A dual-emulsion method for preparing zein microspheres with controllable particle size

Abstract

This invention discloses a method for preparing zein microspheres with controllable particle size using a double emulsion method, comprising the following steps: (1) dispersing and dissolving zein in an aqueous solution of an organic solvent to obtain an aqueous phase; (2) adding a surfactant to mineral oil and premixing evenly to obtain an oil phase, then adding the aqueous phase from step (1) to the oil phase and stirring to obtain a first emulsion; (3) adding the first emulsion prepared in step (2) to water and stirring to obtain a second emulsion, and waiting for the protein to self-assemble to obtain microspheres with controllable particle size. The method of this invention is simple to operate, and the obtained zein microspheres have a particle size range of 0.7-1000 μm, which can be used as biological carriers or adsorbents, and have a wide range of applications.

Description

Technical Field

[0001] This invention belongs to the field of materials technology and relates to a method for preparing zein microspheres. Background Technology Zein is a plant protein found in corn kernels and is a major component of the protein in corn germ. It contains a large number of hydrophobic amino acids and some hydrophilic amino acids, thus possessing unique solubility properties. It is insoluble in water and anhydrous alcohols, with an optimal solubility range of 60%-95% by volume in alcohol solutions. It is also soluble in acetone aqueous solution, strong alkalis, sodium dodecyl sulfate (SDS), high-concentration urea, propylene glycol, and acetic acid. Due to its unique amino acid composition, zein can self-assemble under specific conditions.

[0002] Zeatin has a very wide range of applications. Its protein molecules have strong disulfide bonds and hydrophobic interactions, allowing for the easy preparation of zeatin membranes using simple solvent evaporation. Through compounding with other materials, thin films with good mechanical properties and non-toxic, biodegradable properties can be easily obtained. Zeatin can also encapsulate hydrophobic and unstable drugs, protecting their structure and enabling slow release. Furthermore, zeatin can be processed into fibers using electrospinning technology, and through chemical modification and material compounding, high-quality fiber products can be obtained.

[0003] Most commonly used methods for preparing zein microspheres involve antisolvent self-assembly, where a zein alcohol solution is directly poured into water. This method produces microspheres with uncontrollable particle size, only nanometer-sized particles, making collection difficult and resulting in significant raw material loss. Currently reported methods for preparing micron-sized zein microspheres include: ultrasonic-assisted antisolvent method, which requires ultrasound and a semi-permeable membrane, is costly, and limits particle size to within 10 micrometers (Chemical Engineering Journal, Volume 284, 15 January 2016, Pages 1094-1105); and emulsion solvent evaporation method, which can prepare micron-sized microspheres, but requires reduced pressure equipment, and the microspheres tend to aggregate during evaporation, resulting in low yield (Mater. Chem. Front., 2021, 5, 3897-3902). We have developed a novel dual-emulsion method for the rapid, room-temperature preparation of zein microspheres with a wide size range (approximately 0.7-1000 micrometers). The particle size of the microspheres is controllable, and the method avoids the use of expensive equipment and highly toxic reagents. Specifically, the oil phase of the first emulsion acts as a liquid film, allowing the external aqueous phase to diffuse into the internal aqueous phase. Ethanol from the internal aqueous phase diffuses into the external aqueous phase. As the internal ethanol concentration gradually decreases, zein precipitates and self-assembles into microspheres. Summary of the Invention

[0004] On one hand, this invention provides a method for preparing zein microspheres with controllable particle size, comprising the following steps: (1) Dissolve a certain amount of zein in a 60%-95% ethanol aqueous solution. Alternatively, the protein solution can also be dissolved using solvents such as acetic acid or acetone, with the proportion of organic solvents adjusted accordingly. The concentration range is 1-20 wt%, preferably 6-12 wt%. The amount of zein added in this step will affect the final size of the zein microspheres; the higher the concentration, the larger the microsphere size.

[0005] (2) Add a certain amount of Tween / Span emulsifier (Tween:Span = 3:1) to the mineral white oil and stir for more than 10 min to fully dissolve it. Pour the corn protein solution prepared in (1) (10-30% of the volume fraction of the oil phase, preferably 15%) into the oil phase and stir for a certain time (range 10-30 min, preferably 15-20 min) to form a stable primary emulsion. The stirring speed will affect the stirring speed in step (3). The smaller the emulsion is dispersed, the wider the stirring speed range will be provided for step (3). The stirring speed selected in this step is related to the size to be produced. Other types of emulsifiers can be selected, and those that can make the emulsion more stable are preferred.

[0006] (3) Pour the prepared first emulsion into a certain amount of stirred water at a speed of 2500-6000 rpm. After stirring for about 30 minutes, all the proteins will self-assemble and precipitate. The certain amount refers to the volume fraction of the first emulsion relative to the water (0-30%). If the volume fraction is too high, it will cause the microspheres to aggregate and the sphericity to decrease. The speed in this step is another factor controlling the size of the microspheres. The faster the speed, the smaller the microspheres prepared. The maximum speed depends on the concentration of the protein solution and the speed of the first emulsion. The speed required to prepare microspheres of about 10 micrometers is about 3000 rpm, under the condition of 6% protein concentration.

[0007] The obtained microspheres were centrifuged, washed, and freeze-dried to obtain zein microspheres with good sphericity and dispersibility, with a particle size of 0.7-1000 micrometers.

[0008] The preparation method of this invention controls the particle size of the prepared corn protein microspheres by controlling the concentration of zein, the stirring rate at different stages, and the ratio of the water and oil phases. The solvent can be an aqueous solution of organic solvents such as ethanol, acetone, or acetic acid. The oil phase can be mineral oil or other alkanes that are immiscible with the internal phase. The emulsifier can be Tween, Span, lecithin, etc., with those offering more stable emulsifying capabilities being preferred.

[0009] The concentration of corn protein selected in the first step of this invention has a significant impact on the particle size of the microspheres. If the concentration is too high, the emulsion will become unstable, forcing the protein to precipitate prematurely, resulting in preparation failure. If the concentration is too low, the prepared microspheres will be nanoscale and difficult to collect.

[0010] In the second step of this invention, an emulsifier is added to stabilize the first emulsion. If the emulsifier dosage is insufficient or its emulsifying ability is weak, the formation of the second emulsion will destabilize the multiple emulsions, causing corn protein to precipitate on the surface of the oil phase. This results in the microspheres agglomerating and significantly reducing their sphericity. Therefore, the addition of an emulsifier is necessary; the amount added should be 5-10% of the oil phase.

[0011] In the third step of this invention, the minimum volume of the external aqueous phase should not be less than the volume of the primary emulsion. The optimal ratio is primary emulsion volume: aqueous phase = 1:3, which can provide sufficient thickness to the liquid film, making the diffusion process smoother, reducing the probability of collision between different droplets, and ensuring particle size and dispersibility.

[0012] The solvents used in this invention are all low-toxicity and non-toxic. The prepared oil phase can be reused after filtering out impurities, and the residual surfactants inside have little impact on the preparation of microspheres. Water and ethanol are both inexpensive solvents, and water can be further recycled after appropriate treatment.

[0013] On the other hand, the corn protein microspheres prepared by the present invention can be used as enzyme carriers, drug and fragrance carriers, and pollutant adsorption treatments; they can also be used in a wider range of fields such as cosmetic microspheres by modifying their groups or adding other materials to prepare composite materials. Attached Figure Description

[0014] Figure 1 These are SEM images of the microparticles prepared according to embodiments of the present invention. Detailed Implementation The following examples are provided to further illustrate the present invention. (1) Take 1 g of zein and dissolve it in 15 ml of 70% (v / v) ethanol aqueous solution. It can be dissolved more quickly and completely under an ultrasonic machine. The dissolution should be complete, and the solution should be kept free of impurities as much as possible.

[0015] (2) Measure 100 ml of white oil (mineral oil), add 7.5 ml of Tween-80 and 2.5 ml of Span-80, and stir at 2000 rpm for about 20 min to mix thoroughly. Slowly add the zein solution prepared in the first step to the oil phase at 3500 rpm for 10 min to stabilize the first emulsion.

[0016] (3) Measure 400 ml of water and add the first emulsion dropwise to the water at a speed of 3000 rpm to form the second emulsion. Stir for about 30 minutes and then stop stirring.

[0017] (4) The prepared reaction solution was centrifuged using a large centrifuge. A small amount of microspheres in the upper oil phase could be obtained by centrifuging after the emulsion was broken by salt. The precipitate was washed with water 4-5 times and then frozen at 0 degrees for 12 h.

[0018] (5) The frozen microspheres were placed in a freeze dryer for drying. The resulting microspheres were then observed under an electron microscope. The results are as follows: Figure 1 As shown, it exhibits good sphericity and good dispersibility.

Claims

1. A method for preparing zein microspheres with controllable particle size using a dual emulsification method, characterized in that, Includes the following steps: (1) Dissolve a certain amount of zein in an aqueous solution of 60%-95% by volume ethanol, or in an aqueous solution of acetic acid, or in an aqueous solution of acetone to obtain an aqueous phase, wherein the protein concentration in the aqueous phase is 1-20 wt%; (2) Add emulsifier Span and Tween to liquid paraffin or white oil and stir. The amount of emulsifier shall not be less than 8% of the volume fraction of the oil phase. Pour the aqueous solution from step (1) into the oil phase and stir to form the first emulsion. (3) Stir the first emulsion and pour it into at least twice the volume of water to form the second emulsion. Continue stirring until the microspheres complete self-assembly.

2. The method for preparing zein microspheres with controllable particle size using a dual emulsification method according to claim 1, characterized in that, The protein concentration in the aqueous phase was 20 wt%.

3. The method for preparing zein microspheres with controllable particle size using a dual emulsification method according to claim 1, characterized in that, The stirring rate affects the particle size of the prepared microspheres. The stirring rate is determined by the stirring speed of the first emulsion and the stirring speed of the second emulsion. To prepare microspheres with a particle size of about 1 micrometer, the stirring speed of the first emulsion is 4000 rpm and the stirring speed of the second emulsion is 3000 rpm.

4. The method for preparing zein microspheres with controllable particle size using a dual emulsification method according to claim 1, characterized in that, The particle size of the microspheres ranges from 0.7 to 1000 micrometers.

5. The method for preparing zein microspheres with controllable particle size using a dual emulsification method according to claim 1, characterized in that, The prepared oil phase can be reused after filtering out impurities, and the residual surfactant inside has little impact on the preparation of microspheres.

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