Method for producing astaxanthin emulsion by rhodotorula

Abstract

The present application relates to the field of microbial technology, and particularly to a method for producing astaxanthin emulsion by Phaffia rhodozyma, which comprises the following steps: super-micro grinding dry Phaffia rhodozyma to extract astaxanthin with a solvent, dissolving the astaxanthin in soybean oil, using whey protein as the water phase, preparing O / W emulsion, adding tannic acid to protect astaxanthin from enzymatic cross-linking, and forming a "multi-layer" interface film through electrostatic assembly and enzymatic cross-linking. The "multi-layer" interface film of chitosan-sodium alginate improves the mechanical strength of the interface film and limits the penetration of oxidants into the internal oil phase to contact astaxanthin. The charge neutralization of sodium alginate grafted with polyethylene glycol prevents the flocculation of astaxanthin emulsion, and the enzymatic cross-linking of chitosan grafted with peptide segments realizes the targeted release of astaxanthin in the intestinal tract. The dosage of tyrosinase and the enzymatic reaction time are controlled to prevent the residual of tyrosinase with oxidizing property, and tannic acid prevents the oxidation of astaxanthin by oxidizing substances generated in the tyrosinase reaction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and specifically relates to a method for producing astaxanthin emulsion by Phaffia rhodozyma. BACKGROUND

[0002] Astaxanthin has excellent antioxidant properties and is widely used in functional food ingredients, and also has many applications in the fields of cosmetics, drugs and health products. However, astaxanthin molecules have unsaturated conjugated double bond structures, poor stability and limited solubility, which seriously restricts the more extensive application of astaxanthin. In order to improve the solubility, stability and bioavailability of astaxanthin, various delivery systems such as emulsion, liposome and nanoparticle have been explored and applied. Among them, the emulsion-based delivery system provides an effective way to improve the water-solubility and bioavailability of hydrophobic bioactive compounds.

[0003] The emulsion delivery system is to disperse astaxanthin oil phase in water phase by using emulsifier to form oil droplets (such as O / W emulsion), which has simple process, low cost and easy to scale production. However, the physical stability of such delivery system is relatively poor, and delamination and coalescence may occur during long-term storage. Moreover, the interface film formed by emulsifier cannot completely block the migration of oxygen, metal ions and other pro-oxidation factors into the oil droplets, and the improvement of astaxanthin oxidation protection is limited. Therefore, it is necessary to construct a "multi-layer" interface film to effectively block oxygen and free radicals. However, the "multi-layer" interface film is usually assembled by electrostatic adsorption, and when such astaxanthin emulsion is added to functional beverages, the salt ions (such as Na + , Cl - ) in functional beverages will greatly weaken the electrostatic attraction between the protective layers of astaxanthin, resulting in delamination, coalescence of the protective structure or leakage of astaxanthin, thereby losing its protective and targeting properties. SUMMARY

[0004] (1) Technical problems to be solved

[0005] The present application aims to provide a method for producing astaxanthin emulsion by Phaffia rhodozyma, to solve the problem that the "multi-layer" interface film of astaxanthin emulsion loses its protective and targeting properties in the salt ion environment of functional beverages through the electrostatic assembly of "multi-layer" interface film.

[0006] (2) Technical solutions

[0007] To achieve the above-mentioned purpose, on the one hand, the present application provides a method for producing astaxanthin emulsion by Phaffia rhodozyma, comprising the following steps:

[0008] S1. Solvent extraction: the dry yeast of Phaffia rhodozyma is ultra-finely ground to 10-20 μm, the obtained powder is added to a solvent for extraction, the supernatant is collected by centrifugation, and astaxanthin is obtained by rotary evaporation; vitamin E and tert-butyl hydroquinone are dissolved in soybean oil, astaxanthin is added, and stirring and dissolution are performed to obtain an astaxanthin soybean oil solution;

[0009] S2. Primary emulsification: whey protein and L-ascorbic acid are dissolved in deionized water, the astaxanthin soybean oil solution is slowly added under high-speed shearing, and pre-homogenization is continuously performed under shearing; the solution is transferred into a high-pressure homogenizer, and cycle homogenization is performed to obtain an O / W emulsion;

[0010] S3. Oxidation protection: tannic acid is dissolved in deionized water to obtain a tannic acid solution, and the tannic acid solution is slowly added to the O / W emulsion while stirring to obtain a tannic acid-O / W emulsion;

[0011] S4. Electrostatic assembly: the tannic acid-O / W emulsion is added to a peptide-chitosan solution, stirring is continuously performed, and adsorption is fully performed; the precipitate is collected by centrifugation, and is redispersed in deionized water to obtain a chitosan-tannic acid-O / W emulsion; the chitosan-tannic acid-O / W emulsion is slowly added to a polyethylene glycol-sodium alginate solution, stirring is continuously performed, and the precipitate is collected by centrifugation and redispersed in a phosphate buffer to obtain a multi-layer polymer-coated O / W emulsion;

[0012] S5. Enzymatic crosslinking: tyrosinase is added to the multi-layer polymer-coated O / W emulsion, stirring is continuously performed, and the obtained product is heat-treated to obtain an astaxanthin emulsion.

[0013] Further, the solvent in S1 is one or more of dichloromethane, chloroform, methanol, ethyl acetate, and acetone; preferably, the solvent is acetone.

[0014] Further, the whey protein solution is a neutral solution with negative electricity, and the concentration is 1-1.5% (w / v); the peptide-chitosan solution is an acidic acetic acid solution with positive electricity, and the concentration is 0.2-0.25% (w / v); and the polyethylene glycol-sodium alginate solution is a neutral solution with negative electricity, and the concentration is 0.1-0.2% (w / v).

[0015] Further, the volume ratio of the astaxanthin soybean oil solution to the whey protein solution in S2 is 1:9.

[0016] Further, the mass ratio of the tannic acid solution to the O / W emulsion in S3 is 1-3:100.

[0017] Further, the preparation method of the peptide-chitosan solution comprises the following steps:

[0018] S11. Chitosan is added to a MES buffer solution, glacial acetic acid is added, and stirring and dissolution are performed to obtain a chitosan solution;

[0019] S12. Dissolving the peptide segment of glycine-glycine-arginine-tyrosine sequence in MES buffer solution, adding EDC·HCl and NHS, activating at room temperature to obtain a peptide segment solution;

[0020] S13. Dropping the peptide segment solution into the chitosan solution, stirring to react, purifying the obtained reaction solution by dialysis, and freeze-drying to obtain a peptide segment-chitosan;

[0021] S14. Adding the peptide segment-chitosan into a 0.5% (w / v) acetic acid solution, continuously stirring to dissolve, and filtering through a water-based microporous filter to obtain a peptide segment-chitosan solution.

[0022] Further, the preparation method of the polyethylene glycol-sodium alginate solution comprises the following steps:

[0023] S21. Dissolving sodium alginate in MES buffer solution, adding EDC·HCl and NHS, and stirring to activate to obtain a sodium alginate solution;

[0024] S22. Dissolving mPEG-NH2 in MES buffer solution, adding into the sodium alginate solution, stirring to react, purifying the obtained reaction solution by dialysis, and freeze-drying to obtain polyethylene glycol-sodium alginate;

[0025] S23. Dissolving the polyethylene glycol-sodium alginate in deionized water, magnetically stirring to dissolve to obtain a polyethylene glycol-sodium alginate solution.

[0026] Further, the amount of tyrosinase added in S5 is 50-75 U / mL.

[0027] By 20 μm ultrafine grinding of the red yeast cell wall, considering toxicity and isomerization rate, acetone is preferably used as a solvent to extract astaxanthin, which is dissolved in soybean oil as an oil phase, and whey protein solution in a neutral environment is used as an aqueous phase, the hydrophobic segment of whey protein is anchored in the oil phase, and the hydrophilic segment extends to the aqueous phase to form an interfacial protein film. Due to the steric hindrance of protein chains and the electrostatic repulsion caused by the negative charge of the whey protein film in a neutral environment, the flocculation of O / W emulsion can be effectively inhibited.

[0028] However, the physical stability of whey protein is poor, the single-layer interfacial film formed cannot completely block the migration of oxygen, metal ions and other pro-oxidation factors to the inside of the oil droplets, and the improvement of astaxanthin oxidation protection is limited. Therefore, electrostatic assembly is adopted to construct a "multi-layer" interfacial film to effectively block oxygen and free radicals. Since chitosan is protonated to -NH3 +, and then added to the negatively charged O / W emulsion, through electrostatic attraction, so that the chitosan is uniformly wrapped on the surface of the O / W emulsion, and the sodium alginate solution is added under neutral conditions, and the negative charge is strong, and through electrostatic attraction, it is uniformly wrapped outside the chitosan layer, so that the emulsion droplets formed by the electrostatic layer-by-layer assembly are uniform in size, and each O / W emulsion is completely wrapped, and the chitosan-sodium alginate not only improves the mechanical strength of the interface film, can effectively resist the aggregation between the droplets and the gravity separation, greatly prolongs the shelf life of the product, but also forms a strong physical barrier, which can greatly limit the penetration of oxygen, free radicals and pro-oxidants (such as metal ions) into the internal oil phase and contact with astaxanthin.

[0029] Some salt ions (such as Na + , Cl - ) will be added to the functional beverage + , Cl - will gather around the negatively charged and positively charged sodium alginate and chitosan respectively, forming an "ion atmosphere", neutralizing the net charge it carries, greatly weakening the electrostatic attraction between chitosan and alginate, making the interface film loose, porous, uneven in thickness and poor in mechanical strength, and even causing structural disintegration, astaxanthin leakage and oxidation failure. Therefore, polyethylene glycol is grafted onto sodium alginate. Polyethylene glycol is a strong hydrophilic segment that can form a thick and solid hydration layer on the surface of the emulsion and enhance steric hindrance, not only effectively shielding the charge neutralization effect of salt ions, but also inhibiting the flocculation of astaxanthin emulsion in beverages. Chitosan is modified by glycine-glycine-arginine-tyrosine sequence peptide, and tyrosinase is added to crosslink the tyrosine as a substrate. The enzyme will specifically "bridge" between the phenolic groups of the polymer chain, forming a covalently crosslinked network around the original electrostatic layer, strongly resisting salt ion interference, and the crosslinked network has better mechanical strength, which can effectively prevent droplet coalescence. At the same time, in the stomach environment, sodium alginate is protonated, the outer barrier is weakened, but the crosslinked network remains stable, in the intestine, trypsin specifically cuts the peptide segment, the crosslinked network disintegrates, sodium alginate completely dissociates, electrostatic repulsion is enhanced, chitosan is deprotonated, the network is relaxed, and the chitosan-sodium alginate protective layer fails, bile salts and lipase emulsify the oil phase, and finally astaxanthin is released and absorbed in the intestine, significantly improving the bioavailability of astaxanthin.

[0030] Since tyrosinase is a kind of oxidoreductase, it will produce excess active oxygen and high-reactivity intermediates in the reaction, which may oxidize astaxanthin, resulting in the decrease of astaxanthin content and the loss of activity, therefore, the amount of tyrosinase and the reaction time need to be controlled in the enzymatic cross-linking stage, and the enzyme needs to be inactivated by heat treatment after the reaction is completed, so as to avoid the residue of tyrosinase in the astaxanthin emulsion. At the same time, in order to further prevent the influence of tyrosinase on astaxanthin, tannic acid is added dropwise at the oil / water interface after the preparation of O / W emulsion to form a dense protective shell as an additional physical barrier. The molecular weight of tyrosinase is large and cannot pass through the protective shell, and the phenolic hydroxyl group of tannic acid molecule is rich, so the high-reactivity intermediates produced by enzymatic reaction will attack tannic acid first, thereby protecting astaxanthin from attack. However, the amount of tannic acid added needs to be controlled. If too much tannic acid is added, the tannic acid molecule contains a large number of phenolic hydroxyl groups, which can strongly adsorb on the interface of whey protein-coated oil droplets, occupying the adsorption sites originally reserved for the subsequent positively charged chitosan. Moreover, tannic acid itself is negatively charged, and if it is adsorbed in excess, it will further enhance the negative charge of the O / W emulsion interface, or form a layer of space and charge barrier, hindering the approach and adsorption of chitosan, resulting in the failure or poor effect of the first layer assembly.

[0031] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present application are:

[0032] 1. The "multi-layer" interface film of chitosan-sodium alginate is constructed by electrostatic assembly, which not only improves the mechanical strength of the interface film and can effectively resist the aggregation and gravity separation between droplets, greatly prolonging the shelf life of the product, but 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 to contact astaxanthin.

[0033] 2. To avoid the salt ions in the functional beverage weakening the electrostatic attraction between chitosan and sodium alginate, the sodium alginate grafted with polyethylene glycol effectively shields the charge neutralization of salt ions and also inhibits the flocculation of astaxanthin emulsion in the beverage. At the same time, the chitosan grafted with peptide segments and subjected to enzymatic cross-linking can strongly resist the interference of salt ions, and the cross-linked network has better mechanical strength, which can effectively prevent the coalescence of droplets. Moreover, the cross-linked network remains stable in the acidic environment of the stomach and disintegrates after the peptide segments are degraded by trypsin in the intestinal tract, realizing the targeted release of astaxanthin in the intestinal tract and thereby improving its bioavailability.

[0034] 3. By controlling the amount of tyrosinase and the reaction time of enzymatic reaction and inactivating the enzyme by heat treatment after the reaction is completed, the residue of tyrosinase with oxidizing property is prevented, and tannic acid is added in the O / W emulsion to prevent the oxidation of astaxanthin by the oxidizing substances produced by the reaction of tyrosinase. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Flow chart for preparing astaxanthin emulsion of Example 1 of the present application. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0037] Example 1: The present embodiment discloses a method for producing astaxanthin emulsion by Rhodotorula rubra, comprising the following steps:

[0038] S1. Solvent extraction: The Rhodotorula rubra dry bacteria are ultra-micro pulverized to 10-20 μm, and the obtained powder is added into acetone at a material-liquid ratio of 1:10 (w / v) for extraction. The supernatant is collected by centrifugation, and the extraction is repeated for 3 times. The supernatants are combined and rotary evaporated to obtain astaxanthin. 0.05 g of vitamin E and 0.01 g of tert-butyl hydroquinone are dissolved in 10 g of soybean oil, 10 mg of astaxanthin is added, and stirred and dissolved to obtain an astaxanthin soybean oil solution;

[0039] S2. Primary emulsification: 1.35 g of whey protein and 0.02 g of L-ascorbic acid are dissolved in deionized water, and the volume is made up to 90 g. Under high-speed shearing, the astaxanthin soybean oil solution is slowly added, and pre-homogenized for 2 min. Then, the solution is transferred into a high-pressure homogenizer, and homogenized for 3 times at 100 MPa to obtain an O / W emulsion;

[0040] S3. Oxidation protection: 0.01 g of tannic acid is dissolved in 10 mL of deionized water to obtain a tannic acid solution. 1 mL of the tannic acid solution is slowly added to 100 g of the O / W emulsion, and stirring is continued for 30 min to obtain a tannic acid-O / W emulsion;

[0041] S4. Electrostatic assembly: 100 g of the tannic acid-O / W emulsion is slowly added to 100 mL of a peptide-chitosan solution, and stirring is continued for 30 min. After sufficient adsorption, the precipitate is collected by centrifugation, and is re-dispersed in 100 mL of deionized water to obtain a chitosan-tannic acid-O / W emulsion. The emulsion is slowly added to 100 mL of a polyethylene glycol-sodium alginate solution, and stirring is continued for 30 min. The precipitate is collected by centrifugation, and is re-dispersed in 100 mL of a phosphate buffer to obtain a multi-layer polymer-coated O / W emulsion;

[0042] S5. Enzymatic cross-linking: Tyrosinase is added to the multi-layer polymer-coated O / W emulsion, and stirring is continued at 25°C for 2 hours. The obtained product is subjected to heat treatment at 80°C for 10 min in a water bath, and the precipitate is collected by centrifugation to obtain an astaxanthin emulsion.

[0043] It should be noted that, as Figure 1 The preparation flow chart of astaxanthin emulsion is shown in the figure. Dry Phaffia rhodozyma is super-micro pulverized, and astaxanthin is extracted by using acetone as a solvent. The astaxanthin is dissolved in soybean oil, mixed with whey protein to prepare an O / W emulsion, and vitamin E, tert-butyl hydroquinone and L-ascorbic acid are added as antioxidants to ensure that astaxanthin is not oxidized and disabled in the preparation process. In order to avoid the influence of tyrosinase used in the enzymatic crosslinking of the peptide-chitosan on the activity of astaxanthin, tannic acid solution is added to the O / W emulsion to form a dense protective shell to prevent the penetration of tyrosinase and oxidizing substances. The obtained tannic acid-O / W emulsion is then used to layer-by-layer assemble peptide-chitosan and polyethylene glycol-sodium alginate through electrostatic interaction. Finally, a crosslinking network is formed through enzymatic crosslinking to obtain astaxanthin emulsion.

[0044] The solvent in S1 is one or more of dichloromethane, chloroform, methanol, ethyl acetate, and acetone; preferably acetone.

[0045] The whey protein solution is a neutral solution with negative electricity, and the concentration is 1.5% (w / v); the peptide-chitosan solution is an acidic acetic acid solution with positive electricity, and the concentration is 0.25% (w / v); and the polyethylene glycol-sodium alginate solution is a neutral solution with negative electricity, and the concentration is 0.2% (w / v).

[0046] The volume ratio of the astaxanthin soybean oil solution to the whey protein solution in S2 is 1:9.

[0047] The mass ratio of the tannic acid solution to the O / W emulsion in S3 is 1:100.

[0048] The preparation method of the peptide-chitosan solution comprises the following steps:

[0049] S11. Chitosan is added to the MES buffer solution, glacial acetic acid is added, and stirring and dissolution are performed to obtain a chitosan solution;

[0050] S12. The peptide segment of the glycine-glycine-arginine-tyrosine sequence is dissolved in the MES buffer solution, EDC·HCl and NHS are added, and room temperature activation is performed to obtain a peptide segment solution;

[0051] S13. The peptide segment solution is added dropwise to the chitosan solution, stirring and reaction are performed, the obtained reaction liquid is purified by dialysis, and freeze-drying is performed to obtain peptide-chitosan;

[0052] S14. The peptide-chitosan is added to a 0.5% (w / v) acetic acid solution, continuous stirring and dissolution are performed, and water-based microporous filtration is performed to obtain a peptide-chitosan solution.

[0053] The preparation method of the polyethylene glycol-sodium alginate solution comprises the following steps:

[0054] S21. Dissolve sodium alginate in MES buffer solution, add EDC·HCl and NHS, stir to activate, to obtain a sodium alginate solution;

[0055] S22. Dissolve mPEG-NH2 in MES buffer solution, add to the sodium alginate solution, stir to react, and the obtained reaction liquid is purified by dialysis, freeze-dried to obtain polyethylene glycol-sodium alginate;

[0056] S23. Dissolve the polyethylene glycol-sodium alginate in deionized water, and magnetically stir to dissolve, to obtain a polyethylene glycol-sodium alginate solution.

[0057] The amount of added tyrosinase in S5 is 50 U / mL.

[0058] Examples 2-5 and Comparative Examples 1-6 are based on Example 1, except that the effects of electrostatic assembly, oxidative protection, and enzymatic cross-linking are different in a method for producing astaxanthin emulsion by a red yeast Xanthophyllomyces dendrorhous, and the difference points are shown in Table 1.

[0059]

[0060] 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 added tyrosinase, and the enzymatic cross-linking reaction time all affect the effects of electrostatic assembly, oxidative protection, and enzymatic cross-linking. If the mass ratio of tannic acid solution to O / W emulsion is too high, the tannic acid will occupy the binding sites of the peptide-chitosan solution on the whey protein, resulting in failure of electrostatic assembly. If the mass ratio is too low, the tannic acid will not be able to protect astaxanthin from oxidation by tyrosinase. At the same time, electrostatic assembly, oxidative protection, and enzymatic cross-linking are indispensable. Electrostatic assembly constructs a “multilayer” interfacial membrane, which improves the mechanical strength of the interfacial membrane. Enzymatic cross-linking improves the stability of the astaxanthin emulsion in a salt ion-containing solution, and the peptide segment is cut in the intestinal tract, achieving precise and efficient release of astaxanthin in the intestinal tract. The addition of tannic acid in the oxidative protection stage prevents the influence of enzymatic cross-linking on the astaxanthin emulsion, and the amount of tannic acid needs to be precisely controlled to prevent it from affecting the effect of electrostatic assembly.

[0061] Comparative Example 7: This comparative example is based on Example 1, except that the astaxanthin emulsion in this comparative example is not subjected to electrostatic assembly.

[0062] A method for producing astaxanthin emulsion by a red yeast Xanthophyllomyces dendrorhous, comprising the following steps:

[0063] S1. Solvent extraction: The dry Phaffia rhodozyma was ultra-micro ground to 10-20 μm, and the obtained powder was added into acetone at a material-to-liquid ratio of 1:10 (w / v) for extraction. The supernatant was collected by centrifugation, and the extraction was repeated for 3 times. The supernatants were combined and rotary evaporated to obtain astaxanthin. 0.05 g of vitamin E and 0.01 g of tert-butyl hydroquinone were dissolved in 10 g of soybean oil, and 10 mg of astaxanthin was added and dissolved by stirring to obtain an astaxanthin soybean oil solution;

[0064] S2. Primary emulsification: 1.35 g of whey protein and 0.02 g of L-ascorbic acid were dissolved in deionized water, and the volume was adjusted to 90 g. The astaxanthin soybean oil solution was slowly added under high-speed shearing, and pre-homogenized for 2 min. The mixture was transferred to a high-pressure homogenizer and homogenized at 100 MPa for 3 cycles to obtain an O / W emulsion.

[0065] S3. Oxidation protection: 0.01 g of tannic acid was dissolved in 10 mL of deionized water to obtain a tannic acid solution. 1 mL of the tannic acid solution was slowly added to 100 g of the O / W emulsion, and stirring was continued for 30 min to obtain a tannic acid-O / W emulsion.

[0066] S4. Enzymatic cross-linking: 100 g of the tannic acid-O / W emulsion, 100 mL of the peptide-chitosan solution, and 100 mL of the polyethylene glycol-sodium alginate solution were mixed and stirred for 30 min. Tyrosinase was added, and the mixture was continuously stirred at 25°C for 2 hours. The obtained product was heat-treated at 80°C for 10 min in a water bath, and the precipitate was collected by centrifugation to obtain an astaxanthin emulsion.

[0067] The other components and preparation methods were the same as in Example 1.

[0068] It should be noted that when the tannic acid-O / W emulsion, the peptide-chitosan solution, and the polyethylene glycol-sodium alginate solution are directly blended without layer-by-layer electrostatic assembly, the enzymatic cross-linking forms a disordered gel or precipitate, rather than a regular and directional arranged film, which results in poor encapsulation efficiency and stability of the final emulsion.

[0069] Comparative Example 8: The comparative example was based on Example 1, except that the peptide-chitosan solution in the comparative example was not grafted with a peptide segment.

[0070] The other components and preparation methods were the same as in Example 1.

[0071] Comparative Example 9: The comparative example was based on Example 1, except that the polyethylene glycol-sodium alginate solution in the comparative example was not grafted with polyethylene glycol.

[0072] The other components and preparation methods were the same as in Example 1.

[0073] Test verification:

[0074] Experiment 1:

[0075] (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.

[0076] (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.

[0077] (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.

[0078]

[0079] 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.

[0080] Experiment 2:

[0081] (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.

[0082] (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.

[0083]

[0084] As shown in Table 3, the stability of astaxanthin emulsion in salt ions and the intestinal release rate, from the data in the table, it can be seen that the particle size increases greatly, the anti-flocculation and anti-aggregation ability in salt ion solution is weaker, the flocculation and precipitation is easier to generate, and the protection effect of single electrostatic assembly or enzymatic crosslinking on astaxanthin is limited, only using electrostatic assembly, the electrostatic attraction between chitosan and sodium alginate will be greatly weakened in salt ion environment, and only using enzymatic crosslinking, due to the direct mixing of peptide-chitosan and polyethylene glycol-sodium alginate, the enzymatic crosslinking forms a disordered gel or precipitate, rather than a regular, directional arrangement of film, which leads to poor wrapping efficiency and stability of the final emulsion, and finally cannot realize accurate and efficient release in the intestinal tract.

[0085] The above only describes the preferred embodiments of the present application and is not intended to limit the protection scope of the present application, and any modifications, equivalent replacements and improvements made by any person skilled in the art within the spirit and principle of the present application shall be included in the protection scope of the present application.

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 product is then heat-treated, and the precipitate is collected by centrifugation to obtain astaxanthin emulsion. The mass ratio of tannic acid solution to O / W emulsion in S3 is 1~3:100; The enzyme-catalyzed cross-linking reaction time in S5 is 1-2 hours. The peptide-chitosan is a peptide-modified chitosan with a glycine-glycine-arginine-tyrosine sequence.

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 using *Pseudomonas aeruginosa* 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 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.

6. 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.

7. 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.

Images