Preparation method of phloretin-loaded azolla filliculoidas protein emulsion

Abstract

The invention discloses a preparation method of a phloretin-loaded azolla filliculoidas protein emulsion, and belongs to the technical field of functional factor delivery, the method comprises the following steps: stirring and dissolving azolla filliculoidas protein with a phosphate buffer solution, and then adding a magnetic microsphere auxiliary agent with the mass 2-3 times that of the azolla filliculoidas protein to obtain an azolla filliculoidas protein water phase; stirring and mixing phloretin and linseed oil to obtain a phloretin oil phase; the rumex hanus protein water phase and the phloretin oil phase are stirred and mixed according to the volume ratio of 9: 1, ultrasonic dispersion is performed after high-speed shearing, finally, a magnetic field is applied to separate and remove the magnetic microsphere additive, a phloretin-loaded rumex hanus protein emulsion is obtained, and the rumex hanus protein emulsion is prepared by adding the magnetic microsphere additive and combining ultrasonic dispersion treatment. The rumex hanus protein emulsion loaded with the phloretin is prepared by modifying the phloretin and improving the low water solubility, the instability and the poor biological accessibility of the phloretin, so that the rumex hanus protein emulsion loaded with the phloretin, which is small in particle size, good in dispersity, good in biocompatibility, high in stability and good in packaging effect, can be prepared, and the solubility and the biological accessibility of the phloretin can be well improved.

Description

Technical Field

[0001] The invention belongs to the technical field of functional factor delivery, and in particular relates to a method for preparing a phloretin-loaded chlorophyll protein emulsion. Background Art

[0002] Phloretin, also known as trihydroxyphenol acetone, 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 anti-inflammatory immunity, anti-oxidation, anti-cardiovascular disease, anti-diabetes, anti-tumor and liver protection, and is a potential natural medicine. However, phloretin is sensitive to light and heat, and has poor water solubility, which causes its stability and bioavailability to be very low. These factors greatly limit the application of phloretin in functional foods and the field of medicine. At present, there are many embedded delivery systems that are studied to solve the application problems of phloretin.

[0003] Among the many encapsulation and delivery systems, cyclodextrin can efficiently load phloretin, but its system is single and is not digested in the small intestine, making it difficult to effectively release phloretin to increase its bioaccessibility. Starch is easily absorbed in the human body, but starch-based emulsifiers usually require further modification, and ordinary modified starch has problems such as large particle size, complex structure, and increased starch resistance to digestion, which limits the practical application of starch-based emulsions.

[0004] Plant protein is similar to animal protein and is easily digested and absorbed by the human body. Protein is a natural amphiphilic substance with emulsifying properties. In an oil-water system, protein can spontaneously migrate to the oil-water interface to form a protein adsorption layer, thereby stabilizing the emulsion. Leaf grass protein contains 18 kinds of amino acids, of which hydrophobic amino acids are as high as 45.52%, which can form a hydrophobic cavity to encapsulate hydrophobic flavonoids. Leaf grass protein also has good solubility and thermal stability. When used in a delivery system, it can improve the encapsulation effect and stability of functional factors.

[0005] However, as a natural macromolecular substance, phyllotoxin has a large molecular weight, a slow interfacial adsorption rate during the emulsion formation process, and severe aggregation. Therefore, effective means are often needed to improve the formation of the droplet interface film and the internal structure of the emulsion. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a phloretin-loaded phloretin protein emulsion, by adding a magnetic microsphere auxiliary agent combined with ultrasonic dispersion treatment, the low water solubility, instability and poor bioaccessibility of phloretin are improved, thereby preparing a phloretin-loaded phloretin protein emulsion with small particle size, good dispersibility, good biocompatibility, high stability and good encapsulation effect, which can greatly improve the solubility and bioaccessibility of phloretin.

[0007] The purpose of the present invention can be achieved through the following technical solutions: A method for preparing a phloretin-loaded phloretin protein emulsion comprises the following steps: Step 1: dissolving the foliar grass protein in a phosphate buffer solution with a pH value of 7.8-8.2 by stirring, and then adding a magnetic microsphere auxiliary agent with a mass of 2-3 times that of the foliar grass protein to obtain a foliar grass protein aqueous phase with a mass fraction of 0.1-0.5% of the foliar grass protein; Step 2: stirring and mixing phloretin and linseed oil to obtain a phloretin oil phase with a phloretin mass concentration of 5 mg / mL; Step three: add the chlorophyll protein aqueous phase and the phloretin oil phase into a stirring kettle at a volume ratio of 9:1, stir and mix at 20-25°C and 300-500r / min for 5-10min, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 10000-15000r / min for 5-10min, ultrasonically disperse at a power of 100-500W for 5-25min, and finally apply an external magnetic field to separate and remove the magnetic microsphere auxiliary agent to obtain a chlorophyll protein emulsion loaded with phloretin, thereby completing the preparation method of the chlorophyll protein emulsion loaded with phloretin.

[0008] Furthermore, the magnetic microsphere additive is prepared by the following steps: Step 1: 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 stirring for 20-30 min, stir and react at 190-200° C. for 8-10 h, cool naturally, apply a magnetic field to separate the product, wash with water 2-3 times, and vacuum dry to obtain ferrosoferric oxide magnetic microspheres.

[0009] Furthermore, the usage 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.

[0010] Step 2: Add ferroferric oxide magnetic microspheres, anhydrous ethanol and deionized water into a reactor and stir to mix, then add ammonia water with a mass concentration of 25% into the reactor, stir at 200-300 r / min for 20-30 min, then drop ethyl orthosilicate into the reactor, stir and react at 35-40°C and 300-500 r / min for 20-24 h, separate the product with an external magnetic field, wash with anhydrous ethanol and deionized water for 2-3 times respectively, and vacuum dry to obtain silica-coated composite magnetic microspheres.

[0011] Furthermore, the dosage ratio of ferroferric oxide magnetic microspheres, anhydrous ethanol, deionized water, ammonia water, and tetraethyl orthosilicate is 0.4-0.5 g: 80 mL: 20 mL: 5 mL: 0.5 mL.

[0012] Step 3: Add silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane and tetrahydrofuran into a reaction kettle, react for 10-12 hours under nitrogen protection and 70-75°C, then transfer the product to n-hexane to precipitate, wash the precipitate with tetrahydrofuran by centrifugation for 2-3 times, and vacuum dry the filter cake to obtain amino composite magnetic microspheres.

[0013] Furthermore, the usage ratio of the silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane and tetrahydrofuran is 1 g: 0.5-0.6 g: 25-30 mL.

[0014] Step 4: Add the amino composite magnetic microspheres and 1,4-dioxane into the reactor, ultrasonically disperse for 5-10 minutes, then add isophthalic acid, hydrophilic amino acids and 3 mol / L acetic acid aqueous solution into the reactor, stir and react at 75-85°C and 200-300 r / min for 20-24 hours, separate the products with an external magnetic field, wash with acetone and anhydrous ethanol for 2-3 times respectively, and vacuum dry to obtain a magnetic microsphere additive.

[0015] Furthermore, the dosage ratio of the amino composite magnetic microspheres, 1,4-dioxane, isophthalic acid dialdehyde, 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.

[0016] Furthermore, the hydrophilic amino acid is any one of serine, threonine, aspartic acid and glutamine.

[0017] Beneficial effects of the present invention: The invention uses phylloprotein as an emulsifier, and prepares a phylloprotein emulsion loaded with phloretin by homogenizing a phylloprotein water phase and a phloretin oil phase. The obtained loaded nanoemulsion has small particle size, good stability and is non-toxic.

[0018] In the preparation process of the phloretin-loaded phylloretin protein emulsion of the present invention, a method of high-speed shearing combined with ultrasonic homogenization is adopted, and the droplets of the prepared phloretin-loaded phylloretin protein emulsion are uniformly dispersed, with small particle size, high stability and high loading efficiency; and 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 also induce the denaturation of protein molecules in the system without using chemical additives, thereby improving the properties of the emulsion. At the same time, the introduction of ultrasonic dispersion can also suppress the change of the droplet charge, so that the thermal stability, storage stability and salt ion stability of the emulsion are all improved to varying degrees.

[0019] The present invention adds a magnetic microsphere auxiliary agent to the phylloprotein aqueous phase, which can play the role of grinding balls and stabilizers in the emulsion, and helps to increase the shearing effect of high-speed shearing and ultrasonic dispersion. The surface of the magnetic microsphere auxiliary agent contains a large number of hydrophilic groups, which can be adsorbed on the oil-water interface to form a protective film to prevent the aggregation between droplets, and the magnetic microsphere auxiliary agent interacts with the hydrophobic cavity of the phylloprotein, which helps to guide the orderly arrangement and assembly of the phylloprotein and phloretin molecules, plays the effect of regular morphology, further enhances the phylloprotein entrapment effect on phloretin and reduces the droplet particle size. The magnetic microsphere auxiliary agent can be effectively separated and removed by an external magnetic field, can be reused repeatedly, and is safe and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings.

[0021] Figure 1 These are laser confocal images of different emulsions in Examples 1 to 5 of the present invention; Figure 2 It is a comparison diagram of different emulsions in Examples 1 to 5 of the present invention and Comparative Example 1 before and after storage; Figure 3 is a bar graph of the loading amounts of different emulsions in Examples 1 to 5 of the present invention and Comparative Example 2; Figure 4 It is a comparison diagram of the particle sizes of different emulsions in Examples 1 to 5 of the present invention and Comparative Example 2; Figure 5 These are laser confocal images of different emulsions in Comparative Examples 2 and 3 of the present invention; Figure 6 It is a comparison diagram of different emulsions in Examples 5 to 9 of the present invention and Comparative Example 3 before and after storage; Figure 7 is a bar graph of the loading amounts of different emulsions in Examples 5 to 9 of the present invention and Comparative Example 3; Figure 8 It is a comparison chart of the particle sizes of different emulsions in Examples 5 to 9 of the present invention and Comparative Example 3. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1: This example provides a phloretin-loaded phloretin protein emulsion, which is prepared by the following method: S1: Add 100L ethylene glycol and 2.5kg ferric chloride hexahydrate into a reactor, stir at 200r / min for 20min, then add 7kg sodium acetate and 0.2kg polyethylene glycol with a molecular weight of 400 into the reactor, continue stirring for 20min, stir the reaction at 190°C for 8h, cool naturally, separate the product with an external magnetic field, wash it with water twice, and dry it in vacuum to obtain ferrosoferric oxide magnetic microspheres.

[0024] S2: Add 0.4 kg of ferrosoferric oxide magnetic microspheres, 80 L of anhydrous ethanol and 20 L of deionized water into a reactor and stir to mix. Then, add 5 L of 25% ammonia water into the reactor and stir at 200 r / min for 20 min. Then, add 0.5 L of tetraethyl orthosilicate dropwise into the reactor and stir to react at 35 °C and 300 r / min for 20 h. Separate the product with an external magnetic field, wash twice with anhydrous ethanol and deionized water respectively, and dry in vacuo to obtain silica-coated composite magnetic microspheres.

[0025] S3: 100 g of silica-coated composite magnetic microspheres, 50 g of 3-aminopropyltriethoxysilane and 2.5 L of tetrahydrofuran were added into a reactor, and the reaction was carried out for 10 h under nitrogen protection at 70° C. The product was then transferred into n-hexane for precipitation. The precipitate was washed twice by centrifugation with tetrahydrofuran, and the filter cake was vacuum dried to obtain amino composite magnetic microspheres.

[0026] S4: 40 g of amino composite magnetic microspheres and 2.5 L of 1,4-dioxane were added to the reactor and ultrasonically dispersed for 5 min. Then, 65 g of isophthalic dialdehyde, 55 g of serine and 90 mL of 3 mol / L acetic acid aqueous solution were added to the reactor. The reaction was stirred at 75 ° C and 250 r / min for 22 h. The product was separated by an external magnetic field, washed twice with acetone and anhydrous ethanol respectively, and vacuum dried to obtain a magnetic microsphere additive.

[0027] S5: Dissolve the chlorophyll protein by stirring in a phosphate buffer solution with a pH value of 7.8, and then add a magnetic microsphere auxiliary agent with a mass fraction of 2 times that of the chlorophyll protein to obtain a chlorophyll protein aqueous phase with a mass fraction of 0.1% of the chlorophyll protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0028] S6: Add the aqueous phase of chlorophyll protein and the oil phase of phloretin into a stirring tank at a volume ratio of 9:1, stir and mix at 20°C and 300 r / min for 5 minutes, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 10,000 r / min for 5 minutes, ultrasonically disperse at a power of 100 W for 5 minutes, and finally apply an external magnetic field to separate and remove the magnetic microsphere additive to obtain a chlorophyll protein emulsion loaded with phloretin.

[0029] Example 2: This example provides a phloretin-loaded phloretin protein emulsion, which is prepared by the following method: S1: Add 100L ethylene glycol and 2.6kg ferric chloride hexahydrate into a reactor, stir at 2200r / min for 22min, then add 7.1kg sodium acetate and 0.2kg polyethylene glycol with a molecular weight of 400 into the reactor, continue stirring for 22min, stir the reaction at 195°C for 8.5h, cool naturally, separate the product with an external magnetic field, wash it with water twice, and vacuum dry it to obtain ferrosoferric oxide magnetic microspheres.

[0030] S2: Add 0.42 kg of ferroferric oxide magnetic microspheres, 80 L of anhydrous ethanol and 20 L of deionized water into a reactor and stir to mix. Then, add 5 L of 25% ammonia water into the reactor and stir at 220 r / min for 22 min. Then, add 0.5 L of tetraethyl orthosilicate dropwise into the reactor and stir to react at 36 °C and 350 r / min for 21 h. Separate the product with an external magnetic field, wash twice with anhydrous ethanol and deionized water respectively, and dry in vacuo to obtain silica-coated composite magnetic microspheres.

[0031] S3: 100 g of silica-coated composite magnetic microspheres, 52 g of 3-aminopropyltriethoxysilane and 2.8 L of tetrahydrofuran were added into a reactor, and the reaction was carried out for 11 h under nitrogen protection and 72 °C. The product was then transferred into n-hexane for precipitation. The precipitate was washed twice by centrifugation with tetrahydrofuran, and the filter cake was vacuum dried to obtain amino composite magnetic microspheres.

[0032] S4: 42 g of amino composite magnetic microspheres and 2.8 L of 1,4-dioxane were added to the reactor and ultrasonically dispersed for 8 min. Then, 68 g of isophthalic dialdehyde, 58 g of threonine and 95 mL of 3 mol / L acetic acid aqueous solution were added to the reactor. The reaction was stirred at 80 ° C and 220 r / min for 21 h. The product was separated by an external magnetic field, washed twice with acetone and anhydrous ethanol respectively, and vacuum dried to obtain a magnetic microsphere additive.

[0033] S5: Dissolve the chlorophyll protein by stirring in a phosphate buffer solution with a pH value of 8, and then add a magnetic microsphere auxiliary agent with a mass fraction of 2.5 times that of the chlorophyll protein to obtain a chlorophyll protein aqueous phase with a mass fraction of 0.2% of the chlorophyll protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0034] S6: Add the aqueous phase of phylloprotein and the oil phase of phloretin into a stirring tank at a volume ratio of 9:1, stir and mix at 22°C and 350 r / min for 6 min, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 12000 r / min for 8 min, ultrasonically disperse at a power of 200 W for 10 min, and finally apply an external magnetic field to separate and remove the magnetic microsphere additive to obtain a phylloprotein emulsion loaded with phloretin.

[0035] Example 3: This example provides a phloretin-loaded phloretin protein emulsion, which is prepared by the following method: S1: Add 100L ethylene glycol and 2.8kg ferric chloride hexahydrate into a reactor, stir at 250r / min for 25min, then add 7.2kg sodium acetate and 0.2kg polyethylene glycol with a molecular weight of 400 into the reactor, continue stirring for 25min, stir the reaction at 195°C for 9h, cool naturally, separate the product with an external magnetic field, wash it with water 3 times, and vacuum dry it to obtain ferrosoferric oxide magnetic microspheres.

[0036] S2: Add 0.45 kg of ferrosoferric oxide magnetic microspheres, 80 L of anhydrous ethanol and 20 L of deionized water into a reactor and stir to mix. Then, add 5 L of 25% ammonia water into the reactor and stir at 250 r / min for 25 min. Then, add 0.5 L of tetraethyl orthosilicate dropwise into the reactor and stir to react at 38 °C and 400 r / min for 22 h. Separate the product with an external magnetic field, wash twice with anhydrous ethanol and deionized water respectively, and dry in vacuo to obtain silica-coated composite magnetic microspheres.

[0037] S3: 100 g of silica-coated composite magnetic microspheres, 55 g of 3-aminopropyltriethoxysilane and 2.8 L of tetrahydrofuran were added into a reactor, and the reaction was carried out for 11 h under nitrogen protection and 72 °C. The product was then transferred into n-hexane for precipitation. The precipitate was washed twice by centrifugation with tetrahydrofuran, and the filter cake was vacuum dried to obtain amino composite magnetic microspheres.

[0038] S4: 45 g of amino composite magnetic microspheres and 2.8 L of 1,4-dioxane were added to the reactor and ultrasonically dispersed for 8 min. Then, 68 g of isophthalic acid, 58 g of aspartic acid and 95 mL of 3 mol / L acetic acid aqueous solution were added to the reactor. The reaction was stirred at 80 ° C and 250 r / min for 22 h. The product was separated by an external magnetic field, washed twice with acetone and anhydrous ethanol respectively, and vacuum dried to obtain a magnetic microsphere additive.

[0039] S5: Dissolve the chlorophyll protein by stirring in a phosphate buffer solution with a pH value of 8, and then add a magnetic microsphere auxiliary agent with a mass fraction of 2.5 times that of the chlorophyll protein to obtain a chlorophyll protein aqueous phase with a mass fraction of 0.3% of the chlorophyll protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0040] S6: Add the chlorophyll protein aqueous phase and the phloretin oil phase into a stirring tank at a volume ratio of 9:1, stir and mix at 23°C and 400 r / min for 8 minutes, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 13000 r / min for 8 minutes, ultrasonically disperse at a power of 300 W for 15 minutes, and finally apply an external magnetic field to separate and remove the magnetic microsphere additive to obtain a chlorophyll protein emulsion loaded with phloretin.

[0041] Example 4: This example provides a phloretin-loaded phloretin protein emulsion, which is prepared by the following method: S1: Add 100L ethylene glycol and 2.8kg ferric chloride hexahydrate into a reactor, stir at 280r / min for 28min, then add 7.3kg sodium acetate and 0.2kg polyethylene glycol with a molecular weight of 400 into the reactor, continue stirring for 28min, stir the reaction at 195°C for 9h, cool naturally, separate the product with an external magnetic field, wash with water 3 times, and vacuum dry to obtain ferrosoferric oxide magnetic microspheres.

[0042] S2: Add 0.48 kg of ferrosoferric oxide magnetic microspheres, 80 L of anhydrous ethanol and 20 L of deionized water into a reactor and stir to mix. Then, add 5 L of 25% ammonia water into the reactor and stir at 280 r / min for 28 min. Then, add 0.5 L of tetraethyl orthosilicate dropwise into the reactor and stir to react at 38 °C and 450 r / min for 23 h. Separate the product with an external magnetic field, wash it with anhydrous ethanol and deionized water for 3 times respectively, and dry it in vacuo to obtain silica-coated composite magnetic microspheres.

[0043] S3: 100 g of silica-coated composite magnetic microspheres, 58 g of 3-aminopropyltriethoxysilane and 2.8 L of tetrahydrofuran were added into a reactor, and the reaction was carried out for 11 h under nitrogen protection and 73 °C. The product was then transferred into n-hexane for precipitation. The precipitate was washed three times by centrifugation with tetrahydrofuran, and the filter cake was vacuum dried to obtain amino composite magnetic microspheres.

[0044] S4: 48 g of amino composite magnetic microspheres and 2.8 L of 1,4-dioxane were added to the reactor and ultrasonically dispersed for 8 min. Then, 68 g of isophthalic dialdehyde, 58 g of glutamine and 98 mL of 3 mol / L acetic acid aqueous solution were added to the reactor. The reaction was stirred at 82 ° C and 280 r / min for 23 h. The product was separated by an external magnetic field, washed with acetone and anhydrous ethanol for 3 times respectively, and vacuum dried to obtain a magnetic microsphere additive.

[0045] S5: Dissolve the chlorophyll protein by stirring in a phosphate buffer solution with a pH value of 8, and then add a magnetic microsphere auxiliary agent with a mass fraction of 2.5 times that of the chlorophyll protein to obtain a chlorophyll protein aqueous phase with a mass fraction of 0.4% of the chlorophyll protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0046] S6: Add the chlorophyll protein aqueous phase and the phloretin oil phase into a stirring tank at a volume ratio of 9:1, stir and mix at 23°C and 450 r / min for 8 minutes, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 14000 r / min for 8 minutes, ultrasonically disperse at a power of 400 W for 20 minutes, and finally apply an external magnetic field to separate and remove the magnetic microsphere additive to obtain a chlorophyll protein emulsion loaded with phloretin.

[0047] Example 5: This example provides a phloretin-loaded phloretin protein emulsion, which is prepared by the following method: S1: Add 100L ethylene glycol and 3kg ferric chloride hexahydrate into a reactor, stir at 300r / min for 30min, then add 7.4kg sodium acetate and 0.2kg polyethylene glycol with a molecular weight of 400 into the reactor, continue stirring for 30min, stir the reaction at 200°C for 10h, cool naturally, separate the product with an external magnetic field, wash it with water 3 times, and vacuum dry it to obtain ferrosoferric oxide magnetic microspheres.

[0048] S2: Add 0.5 kg of ferroferric oxide magnetic microspheres, 80 L of anhydrous ethanol and 20 L of deionized water into a reactor and stir to mix. Then, add 5 L of 25% ammonia water into the reactor and stir at 300 r / min for 30 min. Then, add 0.5 L of tetraethyl orthosilicate dropwise into the reactor and stir to react at 40 °C and 500 r / min for 24 h. Separate the product with an external magnetic field, wash it with anhydrous ethanol and deionized water for 3 times respectively, and dry it in vacuum to obtain silica-coated composite magnetic microspheres.

[0049] S3: 100 g of silica-coated composite magnetic microspheres, 60 g of 3-aminopropyltriethoxysilane and 3 L of tetrahydrofuran were added into a reactor, and the reaction was carried out for 12 h under nitrogen protection at 75 °C. The product was then transferred into n-hexane for precipitation. The precipitate was washed three times by centrifugation with tetrahydrofuran, and the filter cake was vacuum dried to obtain amino composite magnetic microspheres.

[0050] S4: Add 50g of amino composite magnetic microspheres and 3L of 1,4-dioxane into the reactor, ultrasonically disperse for 10min, then add 70g of isophthalic dialdehyde, 60g of serine and 100mL of 3mol / L acetic acid aqueous solution into the reactor, stir and react at 85°C and 300r / min for 24h, separate the product with an external magnetic field, wash with acetone and anhydrous ethanol for 3 times respectively, and vacuum dry to obtain a magnetic microsphere additive.

[0051] S5: Dissolve the chlorophyll protein by stirring in a phosphate buffer solution with a pH value of 8.2, and then add a magnetic microsphere auxiliary agent with a mass fraction of 3 times that of the chlorophyll protein to obtain a chlorophyll protein aqueous phase with a mass fraction of 0.5% of the chlorophyll protein; stir and mix phloretin and linseed oil to obtain a phloretin oil phase with a mass concentration of 5 mg / mL of phloretin.

[0052] S6: Add the chlorophyll protein aqueous phase and the phloretin oil phase into a stirring tank at a volume ratio of 9:1, stir and mix at 25°C and 500 r / min for 10 min, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 15000 r / min for 10 min, ultrasonically disperse at a power of 500 W for 25 min, and finally apply an external magnetic field to separate and remove the magnetic microsphere auxiliary agent to obtain a chlorophyll protein emulsion loaded with phloretin.

[0053] The phloretin-loaded phylloxicam protein emulsions in Examples 1 to 5 were characterized. Figure 1 These are laser confocal images of different emulsions (scale size is 10μm). They are analyzed from left to right. The first column shows the emulsion oil droplets stained green by Nile red, the second column shows the phyllophagous protein stained red by Nile blue, the third column is the overall image, and the fourth column is the local magnified image (scale bar is 2μm).

[0054] Depend on Figure 1 It can be seen that the laser confocal images of the phlorothalynx protein emulsions loaded with phlorothalynx prepared in Examples 1 to 5 with different phlorothalynx contents showed that the green oil droplets were surrounded by the red protein solution, indicating that an oil-in-water emulsion was formed.

[0055] And within the range of foliar grass content <0.3% (Example 1-Example 3), as the foliar grass protein content increases, the droplet size of the emulsion decreases and becomes more evenly distributed. When the foliar grass protein content is 0.3%, the droplet size of the emulsion is the smallest and the distribution is most even.

[0056] Comparative Example 1: Based on Example 1, in step S6, the phlorothalyngo protein aqueous phase is directly replaced by a phosphate buffer solution having a pH value of 7.8, and the other steps remain unchanged to prepare a phlorothalyngo emulsion.

[0057] The phloretin-loaded herbaceous protein emulsions in Examples 1 to 5 and the phloretin emulsion in Comparative Example 1 were bottled and stored for 30 days. The results are as follows: Figure 2 shown.

[0058] Depend on Figure 2 It can be seen that the newly prepared phloretin-loaded phloretin emulsions prepared with different phloretin contents are light yellow overall and have good uniformity. After storage for 30 days, except for the obvious stratification phenomenon in the control group without phloretin, the phloretin-loaded phloretin emulsions prepared with low phloretin content in Examples 1-2 showed a little stratification, while the phloretin-loaded phloretin emulsions in Examples 3-5 were still stable and did not show stratification, indicating that the emulsions loaded with phloretin have better stability.

[0059] Comparative Example 2: Based on Example 1, no magnetic microsphere auxiliary agent is added when preparing the phlorothalyngo protein aqueous phase in step S5, and the other steps remain unchanged to prepare a phlorothalyngo protein emulsion loaded with phlorothalyngo protein.

[0060] The loading amount bar graph of the phloretin-loaded ... Figure 3 ) and particle size comparison chart (see Figure 4) It can be seen that with the increase of the content of chlorophyll protein, the loading amount of chlorophyll protein emulsion on phloretin shows a trend of first increasing and then decreasing. In Example 3, when the content of chlorophyll protein is 0.3%, the loading amount is the largest, reaching 12.45%, and the particle size of the chlorophyll protein emulsion loaded with phloretin in Example 3 is the smallest, and the particle size distribution is the most uniform. In Comparative Example 2, the loading amount of the chlorophyll protein emulsion loaded with phloretin decreases, and the particle size becomes larger, indicating that the addition of magnetic microsphere additives can reduce the particle size of the chlorophyll protein emulsion loaded with phloretin and increase the loading amount of phloretin.

[0061] Example 6: Based on Example 5, the power of ultrasonic dispersion was adjusted to 400 W, and the other steps remained unchanged to prepare a phloretin emulsion.

[0062] Example 7: Based on Example 5, the power of ultrasonic dispersion was adjusted to 300 W, and the other steps remained unchanged to prepare a phloretin emulsion.

[0063] Example 8: Based on Example 5, the power of ultrasonic dispersion was adjusted to 200 W, and the other steps remained unchanged to prepare a phloretin emulsion.

[0064] Example 9: Based on Example 5, the power of ultrasonic dispersion was adjusted to 100 W, and the other steps remained unchanged to prepare a phloretin emulsion.

[0065] Comparative Example 3: Based on Example 5, the phloretin emulsion was prepared without ultrasonic dispersion treatment and the other steps remained unchanged.

[0066] The phlorothalon protein emulsions loaded with phloretin in Comparative Examples 2 and 3 were characterized. Figure 5 The laser confocal images of each emulsion (scale size is 10μm). The droplet sizes of the two emulsions are relatively large. It can be analyzed that the addition of magnetic microsphere additives and ultrasonic assistance are helpful to reduce the droplet size.

[0067] The phloretin-loaded phloretin emulsions in Examples 5 to 9 and Comparative Example 3 were bottled and stored for 30 days. Figure 6 shown.

[0068] Depend on Figure 6 It can be seen that the phloretin-loaded phloretin protein emulsion prepared by the newly prepared ultrasonic power is light yellow overall and has good homogeneity. After storage for 30 days, except for the obvious stratification phenomenon in the bottle without ultrasonic treatment in Comparative Example 3, the phloretin-loaded phloretin protein emulsion prepared by low ultrasonic power in Example 9 showed a little stratification phenomenon, and the phloretin-loaded phloretin protein emulsion prepared in Examples 5-8 was still stable without stratification phenomenon.

[0069] The loading amount bar graph of the phloretin-loaded ... Figure 7 ) and particle size comparison chart (see Figure 8 ) It can be seen that with the increase of ultrasonic power, the loading amount of phloretin by the chlorophyll protein emulsion increases first and then decreases. When the ultrasonic power in Example 6 is 400 W, the loading amount is the largest, and under this ultrasonic dispersion power, the particle size of the chlorophyll protein emulsion loaded with phloretin is the smallest and the particle size distribution is the most uniform.

[0070] Determination of the particle size of the phloretin-loaded herbivorous protein emulsion: The particle size of the phloretin-loaded herbivorous protein emulsion was determined using a dynamic light scattering instrument (ZSU3100, Malvern Instruments Ltd., UK).

[0071] Morphological observation of phloretin-loaded herbivorous protein emulsion: The morphology of phloretin-loaded herbivorous protein emulsion was observed using a Leica laser scanning confocal microscope (STELLARIS 5, Germany).

[0072] Determination of the loading rate of the phloretin-loaded chlorophyll protein emulsion: The loading rate of the phloretin-loaded chlorophyll protein emulsion was determined using a fully automatic multifunctional microplate reader (HH3500, Pontyclum, UK).

[0073] It should be noted that, in this article, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0074] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a phloretin-loaded herbaceous plant protein emulsion, characterized in that: The steps include: Step 1: dissolving the foliar grass protein in a phosphate buffer solution with a pH value of 7.8-8.2 by stirring, and then adding a magnetic microsphere auxiliary agent with a mass of 2-3 times that of the foliar grass protein to obtain a foliar grass protein aqueous phase with a mass fraction of 0.1-0.5% of the foliar grass protein; Step 2: stirring and mixing phloretin and linseed oil to obtain a phloretin oil phase with a phloretin mass concentration of 5 mg / mL; Step three: add the chlorophyll protein aqueous phase and the phloretin oil phase into a stirring kettle at a volume ratio of 9:1, stir and mix at 20-25°C and 300-500r / min for 5-10min, then transfer to a high-speed shear dispersing emulsifier, high-speed shear at a shear rate of 10000-15000r / min for 5-10min, ultrasonically disperse at a power of 100-500W for 5-25min, finally apply an external magnetic field to separate and remove the magnetic microsphere auxiliary agent to obtain a chlorophyll protein emulsion loaded with phloretin, thereby completing the preparation method of the chlorophyll protein emulsion loaded with phloretin.

2. The method for preparing a phloretin-loaded herbaceous plant protein emulsion according to claim 1, characterized in that: The magnetic microsphere additive is prepared by the following steps: The aminated composite magnetic microspheres and 1,4-dioxane are added to a reactor, ultrasonically dispersed for 5-10 minutes, and then isophthalic acid, hydrophilic amino acids and an acetic acid aqueous solution with a molar concentration of 3 mol / L are added to the reactor. The reaction is stirred at 75-85° C. and 200-300 r / min for 20-24 hours. The product is separated by an external magnetic field, washed with acetone and anhydrous ethanol for 2-3 times respectively, and vacuum dried to obtain a magnetic microsphere additive.

3. The method for preparing a phloretin-loaded herbaceous plant protein emulsion according to claim 2, characterized in that: The dosage ratio of the amino composite magnetic microspheres, 1,4-dioxane, isophthalic acid dialdehyde, 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.

4. The method for preparing a phloretin-loaded herbaceous plant protein emulsion according to claim 2, characterized in that: The hydrophilic amino acid is any one of serine, threonine, aspartic acid and glutamine.

5. The method for preparing a phloretin-loaded phylloprotein emulsion according to claim 2, characterized in that: The amino composite magnetic microspheres are prepared by the following steps: The silica-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane and tetrahydrofuran were added into a reaction kettle, and the reaction was carried out for 10-12 hours under nitrogen protection and 70-75° C., and then the product was transferred into n-hexane to precipitate, and the precipitate was centrifugally washed with tetrahydrofuran for 2-3 times, and the filter cake was vacuum dried to obtain the amino composite magnetic microspheres.

6. The method for preparing a phloretin-loaded herbaceous plant protein emulsion according to claim 5, characterized in that: The dosage ratio of the silicon dioxide-coated composite magnetic microspheres, 3-aminopropyltriethoxysilane and tetrahydrofuran is 1 g: 0.5-0.6 g: 25-30 mL.

7. The method for preparing a phloretin-loaded herb protein emulsion according to claim 5, characterized in that: The silica-coated composite magnetic microspheres are prepared by the following steps: Add ferroferric oxide magnetic microspheres, anhydrous ethanol and deionized water into a reactor and stir to mix, then add ammonia water with a mass concentration of 25% into the reactor, stir at 200-300 r / min for 20-30 min, then drop ethyl orthosilicate into the reactor, stir and react at 35-40° C. and 300-500 r / min for 20-24 h, separate the product with an external magnetic field, wash with anhydrous ethanol and deionized water for 2-3 times respectively, and vacuum dry to obtain silica-coated composite magnetic microspheres.

8. The method for preparing a phloretin-loaded herbaceous plant protein emulsion according to claim 7, characterized in that: The dosage ratio of the ferroferric oxide magnetic microspheres, anhydrous ethanol, deionized water, ammonia water and ethyl orthosilicate is 0.4-0.5 g: 80 mL: 20 mL: 5 mL: 0.5 mL.

9. The method for preparing a phloretin-loaded phylloprotein emulsion according to claim 7, characterized in that: The ferroferric oxide magnetic 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 stirring for 20-30 min, stir and react at 190-200° C. for 8-10 h, cool naturally, separate the product with an external magnetic field, wash with water 2-3 times, and vacuum dry to obtain ferrosoferric oxide magnetic microspheres.

10. The method for preparing a phloretin-loaded phylloprotein emulsion according to claim 9, characterized in that: The usage 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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