Preparation method of phloretin-loaded Portulaca oleracea L. protein emulsion

Abstract

The present invention discloses a preparation method of a phyllodium album protein emulsion loaded with phloretin, belonging to the technical field of functional factor delivery. The phyllodium album protein is stirred and dissolved with phosphate buffer solution, and then a magnetic microsphere auxiliary agent with a mass 2-3 times that of the phyllodium album protein is added to obtain a phyllodium album protein aqueous phase; phloretin and linseed oil are stirred and mixed to obtain a phloretin oil phase; the phyllodium album protein aqueous phase and the phloretin oil phase are stirred and mixed according to a volume ratio of 9:1, ultrasonically dispersed after high-speed shearing, and finally the magnetic microsphere auxiliary agent is removed by an external magnetic field separation to obtain a phyllodium album protein emulsion loaded with phloretin. By adding the magnetic microsphere auxiliary agent and combining ultrasonic dispersion treatment, the low water solubility, instability and poor bioavailability of phloretin are improved, so as to prepare a phyllodium album protein emulsion loaded with phloretin with a smaller particle size, good dispersion, good biocompatibility, high stability and good encapsulation effect, which can well improve the solubility and bioavailability of phloretin.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional factor delivery, and particularly relates to a preparation method of a portulacain-containing portulaca oleracea protein emulsion. Background Art

[0002] Portulacain, also known as trihydroxyphenylacetone, is mainly derived from the roots and peels of juicy fruit plants such as pears, apples, and strawberries. It has a wide range of pharmacological activities in aspects such as anti-inflammatory immunity, antioxidant, anti-cardiovascular disease, anti-diabetes, anti-tumor, and liver protection, and is a potential natural medicine. However, portulacain is sensitive to light and heat and has poor water solubility, resulting in very low stability and bioavailability. These factors greatly limit the application of portulacain in the fields of functional foods and pharmaceuticals. Currently, numerous embedding delivery systems have been studied to solve the application problems of portulacain.

[0003] Among numerous embedding delivery systems, cyclodextrin can efficiently load portulacain, but its system is single and it is not digested in the small intestine, making it difficult to effectively release portulacain to improve its bioaccessibility. Starch is easily absorbed in the human body, but starch-based emulsifiers usually require further modification treatment, and common modified starches have problems such as large particle size, complex structure, and increased starch resistance to digestion, which limit the practical application of starch-based emulsions.

[0004] Plant proteins are similar to animal proteins, are easily digested and absorbed by the human body, and proteins are natural amphiphilic substances with emulsifying properties. In an oil-water system, proteins can spontaneously migrate to the oil-water interface to form a protein adsorption layer, thereby playing a role in stabilizing the emulsion. Portulaca oleracea protein contains 18 kinds of amino acids, among which the hydrophobic amino acids are as high as 45.52%, and can form hydrophobic cavities to encapsulate hydrophobic flavonoid compounds. And portulaca oleracea protein has good solubility and thermal stability, and when used in a delivery system, it can improve the encapsulation effect and stability of functional factors.

[0005] However, as a natural macromolecular substance, portulaca oleracea protein has a large molecular weight, slow interfacial adsorption rate during the formation of the emulsion, and serious aggregation. Therefore, effective means are often needed to improve the formation of the droplet interfacial film and the internal structure of the emulsion. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of a portulacain-containing portulaca oleracea protein emulsion. By adding a magnetic microsphere auxiliary agent and combining ultrasonic dispersion treatment, the low water solubility, instability, and poor bioaccessibility of portulacain are improved, so as to prepare a portulacain-containing portulaca oleracea protein emulsion with small particle size, good dispersibility, good biocompatibility, high stability, and good encapsulation effect, which can well improve the solubility and bioaccessibility of portulacain.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] A preparation method of a phyllodium album protein emulsion loaded with phloretin, comprising the following steps:

[0009] Step 1: Dissolve phyllodium album protein with a phosphate buffer solution having a pH value of 7.8 - 8.2 by stirring, and then add a magnetic microsphere auxiliary agent with a mass 2 - 3 times that of the phyllodium album protein to obtain a phyllodium album protein aqueous phase with a mass fraction of phyllodium album protein of 0.1 - 0.5%;

[0010] Step 2: Stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of phloretin of 5 mg / mL;

[0011] Step 3: Add the phyllodium album protein aqueous phase and the phloretin oil phase to a stirring kettle according to a volume ratio of 9:1, stir and mix at 20 - 25°C and 300 - 500 r / min for 5 - 10 min, then transfer to a high - speed shear dispersion emulsifier, perform high - speed shearing at a shear rate of 10000 - 15000 r / min for 5 - 10 min, perform ultrasonic dispersion at a power of 100 - 500 W for 5 - 25 min, and finally separate and remove the magnetic microsphere auxiliary agent by applying an external magnetic field to obtain a phyllodium album protein emulsion loaded with phloretin, thus completing the preparation method of the phyllodium album protein emulsion loaded with phloretin.

[0012] Furthermore, the magnetic microsphere auxiliary agent is prepared by the following steps:

[0013] Step 1: Add ethylene glycol and ferric chloride hexahydrate to a reaction kettle, stir at 200 - 300 r / min for 20 - 30 min, then add sodium acetate and polyethylene glycol with a molecular weight of 400 to the reaction kettle, continue to stir for 20 - 30 min, stir and react at 190 - 200°C for 8 - 10 h, naturally cool, separate the product by applying an external magnetic field, wash 2 - 3 times with water, and perform vacuum drying to obtain magnetic iron oxide microspheres.

[0014] Furthermore, the dosage ratio of ethylene glycol, ferric chloride hexahydrate, sodium acetate and polyethylene glycol is 100 mL: 2.5 - 3 g: 7 - 7.4 g: 0.2 g.

[0015] Step 2: Add the magnetic iron oxide microspheres, absolute ethanol and deionized water to a reaction kettle and stir and mix, then add ammonia water with a mass concentration of 25% to the reaction kettle, stir at 200 - 300 r / min for 20 - 30 min, then dropwise add tetraethyl orthosilicate to the reaction kettle, stir and react at 35 - 40°C and 300 - 500 r / min for 20 - 24 h, separate the product by applying an external magnetic field, wash 2 - 3 times with absolute ethanol and deionized water respectively, and perform vacuum drying to obtain silica - coated composite magnetic microspheres.

[0016] Furthermore, the dosage ratio of magnetite magnetic microspheres, absolute ethanol, deionized water, ammonia water, and tetraethyl orthosilicate is 0.4 - 0.5 g : 80 mL : 20 mL : 5 mL : 0.5 mL.

[0017] Step 3: Add the silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane, and tetrahydrofuran into a reaction kettle, react for 10 - 12 h under nitrogen protection and at 70 - 75 °C, then transfer the product to n-hexane to precipitate, wash the precipitate with tetrahydrofuran by centrifugation 2 - 3 times, and vacuum-dry the filter cake to obtain amino-functionalized composite magnetic microspheres.

[0018] Furthermore, the dosage ratio of the silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane, and tetrahydrofuran is 1 g : 0.5 - 0.6 g : 25 - 30 mL.

[0019] Step 4: Add the amino-functionalized composite magnetic microspheres and 1,4-dioxane into a reaction kettle, ultrasonically disperse for 5 - 10 min, then add isophthalaldehyde, hydrophilic amino acid, and an aqueous acetic acid solution with a molar concentration of 3 mol / L into the reaction kettle, stir and react for 20 - 24 h under the conditions of 75 - 85 °C and 200 - 300 r / min, separate the product by an external magnetic field, wash it with acetone and absolute ethanol 2 - 3 times respectively, and vacuum-dry to obtain the magnetic microsphere additive.

[0020] Furthermore, the dosage ratio of the amino-functionalized composite magnetic microspheres, 1,4-dioxane, isophthalaldehyde, hydrophilic amino acid, and aqueous acetic acid solution is 0.4 - 0.5 g : 25 - 30 mL : 0.65 - 0.7 g : 0.55 - 0.6 g : 0.9 - 1 mL.

[0021] Furthermore, the hydrophilic amino acid is any one of serine, threonine, aspartic acid, and glutamine.

[0022] Advantages of the present invention:

[0023] The present invention uses Rumex patientia protein as an emulsifier, and prepares a Rumex patientia protein emulsion loaded with phloretin by mixing and homogenizing the aqueous phase of Rumex patientia protein and the oil phase of phloretin. The obtained loaded nanoemulsion has small particle size, good stability, and is non-toxic.

[0024] In the preparation process of the phyllodium protein emulsion loaded with phloretin of the present invention, a method combining high-speed shearing and ultrasonic homogenization is adopted. The prepared phyllodium protein emulsion loaded with phloretin has uniformly dispersed droplets, small particle size, high stability and high loading efficiency; moreover, the preparation process is simple and the biocompatibility is good. Compared with traditional high-pressure homogenization, ultrasonic dispersion, as a high-energy emulsification method, is environmentally friendly and can induce the denaturation of protein molecules in the system without using chemical additives, thereby improving the emulsion properties. At the same time, the introduction of ultrasonic dispersion can also inhibit the change of droplet charge, and improve the thermal stability, storage stability and salt ion stability of the emulsion to varying degrees.

[0025] In the aqueous phase of the phyllodium protein of the present invention, magnetic microsphere additives are added, which can act as grinding balls and stabilizers in the emulsion, and help to increase the shearing effect of high-speed shearing and ultrasonic dispersion. The surface of the magnetic microsphere additives contains a large number of hydrophilic groups, which can adsorb on the oil-water interface to form a protective film to prevent the coalescence of droplets. And the magnetic microsphere additives interact with the hydrophobic cavities of phyllodium protein, which helps to guide the orderly arrangement and assembly of phyllodium protein and phloretin molecules, achieving the effect of shaping the morphology, further enhancing the encapsulation effect of phyllodium protein on phloretin and reducing the droplet size. The magnetic microsphere additives can be effectively separated and removed by an external magnetic field, can be reused repeatedly, and are safe and environmentally friendly. Description of the Drawings

[0026] The present invention will be further described below with reference to the drawings.

[0027] Figure 1 are the laser confocal pictures of different emulsions in Examples 1-5 of the present invention;

[0028] Figure 2 are the comparison pictures of different emulsions before and after physical storage in Examples 1-5 and Comparative Example 1 of the present invention;

[0029] Figure 3 are the bar graphs of the loading amounts of different emulsions in Examples 1-5 and Comparative Example 2 of the present invention;

[0030] Figure 4 are the particle size comparison pictures of different emulsions in Examples 1-5 and Comparative Example 2 of the present invention;

[0031] Figure 5 are the laser confocal pictures of different emulsions in Comparative Examples 2-3 of the present invention;

[0032] Figure 6 are the comparison pictures of different emulsions before and after physical storage in Examples 5-9 and Comparative Example 3 of the present invention;

[0033] Figure 7It is a bar chart of the loading amounts of different emulsions in Example 5 - Example 9 and Comparative Example 3 of the present invention;

[0034] Figure 8 It is a comparison chart of the particle sizes of different emulsions in Example 5 - Example 9 and Comparative Example 3 of the present invention. Detailed implementation manners

[0035] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0036] Example 1: This example provides a Portulaca oleracea protein emulsion loaded with phloretin, which is prepared by the following method:

[0037] S1: Add 100 L of ethylene glycol and 2.5 kg of ferric chloride hexahydrate into a reaction kettle, stir for 20 min under the condition of 200 r / min, then add 7 kg of sodium acetate and 0.2 kg of polyethylene glycol with a molecular weight of 400 into the reaction kettle, continue to stir for 20 min, and stir and react for 8 h under the condition of 190 °C. Naturally cool, separate the product by an external magnetic field, wash it twice with water, and dry it under vacuum to obtain magnetic iron oxide microspheres.

[0038] S2: Add 0.4 kg of magnetic iron oxide microspheres, 80 L of absolute ethanol and 20 L of deionized water into a reaction kettle and stir to mix. Then add 5 L of ammonia water with a mass concentration of 25% into the reaction kettle, stir at 200 r / min for 20 min, and then dropwise add 0.5 L of tetraethyl orthosilicate into the reaction kettle. Stir and react for 20 h under the conditions of 35 °C and 300 r / min. Separate the product by an external magnetic field, wash it twice with absolute ethanol and deionized water respectively, and dry it under vacuum to obtain silica-coated composite magnetic microspheres.

[0039] S3: Add 100 g of silica-coated composite magnetic microspheres, 50 g of 3-aminopropyltriethoxysilane and 2.5 L of tetrahydrofuran into a reaction kettle, react under the conditions of nitrogen protection and 70 °C for 10 h. Then transfer the product to n-hexane to precipitate, wash the precipitate twice by centrifugation with tetrahydrofuran, and dry the filter cake under vacuum to obtain amino-functionalized composite magnetic microspheres.

[0040] S4: Add 40 g of amino-functionalized composite magnetic microspheres and 2.5 L of 1,4-dioxane into a reaction kettle, ultrasonically disperse for 5 min, then add 65 g of isophthalaldehyde, 55 g of serine and 90 mL of acetic acid aqueous solution with a molar concentration of 3 mol / L into the reaction kettle, stir and react at 75 °C and 250 r / min for 22 h, separate the product by an external magnetic field, wash it twice with acetone and absolute ethanol respectively, and dry it under vacuum to obtain a magnetic microsphere auxiliary agent.

[0041] S5: Stir and dissolve the Portulaca oleracea L. protein with a phosphate buffer solution at pH 7.8, then add a magnetic microsphere auxiliary agent with a mass twice that of the Portulaca oleracea L. protein to obtain an aqueous phase of Portulaca oleracea L. protein with a mass fraction of 0.1% of the Portulaca oleracea L. protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a phloretin mass concentration of 5 mg / mL.

[0042] S6: Add the aqueous phase of Portulaca oleracea L. protein and the phloretin oil phase into a stirring kettle according to a volume ratio of 9:1, stir and mix at 20 °C and 300 r / min for 5 min, then transfer it to a high-speed shear dispersion emulsifier, perform high-speed shearing at a shearing rate of 10000 r / min for 5 min, ultrasonically disperse at a power of 100 W for 5 min, and finally separate and remove the magnetic microsphere auxiliary agent by an external magnetic field to obtain a Portulaca oleracea L. protein emulsion loaded with phloretin.

[0043] Example 2: This example provides a Portulaca oleracea L. protein emulsion loaded with phloretin, which is prepared by the following method:

[0044] S1: Add 100 L of ethylene glycol and 2.6 kg of ferric chloride hexahydrate into a reaction kettle, stir at 2200 r / min for 22 min, then add 7.1 kg of sodium acetate and 0.2 kg of polyethylene glycol with a molecular weight of 400 into the reaction kettle, continue to stir for 22 min, stir and react at 195 °C for 8.5 h, cool naturally, separate the product by an external magnetic field, wash it twice with water, and dry it under vacuum to obtain magnetic iron oxide microspheres.

[0045] S2: Add 0.42 kg of magnetic iron oxide microspheres, 80 L of absolute ethanol and 20 L of deionized water into a reaction kettle and stir and mix, then add 5 L of ammonia water with a mass concentration of 25% into the reaction kettle, stir at 220 r / min for 22 min, then dropwise add 0.5 L of tetraethyl orthosilicate into the reaction kettle, stir and react at 36 °C and 350 r / min for 21 h, separate the product by an external magnetic field, wash it twice with absolute ethanol and deionized water respectively, and dry it under vacuum to obtain silica-coated composite magnetic microspheres.

[0046] S3: Add 100 g of silica-coated composite magnetic microspheres, 52 g of 3-aminopropyltriethoxysilane, and 2.8 L of tetrahydrofuran into a reaction kettle, react for 11 h under nitrogen protection at 72 °C, then transfer the product to n-hexane to precipitate, wash the precipitate twice by centrifugation with tetrahydrofuran, and vacuum dry the filter cake to obtain amino-functionalized composite magnetic microspheres.

[0047] S4: Add 42 g of amino-functionalized composite magnetic microspheres and 2.8 L of 1,4-dioxane into a reaction kettle, ultrasonically disperse for 8 min, then add 68 g of isophthalaldehyde, 58 g of threonine, and 95 mL of acetic acid aqueous solution with a molar concentration of 3 mol / L into the reaction kettle, stir and react for 21 h at 80 °C and 220 r / min, separate the product by an external magnetic field, wash twice with acetone and absolute ethanol respectively, and vacuum dry to obtain magnetic microsphere additives.

[0048] S5: Stir and dissolve the Portulaca oleracea L. protein with a phosphate buffer solution with a pH value of 8, then add magnetic microsphere additives with a mass 2.5 times that of the Portulaca oleracea L. protein to obtain an aqueous phase of Portulaca oleracea L. protein with a mass fraction of 0.2% of the Portulaca oleracea L. protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a phloretin mass concentration of 5 mg / mL.

[0049] S6: Add the aqueous phase of Portulaca oleracea L. protein and the phloretin oil phase into a stirring kettle according to a volume ratio of 9:1, stir and mix for 6 min at 22 °C and 350 r / min, then transfer to a high-speed shear dispersion emulsifier, perform high-speed shearing for 8 min at a shear rate of 12000 r / min, perform ultrasonic dispersion for 10 min at a power of 200 W, and finally separate and remove the magnetic microsphere additives by an external magnetic field to obtain a Portulaca oleracea L. protein emulsion loaded with phloretin.

[0050] Example 3: This example provides a Portulaca oleracea L. protein emulsion loaded with phloretin, which is prepared by the following method:

[0051] S1: Add 100 L of ethylene glycol and 2.8 kg of ferric chloride hexahydrate into a reaction kettle, stir for 25 min at 250 r / min, then add 7.2 kg of sodium acetate and 0.2 kg of polyethylene glycol with a molecular weight of 400 into the reaction kettle, continue to stir for 25 min, stir and react for 9 h at 195 °C, naturally cool, wash three times with water after separating the product by an external magnetic field, and vacuum dry to obtain magnetite magnetic microspheres.

[0052] S2: Add 0.45 kg of magnetic iron oxide microspheres, 80 L of absolute ethanol, and 20 L of deionized water into a reaction kettle, stir and mix them. Then add 5 L of ammonia water with a mass concentration of 25% into the reaction kettle, stir at 250 r / min for 25 min. Then add 0.5 L of tetraethyl orthosilicate dropwise into the reaction kettle, stir and react at 38 °C and 400 r / min for 22 h. Separate the product by an external magnetic field, wash it twice with absolute ethanol and deionized water respectively, and dry it under vacuum to obtain silica-coated composite magnetic microspheres.

[0053] S3: Add 100 g of silica-coated composite magnetic microspheres, 55 g of 3-aminopropyltriethoxysilane, and 2.8 L of tetrahydrofuran into a reaction kettle, react at 72 °C under nitrogen protection for 11 h. Then transfer the product to n-hexane to precipitate, wash the precipitate twice by centrifugation with tetrahydrofuran, and dry the filter cake under vacuum to obtain amino-functionalized composite magnetic microspheres.

[0054] S4: Add 45 g of amino-functionalized composite magnetic microspheres and 2.8 L of 1,4-dioxane into a reaction kettle, disperse them by ultrasonic wave for 8 min. Then add 68 g of isophthalaldehyde, 58 g of aspartic acid, and 95 mL of acetic acid aqueous solution with a molar concentration of 3 mol / L into the reaction kettle, stir and react at 80 °C and 250 r / min for 22 h. Separate the product by an external magnetic field, wash it twice with acetone and absolute ethanol respectively, and dry it under vacuum to obtain magnetic microsphere additives.

[0055] S5: Stir and dissolve the Portulaca oleracea L. protein with a phosphate buffer solution with a pH value of 8, and then add magnetic microsphere additives with a mass 2.5 times that of the Portulaca oleracea L. protein to obtain an aqueous phase of Portulaca oleracea L. protein with a mass fraction of 0.3% of the Portulaca oleracea L. protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0056] S6: Add the aqueous phase of Portulaca oleracea L. protein and the phloretin oil phase into a stirring kettle according to a volume ratio of 9:1, stir and mix at 23 °C and 400 r / min for 8 min, then transfer it to a high-speed shear dispersion emulsifier, carry out high-speed shearing at a shear rate of 13000 r / min for 8 min, carry out ultrasonic dispersion at a power of 300 W for 15 min, and finally separate and remove the magnetic microsphere additives by an external magnetic field to obtain a Portulaca oleracea L. protein emulsion loaded with phloretin.

[0057] Example 4: This example provides a Portulaca oleracea L. protein emulsion loaded with phloretin, which is prepared by the following method:

[0058] S1: Add 100 L of ethylene glycol and 2.8 kg of ferric chloride hexahydrate into the reaction kettle, stir for 28 min under the condition of 280 r / min, then add 7.3 kg of sodium acetate and 0.2 kg of polyethylene glycol with a molecular weight of 400 into the reaction kettle, continue to stir for 28 min, stir and react for 9 h under the condition of 195 °C, cool naturally, separate the product by external magnetic field, wash it with water 3 times, and dry it under vacuum to obtain magnetic iron oxide microspheres.

[0059] S2: Add 0.48 kg of magnetic iron oxide microspheres, 80 L of absolute ethanol and 20 L of deionized water into the reaction kettle and stir to mix. Then add 5 L of ammonia water with a mass concentration of 25% into the reaction kettle, stir at 280 r / min for 28 min, and then dropwise add 0.5 L of tetraethyl orthosilicate into the reaction kettle. Stir and react for 23 h under the conditions of 38 °C and 450 r / min. Separate the product by external magnetic field, wash it 3 times with absolute ethanol and deionized water respectively, and dry it under vacuum to obtain silica-coated composite magnetic microspheres.

[0060] S3: Add 100 g of silica-coated composite magnetic microspheres, 58 g of 3-aminopropyltriethoxysilane and 2.8 L of tetrahydrofuran into the reaction kettle, react for 11 h under the protection of nitrogen and at 73 °C. Then transfer the product to n-hexane to precipitate, wash the precipitate 3 times by centrifugation with tetrahydrofuran, and dry the filter cake under vacuum to obtain amino-functionalized composite magnetic microspheres.

[0061] S4: Add 48 g of amino-functionalized composite magnetic microspheres and 2.8 L of 1,4-dioxane into the reaction kettle, disperse them by ultrasonic wave for 8 min. Then add 68 g of isophthalaldehyde, 58 g of glutamine and 98 mL of acetic acid aqueous solution with a molar concentration of 3 mol / L into the reaction kettle. Stir and react for 23 h under the conditions of 82 °C and 280 r / min. Separate the product by external magnetic field, wash it 3 times with acetone and absolute ethanol respectively, and dry it under vacuum to obtain magnetic microsphere additives.

[0062] S5: Stir and dissolve the Portulaca oleracea L. protein with a phosphate buffer solution with a pH value of 8, and then add magnetic microsphere additives with a mass 2.5 times that of the Portulaca oleracea L. protein to obtain an aqueous phase of Portulaca oleracea L. protein with a mass fraction of 0.4% of the Portulaca oleracea L. protein; Stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0063] S6: Add the aqueous phase of Portulaca oleracea L. protein and the phloretin oil phase into the stirring kettle according to a volume ratio of 9:1, stir and mix at 23 °C and 450 r / min for 8 min, then transfer it to a high-speed shear dispersion emulsifier, carry out high-speed shearing at a shear rate of 14000 r / min for 8 min, carry out ultrasonic dispersion at a power of 400 W for 20 min, and finally separate and remove the magnetic microsphere additives by external magnetic field to obtain a Portulaca oleracea L. protein emulsion loaded with phloretin.

[0064] Example 5: This example provides a Portulaca oleracea L. protein emulsion loaded with phloretin, which is prepared by the following method:

[0065] S1: Add 100 L of ethylene glycol and 3 kg of ferric chloride hexahydrate to the reaction kettle, stir for 30 min under the condition of 300 r / min, then add 7.4 kg of sodium acetate and 0.2 kg of polyethylene glycol with a molecular weight of 400 to the reaction kettle, continue to stir for 30 min, stir and react for 10 h under the condition of 200 °C, naturally cool, separate the product by an external magnetic field, wash it 3 times with water, and dry it under vacuum to obtain magnetic iron oxide microspheres.

[0066] S2: Add 0.5 kg of magnetic iron oxide microspheres, 80 L of absolute ethanol and 20 L of deionized water to the reaction kettle and stir to mix. Then add 5 L of ammonia water with a mass concentration of 25% to the reaction kettle, stir at 300 r / min for 30 min, and then dropwise add 0.5 L of tetraethyl orthosilicate to the reaction kettle. Stir and react for 24 h under the conditions of 40 °C and 500 r / min. Separate the product by an external magnetic field, wash it 3 times with absolute ethanol and deionized water respectively, and dry it under vacuum to obtain silica-coated composite magnetic microspheres.

[0067] S3: Add 100 g of silica-coated composite magnetic microspheres, 60 g of 3-aminopropyltriethoxysilane and 3 L of tetrahydrofuran to the reaction kettle, react for 12 h under the protection of nitrogen and at 75 °C. Then transfer the product to n-hexane to precipitate, wash the precipitate 3 times by centrifugation with tetrahydrofuran, and dry the filter cake under vacuum to obtain amino-functionalized composite magnetic microspheres.

[0068] S4: Add 50 g of amino-functionalized composite magnetic microspheres and 3 L of 1,4-dioxane to the reaction kettle, ultrasonically disperse for 10 min, then add 70 g of isophthalaldehyde, 60 g of serine and 100 mL of acetic acid aqueous solution with a molar concentration of 3 mol / L to the reaction kettle. Stir and react for 24 h under the conditions of 85 °C and 300 r / min. Separate the product by an external magnetic field, wash it 3 times with acetone and absolute ethanol respectively, and dry it under vacuum to obtain magnetic microsphere additives.

[0069] S5: Stir and dissolve Portulaca oleracea L. protein with phosphate buffer solution with a pH value of 8.2, and then add magnetic microsphere additives with a mass 3 times that of Portulaca oleracea L. protein to obtain an aqueous phase of Portulaca oleracea L. protein with a mass fraction of 0.5% of Portulaca oleracea L. protein; stir and mix phloretin and linseed oil to obtain an oil phase of phloretin with a mass concentration of 5 mg / mL.

[0070] S6: Add the water phase of Rumex patientia protein and the oil phase of phloretin into a stirring kettle at a volume ratio of 9:1, stir and mix for 10 min under the conditions of 25 °C and 500 r / min, then transfer it to a high-speed shear dispersion emulsifier, and perform high-speed shearing for 10 min at a shear rate of 15000 r / min, and perform ultrasonic dispersion for 25 min at a power of 500 W. Finally, remove the magnetic microsphere auxiliary agent by external magnetic field separation to obtain the Rumex patientia protein emulsion loaded with phloretin.

[0071] Characterize the Rumex patientia protein emulsions loaded with phloretin in Examples 1 - 5. Figure 1 Figure 5 shows the laser confocal pictures of different emulsions (scale size is 10 μm). Analyze from left to right. The first column shows that the emulsion oil droplets are stained green by Nile red, the second column shows that the Rumex patientia protein is stained red by Nile blue, the third column is the overall picture, and the fourth column is the partial enlarged picture (scale bar is 2 μm).

[0072] From Figure 1 It can be seen that the laser confocal pictures of the Rumex patientia protein emulsions loaded with phloretin prepared with different Rumex patientia contents in Examples 1 - 5 show that the green oil droplets are surrounded by the red protein solution, indicating the formation of an oil-in-water emulsion.

[0073] And within the range of Rumex patientia content < 0.3% (Examples 1 - 3), as the content of Rumex patientia protein increases, the droplet size of the emulsion decreases and becomes more evenly distributed. When the content of Rumex patientia protein is 0.3%, the droplet size of the emulsion is the smallest and the distribution is the most uniform.

[0074] Comparative Example 1: On the basis of Example 1, in step S6, the water phase of Rumex patientia protein is directly replaced with a phosphate buffer solution with a pH value of 7.8, and the remaining steps remain unchanged to prepare a phloretin emulsion.

[0075] Bottle and store the Rumex patientia protein emulsions loaded with phloretin in Examples 1 - 5 and the phloretin emulsion in Comparative Example 1 for 30 days, and the results are as Figure 2 shown.

[0076] From Figure 2 It can be seen that the freshly prepared Rumex patientia protein emulsions loaded with phloretin prepared with different Rumex patientia contents are overall faintly yellow and have good homogeneity. After storing for 30 days, except for the control group without Rumex patientia protein showing obvious stratification, and the Rumex patientia protein emulsions loaded with phloretin prepared with low Rumex patientia protein content in Examples 1 - 2 showing a little stratification, the Rumex patientia protein emulsions loaded with phloretin in Examples 3 - 5 are still stable and do not show stratification, indicating that the emulsion after being encapsulated by Rumex patientia protein has better stability.

[0077] Comparative Example 2: On the basis of Example 1, magnetic microsphere additives were not added when preparing the aqueous phase of Portulaca oleracea protein, and the remaining steps remained unchanged, and a Portulaca oleracea protein emulsion loaded with phloretin was prepared.

[0078] According to the bar chart of the loading amount of the Portulaca oleracea protein emulsion loaded with phloretin in Examples 1-5 and Comparative Example 2 (please refer to Figure 3 ), and the particle size comparison chart (please refer to Figure 4 ), it can be seen that as the content of Portulaca oleracea protein increases, the loading amount of phloretin in the Portulaca oleracea protein emulsion first increases and then decreases. In Example 3, when the content of Portulaca oleracea protein was 0.3%, the loading amount was the largest, reaching 12.45%, and the particle size of the Portulaca oleracea protein emulsion loaded with phloretin in Example 3 was the smallest and the particle size distribution was the most uniform. The loading amount of the Portulaca oleracea protein emulsion loaded with phloretin in Comparative Example 2 decreased, and the particle size increased, indicating that adding magnetic microsphere additives can reduce the particle size of the Portulaca oleracea protein emulsion loaded with phloretin and increase the loading amount of phloretin.

[0079] Example 6: On the basis of Example 5, the power of ultrasonic dispersion was adjusted to 400 W, and the remaining steps remained unchanged, and a phloretin emulsion was prepared.

[0080] Example 7: On the basis of Example 5, the power of ultrasonic dispersion was adjusted to 300 W, and the remaining steps remained unchanged, and a phloretin emulsion was prepared.

[0081] Example 8: On the basis of Example 5, the power of ultrasonic dispersion was adjusted to 200 W, and the remaining steps remained unchanged, and a phloretin emulsion was prepared.

[0082] Example 9: On the basis of Example 5, the power of ultrasonic dispersion was adjusted to 100 W, and the remaining steps remained unchanged, and a phloretin emulsion was prepared.

[0083] Comparative Example 3: On the basis of Example 5, ultrasonic dispersion treatment was not carried out, and the remaining steps remained unchanged, and a phloretin emulsion was prepared.

[0084] The Portulaca oleracea protein emulsions loaded with phloretin in Comparative Example 2 - Comparative Example 3 were characterized. Figure 5 are the laser confocal pictures of each emulsion (the scale size is 10 μm). The droplet sizes of both emulsions are relatively large. It can be analyzed that adding magnetic microsphere additives and ultrasonic assistance are both helpful for reducing the droplet size.

[0085] The Portulaca oleracea protein emulsions loaded with phloretin in Examples 5 - 9 and Comparative Example 3 were bottled and stored for 30 days, and the results are as Figure 6 shown.

[0086] From Figure 6It can be seen that the freshly prepared Portulaca oleracea protein emulsion loaded with phloretin is overall light yellow and has good homogeneity. After 30 days of storage, obvious stratification occurred in the bottle of Comparative Example 3 that was not ultrasonically treated, and slight stratification occurred in the Portulaca oleracea protein emulsion loaded with phloretin prepared at a low ultrasonic power in Example 9. The Portulaca oleracea protein emulsions loaded with phloretin prepared in Examples 5 - 8 remained stable and did not show stratification.

[0087] According to the histogram of the loading amount of the Portulaca oleracea protein emulsion loaded with phloretin in Examples 5 - 9 and Comparative Example 3 (please refer to Figure 7 ), and the particle size comparison chart (please refer to Figure 8 ), it can be seen that as the ultrasonic power increases, the loading amount of the Portulaca oleracea protein emulsion on phloretin first increases and then decreases. When the ultrasonic power in Example 6 is 400 W, the loading amount is the largest, and at this ultrasonic dispersion power, the particle size of the Portulaca oleracea protein emulsion loaded with phloretin is the smallest and the particle size distribution is the most uniform.

[0088] Measurement of the particle size of the Portulaca oleracea protein emulsion loaded with phloretin: The particle size of the Portulaca oleracea protein emulsion loaded with phloretin was measured using a dynamic light scattering instrument (ZSU3100, Malvern Instruments Ltd., UK).

[0089] Observation of the morphology of the Portulaca oleracea protein emulsion loaded with phloretin: The morphology of the Portulaca oleracea protein emulsion loaded with phloretin was observed using a Leica laser scanning confocal microscope (STELLARIS 5, Germany).

[0090] Measurement of the loading rate of the Portulaca oleracea protein emulsion loaded with phloretin: The loading rate of the Portulaca oleracea protein emulsion loaded with phloretin was measured using a fully automatic multifunctional microplate reader (HH3500, Pontyclum Co., UK).

[0091] It should be noted that in this article, terms such as "including", "comprising" or any other variant thereof are intended to cover non - exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or elements inherent to such process, method, article or device.

[0092] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a phloretin-loaded Rumex patientia protein emulsion, characterized in that It includes the following steps: Step 1: Dissolve the protein of Rumex patientia L. in a phosphate buffer solution with a pH value of 7.8 - 8.2 by stirring, and then add a magnetic microsphere auxiliary agent with a mass 2 - 3 times that of the protein of Rumex patientia L. to obtain an aqueous phase of the protein of Rumex patientia L. with a mass fraction of 0.1 - 0.5% of the protein of Rumex patientia L.; Step 2: Stir and mix phloretin and linseed oil to obtain an oil phase of phloretin with a mass concentration of 5 mg / mL of phloretin; Step 3: Add the aqueous phase of the protein of Rumex patientia L. and the oil phase of phloretin to a stirring kettle according to a volume ratio of 9:1, stir and mix at 20 - 25 °C and 300 - 500 r / min for 5 - 10 min, then transfer it to a high-speed shear dispersion emulsifier, perform high-speed shearing at a shear rate of 10000 - 15000 r / min for 5 - 10 min, perform ultrasonic dispersion at a power of 100 - 500 W for 5 - 25 min, and finally separate and remove the magnetic microsphere auxiliary agent by applying an external magnetic field to obtain an emulsion of the protein of Rumex patientia L. loaded with phloretin, thus completing the preparation method of the emulsion of the protein of Rumex patientia L. loaded with phloretin; The magnetic microsphere auxiliary agent is prepared through the following steps: Add the amino-functionalized composite magnetic microspheres and 1,4-dioxane to a reaction kettle, perform ultrasonic dispersion for 5 - 10 min, then add phthalaldehyde, hydrophilic amino acid and an acetic acid aqueous solution with a molar concentration of 3 mol / L to the reaction kettle, stir and react at 75 - 85 °C and 200 - 300 r / min for 20 - 24 h, separate the product by applying an external magnetic field, wash it 2 - 3 times with acetone and absolute ethanol respectively, and perform vacuum drying to obtain the magnetic microsphere auxiliary agent; The amino-functionalized composite magnetic microspheres are prepared through the following steps: Add the silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane and tetrahydrofuran to a reaction kettle, react at 70 - 75 °C under nitrogen protection for 10 - 12 h, then transfer the product to n-hexane to precipitate, wash the precipitate 2 - 3 times by centrifugation with tetrahydrofuran, and perform vacuum drying on the filter cake to obtain the amino-functionalized composite magnetic microspheres; The silica-coated composite magnetic microspheres are prepared through the following steps: Add the magnetic microspheres of ferric oxide, absolute ethanol and deionized water to a reaction kettle and stir and mix, then add ammonia water with a mass concentration of 25% to the reaction kettle, stir at 200 - 300 r / min for 20 - 30 min, then dropwise add tetraethyl orthosilicate to the reaction kettle, stir and react at 35 - 40 °C and 300 - 500 r / min for 20 - 24 h, separate the product by applying an external magnetic field, wash it 2 - 3 times with absolute ethanol and deionized water respectively, and perform vacuum drying to obtain the silica-coated composite magnetic microspheres.

2. The preparation method of a portulaca oleracea protein emulsion loaded with phloretin according to claim 1, characterized in that, The dosage ratio of the amino-functionalized composite magnetic microspheres, 1,4-dioxane, phthalaldehyde, hydrophilic amino acid and acetic acid aqueous solution is 0.4 - 0.5 g: 25 - 30 mL: 0.65 - 0.7 g: 0.55 - 0.6 g: 0.9 - 1 mL.

3. The preparation method of a Portulaca oleracea L. protein emulsion loaded with phloretin according to claim 1, characterized in that, The hydrophilic amino acid is any one of serine, threonine, aspartic acid and glutamine.

4. The preparation method of a Portulaca oleracea L. protein emulsion loaded with phloretin according to claim 1, characterized in that, The dosage ratio of the silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane and tetrahydrofuran is 1 g: 0.5 - 0.6 g: 25 - 30 mL.

5. The preparation method of a Portulaca oleracea protein emulsion loaded with phloretin according to claim 1, wherein, The dosage ratio of the magnetic iron oxide microspheres, absolute ethanol, deionized water, ammonia water and tetraethyl orthosilicate is 0.4 - 0.5 g: 80 mL: 20 mL: 5 mL: 0.5 mL.

6. The preparation method of a Portulaca oleracea L. protein emulsion loaded with phloretin according to claim 1, characterized in that, The magnetic iron oxide microspheres are prepared by the following steps: Add ethylene glycol and ferric chloride hexahydrate into a reaction kettle, stir at 200 - 300 r / min for 20 - 30 min, then add sodium acetate and polyethylene glycol with a molecular weight of 400 into the reaction kettle, continue to stir for 20 - 30 min, stir and react at 190 - 200 °C for 8 - 10 h, naturally cool, separate the product by an external magnetic field, wash 2 - 3 times with water, and dry in vacuum to obtain the magnetic iron oxide microspheres.

7. The preparation method of a Portulaca oleracea L. protein emulsion loaded with phloretin according to claim 6, characterized in that, The dosage ratio of the ethylene glycol, ferric chloride hexahydrate, sodium acetate and polyethylene glycol is 100 mL: 2.5 - 3 g: 7 - 7.4 g: 0.2 g.

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