Vegetable protein microspheres and controllable preparation method thereof

Abstract

The invention discloses a vegetable protein microsphere and a controllable preparation method thereof. The preparation method comprises the following steps: dissolving Ca-EDTA in deionized water, and adding sodium alginate to obtain a solution A; dissolving zein in an organic solvent to obtain a solution B; a mixed solution of the solution A and the solution B is a dispersion phase W; preparing an oil solution of a lipophilic surfactant as a continuous phase O; dissolving a lipophilic surfactant and glacial acetic acid in an oily solvent to obtain a solution which is a pre-gel phase P; dissolving calcium chloride in deionized water, and adding a hydrophilic surfactant to obtain a solution which is a collection solution Wo; the W, the O and the P respectively pass through a glass capillary device and are sheared to obtain pre-gelatinized ALG-ZEIN microspheres, the pre-gelatinized ALG-ZEIN microspheres are received in the Wo to be gelatinized and cleaned, and then the plant protein microspheres are obtained. According to the vegetable protein microspheres prepared by the invention, hydrophobic natural macromolecules can provide adhesion sites which can be recognized by cell surface integrin, and the vegetable protein microspheres have good biodegradability and biocompatibility; the content of the plant protein can be used for adjusting the mechanical property and the hydrophilic-hydrophobic property of the microspheres.

Description

Technical Field

[0001] The invention relates to the technical field of degradable microspheres and their application in the field of cell proliferation, and in particular to plant protein microspheres and a controllable preparation method thereof. Background Art

[0002] In recent years, microspheres have received extensive attention in the field of cell culture and regenerative medicine. These microspheres are a type of biologically functional microspherical scaffold with particle sizes ranging from tens of microns to hundreds of microns. As a new type of cell scaffold, microspheres have many advantages that other scaffolds cannot match, such as providing a high surface area, carrying a large number of cells, and maintaining cell phenotype and differentiation ability. At present, a variety of biomaterials, including bioactive inorganic materials, natural and synthetic polymers and their composites, have been developed for the preparation of cell culture microspheres. Among them, calcium alginate, as a natural macromolecular material, frequently appears as a matrix material. The conditions for the formation of calcium alginate gel are mild, and the preparation process does not involve chemicals that are detrimental to cell activity. Therefore, calcium alginate is often used for surface loading / encapsulation of cells to achieve in vitro cell expansion and cell delivery. However, calcium alginate itself does not contain adhesion sites that can be recognized by cell surface integrins, which limits the cell's ability to attach. The introduction of hydrophobic polymers may provide an environment for cell proliferation. At the same time, protein macromolecules from natural sources can form better interactions with cells due to their interactions with membrane receptors. Over the years, interfacial properties have been recognized to play an increasingly important role in a wide range of cellular functions, from cell adhesion to mechanics. The hydrophobicity of functional substrates of biomacromolecules shows a strong correlation with observed cellular behaviors.

[0003] The traditional preparation methods of microspheres mainly include emulsification, spray drying and supercritical CO 2 Methods, etc. These different preparation methods will cause differences in the properties of the microspheres, such as morphology, internal structure and cell adhesion rate, and these differences directly affect whether the microspheres can effectively promote cell adhesion and proliferation and their subsequent applications. Therefore, it is crucial to select and optimize the method for preparing microspheres. The more uniform the diameter of the microspheres, the more helpful it is to accurately evaluate the cell response to them. In addition, microspheres with hydrophobic plant protein can improve the mechanical properties and hydrophilicity of the microspheres by regulating the content of plant protein, increase the initial adhesion ability of the microspheres to cells, and the plant protein does not affect the biological activity, has biocompatibility, and is degradable during the cell culture process. Therefore, it has significant application advantages in cell culture amplification and culturing artificial meat. Therefore, the present invention provides a plant protein microsphere and a controllable preparation method thereof. Summary of the invention

[0004] The object of the present invention is to provide a plant protein microsphere and a controllable preparation method thereof. The plant protein microsphere of the present invention can be used for cell expansion and the culture process of artificial meat. The present invention preferably uses alginate that meets production requirements and has biocompatibility, and hydrophobic plant protein, and promotes the adhesion of microspheres to cells through a reasonable component ratio, thereby improving the proliferation efficiency and realizing cell expansion. The preparation method of the present invention uses oil-in-water emulsion and endogenous and exogenous calcium cross-linking method as principles, and through a specific glass capillary device, tube orifice diameter and flow rate control, as well as regulation of plant protein content, the microspheres have single dispersion, suitable hydrophobicity and mechanical strength.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A plant protein microsphere, wherein the plant protein microsphere is composed of calcium alginate as a main framework component and zein as a functional component, and has a uniform particle size; calculated based on the total mass percentage (dry weight) of the plant protein microsphere, the mass percentage of the main framework component calcium alginate is 80% to 90%, and the mass percentage of the functional component zein is 10% to 20%.

[0007] The main framework component of the above-mentioned plant protein microspheres is calcium alginate, and the functional component is hydrophobic zein. Calcium alginate serves as the main material of the microspheres to support the integrity of the microspheres. At the same time, with the help of the hydrophobicity of zein, on the one hand, it can reduce the initial water absorption of the microspheres and reduce the swelling degree of the microspheres in the physiological environment of the cells. On the other hand, the improvement of hydrophobicity is conducive to the adhesion of cells, thereby increasing the cell proliferation rate. With the help of calcium alginate and zein as microsphere components, the microspheres become good tissue engineering scaffolds, which are suitable for practical directions such as cell expansion and cultivation of artificial meat.

[0008] Preferably, the content of guluronic acid in the calcium alginate is greater than 60% by mass.

[0009] Preferably, the zein is α-zein, which can be dissolved in an alcohol aqueous solution with a volume fraction of 60% to 95%.

[0010] The present application also claims a controllable preparation method of plant protein microspheres, comprising the following steps:

[0011] (1) dissolving calcium disodium ethylenediaminetetraacetate (Ca-EDTA) in deionized water to obtain a calcium disodium ethylenediaminetetraacetate (Ca-EDTA) aqueous solution, and then adding sodium alginate thereto and stirring until dissolved to obtain a solution A;

[0012] (2) dissolving zein in an organic solvent to obtain solution B;

[0013] (3) mixing solution A obtained in step (1) with solution B obtained in step (2), heating and stirring until the mixture is uniformly mixed, and the resulting mixed solution is used as dispersed phase W;

[0014] (4) preparing an oil solution of a lipophilic surfactant and using it as a continuous phase O;

[0015] (5) dissolving a lipophilic surfactant and glacial acetic acid in an oily solvent, and the resulting solution is used as a pre-gel phase P;

[0016] (6) dissolving calcium chloride in deionized water, and then adding a hydrophilic surfactant thereto, and the resulting solution is used as the collection liquid Wo;

[0017] (7) passing the dispersed phase W, the continuous phase O and the pre-gel phase P through a glass capillary device at set flow rates, respectively, and obtaining pre-gelled calcium alginate-zein (ALG-ZEIN) microspheres after shearing;

[0018] (8) The pre-gelled calcium alginate-zein (ALG-ZEIN) microspheres obtained in step (7) are received in the collection liquid Wo of step (6), gelled under low-speed stirring, and washed with deionized water to obtain the plant protein microspheres.

[0019] Preferably, the diameter of the plant protein microspheres is regulated by the flow rates of the dispersed phase W and the continuous phase O or the size of the dispersed phase and the continuous phase glass capillary microchannel or the pipeline combination; the flow rate of the dispersed phase W is 0.2mL / h~0.6mL / h, and the flow rate of the continuous phase O is 1mL / h~10mL / h; the diameter of the channel I through which the dispersed phase passes is 100μm~500μm, and the diameter of the channel II through which the continuous phase O passes is 400μm~800μm; the pipeline combination is selected from one of the Y-type, T-type, cross-type, co-flow type, and flow focusing type.

[0020] Preferably, the mechanical properties and hydrophilicity of the plant protein microspheres are regulated by the concentrations of solution A and solution B and the volume ratio during mixing; the concentration of calcium disodium ethylenediaminetetraacetate (Ca-EDTA) in solution A is 20mM to 70mM, and the concentration of sodium alginate is 0.2wt% to 2.5wt%; the concentration of zein in solution B is 0.3wt% to 3.0wt%.

[0021] Preferably, the flow rate of the pregel phase P is 15 mL / h to 25 mL / h; the concentration of acetic acid in the pregel phase P is 0.5 wt% to 2.5 wt%; and the concentration of calcium chloride in the collected liquid Wo is 1 wt% to 8 wt%.

[0022] Preferably, the oily solvent is selected from one of oleic acid, liquid paraffin, and soybean oil; the organic solvent is selected from one of 60% to 80% ethanol aqueous solution, a strong base, and sodium dodecyl sulfate.

[0023] Preferably, the lipophilic surfactant is selected from one of Span 85 and Span 80; the concentration of the lipophilic surfactant in the continuous phase O and the pregel phase P is 1wt% to 5wt%; the hydrophilic surfactant is selected from one of Tween 80, polyvinyl alcohol (PVA), and polyvinyl pyrrolidone (PVP); the concentration of the hydrophilic surfactant in the collected liquid Wo is 1wt% to 5wt%

[0024] Preferably, in step (8), the rotation speed of the low-speed stirring is 100 to 400 rpm, and the time is 1 to 12 hours.

[0025] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0026] 1. The present invention uses two natural macromolecular components with biocompatibility and biodegradability as the supporting material and functional material of the microspheres, and through the reasonable composition ratio of the two natural components, the microspheres have a stable spherical structure and suitable properties for cell adhesion and proliferation;

[0027] 2. The present invention utilizes the shear force and surface tension between microfluids to prepare droplets with uniform particle size, mixes two natural ingredients, and disperses the plant protein in the sodium alginate solution; and based on the principle of the endogenous and exogenous calcium cross-linking method, forms gel microspheres with calcium alginate as the main sphere and plant protein dispersed therein. The microspheres have uniform particle size and good mechanical properties, and the introduction of hydrophobic plant protein improves the adhesion of the microspheres to cells, thereby improving cell proliferation, which is significantly superior to the methods of the prior art;

[0028] 3. The plant protein microspheres of the present invention can be used in the process of cell expansion and artificial meat culture. The present invention preferably uses alginate that meets production requirements and has biocompatibility, and hydrophobic plant protein, and promotes the adhesion of microspheres to cells through a reasonable component ratio, thereby improving proliferation efficiency and achieving cell expansion; the preparation method of the present invention uses oil-in-water emulsion and endogenous and exogenous calcium cross-linking method as principles, and through a specific glass capillary device, orifice diameter and flow rate control, as well as regulation of plant protein content, the microspheres have monodispersity, suitable hydrophobicity and mechanical strength;

[0029] 4. The plant protein microspheres prepared by the present invention have hydrophobic natural macromolecules that can provide adhesion sites recognizable by cell surface integrins, and have good biodegradability and biocompatibility, can achieve the adhesion and proliferation of mouse myoblasts (C2C12), and have the feasibility of large-scale industrial cell expansion. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be made based on these drawings without paying any creative work.

[0031] Figure 1 Schematic diagram of a glass capillary device according to Example 1 of the present invention;

[0032] Figure 2 This is an optical microscope image of the plant protein microspheres of Example 1 of the present invention;

[0033] Figure 3 This is a size distribution diagram of the plant protein microspheres of Example 1 of the present invention;

[0034] Figure 4 This is an optical microscope image of the plant protein microspheres of Example 2 of the present invention;

[0035] Figure 5 This is a size distribution diagram of the plant protein microspheres of Example 2 of the present invention;

[0036] Figure 6 This is a laser confocal microscope image of the plant protein microspheres of Example 2 of the present invention;

[0037] Figure 7 This is an optical microscope image of the calcium alginate microspheres of Comparative Example 1 of the present invention;

[0038] Figure 8 This is a size distribution diagram of calcium alginate microspheres of Comparative Example 1 of the present invention;

[0039] Fig. 9 The infrared spectrum detection diagram of the microspheres of Example 2 and Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0040] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.

[0041] The present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0042] Example 1

[0043] See attached Figure 1 -Attached Figure 3 This embodiment provides a plant protein microsphere and a controllable preparation method thereof, comprising the following steps:

[0044] (1) Ca-EDTA was dissolved in deionized water to form a 50 mM Ca-EDTA aqueous solution, and sodium alginate was added thereto and stirred until dissolved to obtain a 1 wt % solution A;

[0045] (2) dissolving zein in 70% ethanol solution to obtain a 1 wt% zein-ethanol aqueous solution as solution B;

[0046] (3) mixing solution A obtained in step (1) and solution B obtained in step (2) at a volume ratio of 9:1, heating and stirring until the mixture is uniformly mixed, and the resulting mixed solution is used as dispersed phase W;

[0047] (4) adding 2 wt% Span 80 to oleic acid as the continuous phase O;

[0048] (5) adding 2 wt % Span 80 and 2 wt % glacial acetic acid to oleic acid as pre-gel phase P;

[0049] (6) dissolving calcium chloride in deionized water to prepare a 2 wt % calcium chloride aqueous solution, and then adding 2 % Tween 80 thereto to prepare a collection solution Wo;

[0050] (7) Inject the dispersed phase W into a glass capillary device (such as Figure 1 ) channel I, the diameter of channel I was set to 200 μm, and the injection rate was 0.4 mL / h; the continuous phase O was injected into channel II of the fluidic device, the diameter of channel II was set to 550 μm, and the injection rate was 5 mL / h; the pregel phase P was injected into channel III of the microfluidic device, and the injection rate was 15 mL / h; the shear effect of the fluid and Ca 2+ Pregelled calcium alginate-zein (ALG-ZEIN) microspheres were obtained after shearing by double cross-linking of inside and outside.

[0051] (8) The pre-gelled calcium alginate-zein (ALG-ZEIN) microspheres obtained in step (7) are received in the collection liquid Wo of step (6), stirred at 400 rpm for 5 h in the collection liquid Wo to make them completely gelled, and repeatedly washed with deionized water five times to obtain plant protein microspheres of uniform size.

[0052] Example 2

[0053] See attached Figure 4 -Attached Figure 6 And attached Fig. 9 This embodiment provides a plant protein microsphere and a controllable preparation method thereof, comprising the following steps:

[0054] (1) Ca-EDTA was dissolved in deionized water to form a 50 mM Ca-EDTA aqueous solution, and sodium alginate was added thereto and stirred until dissolved to obtain a 1 wt % solution A;

[0055] (2) dissolving zein in 70% ethanol solution to obtain a 1 wt% zein-ethanol aqueous solution as solution B;

[0056] (3) mixing solution A obtained in step (1) and solution B obtained in step (2) at a volume ratio of 4:1, heating and stirring until the mixture is uniformly mixed, and the resulting mixed solution is used as dispersed phase W;

[0057] (4) adding 2 wt% Span 80 to oleic acid as the continuous phase O;

[0058] (5) adding 2 wt % Span 80 and 2 wt % glacial acetic acid to oleic acid as pre-gel phase P;

[0059] (6) dissolving calcium chloride in deionized water to prepare a 2 wt % calcium chloride aqueous solution, and then adding 2 % Tween 80 thereto to prepare a collection solution Wo;

[0060] (7) The dispersed phase W is injected into channel I of the glass capillary device, the diameter of channel I is set to 200 μm, and the injection rate is 0.4 mL / h; the continuous phase O is injected into channel II of the fluidic device, the diameter of channel II is set to 550 μm, and the injection rate is 5 mL / h; the pregel phase P is injected into channel III of the microfluidic device, and the injection rate is 15 mL / h; the shear effect of the fluid and Ca 2+ Pregelled calcium alginate-zein (ALG-ZEIN) microspheres were obtained after shearing by double cross-linking of inside and outside.

[0061] (8) The pre-gelled calcium alginate-zein (ALG-ZEIN) microspheres obtained in step (7) are received in the collection liquid Wo of step (6), stirred at 400 rpm for 5 h in the collection liquid Wo to make them completely gelled, and repeatedly washed with deionized water five times to obtain plant protein microspheres of uniform size.

[0062] Comparative Example 1

[0063] See attached Figure 7 -Attached Fig. 9 This comparative example provides a microsphere and a controllable preparation method thereof. This comparative example is a preparation method of calcium alginate (Ca-ALG) hydrogel microspheres, comprising the following steps:

[0064] (1) Ca-EDTA is dissolved in deionized water to form a 50 mM Ca-EDTA aqueous solution, and sodium alginate is added thereto and stirred until dissolved to obtain a 1 wt % solution as the dispersed phase W;

[0065] (2) adding 2 wt% Span 80 to oleic acid as the continuous phase O;

[0066] (3) adding 2 wt% Span 80 and 2 wt% glacial acetic acid to oleic acid as pre-gel phase P;

[0067] (4) dissolving calcium chloride in deionized water to prepare a 2 wt % calcium chloride aqueous solution, and then adding 2 % Tween 80 thereto to prepare a collection solution Wo;

[0068] (5) The dispersed phase W is injected into channel I of the glass capillary device, the diameter of channel I is set to 200 μm, and the injection rate is 0.4 mL / h; the continuous phase O is injected into channel II of the fluidic device, the diameter of channel II is set to 550 μm, and the injection rate is 5 mL / h; the pregel phase P is injected into channel III of the microfluidic device, and the injection rate is 15 mL / h;

[0069] (6) Using the shearing effect of the fluid and Ca 2+ The pre-gelled microspheres were obtained after shearing by the double cross-linking effect of the inside and outside, received in the collecting liquid Wo, stirred at 400 rpm for 5 h in the collecting liquid to make it completely gelled, and repeatedly washed five times with deionized water to obtain calcium alginate (Ca-ALG) microspheres with uniform size.

[0070] The above examples and comparative examples can be seen in conjunction with the accompanying drawings. The optical microscope image of the plant protein microspheres prepared in Example 1 is shown in FIG. Figure 2 The wet ALG-ZEIN microspheres are light yellow and translucent as a whole; the size distribution diagram of the prepared plant protein microspheres can be found in Figure 3 , the diameter of ALG-ZEIN microspheres is about 285.59 μm, CV < 5%, showing a high degree of monodispersity;

[0071] The optical microscope image of the plant protein microspheres prepared in Example 2 is shown in Figure 4 The wet ALG-ZEIN microspheres are light yellow in color as a whole, and the color is darker than that of Example 1, indicating an increase in the content of zein. The size distribution diagram of the prepared plant protein microspheres is shown in Figure 5 The diameter of ALG-ZEIN microspheres is about 272.63 μm, and CV <5%, showing a high degree of monodispersity. Under the same flow rate conditions, compared with Example 1, the diameter of the microspheres is reduced, indicating that the increase in the content of zein reduces the initial water absorption of the hydrogel microspheres; the laser confocal microscopy image of the prepared plant protein microspheres can be found in Figure 6 , zein was dispersed in the microspheres;

[0072] The optical microscope image of calcium alginate (Ca-ALG) microspheres prepared in Comparative Example 1 is shown in Figure 7 Calcium alginate (Ca-ALG) microspheres are transparent as a whole; the size distribution diagram of calcium alginate microspheres can be found in Figure 8 , the diameter of calcium alginate (Ca-ALG) microspheres is about 311.77 μm, CV<5%, and also shows a high degree of monodispersity. Compared with Examples 1 and 2, the diameter of the microspheres is the largest, indicating that when there is no zein, the initial water absorption of the microspheres is the largest; see Fig. 9 , it can be determined that Example 2 contains zein.

[0073] In summary, the present invention uses two natural macromolecular components with biocompatibility and biodegradability as supporting materials and functional materials of microspheres, and through the reasonable composition ratio of the two natural components, the microspheres have a stable spherical structure and suitable cell adhesion and proliferation properties; the shear force and surface tension between microfluids are used to prepare droplets with uniform particle size, and the two natural components are mixed to disperse the plant protein in the sodium alginate solution; and based on the principle of endogenous and exogenous calcium cross-linking method, gel microspheres with calcium alginate as the main sphere and plant protein dispersed therein are formed, the microspheres have uniform particle size, good mechanical properties, and the hydrophobic plant protein is introduced to improve The adhesion of the microspheres to the cells, thereby improving cell proliferation, is significantly superior to the methods of the prior art; the plant protein microspheres of the present invention can be used for cell expansion and the culture process of artificial meat. The present invention preferably uses alginate that meets production requirements and is biocompatible, and hydrophobic plant protein, and promotes the adhesion of the microspheres to the cells through a reasonable component ratio, thereby improving the proliferation efficiency and achieving cell expansion; the preparation method of the present invention uses oil-in-water emulsion and endogenous and exogenous calcium cross-linking method as principles, and through a specific glass capillary device, tube orifice diameter and flow rate control, as well as regulation of the plant protein content, the microspheres have single dispersion, suitable hydrophobicity and mechanical strength.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A plant protein microsphere, characterized in that: The plant protein microspheres are composed of calcium alginate as the main framework component and zein as the functional component, and have uniform particle size. Calculated based on the total mass percentage of the plant protein microspheres, the mass percentage of the main framework component calcium alginate is 80% to 90%, and the mass percentage of the functional component zein is 10% to 20%.

2. The plant protein microsphere according to claim 1, characterized in that: Calculated by mass percentage, the content of guluronic acid in the calcium alginate is greater than 60%.

3. The plant protein microsphere according to claim 1, characterized in that: The zein is α-zein, which can be dissolved in an alcohol aqueous solution with a volume fraction of 60% to 95%.

4. The controllable preparation method of plant protein microspheres according to any one of claims 1 to 3, characterized in that: The steps include: (1) dissolving calcium disodium ethylenediaminetetraacetate in deionized water to obtain an aqueous solution of calcium disodium ethylenediaminetetraacetate, and then adding sodium alginate thereto and stirring until dissolved to obtain a solution A; (2) dissolving zein in an organic solvent to obtain solution B; (3) mixing solution A obtained in step (1) with solution B obtained in step (2), heating and stirring until the mixture is uniformly mixed, and the resulting mixed solution is used as dispersed phase W; (4) preparing an oil solution of a lipophilic surfactant and using it as a continuous phase O; (5) dissolving a lipophilic surfactant and glacial acetic acid in an oily solvent, and the resulting solution is used as a pre-gel phase P; (6) dissolving calcium chloride in deionized water, and then adding a hydrophilic surfactant thereto, and the resulting solution is used as the collection liquid Wo; (7) passing the dispersed phase W, the continuous phase O and the pre-gel phase P through a glass capillary device at set flow rates, respectively, and obtaining pre-gel calcium alginate-zein microspheres after shearing; (8) The pre-gelled calcium alginate-zein microspheres obtained in step (7) are received in the collection liquid Wo of step (6), gelled under low-speed stirring, and washed with deionized water to obtain the plant protein microspheres.

5. The controllable preparation method of plant protein microspheres according to claim 4, characterized in that: The diameter of the plant protein microspheres is regulated by the flow rates of the dispersed phase W and the continuous phase O or the size of the dispersed phase and the continuous phase glass capillary microchannel or the pipeline combination; the flow rate of the dispersed phase W is 0.2mL / h to 0.6mL / h, and the flow rate of the continuous phase O is 1mL / h to 10mL / h; the diameter of the channel I through which the dispersed phase passes is 100μm to 500μm, and the diameter of the channel II through which the continuous phase O passes is 400μm to 800μm; the pipeline combination is selected from one of the Y-type, T-type, cross-type, co-flow type, and flow focusing type.

6. The controllable preparation method of plant protein microspheres according to claim 4, characterized in that: The mechanical properties and hydrophilicity of the plant protein microspheres are regulated by the concentrations of solution A and solution B and the volume ratio during mixing; the concentration of calcium disodium ethylenediaminetetraacetate in solution A is 20mM to 70mM, and the concentration of sodium alginate is 0.2wt% to 2.5wt%; the concentration of zein in solution B is 0.3wt% to 3.0wt%.

7. The controllable preparation method of plant protein microspheres according to claim 4, characterized in that: The flow rate of the pre-gel phase P is 15 mL / h to 25 mL / h; the concentration of acetic acid in the pre-gel phase P is 0.5 wt% to 2.5 wt%; and the concentration of calcium chloride in the collected liquid Wo is 1 wt% to 8 wt%.

8. The controllable preparation method of plant protein microspheres according to claim 4, characterized in that: The oily solvent is selected from one of oleic acid, liquid paraffin and soybean oil; the organic solvent is selected from one of 60% to 80% ethanol aqueous solution, strong alkali and sodium dodecyl sulfate.

9. The controllable preparation method of plant protein microspheres according to claim 4, characterized in that: The lipophilic surfactant is selected from one of Span 85 and Span 80; the concentration of the lipophilic surfactant in the continuous phase O and the pregel phase P is 1wt% to 5wt%; the hydrophilic surfactant is selected from one of Tween 80, polyvinyl alcohol, and polyvinyl pyrrolidone; the concentration of the hydrophilic surfactant in the collected liquid Wo is 1wt% to 5wt%.

10. The controllable preparation method of plant protein microspheres according to claim 4, characterized in that: In step (8), the rotation speed of the low-speed stirring is 100 to 400 rpm, and the time is 1 to 12 hours.

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