CN110693036A - Method for preparing nano particles by self-assembling zein driven by ultrasonic-assisted dialysis - Google Patents
Method for preparing nano particles by self-assembling zein driven by ultrasonic-assisted dialysis Download PDFInfo
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- 229940093612 zein Drugs 0.000 title claims abstract description 162
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 119
- 238000000502 dialysis Methods 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 37
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- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims abstract description 45
- 238000002604 ultrasonography Methods 0.000 claims abstract description 19
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 14
- 238000001338 self-assembly Methods 0.000 claims abstract description 10
- 102000011632 Caseins Human genes 0.000 claims description 62
- 108010076119 Caseins Proteins 0.000 claims description 62
- 229940080237 sodium caseinate Drugs 0.000 claims description 62
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
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- 229910021641 deionized water Inorganic materials 0.000 claims description 21
- 239000000463 material Substances 0.000 claims description 20
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- 238000007789 sealing Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 238000003860 storage Methods 0.000 abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 14
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- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 8
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/185—Vegetable proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/14—Vegetable proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
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.
Description
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.
Example 1
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) The material container is placed at room temperature for dialysis for 12h
(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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm. The particle size of fresh zein nanoparticles without ultrasonic treatment is 256.62 +/-6.78 nm, PDI is 0.13 +/-0.07, the potential is-30.78 +/-0.42 mv, the particle size of the zein nanoparticles stored for 15 days is 272.45 +/-9.65 nm, the PDI is 0.18 +/-0.07, and the potential is-20.46 +/-2.93 mv. During storage, the particle size of the non-sonicated binary zein nanoparticles showed a tendency to increase in particle size, with an increase in PDI but not significantly, with a shift in potential towards a decrease.
Example 2
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency at 20KHz, the ultrasonic power at 300w, the ultrasonic time at 60min, the ultrasonic mode at a continuous mode, and the constant-temperature water bath temperature at 25 deg.C, taking out the dialysis bag, and adding 400mL of deionized water again to dialyze at room temperature for 11 h.
(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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm. Fresh zein nanoparticles sonicated at 20KHz had particle size of 239.90 + -8.57 nm, PDI of 0.11 + -0.04, potential of-37.78 + -0.38 mv, particle size of 254.75 + -12.10 nm, PDI of 0.16 + -0.06, potential of-31.44 + -2.31 mv after 15 days of zein nanoparticle storage. 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 storage, the particle size of the binary zein nanoparticles shows a trend of increasing the particle size, PDI is increased but is not obvious, potential is transferred to a direction of reducing, but the particle size and the degree of potential transfer are smaller than those of the binary zein nanoparticles which are not subjected to ultrasonic treatment, so that the storage stability of the binary nanocomposite is improved.
Example 3
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency at 28KHz, the ultrasonic power at 300w, the ultrasonic time at 60min, the ultrasonic mode at a continuous mode, and the constant-temperature water bath temperature at 25 deg.C, taking out the dialysis bag, and adding 400mL of deionized water again to dialyze at room temperature for 11 h.
(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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm. Fresh zein nanoparticles treated with 28KHz sonication had a particle size of 239.35 + -2.44 nm, PDI of 0.14 + -0.03, a potential of-31.13 + -4.83 mv, zein nanoparticles stored for 15 days had a particle size of 264.82 + -2.84 nm, PDI of 0.15 + -0.05, and a potential of-27.77 + -1.21 mv. 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 is increased, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with example 2, the particle diameter and PDI values are close, and the potential is decreased. During the storage process, the particle size of the binary zein nanoparticles treated by 28KHz ultrasound shows a trend of increasing the particle size, PDI is increased but is not obvious, the potential is transferred to the direction of decreasing, but the particle size and the degree of potential transfer are smaller than those of the binary zein nanoparticles which are not treated by ultrasound, so that the storage stability of the binary nano composite is improved.
Example 4
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency at 40KHz, the ultrasonic power at 300w, the ultrasonic time at 60min, the ultrasonic mode at a continuous mode, and the constant-temperature water bath temperature at 25 deg.C, taking out the dialysis bag, and adding 400mL of deionized water again to dialyze at room temperature for 11 h.
(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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm. Fresh zein nanoparticles treated with 40KHz ultrasound have a particle size of 231.20 + -15.75 nm, PDI of 0.14 + -0.01, a potential of-32.60 + -1.32 mv, zein nanoparticles stored for 15 days have a particle size of 267.93 + -3.44 nm, PDI of 0.19 + -0.03, and a potential of-26.01 + -2.08 mv. Compared with example 1, the particle size is significantly reduced, PDI is smaller, 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. Compared with examples 2 and 3, the nano-particles obtained after increasing the frequency have smaller particle size. In the storage process, the particle size of the binary zein nanoparticles treated by 40KHz ultrasound shows a trend of increasing the particle size, PDI is increased but is not obvious, the potential is transferred towards the direction of reduction, but the particle size and the degree of potential transfer are both smaller than those of the binary zein nanoparticles which are not treated by ultrasound, so that the storage stability of the binary nano composite is improved.
Example 5
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency to be 20KHz +28KHz, the ultrasonic power to be 300w, the ultrasonic time to be 60min, the ultrasonic mode to be continuous, the constant temperature water bath temperature to be 25 ℃, taking out the dialysis bag, and adding 400mL of deionized water again to dialyze for 11h 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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm. Fresh zein nanoparticles subjected to 20KHz +28KHz ultrasonic treatment have a particle size of 242.92 + -1.28 nm, a PDI of 0.13 + -0.03, a potential of-34.84 + -0.59 mv, and zein nanoparticles stored for 15 days have a particle size of 249.16 + -2.62 nm, a PDI of 0.11 + -0.03, and a potential of-31.65 + -2.37 mv. 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 is increased, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with the examples 1, 2, 3 and 4, the particle size of the nanoparticle obtained after the 20KHz +28KHz double-frequency treatment and the amplitude change before and after potential storage are small, and the storage stability of the nanoparticle is enhanced. In the storage process, the particle size of the binary zein nanoparticles treated by ultrasonic of 20KHz +28KHz shows a trend of increasing the particle size, the PDI changes insignificantly, the potential is transferred towards the direction of reducing, but the particle size and the degree of potential transfer are both smaller than those of the binary zein nanoparticles which are not treated by ultrasonic, so that the storage stability of the binary nanocomposite is improved.
Example 6
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency at 28KHz +40KHz, the ultrasonic power at 300w, the ultrasonic time at 60min, the ultrasonic mode at a continuous mode, and the constant-temperature water bath temperature at 25 deg.C, taking out the dialysis bag, and adding 400mL deionized water again to dialyze at room temperature for 11 h.
(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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm.
The particle size of fresh zein nanoparticles subjected to 28KHz +40KHz ultrasonic treatment is 236.62 +/-3.26 nm, PDI is 0.13 +/-0.02, the potential is-33.47 +/-1.38 mv, the particle size of zein nanoparticles stored for 15 days is 242.61 +/-3.15 nm, PDI is 0.1 +/-0.06, and the potential is-32.00 +/-1.01 mv. Compared with example 1, the particle size is significantly reduced, PDI is smaller, 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. Compared with the examples 2, 3 and 5, the particle size of the nano-particles obtained after the double-frequency treatment of 28KHz and 40KHz is smaller. Compared with the examples 2, 3, 4 and 5, the particle size of the nanoparticles obtained after the 28KHz +40KHz double-frequency treatment and the amplitude change before and after potential storage are small, and the storage stability of the nanoparticles is enhanced. In the storage process, the particle size of the binary zein nanoparticles treated by ultrasonic of 28KHz +40KHz shows a trend of increasing the particle size, the PDI changes insignificantly, the potential is transferred towards the direction of reducing, but the particle size and the degree of potential transfer are both smaller than those of the binary zein nanoparticles which are not treated by ultrasonic, so that the storage stability of the binary nano composite is improved.
Example 7
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency to be 20KHz +40KHz, the ultrasonic power to be 300w, the ultrasonic time to be 60min, the ultrasonic mode to be continuous, the constant temperature water bath temperature to be 25 ℃, taking out the dialysis bag, and adding 400mL of deionized water again to dialyze for 11h 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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm. Fresh zein nanoparticles subjected to 20KHz +40KHz ultrasonic treatment have a particle size of 225.94 + -45.84 nm, a PDI of 0.17 + -0.06, and a potential of-29.97 + -1.49 mv, and zein nanoparticles stored for 15 days have a particle size of 226.02 + -11.28 nm, a PDI of 0.19 + -0.08, and a potential of-28.50 + -0.60 mv. 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 is increased, indicating that ultrasound promotes the formation of uniform and stable binary zein nanoparticles. Compared with examples 2, 3, 4, 5 and 6, the double-frequency ultrasonic treatment is superior to but single-frequency ultrasonic treatment, and the particle size of the nanoparticles obtained after double-frequency treatment of 20KHz +40KHz is smaller. Compared with the examples 2, 3, 4, 5 and 6, the particle size of the nanoparticles obtained after the 28KHz +40KHz double-frequency treatment and the amplitude change before and after potential storage are small, and the storage stability of the nanoparticles is enhanced. In the storage process, the particle size of the binary zein nanoparticles treated by ultrasonic of 20KHz +40KHz shows a trend of increasing the particle size, the PDI changes insignificantly, the potential is transferred towards the direction of reducing, but the particle size and the degree of potential transfer are both smaller than those of the binary zein nanoparticles which are not treated by ultrasonic, so that the storage stability of the binary nano composite is improved.
Example 8
(1) Dissolving a certain mass of zein and sodium caseinate in an 80% (v/v) propylene glycol aqueous solution, wherein the mass ratio of the zein to the sodium caseinate is 2%, uniformly stirring the zein and the sodium caseinate by magnetic force, visually observing the zein and the sodium caseinate without obvious precipitation, and centrifuging the binary mixed solution at 5000rpm for 10min to remove residual large particles to obtain the binary mixed solution of the zein and the sodium caseinate.
(2) 20mL of binary mixed solution of zein and sodium caseinate is measured and put into a dialysis bag which is subjected to activation treatment, has the length of 45cm, the width of 34mm and the molecular weight cutoff of 8000-14000, the dialysis bag is sealed and put into a material container, and 400mL of deionized water is added.
(3) Placing the material container in an ultrasonic tank, setting the ultrasonic frequency to be 20KHz +28KHz +40KHz, the ultrasonic power to be 300w, the ultrasonic time to be 60min, the ultrasonic mode to be continuous, the constant temperature water bath temperature to be 25 ℃, taking out the dialysis bag, and adding 400mL of deionized water again to dialyze for 11h 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.
(5) And (3) measuring turbidity: fresh samples were taken and the absorbance values of the samples were measured at a wavelength of 500nm using a UV-vis UV-Vis spectrophotometer to characterize the turbidity of the samples. The measurements were performed in triplicate for each sample at room temperature and the results are expressed as mean values.
(6) Measurement of particle diameter and Zeta potential: and measuring the particle size, PDI and Zeta potential of the zein binary nanoparticles by adopting 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 a suitable concentration prior to measurement to avoid multiple scattering effects. All measurements were averaged in triplicate.
(7) Fluorescence spectrometry: for selective excitation of tryptophan residues, 280nm is used as the excitation wavelength, the scanning range is 290-450nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are both set to be 10 nm.
Fresh zein nanoparticles subjected to ultrasonic treatment of 20KHz +28KHz +40KHz have a particle size of 239.09 + -3.11 nm, a PDI of-34.52 + -2.62 mv, and zein nanoparticles stored for 15 days have a particle size of 242.48 + -5.13 nm and a PDI of-28.74 + -5.84 mv. 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 is increased, 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 the examples 2, 3 and 4, the particle size of the nanoparticles obtained after 28KHz +40KHz double-frequency treatment has small change with the amplitude before and after potential storage, and the storage stability of the nanoparticles is enhanced. In the storage process, the particle size of the binary zein nanoparticles processed by ultrasonic of 20KHz +28KHz +40KHz shows a trend of increasing the particle size, the PDI changes insignificantly, the potential is transferred towards the direction of reducing, but the particle size and the potential transfer degree are both smaller than those of the binary zein nanoparticles which are not processed by ultrasonic, and the storage stability of the binary nanocomposite is improved.
Table 1 shows the particle size, PDI and Zeta potential of nanoparticles for different storage times.
The particle size of the zein nanoparticles subjected to ultrasonic-assisted dialysis treatment is obviously reduced, the PDI value is small, the particle size distribution is narrow, the particle size distribution is uniform, the potential is increased, and uniform and stable nanoparticles are formed. The ultrasonic and dialysis biphase physical driving force opens the high-level structure in the zein, exposes more aromatic amino acid residues in the zein, and increases the fluorescence intensity of the zein colloid solution. The nanoparticles subjected to auxiliary dialysis treatment by different combined frequencies show different physical properties, wherein the particle size of the zein nanoparticles subjected to ultrasonic auxiliary dialysis treatment at 20KHz +40KHz is 225.94 +/-45.84 nm, the PDI is 0.17 +/-0.06, the potential is-29.97 +/-1.49 mv, the particle size of the zein nanoparticles stored for 15 days is 226.02 +/-11.28 nm, the PDI is 0.19 +/-0.08, the potential is-28.50 +/-0.60 mv, the particle size is the smallest, the fluorescence intensity is the strongest, the optimal auxiliary frequency is selected, the particle size potential change degree is small during storage, and the storage stability is good.
TABLE 1 nanoparticle size, PDI, Zeta potential map for different storage times
Claims (4)
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 ℃.
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