CN110693036A - Ultrasound-assisted dialysis-driven zein self-assembly to prepare nanoparticles - Google Patents

Abstract

The invention discloses a method for preparing nano-particles by ultrasonic-assisted dialysis-driven zein self-assembly, and belongs to the technical field of functional food. The invention uses combined frequency ultrasonic-assisted dialysis to prepare nanoparticles, and uses ultrasonic-assisted dialysis in the early stage, and only uses dialysis in the later stage. The present invention has the following characteristics: firstly, the additive propylene glycol (GRAS) in alcohol-free food is used, which avoids the use of alcohol, reduces the risk of inflammability and explosion, and reduces the production cost. The second is to use a two-phase physical driving force (ultrasonic and concentration difference) to promote the formation of uniform and smaller-sized nanoparticles during solvent removal. The third is to avoid the use of acid-base solutions, and the reaction process is easy to achieve automated production. Finally, different combined frequencies of ultrasound can significantly reduce the particle size of the nanoparticles after ultrasonic treatment, and improve the storage stability of the nanoparticles, providing a new method for the delivery system of biologically active compounds.

Description

Ultrasound-assisted dialysis-driven zein self-assembly to prepare nanoparticles

technical field

The invention belongs to the technical field of functional foods, and in particular relates to a new method for driving zein self-assembly to form nanoparticles by using ultrasonic-assisted dialysis.

Background technique

Particles derived from organic biopolymers and with a particle size reaching the nanometer level are called nanoparticles. Nanoparticles have small particle size, high hydrophilicity, high environmental adaptability, and high absorption and utilization in gastrointestinal digestion. , which improves the bioavailability of the encapsulated active substances, so nanoparticles are widely used in delivery systems loaded with lipophilic and hydrophobic nutrients.

Zein is the main storage protein in maize endosperm and the main by-product of maize in the food industry. It is composed of polypeptides with different molecular weights and solubility, and can be divided into four types according to the solubility and amino acid sequence: α-zein (19 and 22KD), β-zein (14KD), δ-zein (16 and 27KD), gamma-zein (10KD). Alpha-zein accounted for the highest proportion, followed by gamma-zein. Zein has unique natural physicochemical properties, poor water solubility, but can be dissolved in high concentration alcohol solution. Food-grade nanoparticles can be prepared using the unique solubility of zein, and zein is recognized as a safe food ingredient (GRAS). Zein with a high proportion of lipophilic amino acid residues (≥50%) is a highly hydrophobic protein and therefore has the potential to deliver non-polar bioactive compounds.

Zein nanoparticles are highly sensitive to the processing environment, so biopolymers need to be added to form a protective shell on their hydrophobic surface to avoid aggregation and precipitation of nanoparticles. In addition, the added biopolymer also has good storage stability, anti-degradation stability of the encapsulated substance, and high encapsulation efficiency to the core-shell structure of the formed nanoparticles. As a small molecule surfactant, sodium caseinate has an amphiphilic group, which can reduce the surface hydrophobicity of zein, increase electrostatic attraction and steric stability, prevent the aggregation of zein, and can be used as an anti-solvent In the food-grade nanoparticle delivery system formed by methods such as method, pH cycle driving, ultrasonic-assisted dialysis, etc.

The principle of ultrasound-assisted dialysis-driven self-assembly of zein nanoparticles is to exploit the different solubility of zein in different polar solvents. Zein is first dissolved in the solvent, and under the assistance of ultrasound, the zein solution is dispersed, and the solvent is mass-transferred with the strong polar solvent water under the driving force of the concentration difference and ultrasound, thereby continuously reducing the concentration of the solvent , zein gradually formed nano-microspheres, and under the action of ultrasonic mechanical effect and cavitation, zein nanoparticles with uniform and stable size and smaller particle size were formed.

The methods for preparing zein self-assembled nanoparticles include anti-solvent method, pH cycle driving method, etc. Ethanol is usually used in the anti-solvent method. Ethanol cannot be used in alcohol-free food and is flammable. It is very unfavorable for industrial production processes. The particle size depends on the volume of anti-solvent added dropwise. Production costs are not conducive to automated production processes. The pH cycle driving method avoids the use of ethanol, but requires precise adjustment of pH, the use of a large amount of acid-base solution, and the adjustment process is a dynamic change process with many influencing factors.

In order to solve these problems, the present invention uses combined frequency ultrasonic-assisted dialysis to prepare nanoparticles. First, the additive propylene glycol (GRAS) in alcohol-free food is used, which avoids the use of alcohol, reduces the risk of inflammability and explosion, and reduces production costs. . The second is to use a two-phase physical driving force (ultrasonic and concentration difference) to promote the formation of uniform and smaller-sized nanoparticles during solvent removal. The third is to avoid the use of acid-base solutions, and the reaction process is easy to achieve automated production. Finally, different combined frequencies of ultrasonics significantly reduce the particle size of the nanoparticles after ultrasonic treatment, and improve the storage stability of nanoparticles, provide a new method for the delivery system of biologically active compounds, and improve the environmental tolerance. The weak bioavailability of nutrients expands the application scope of lipophilic bioactive compounds in functional foods.

SUMMARY OF THE INVENTION

The purpose of the present invention is to use biphasic physical driving force (ultrasonic and concentration difference) to form zein nanoparticles with uniform, stable and smaller particle size. Ultrasonic-assisted dialysis is used in the early stage, and only dialysis is used in the later stage. And avoiding the use of ethanol, using the food-grade additive propylene glycol as a solvent, opens up new ways to drive the self-assembly of zein nanoparticles.

The technical scheme of the present invention is as follows:

The method for preparing nanoparticles by ultrasonic-assisted dialysis-driven zein self-assembly is carried out according to the following steps:

(1) Weigh a certain mass of zein and sodium caseinate and dissolve them in an aqueous propylene glycol solution, stir the stock solution evenly with a magnetic force and see no obvious precipitation, and remove insoluble large particles by centrifugation to obtain zein and caseinic acid. A binary mixed solution of sodium.

(2) Put the binary mixed solution of zein and sodium caseinate into a dialysis bag of a certain length, seal the dialysis bag and put it into the material container, and add deionized water; The ratio of the binary mixed solution of sodium caseinate to deionized water is 1:20;

(3) Put the material container in the ultrasonic tank, set the ultrasonic frequency, ultrasonic power, ultrasonic time, ultrasonic mode and constant temperature water bath, ultrasonic for 60min, take out the dialysis bag, re-add deionized water equal to the volume of step (2) at room temperature dialysis under conditions.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

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

Wherein the specification of the dialysis bag in step (2) is 34mm (width), the molecular weight cut off is 8000-14000, the used dialysis bag is activated and the used length is 45cm.

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

Compared with the prior art, the advantages and technical effects of the present invention are:

1. The present invention uses a biphasic physical driving force to facilitate the formation of zein nanoparticles. Ultrasonic assisted treatment is added during the dialysis process. Ultrasound can open the internal structure of zein to make it loose, expose the internal chromophore, and react more fully, thereby combining with sodium caseinate to form a tighter form. structure to reduce the size of composite nanoparticles. And the added ultrasound during dialysis helps to increase the mass transfer rate and promote the formation of composite nanoparticles.

2. The use of ethanol is avoided in the present invention, thereby reducing the cost of removing ethanol and facilitating the development of alcohol-free food, further reducing the risk of inflammable and explosive, and without using organic reagents in the operation process, the whole process is simple and easy to operate, and has Facilitates the continuous production of delivery systems for embedding biologically active compounds.

3. The zein composite nanoparticles formed by the new method of ultrasonic-assisted dialysis in the present invention not only have small particle size, uniform and stable particle size, but also can improve the storage stability of the composite nanoparticles.

4. The present invention improves the utilization rate of corn starch by-products, provides a new method for the delivery system of bioactive compounds, and improves the bioavailability of nutrients with weaker environmental tolerance, and expands the lipophilic bioactive compounds in the bioactive compounds. The scope of application of functional food.

Description of drawings

Fig. 1 is the process flow diagram of ultrasonic-assisted dialysis-driven zein self-assembly to prepare nanoparticles;

Figure 2 is the fluorescence spectra of nanoparticles for different combined frequencies of ultrasound-assisted dialysis.

Detailed ways

The technical solutions of the present invention are further described in detail below in conjunction with specific examples and data. However, these examples are not intended to limit the technical solutions of the present invention, but are merely illustrative.

Example 1

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container at room temperature for 12h dialysis

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm. The particle size of fresh zein nanoparticles without sonication was 256.62 ± 6.78 nm, the PDI was 0.13 ± 0.07, the potential was -30.78 ± 0.42 mv, and the particle size of zein nanoparticles stored for 15 days 272.45±9.65nm, PDI is 0.18±0.07 potential is -20.46±2.93mv. During the storage process, the particle size of the unsonicated binary zein nanoparticles showed an increasing trend, the PDI increased but not significantly, and the potential shifted to a decreasing direction.

Example 2

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 20KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, the temperature of the constant temperature water bath to be 25°C, take out the dialysis bag, and re-add 400mL of deionized Water was dialyzed at room temperature for 11 h.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm. The particle size of fresh zein nanoparticles subjected to 20KHz sonication was 239.90 ± 8.57 nm, the PDI was 0.11 ± 0.04, the potential was -37.78 ± 0.38 mv, and the particle size of zein nanoparticles stored for 15 days 254.75±12.10nm, PDI was 0.16±0.06, and potential was -31.44±2.31mv. Compared with Example 1, the particle size is significantly reduced, the PDI is reduced, the particle size distribution is narrower, the particle size distribution is uniform, and the potential is increased, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. During the storage process, the particle size of binary zein nanoparticles showed a trend of increasing particle size, PDI increased but not significant, and the potential shifted to a decreasing direction, but the degree of particle size and potential shift were the same. Smaller than unsonicated binary zein nanoparticles, improving the storage stability of the binary nanocomposite.

Example 3

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 28KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, and the temperature of the constant temperature water bath to be 25°C, take out the dialysis bag, and re-add 400mL of deionized Water was dialyzed at room temperature for 11 h.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm. The particle size of fresh zein nanoparticles subjected to 28KHz sonication was 239.35 ± 2.44 nm, the PDI was 0.14 ± 0.03, the potential was -31.13 ± 4.83 mv, and the particle size of zein nanoparticles stored for 15 days was 264.82±2.84nm, PDI was 0.15±0.05, and the potential was -27.77±1.21mv. Compared with Example 1, the particle size is significantly reduced, the PDI is smaller, the particle size distribution is narrower, the particle size distribution is uniform, and the potential increases, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with Example 2, the particle size and PDI value are close, and the potential decreases. During the storage process, the particle size of the 28KHz ultrasonic-treated binary zein nanoparticles showed a trend of increasing particle size, PDI increased but not significantly, and the potential shifted to a decreasing direction, but the particle size and potential The degree of transfer is smaller than that of the unsonicated binary zein nanoparticles, which improves the storage stability of the binary nanocomposite.

Example 4

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 40KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, the temperature of the constant temperature water bath to be 25°C, take out the dialysis bag, and re-add 400mL of deionized Water was dialyzed at room temperature for 11 h.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm. The particle size of fresh zein nanoparticles subjected to 40KHz sonication was 231.20 ± 15.75 nm, the PDI was 0.14 ± 0.01, the potential was -32.60 ± 1.32 mv, and the particle size of zein nanoparticles stored for 15 days was 267.93±3.44nm, PDI was 0.19±0.03, and the potential was -26.01±2.08mv. Compared with Example 1, the particle size is significantly reduced, the PDI is smaller, the particle size distribution is narrower, the particle size distribution is uniform, and the potential increases, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with Examples 2 and 3, the nanoparticle size obtained after increasing the frequency is smaller. During the storage process, the particle size of the 40KHz ultrasonic-treated binary zein nanoparticles showed a trend of increasing particle size, PDI increased but not significantly, and the potential shifted to a decreasing direction, but the particle size and potential The degree of transfer is smaller than that of the unsonicated binary zein nanoparticles, which improves the storage stability of the binary nanocomposite.

Example 5

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 20KHz+28KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, and the temperature of the constant temperature water bath to be 25°C, take out the dialysis bag, and add 400mL of Dialysis was performed against deionized water for 11 h at room temperature.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm. The particle size of fresh zein nanoparticles subjected to 20KHz+28KHz ultrasonic treatment was 242.92±1.28nm, the PDI was 0.13±0.03, and the potential was -34.84±0.59mv. The zein nanoparticles stored for 15 days had a The particle size was 249.16±2.62nm, the PDI was 0.11±0.03, and the potential was -31.65±2.37mv. Compared with Example 1, the particle size is significantly reduced, the PDI is smaller, the particle size distribution is narrower, the particle size distribution is uniform, and the potential increases, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with Examples 1, 2, 3, and 4, the particle size of the nanoparticles obtained after the 20KHz+28KHz dual-frequency treatment and the amplitude before and after potential storage are small, which enhances the storage stability of the nanoparticles. During storage, the particle size of binary zein nanoparticles treated with 20KHz+28KHz ultrasound showed a trend of increasing particle size, PDI did not change significantly, and the potential shifted to a decreasing direction, but the particle size and potential The degree of transfer is smaller than that of the unsonicated binary zein nanoparticles, which improves the storage stability of the binary nanocomposite.

Example 6

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 28KHz+40KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, and the temperature of the constant temperature water bath to be 25°C, take out the dialysis bag, and add 400mL of Dialysis was performed against deionized water for 11 h at room temperature.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm.

The particle size of fresh zein nanoparticles subjected to 28KHz+40KHz ultrasonic treatment was 236.62±3.26nm, the PDI was 0.13±0.02, the potential was -33.47±1.38mv, and the zein nanoparticles were stored for 15 days. The particle size was 242.61±3.15nm, the PDI was 0.1±0.06, and the potential was -32.00±1.01mv. Compared with Example 1, the particle size is significantly reduced, the PDI is smaller, the particle size distribution is narrower, the particle size distribution is uniform, and the potential increases, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with Examples 2, 3, and 5, the particle size of nanoparticles obtained after 28KHz+40KHz dual-frequency treatment is smaller. Compared with Examples 2, 3, 4, and 5, the particle size of the nanoparticles obtained after the 28KHz+40KHz dual-frequency treatment and the amplitude before and after potential storage are small, which enhances the storage stability of the nanoparticles. During storage, the particle size of 28KHz+40KHz ultrasonic-treated binary zein nanoparticles showed a trend of increasing particle size, PDI did not change significantly, and the potential shifted to a decreasing direction, but the particle size and potential shifted. The extent of zein nanoparticles is smaller than that of the unsonicated binary zein nanoparticles, which improves the storage stability of the binary nanocomposites.

Example 7

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 20KHz+40KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, and the temperature of the constant temperature water bath to be 25°C, take out the dialysis bag, and add 400mL of Dialysis was performed against deionized water for 11 h at room temperature.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm. The particle size of fresh zein nanoparticles subjected to 20KHz+40KHz sonication was 225.94±45.84nm, the PDI was 0.17±0.06, the potential was -29.97±1.49mv, and the zein nanoparticles stored for 15 days were The particle size was 226.02±11.28nm, the PDI was 0.19±0.08, and the potential was -28.50±0.60mv. Compared with Example 1, the particle size is significantly reduced, the PDI is smaller, the particle size distribution is narrower, the particle size distribution is uniform, and the potential increases, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with Examples 2, 3, 4, 5, and 6, dual-frequency ultrasonic treatment is better than single-frequency ultrasonic treatment, and the nanoparticles obtained after dual-frequency 20KHz+40KHz have smaller particle size. Compared with Examples 2, 3, 4, 5, and 6, the particle size of the nanoparticles obtained after the 28KHz+40KHz dual-frequency treatment and the amplitude before and after potential storage are small, which enhances the storage stability of the nanoparticles. During storage, the particle size of 20KHz+40KHz ultrasonic-treated binary zein nanoparticles showed a trend of increasing particle size, PDI did not change significantly, and the potential shifted to a decreasing direction, but the particle size and potential shifted. The extent of zein nanoparticles is smaller than that of the unsonicated binary zein nanoparticles, which improves the storage stability of the binary nanocomposites.

Example 8

(1) Dissolve a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, the mass ratio of zein and sodium caseinate is both 2%, and the magnetic Stir well and see no obvious precipitation. The binary mixed solution is centrifuged at 5000 rpm for 10 min to remove the remaining large particles to obtain a binary mixed solution of zein and sodium caseinate.

(2) Measure the binary mixed solution of zein and sodium caseinate of 20mL, put into the dialysis bag with a length of 45cm and a width of 34mm after activation treatment, and a molecular weight cut-off of 8000-14000, and seal the dialysis bag. Put into the material container and add 400 mL of deionized water.

(3) Place the material container in the ultrasonic tank, set the ultrasonic frequency to 20KHz+28KHz+40KHz, the ultrasonic power to 300w, the ultrasonic time to 60min, the ultrasonic mode to be continuous, and the temperature of the constant temperature water bath to be 25°C. Take out the dialysis bag and add it again. Dialysis was performed against 400 mL of deionized water for 11 h at room temperature.

(4) After the dialysis, take out a fresh sample to obtain a zein composite nanoparticle colloidal dispersion. A part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48h.

(5) Turbidity measurement: Take a fresh sample and measure the absorbance value of the sample at a wavelength of 500 nm using a UV-vis ultraviolet-visible spectrophotometer to characterize the turbidity of the sample. Measured at room temperature, each sample was measured three times, and the results were expressed as an average value.

(6) Determination of particle size and Zeta potential: The particle size, PDI and Zeta potential of zein binary nanoparticles were determined by dynamic light scattering at room temperature. The particle size of each sample was calculated by the instrument based on the Stokes-Einstein equation, while the surface potential was calculated based on the Smoluchowski model, and the nanoparticle dispersion was diluted to an appropriate concentration before measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.

(7) Determination of fluorescence spectrum: In order to selectively excite tryptophan residues, the excitation wavelength is 280 nm, the scanning range is 290-450 nm, the scanning speed is 1000 nm/min, the scanning interval is 1 nm, and the excitation bandwidth and emission bandwidth are both set Set to 10nm.

The particle size of the fresh zein nanoparticles subjected to 20KHz+28KHz+40KHz ultrasonic treatment is 239.09±3.11nm, the PDI is, the potential is -34.52±2.62mv, and the grain size of the zein nanoparticles stored for 15 days. The diameter is 242.48±5.13nm, the PDI is 242.48±5.13nm, and the potential is -28.74±5.84mv. Compared with Example 1, the particle size is significantly reduced, the PDI is smaller, the particle size distribution is narrower, the particle size distribution is uniform, and the potential increases, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with Examples 2, 3, and 5, the obtained nanoparticles have smaller particle size. Compared with Examples 2, 3, and 4, the particle size of the nanoparticles obtained after the 28KHz+40KHz dual-frequency treatment and the amplitude before and after potential storage have small changes, which enhances the storage stability of the nanoparticles. During storage, the particle size of 20KHz+28KHz+40KHz ultrasonic-treated binary zein nanoparticles showed a trend of increasing particle size, PDI did not change significantly, and the potential shifted to a decreasing direction, but the particle size and The degree of potential transfer is smaller than that of the unsonicated binary zein nanoparticles, which improves the storage stability of the binary nanocomposite.

Table 1 is the nanoparticle size, PDI, and Zeta potential maps for different storage times.

After ultrasonic-assisted dialysis treatment, the particle size of zein nanoparticles was significantly reduced, the PDI value was smaller, the particle size distribution was narrow, the particle size distribution was uniform, and the potential increased, forming uniform and stable nanoparticles. The biphasic physical driving force of ultrasound and dialysis opened the internal higher-order structure of zein, exposing more aromatic amino acid residues inside the protein, and increasing the fluorescence intensity of the zein colloid solution. The nanoparticles treated with different combined frequencies of assisted dialysis showed different physical properties. The particle size of zein nanoparticles treated by 20KHz+40KHz ultrasonic assisted dialysis was 225.94±45.84nm, the PDI was 0.17±0.06, and the potential was -29.97±29.97± 1.49mv, the particle size of the zein nanoparticles stored for 15 days is 226.02±11.28nm, the PDI is 0.19±0.08, the potential is -28.50±0.60mv, the particle size is the smallest, and the fluorescence intensity is the strongest, which is the best choice Auxiliary frequency, and the degree of particle size potential change during storage is small, and the storage stability is good.

Table 1 Nanoparticle size, PDI and Zeta potential diagrams of different storage times

Claims (4)

1. Ultrasound-assisted dialysis drives the method for zein self-assembly to prepare nanoparticles, characterized in that carrying out according to the following steps: (1) Weigh a certain mass of zein and sodium caseinate and dissolve them in an aqueous propylene glycol solution, stir the stock solution evenly with a magnetic force, and see no obvious precipitation. Centrifuge to remove insoluble large particles to obtain zein and caseinic acid. A binary mixed solution of sodium; (2) Put the binary mixed solution of zein and sodium caseinate into a dialysis bag of a certain length, seal the dialysis bag and put it into the material container, and add deionized water; The ratio of the binary mixed solution of sodium caseinate to deionized water is 1:20; (3) Put the material container in the ultrasonic tank, set the ultrasonic frequency, ultrasonic power, ultrasonic time, ultrasonic mode and constant temperature water bath, ultrasonic for 60min, take out the dialysis bag, re-add deionized water equal to the volume of step (2) at room temperature dialysis under conditions; (4) After the dialysis, fresh samples were taken out to obtain a zein composite nanoparticle colloidal dispersion; part of the samples were refrigerated at 4°C, and another part of the colloidal solution was vacuum freeze-dried for 48 hours. 2. The method for preparing nanoparticle by ultrasonic-assisted dialysis-driven zein self-assembly according to claim 1, wherein the mass ratio of zein in step (1) and sodium caseinate is 2%, the concentration of propylene glycol aqueous solution is 80% (v/v), and the binary mixed solution is centrifuged at 5000 rpm for 10 min to remove residual large particles. The mixed solution is prepared at room temperature. 3. the method for preparing nanoparticle by ultrasonic-assisted dialysis-driven zein self-assembly according to claim 1, is characterized in that wherein the specification of the dialysis bag in the step (2) is 34mm (width), and the molecular weight cut-off is 8000- 14000, the dialysis bag used was activated and used with a length of 45 cm. 4. the method for preparing nanoparticle by ultrasonic-assisted dialysis-driven zein self-assembly according to claim 1, is characterized in that wherein in step (3), ultrasonic frequency is combined frequency ultrasonic, is respectively 20KHz, 28KHz, 40KHz, 20KHz +28KHz, 28KHz+40KHz, 20KHz+40KHz, 20KHz+28KHz+40KHz, the ultrasonic power is 300W, the ultrasonic mode is continuous ultrasonic, and the temperature of the constant temperature water bath is 25℃.

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