阅读说明：本技术 超声辅助透析驱动玉米醇溶蛋白自组装制备纳米颗粒方法 (Method for preparing nano particles by self-assembling zein driven by ultrasonic-assisted dialysis ) 是由 周存山 余筱洁 陈慧琳 张磊 宋晓倩 李墨 于 2019-09-18 设计创作，主要内容包括：本发明公开了超声辅助透析驱动玉米醇溶蛋白自组装制备纳米颗粒方法,属于功能性食品技术领域。本发明使用组合频率超声辅助透析来制备纳米颗粒,前期使用超声辅助透析,后期只使用透析。本发明特点在于：一是使用了无酒精食品中的添加剂丙二醇(GRAS),避免使用酒精,降低了易燃易爆的风险,减少了生产成本。二是在去除溶剂时利用双相物理驱动力(超声与浓度差)促进形成均匀、尺寸较小的纳米颗粒。三是避免使用酸碱溶液,反应过程易于实现自动化生产。最终不同组合频率超声对形成经过超声处理后的纳米颗粒粒径明显减小,并且提高了纳米粒子的储藏稳定性,为生物活性化合物的递送系统提供新方法。(The invention discloses a method for preparing nanoparticles by driving self-assembly of zein through ultrasonic-assisted dialysis, and belongs to the technical field of functional foods. The invention uses the combined frequency ultrasonic auxiliary dialysis to prepare the nano particles, the ultrasonic auxiliary dialysis is used in the early stage, and only the dialysis is used in the later stage. The invention is characterized in that: firstly, the additive propylene Glycol (GRAS) in alcohol-free food is used, alcohol is avoided, the risk of flammability and explosiveness is reduced, and the production cost is reduced. And secondly, the two-phase physical driving force (ultrasound and concentration difference) is utilized to promote the formation of uniform and small-sized nano particles when the solvent is removed. Thirdly, acid-base solution is avoided, and the reaction process is easy to realize automatic production. Finally, the particle size of the nanoparticles after ultrasonic treatment is obviously reduced by ultrasonic treatment with different combined frequencies, the storage stability of the nanoparticles is improved, and a new method is provided for a delivery system of the bioactive compound.)

1. The method for preparing the nano-particles by driving self-assembly of the zein through ultrasonic-assisted dialysis is characterized by comprising the following steps of:

(1) weighing a certain mass of zein and sodium caseinate, dissolving the zein and the sodium caseinate in a propylene glycol aqueous solution, magnetically stirring the stock solution uniformly, visually observing the mixture without obvious precipitation, and centrifuging to remove insoluble large particles to obtain a binary mixed solution of the zein and the sodium caseinate;

(2) putting a binary mixed solution of zein and sodium caseinate into a dialysis bag with a certain length, sealing the dialysis bag, putting the dialysis bag into a material container, and adding deionized water; the proportion of the binary mixed solution of zein and sodium caseinate to deionized water is 1: 20;

(3) placing the material container in an ultrasonic tank, setting ultrasonic frequency, ultrasonic power, ultrasonic time, ultrasonic mode and constant-temperature water bath, carrying out ultrasonic treatment for 60min, taking out the dialysis bag, and adding deionized water with the volume equal to that of the step (2) again to carry out dialysis at room temperature;

(4) after the dialysis is finished, taking out a fresh sample to obtain zein composite nanoparticle colloid dispersion liquid; refrigerating a part of the sample in a refrigerator at 4 ℃, and carrying out vacuum freeze drying on another part of the colloidal solution for 48 h.

2. The method for preparing nanoparticles by self-assembly of zein driven by ultrasonic-assisted dialysis as claimed in claim 1, wherein the mass ratio of zein to sodium caseinate in step (1) is 2%, the concentration of the propylene glycol aqueous solution is 80% (v/v), and the preparation of the mixed solution by centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles is performed at room temperature.

3. The method for preparing nanoparticles by driving self-assembly of zein through ultrasonic-assisted dialysis as claimed in claim 1, wherein the dialysis bag in step (2) has a specification of 34mm (width), a molecular weight cut-off of 8000-14000, and a activated dialysis bag with a length of 45 cm.

4. The method for preparing nanoparticles by driving self-assembly of zein through ultrasonic-assisted dialysis as claimed in claim 1, wherein the ultrasonic frequency in step (3) is combined frequency ultrasound, which is 20KHz, 28KHz, 40KHz, 20KHz +28KHz, 28KHz +40KHz, 20KHz +28KHz +40KHz, the ultrasonic power is 300W, the ultrasonic mode is continuous ultrasonic, and the temperature of the thermostatic water bath is 25 ℃.

Technical Field

The invention belongs to the technical field of functional foods, and particularly relates to a novel method for driving zein to self-assemble to form nanoparticles by utilizing ultrasonic-assisted dialysis.

Background

Particles derived from organic biopolymers and having a particle size of the order of nanometers are called nanoparticles, which have a small particle size, high hydrophilicity, high environmental compatibility, and a high absorption and utilization rate in gastrointestinal digestion, and improve the bioavailability of encapsulated active substances, and thus are widely used in delivery systems loaded with lipophilic hydrophobic nutrients.

Zein is the main storage protein in corn endosperm and is the main byproduct of corn in the production of food industry. It is composed of polypeptides with different molecular weights and solubilities, and can be divided into four types according to the difference of solubilities and amino acid sequences: alpha-zein (19 and 22kD), beta-zein (14kD), delta-zein (16 and 27kD), and gamma-zein (10 kD). The alpha-zein has the highest proportion, and the gamma-zein has the second highest proportion. Zein has unique natural physicochemical properties and poor water solubility, but can be dissolved in high-concentration alcoholic solutions. Food grade nanoparticles can be prepared using the unique solubility of zein, and zein is generally recognized as a safe food ingredient (GRAS). Zein has a high proportion of lipophilic amino acid residues (more than or equal to 50 percent) and belongs to high-hydrophobicity protein, so that the zein has the potential of delivering non-polar bioactive compounds.

Zein nanoparticles are highly sensitive to the processing environment, and therefore, biopolymers need to be added to form a protective shell on the hydrophobic surface of the zein nanoparticles, so that aggregation and precipitation of the nanoparticles are avoided. In addition, the added biopolymer also has good storage stability, stability of the encapsulated substance against degradation, and high encapsulation efficiency for the core-shell structure of the formed nanoparticle. Sodium caseinate is used as a micromolecular surfactant, has amphiphilic groups, can reduce the surface hydrophobicity of zein, increases the electrostatic attraction and the space stability, prevents the zein from aggregating, and can be used in a food-grade nanoparticle delivery system formed by an anti-solvent method, a pH circulation driving method, an ultrasonic auxiliary dialysis method and other methods.

The principle of driving self-assembly of zein nanoparticles by ultrasonic-assisted dialysis is to utilize the different solubilities of zein in solvents with different polarities. Zein is firstly dissolved in a solvent, the zein solution is dispersed under the action of ultrasonic assistance, the solvent and strong polar solvent water carry out mass transfer under the driving force of concentration difference and ultrasonic, so that the concentration of the solvent is continuously reduced, the zein gradually forms nano microspheres, and zein nano particles which are uniform, stable and small in particle size are formed under the action of ultrasonic mechanical effect and cavitation.

The method for preparing the zein self-assembly nano particles comprises an anti-solvent method, a pH cycle driving method and the like. Ethanol is generally used in an anti-solvent method, the ethanol cannot be applied to alcohol-free food, is inflammable and is very unfavorable for the industrial production process, the particle size depends on the volume of the dropwise added anti-solvent, the solvent removal rotary evaporation operation process is complicated, the production cost is increased, and the automatic production process is not facilitated. The pH circulation driving method avoids using ethanol, but needs to accurately regulate and control the pH, uses a large amount of acid-base solution, and the regulation process belongs to a dynamic change process and has more influence factors.

In order to solve the problems, the invention uses the combined frequency ultrasonic assisted dialysis to prepare the nano particles, firstly, the additive propylene Glycol (GRAS) in alcohol-free food is used, alcohol is avoided, the risk of flammability and explosiveness is reduced, and the production cost is reduced. And secondly, the two-phase physical driving force (ultrasound and concentration difference) is utilized to promote the formation of uniform and small-sized nano particles when the solvent is removed. Thirdly, acid-base solution is avoided, and the reaction process is easy to realize automatic production. Finally, the particle size of the nanoparticles after ultrasonic treatment is obviously reduced by ultrasonic treatment with different combined frequencies, the storage stability of the nanoparticles is improved, a new method is provided for a delivery system of the bioactive compound, the bioavailability of nutrients with weak environmental tolerance is improved, and the application range of the lipophilic bioactive compound in functional foods is expanded.

Disclosure of Invention

The invention aims to form uniform and stable zein nanoparticles with small particle size by using a two-phase physical driving force (ultrasound and concentration difference), wherein ultrasound-assisted dialysis is used in the early stage, and only dialysis is used in the later stage. And ethanol is avoided, food-grade additive propylene glycol is used as a solvent, and a new method for driving the self-assembly of the zein nanoparticles is developed.

The technical scheme of the invention is as follows:

the method for preparing the nano-particles by driving self-assembly of the zein through ultrasonic-assisted dialysis comprises the following steps:

(1) weighing a certain mass of zein and sodium caseinate, dissolving the zein and the sodium caseinate in a propylene glycol aqueous solution, magnetically stirring the stock solution uniformly, visually observing the stock solution without obvious precipitation, and centrifuging to remove insoluble large particles to obtain a binary mixed solution of the zein and the sodium caseinate.

(2) Putting a binary mixed solution of zein and sodium caseinate into a dialysis bag with a certain length, sealing the dialysis bag, putting the dialysis bag into a material container, and adding deionized water; the proportion of the binary mixed solution of zein and sodium caseinate to deionized water is 1: 20;

(3) and (3) placing the material container in an ultrasonic tank, setting ultrasonic frequency, ultrasonic power, ultrasonic time, ultrasonic mode and constant-temperature water bath, carrying out ultrasonic treatment for 60min, taking out the dialysis bag, and adding deionized water with the volume equal to that of the step (2) again to carry out dialysis at room temperature.

(4) And after the dialysis is finished, taking out a fresh sample to obtain the zein composite nanoparticle colloid dispersion liquid. Refrigerating a part of the sample in a refrigerator at 4 ℃, and carrying out vacuum freeze drying on another part of the colloidal solution for 48 h.

Wherein the mass ratio of the zein to the sodium caseinate in the step (1) is 2%, the concentration of the propylene glycol aqueous solution is 80% (v/v), and the binary mixed solution is prepared at room temperature by centrifuging at 5000rpm for 10min to remove residual large-particle mixed solution.

Wherein the dialysis bag in step (2) has a size of 34mm (width), a molecular weight cut-off of 8000-.

Wherein the ultrasonic frequency in the step (3) is combined frequency ultrasonic, which is respectively 20KHz, 28KHz, 40KHz, 20KHz +28KHz, 28KHz +40KHz, 20KHz +28KHz +40KHz, the ultrasonic power is 300W, the ultrasonic mode is continuous ultrasonic, and the temperature of the thermostatic water bath is 25 ℃.

Compared with the prior art, the invention has the advantages and the technical effects that:

1. the invention uses a two-phase physical driving force and is beneficial to the formation of zein nano particles. Ultrasonic auxiliary treatment is added in the dialysis process, the structure inside the zein can be opened by ultrasonic to be loosened, the chromophoric group inside the zein is exposed, the reaction is more sufficient, and the zein is combined with sodium caseinate to form a more compact structure, so that the size of the composite nano particles is reduced. And the ultrasound added in the dialysis process is helpful to improve the mass transfer rate and promote the formation of the composite nano particles.

2. The method avoids using ethanol, thereby reducing the cost of removing the ethanol, being beneficial to the development of alcohol-free food, further reducing the risk of flammability and explosiveness, not using organic reagents in the operation process, having simple and easy operation in the whole process, and being beneficial to the continuous production of a delivery system for embedding the bioactive compounds.

3. The zein composite nano particles formed by the novel ultrasonic-assisted dialysis method have small particle size, uniformity and stability, and can improve the storage stability of the composite nano particles.

4. The invention improves the utilization rate of the corn starch by-product, provides a new method for a delivery system of the bioactive compound, improves the bioavailability of the nutrient with weak environmental tolerance, and enlarges the application range of the lipophilic bioactive compound in functional food.

Drawings

FIG. 1 is a flow chart of a process for preparing nanoparticles by self-assembly of zein driven by ultrasonic-assisted dialysis;

FIG. 2 is a fluorescence spectrum of nanoparticles for ultrasound-assisted dialysis at different combined frequencies.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the specific examples and data. However, these examples are not intended to limit the technical solution of the present invention, and are only illustrative.