion. S4. Electrostatic assembly: 100g of tannic acid-O / W emulsion was slowly added dropwise to 100mL of peptide-chitosan solution and stirred continuously for 30min to allow for full adsorption. The precipitate was collected by centrifugation and redispersed in 100mL of deionized water to obtain chitosan-tannic acid-O / W emulsion. The emulsion was then slowly added to 100mL of polyethylene glycol-sodium alginate solution and stirred continuously for 30min. The precipitate was collected by centrifugation and redispersed in 100mL of phosphate buffer to obtain multilayer polymer-coated O / W emulsion. S5. Enzymatic cross-linking: Tyrosinase was added to the O / W emulsion coated with multilayer polymer, and the reaction was carried out by stirring at 25°C for 2 hours. The product was then heat-treated in a water bath at 80°C for 10 minutes, and the precipitate was collected by centrifugation to obtain astaxanthin emulsion.

[0023] It should be noted that, as Figure 1The diagram shows the preparation process of the astaxanthin emulsion of this invention. Astaxanthin is extracted from dried Rhodopseudomonas erythropoiesis-1,000 yeast using acetone as a solvent after ultrafine pulverization. The extracted astaxanthin is then dissolved in soybean oil and mixed with whey protein to prepare an O / W emulsion. Vitamin E, tert-butylhydroquinone, and L-ascorbic acid are added as antioxidants to ensure that the astaxanthin is not oxidized and degraded during the preparation process. To avoid the tyrosinase used in the peptide-chitosan enzymatic cross-linking affecting the astaxanthin activity, a tannic acid solution is added to the O / W emulsion to form a dense protective shell, preventing the penetration of tyrosinase and oxidizing substances. The resulting tannic acid-O / W emulsion is then assembled layer by layer with peptide-chitosan and polyethylene glycol-sodium alginate through electrostatic interactions. Finally, enzymatic cross-linking is used to form a cross-linked network, yielding the astaxanthin emulsion.

[0024] The solvent in S1 is one or more of dichloromethane, chloroform, methanol, ethyl acetate, and acetone; preferably acetone.

[0025] The whey protein solution is a neutral solution with a negative charge and a concentration of 1.5% (w / v); the peptide-chitosan solution is an acidic acetic acid solution with a positive charge and a concentration of 0.25% (w / v); and the polyethylene glycol-sodium alginate solution is a neutral solution with a negative charge and a concentration of 0.2% (w / v).

[0026] The volume ratio of astaxanthin soybean oil solution to whey protein solution in S2 is 1:9.

[0027] The mass ratio of tannic acid solution to O / W emulsion in S3 is 1:100.

[0028] The preparation method of the peptide-chitosan solution includes the following steps: S11. Add chitosan to MES buffer, add glacial acetic acid, stir to dissolve, and obtain chitosan solution; S12. Dissolve the peptide with the glycine-glycine-arginine-tyrosine sequence in MES buffer, add EDC·HCl and NHS, and activate at room temperature to obtain a peptide solution. S13. Add the peptide solution dropwise to the chitosan solution, stir the reaction, dialyze the resulting reaction solution for purification, freeze-dry to obtain peptide-chitosan; S14. Add the peptide-chitosan to a 0.5% (w / v) acetic acid solution, stir continuously to dissolve, and filter through an aqueous microporous membrane to obtain a peptide-chitosan solution.

[0029] The preparation method of the polyethylene glycol-sodium alginate solution includes the following steps: S21. Dissolve sodium alginate in MES buffer, add EDC·HCl and NHS, stir to activate, and obtain sodium alginate solution; S22. Dissolve mPEG-NH2 in MES buffer, add it to sodium alginate solution, stir to react, dialyze to purify the reaction solution, freeze dry to obtain polyethylene glycol-sodium alginate; S23. Dissolve polyethylene glycol-sodium alginate in deionized water and stir magnetically to obtain a polyethylene glycol-sodium alginate solution.

[0030] The amount of tyrosinase added in S5 is 50 U / mL.

[0031] Examples 2-5 and Comparative Examples 1-6 are based on Example 1, but differ from Example 1 in that the electrostatic assembly, oxidative protection, and enzymatic cross-linking effects are different in a method for producing astaxanthin emulsion with Rhodotorula rubra. The differences are shown in Table 1.

[0032]

[0033] As shown in Table 1, the concentrations of whey protein solution, peptide-chitosan solution, and polyethylene glycol-sodium alginate solution, the mass ratio of tannic acid solution to O / W emulsion, the amount of tyrosinase added, and the enzymatic cross-linking reaction time all affect the effectiveness of electrostatic assembly, oxidative protection, and enzymatic cross-linking. If the mass ratio of tannic acid solution to O / W emulsion is too high, electrostatic assembly will fail because tannic acid will occupy the binding sites of peptide-chitosan solution on whey protein. If it is too low, it will not protect astaxanthin from oxidation by tyrosinase. Simultaneously, electrostatic assembly, oxidative protection, and enzymatic cross-linking are all indispensable. Electrostatic assembly constructs a multi-layered interfacial membrane, improving its mechanical strength. Enzymatic cross-linking improves the stability of astaxanthin emulsion in salt-containing ionic solutions and enables precise and efficient release of astaxanthin in the intestine through peptide cleavage. Adding tannic acid during the oxidative protection stage prevents the enzymatic cross-linking stage from affecting the astaxanthin emulsion. At the same time, the amount of tannic acid needs to be precisely controlled to prevent it from affecting the electrostatic assembly effect.

[0034] Comparative Example 7: This comparative example is based on Example 1, but differs from Example 1 in that the astaxanthin emulsion described in this comparative example is not electrostatically assembled.

[0035] A method for producing astaxanthin emulsion using *Pseudomonas aeruginosa* includes the following steps: S1. Solvent extraction: The dried Rhodopseudomonas erythropoiesis was ultra-finely pulverized to 10-20 μm. The powder was added to acetone at a material-to-liquid ratio of 1:10 (w / v) for extraction. The supernatant was collected by centrifugation. The extraction was repeated 3 times. The supernatants were combined and evaporated by rotary evaporation to obtain astaxanthin. 0.05 g of vitamin E and 0.01 g of tert-butylhydroquinone were dissolved in 10 g of soybean oil. 10 mg of astaxanthin was added and stirred to dissolve to obtain an astaxanthin soybean oil solution. S2. Primary emulsification: Dissolve 1.35g whey protein and 0.02g L-ascorbic acid in deionized water, bring the volume to 90g, and slowly add astaxanthin soybean oil solution under high-speed shearing. Continue shearing for 2 minutes for pre-homogenization, then transfer to a high-pressure homogenizer and cycle homogenize 3 times at 100MPa to obtain O / W emulsion. S3. Oxidative protection: Dissolve 0.01g tannic acid in 10mL deionized water to obtain a tannic acid solution. Slowly add 1mL of the tannic acid solution to 100g O / W emulsion and continue stirring for 30min to obtain a tannic acid-O / W emulsion. S4. Enzymatic cross-linking: Mix 100g tannic acid-O / W emulsion, 100mL peptide-chitosan solution, and 100mL polyethylene glycol-sodium alginate solution, stir continuously for 30min, add tyrosinase, and stir continuously at 25℃ for 2 hours. The resulting product is heat-treated in a water bath at 80℃ for 10min, and the precipitate is collected by centrifugation to obtain astaxanthin emulsion.

[0036] The other components and preparation methods are the same as in Example 1.

[0037] It should be noted that when tannic acid-O / W emulsion, peptide-chitosan solution and polyethylene glycol-sodium alginate solution are directly blended without layer-by-layer electrostatic assembly, the enzymatic cross-linking forms disordered gel blocks or precipitates instead of regular, oriented films. This results in poor encapsulation efficiency and stability of the final emulsion.

[0038] Comparative Example 8: This comparative example is based on Example 1, but differs from Example 1 in that the peptide-chitosan solution described in this comparative example is not grafted with peptides.

[0039] The other components and preparation methods are the same as in Example 1.

[0040] Comparative Example 9: This comparative example is based on Example 1, but differs from Example 1 in that the polyethylene glycol-sodium alginate solution in this comparative example is not grafted with polyethylene glycol.

[0041] The other components and preparation methods are the same as in Example 1.

[0042] Experimental verification: Experiment 1: (1) Zeta potential change: In the preparation of astaxanthin, samples of tannic acid-O / W emulsion, chitosan-tannic acid-O / W emulsion, and multilayer polymer-coated O / W emulsion were taken and diluted with deionized water to measure the Zeta potential. The Zeta potential was tested to see if it conformed to the change law of "negative → positive → negative". If it did, it indicated that the electrostatic assembly was successful.

[0043] (2) Physical stability: The astaxanthin emulsions prepared in Examples 1-5 and Comparative Examples 1-7 were stored at 4°C for 30 days, and the presence of layering, oil separation, and flocculation was observed.

[0044] (3) Chemical stability: The astaxanthin emulsions prepared in Examples 1-5 and Comparative Examples 1-7 were stored at 40°C under accelerated oxidation conditions for 30 days. The emulsions were broken up, astaxanthin was extracted, and its content was detected by HPLC. The retention rate of astaxanthin was calculated.

[0045]

[0046] Table 2 shows the Zeta potential changes, physical stability, and chemical stability of the astaxanthin emulsion. The data shows that the Zeta potential exhibits a "negative→positive→negative" change pattern, indicating successful electrostatic assembly, and the corresponding astaxanthin emulsion shows good physical stability. Comparing Example 1 with Comparative Examples 4-6, it can be seen that chemical stability is affected not only by electrostatic assembly but also by the mass of tannic acid added during the oxidative protection stage, the amount of tyrosinase added during the enzymatic cross-linking stage, and the reaction time.

[0047] Experiment 2: (1) Salt ion stability test: Prepare a phosphate buffer containing NaCl (15mM) and sucrose to obtain a simulated beverage matrix. Mix the astaxanthin emulsion prepared in each example and comparative example with the simulated beverage matrix at a ratio of 1:9 and store at 25°C for 14 days. Take samples on the 1st and 14th days to detect the average particle size (Dz) and calculate the particle size growth.

[0048] (2) In vitro intestinal release rate: The astaxanthin emulsions prepared in each example and the comparative example were incubated in simulated gastric juice (containing pepsin, pH 2.0) for 2 hours, and the gastric digests were transferred into simulated intestinal juice (containing trypsin and bile salts, pH 7.0) for 4 hours. The release rate of astaxanthin was then detected.

[0049]

[0050] Table 3 shows the stability and intestinal release rate of astaxanthin emulsion in salt ions. The data indicates that larger particle sizes result in weaker anti-flocculation and anti-aggregation capabilities in salt ion solutions, making flocculation and precipitation more likely. Furthermore, single electrostatic assembly or enzymatic cross-linking offers limited protection for astaxanthin. Electrostatic assembly alone significantly weakens the electrostatic attraction between chitosan and sodium alginate in a salt ion environment. Enzymatic cross-linking, due to the direct mixing of peptide-chitosan and polyethylene glycol-sodium alginate, forms disordered gel fragments or precipitates rather than a well-ordered, oriented film. This leads to poor encapsulation efficiency and stability of the final emulsion, ultimately preventing precise and efficient release into the intestines.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for producing astaxanthin emulsion using *Pseudomonas aeruginosa*, characterized in that, Includes the following steps: S1. Solvent extraction: The dried Rhodotorula rubra yeast was ultra-finely pulverized to 10~20μm, and the obtained powder was extracted with solvent. The supernatant was collected by centrifugation and rotary evaporation to obtain astaxanthin. Vitamin E and tert-butylhydroquinone were dissolved in soybean oil, and astaxanthin was added and stirred to dissolve to obtain astaxanthin soybean oil solution. S2. Primary emulsification: Dissolve whey protein and L-ascorbic acid in deionized water, slowly add astaxanthin soybean oil solution under high-speed shearing, continue shearing pre-homogenization, transfer to high-pressure homogenizer, and cycle homogenize to obtain O / W emulsion; S3. Oxidative protection: Dissolve tannic acid in deionized water to obtain a tannic acid solution. Slowly add the tannic acid solution dropwise to the O / W emulsion and continue stirring to obtain a tannic acid-O / W emulsion. S4. Electrostatic assembly: Tannic acid-O / W emulsion was added to peptide-chitosan solution, stirred continuously, fully adsorbed, centrifuged to collect the precipitate, and redispersed in deionized water to obtain chitosan-tannic acid-O / W emulsion. Polyethylene glycol-sodium alginate solution was slowly added, stirred continuously, centrifuged to collect the precipitate, and redispersed in phosphate buffer to obtain multilayer polymer-coated O / W emulsion. S5. Enzymatic cross-linking: Tyrosinase is added to the O / W emulsion coated with multilayer polymer, and the reaction is continuously stirred for 1-2 hours. The resulting product is heat-treated, and the precipitate is collected by centrifugation to obtain astaxanthin emulsion.

2. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The solvent in S1 is one or more of dichloromethane, chloroform, methanol, ethyl acetate, and acetone.

3. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The whey protein solution is a neutral solution with a negative charge and a concentration of 1-1.5% (w / v); the peptide-chitosan solution is an acidic acetic acid solution with a positive charge and a concentration of 0.2-0.25% (w / v); the polyethylene glycol-sodium alginate solution is a neutral solution with a negative charge and a concentration of 0.1-0.2% (w / v).

4. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The volume ratio of astaxanthin soybean oil solution to whey protein solution in S2 is 1:

9.

5. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The mass ratio of tannic acid solution to O / W emulsion in S3 is 1~3:

100.

6. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The preparation method of the peptide-chitosan solution includes the following steps: S11. Add chitosan to MES buffer, add glacial acetic acid, stir to dissolve, and obtain chitosan solution; S12. Dissolve the peptide with the glycine-glycine-arginine-tyrosine sequence in MES buffer, add EDC·HCl and NHS, and activate at room temperature to obtain a peptide solution. S13. Add the peptide solution dropwise to the chitosan solution, stir the reaction, dialyze the resulting reaction solution for purification, freeze-dry to obtain peptide-chitosan; S14. Add the peptide-chitosan to a 0.5% (w / v) acetic acid solution, stir continuously to dissolve, and filter through an aqueous microporous membrane to obtain a peptide-chitosan solution.

7. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The preparation method of the polyethylene glycol-sodium alginate solution includes the following steps: S21. Dissolve sodium alginate in MES buffer, add EDC·HCl and NHS, stir to activate, and obtain sodium alginate solution; S22. Dissolve mPEG-NH2 in MES buffer, add it to sodium alginate solution, stir to react, dialyze to purify the reaction solution, freeze dry to obtain polyethylene glycol-sodium alginate; S23. Dissolve polyethylene glycol-sodium alginate in deionized water and stir magnetically to obtain a polyethylene glycol-sodium alginate solution.

8. The method for producing astaxanthin emulsion from *Pharbitis repens* according to claim 1, characterized in that, The amount of tyrosinase added in S5 is 50~75U / mL.

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