CN109797447B - A kind of preparation method of zein nanofibers - Google Patents
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Abstract
本发明公开了一种玉米醇溶蛋白纳米纤维的制备方法，属于蛋白纳米纤维技术领域。所述玉米醇溶蛋白纳米纤维的制备方法为：将玉米醇溶蛋白溶于冰乙酸中，置于40~80℃的水浴中加热5~120 min，得到玉米醇溶蛋白乙酸溶液；将4~15℃去离子水滴入40~80℃的玉米醇溶蛋白乙酸溶液中，得到玉米醇溶蛋白乙酸水溶液；将玉米醇溶蛋白乙酸水溶液真空冷冻干燥，得到玉米醇溶蛋白纳米纤维。电镜结果表明，该纤维呈细丝状，直径为50‑300 nm，纤维间互相交联形成三位网络结构。本方法低耗节能、工艺简单、易于操作、便于工业化生产，本发明还为玉米醇溶蛋白纤维在面团特性改良、仿生肉制品中的应用奠定技术基础。
The invention discloses a preparation method of zein nanofibers, which belongs to the technical field of protein nanofibers. The preparation method of the zein nanofibers is as follows: dissolving zein in glacial acetic acid, placing it in a water bath at 40-80° C. and heating for 5-120 min to obtain a zein-acetic acid solution; dissolving 4- Deionized water at 15°C is dropped into the zein acetic acid solution at 40-80°C to obtain the zein acetic acid aqueous solution; the zein acetic acid aqueous solution is vacuum freeze-dried to obtain the zein nanofibers. The electron microscope results showed that the fibers were filamentous with a diameter of 50-300 nm, and the fibers were cross-linked to form a three-dimensional network structure. The method has low energy consumption, simple process, easy operation and convenient industrial production, and the invention also lays a technical foundation for the application of zein fiber in dough property improvement and biomimetic meat products.
Description
Technical Field
The invention belongs to the technical field of protein nanofibers, and particularly relates to a preparation method of zein nanofibers.
Background
Zein is natural protein extracted from corn starch processing byproducts, and is one of high value-added products comprehensively developed and utilized by corn byproducts. Unlike wheat protein, zein is a non-allergenic protein, has unique self-assembly characteristics, film-forming properties, biocompatibility and degradability, and is a well-known safe food-grade raw material. The zein contains more than 50% of nonpolar amino acid, has strong hydrophobicity, is insoluble in pure water or pure alcohol, and can be dissolved in 60% -95% (v/v) ethanol water solution. Current research on zein has focused on the preparation of spherical nanoparticles by an anti-solvent precipitation process in aqueous ethanol, particle stabilizers commonly used to construct delivery vehicles and emulsions for bioactive substances, and the like.
Molecular self-assembly is the process by which building blocks spontaneously form aggregates with specific structures and functions through non-covalent interactions under certain conditions. The highly ordered fiber structure can be formed by taking protein as a structural unit through molecular self-assembly. Currently, the mechanism of the self-assembly of a few pathogenic proteins into fibers (amyloid fibers) is systematically and intensively studied. Some water-soluble food-derived proteins also aggregate to form fibrous structures, such as β -lactoglobulin, ovalbumin, bovine serum albumin, soy protein, and the like, which are typically self-assembled by heating above the denaturation temperature under conditions of low pH and low ionic strength. The zein fiber can be prepared from the zein ethanol aqueous solution by an electrostatic spinning method, has excellent physical and mechanical properties, is commonly used as biomedical materials such as biological membrane materials, tissue engineering scaffolds and the like, and has good application prospect in the field of in-vitro renewable high-performance silk fibroin fiber materials. However, the electrostatic spinning method requires special equipment, the process is complicated, and the spinning effect is easily affected by the voltage value, the spinning distance, the propelling speed of the injection pump, the temperature and humidity of the spinning environment, and the like. How to construct a new method which is simple in process and easy to operate for the preparation of zein nanofiber based on a molecular self-assembly fibrosis mechanism is a problem which needs to be solved at present.
The acetic acid is a common reagent for extracting zein from the corn gluten meal, and compared with an ethanol water solution, the zein has better solubility in the acetic acid, and a molecular chain is completely stretched and exists in a monomer form. Zein has poor water-swelling properties due to its strong hydrophobicity, and cannot form a dough. Prior studies have indicated that zein dissolved in acetic acid can form a viscoelastic dough with a network structure upon stirring and kneading. However, researchers have concentrated on improving the dough rheology of zein on a macroscopic level, ignoring zein fibers.
Disclosure of Invention
The invention firstly purifies the commercial zein to obtain high-purity alpha-zein, researches the solubility of the zein in different acetic acid water solutions, and provides a brand-new method for preparing zein nanofibers by utilizing acetic acid, water solutions and temperature changes by adopting a top-down molecular self-assembly method based on the molecular engineering principle according to the dissolution characteristic of the zein in the acetic acid water solution in the later stage, wherein the preparation method of the zein nanofibers comprises the following steps: dissolving zein in glacial acetic acid, and heating in a water bath at 40-80 ℃ for 5-120 min to obtain a zein acetic acid solution; dripping deionized water at 4-15 ℃ into a zein acetic acid solution at 40-80 ℃ to obtain a zein acetic acid aqueous solution; and (3) carrying out vacuum freeze drying on the zein acetic acid aqueous solution to obtain the zein nano fiber.
Further, the mixing ratio of the zein and the glacial acetic acid is as follows: 0.1 to 10 wt%.
Further, the vacuum freeze-drying conditions are as follows: the precooling time is 4-12 h, the precooling temperature is-40 to-60 ℃, the freeze-drying time is 24-72 h, and the vacuum degree is 10-40 pa.
Furthermore, the mixing volume ratio of the deionized water to the zein acetic acid solution is 5: 1-1000: 1.
Further, the zein is alpha-zein.
Further, the extraction method of the alpha-zein comprises the following steps:
dissolving zein in an 85-95% (v/v) ethanol water solution, performing vortex to completely dissolve the zein, and placing the zein at the temperature of 4 +/-1 ℃; and carrying out low-temperature centrifugal treatment; taking supernatant after low-temperature centrifugation, and keeping the supernatant at 4 +/-1 ℃;
dripping deionized water at 15 +/-1 ℃ into the supernatant after low-temperature centrifugation, completely mixing, and performing secondary centrifugation treatment at 15 +/-1 ℃;
and (4) carrying out vacuum freeze drying on the precipitate after the secondary centrifugation treatment to obtain the alpha-zein.
Wherein, the low-temperature centrifugal treatment and the secondary centrifugal treatment are both carried out for 20-40 min by adopting a centrifugal force of 8000-12000 g. The vacuum freeze-drying conditions are as follows: the precooling time is 4-12 h, the precooling temperature is-40 to-60 ℃, the freeze-drying time is 24-72 h, and the vacuum degree is 10-40 pa.
Advantageous effects
The inventor firstly purifies the commercial zein to obtain the high-purity alpha-zein and explores the solubility of the alpha-zein in different acetic acid aqueous solutions. Zein is not completely soluble in 10% and 20% acetic acid aqueous solutions, whereas in 30% and above acetic acid aqueous solutions, the solubility is good, and as the acetic acid concentration increases, the zein solution becomes clearer and brighter to the naked eye, as shown in fig. 1.
According to the invention, by observing the form of the zein in the acetic acid solution, the form of the zein in the acetic acid solution is different from the form of spherical zein nanoparticles in the ethanol water solution, specifically shown in fig. 2, the zein in the acetic acid water solution can form mutually cross-linked filament-shaped nanofibers through molecular self-assembly, experimental parameters such as zein concentration and acetic acid concentration are changed, and the form and the size of the zein are changed, specifically shown in fig. 3 and 4. The AFM results also confirm that the method provided by the present invention successfully produces zein nanofibers, as shown in fig. 5. Experimental results show that the zein fiber prepared by the hot-melt cold-reaction method in the acetic acid aqueous solution has feasibility and practicability.
According to the dissolution characteristic of zein in an acetic acid aqueous solution, based on the principle of molecular engineering, the zein hydrophilic/hydrophobic characteristic is adjusted by adopting a top-down molecular self-assembly method and changing experimental parameters such as acetic acid concentration, zein concentration, heat treatment time and temperature, ionic strength, stirring rate, dropping sequence and the like, the competition and balance of intramolecular/intermolecular microscopic interaction force are regulated, and a zein fiberization molecular mechanism is disclosed. Meanwhile, a hot melt cold reaction method is constructed by combining the glass transition characteristics of the zein nanofiber. The molecular mechanism of zein fibrosis is analyzed, so that an important technical means can be provided for preparing novel food ingredients with various structures and rich functions. Meanwhile, the zein nanofiber is expected to become a new-generation functional food ingredient to be applied to the food industry, and enriches the varieties of the existing food ingredients. In addition, compared with the electrostatic spinning method, the method has the advantages of low energy consumption, energy conservation, simple process, easy operation and convenient industrial production, and lays a technical foundation for the application of the zein fiber in dough property improvement and bionic meat products.
Description of the drawings:
FIG. 1: dissolution profile of alpha-zein in aqueous acetic acid.
FIG. 2: scanning electron micrographs of zein nanoparticles.
FIG. 3: scanning electron microscope images of the nanofibers with a mixing ratio of zein to glacial acetic acid of 1.0wt% and a mixing volume ratio of deionized water to zein acetic acid solution of 5: 1.
FIG. 4: scanning electron microscope images of the nanofibers with the mixing ratio of zein to glacial acetic acid being 1.0wt% and the mixing volume ratio of deionized water to zein acetic acid solution being 10: 1.
FIG. 5: an atomic force microscope image of the nanofiber with the mixing ratio of the zein to the glacial acetic acid being 1.0wt% and the mixing volume ratio of the deionized water to the zein acetic acid solution being 100: 1.
Detailed Description
The invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope, which after reading the present invention, is susceptible of modification in various equivalent forms by those skilled in the art, all falling within the scope of the invention as defined in the appended claims.
In the present invention, zein was purchased from Sigma, CAS No.: 9010-66-6, and all other reagents are food grade.
Example 1
A preparation method of alpha-zein nano fibers comprises the following steps:
a) commercially available zein 5.0 g was weighed on an analytical balance, dissolved in 20 mL of 90% (v/v) aqueous ethanol, vortexed to dissolve completely, and then allowed to stand at 4. + -. 1 ℃ for 12 h.
b) And (c) centrifuging the solution after standing for 12 hours in the step a by adopting a centrifugal force of 10000 g at 4 +/-1 ℃ through a low-temperature refrigerated centrifuge for 20 minutes.
c) And (3) subpackaging the centrifuged supernatant into a centrifugal tube, keeping the temperature of the centrifugal tube at 4 +/-1 ℃, then dropwise adding 15 +/-1 ℃ deionized water into the centrifugal tube, and then carrying out secondary centrifugation for 20min at 15 +/-1 ℃ by adopting 10000 g of centrifugal force.
d) And finally, carrying out vacuum freeze drying on the centrifuged precipitate to obtain the high-purity alpha-zein, wherein the vacuum freeze drying conditions are as follows: the precooling time is 4h, the precooling temperature is-40 ℃, the freeze-drying time is 24h, and the vacuum degree is 10 pa.
e) Dissolving alpha-zein in glacial acetic acid according to the proportion of 0.1 wt%, and heating in water bath at 40 ℃ for 5min to obtain an alpha-zein acetic acid solution.
f) And (2) dripping deionized water at 4 ℃ into an alpha-zein acetic acid solution at 40 ℃ to obtain the alpha-zein acetic acid aqueous solution, wherein the mixing volume ratio of the deionized water to the alpha-zein acetic acid solution is 5: 1.
g) And f, carrying out vacuum freeze drying on the zein acetic acid aqueous solution obtained in the step f, wherein the vacuum freeze drying conditions are as follows: precooling for 4h, wherein the precooling temperature is-40 ℃, the freeze-drying time is 24h, and the vacuum degree is 10pa, so that the alpha-zein nanofiber is obtained.
Scanning electron microscope results show that the fiber is in a filament shape, the diameter of the fiber is 50-300 nm, and the fibers are mutually crosslinked to form a three-dimensional network structure. Compared with the electrostatic spinning method commonly used for preparing the zein fiber, the method has the advantages of low energy consumption, energy conservation, simple process, easy operation and convenient industrial production, not only establishes a new method for preparing the zein nanofiber, but also lays a technical foundation for the application of the zein fiber in dough property improvement and bionic meat products.
Example 2
A preparation method of alpha-zein nano fibers comprises the following steps:
a) commercially available zein (1.0 g) was weighed on an analytical balance, dissolved in 5mL of 90% (v/v) aqueous ethanol, vortexed to dissolve completely, and then allowed to stand at 4. + -. 1 ℃ for 4 h.
b) And (c) centrifuging the solution after standing for 4 hours in the step a for 40 minutes by using a low-temperature refrigerated centrifuge at the temperature of 4 +/-1 ℃ by adopting a centrifugal force of 8000 g.
c) And (3) subpackaging the centrifuged supernatant into a centrifugal tube, keeping the temperature of the centrifugal tube at 4 +/-1 ℃, then dropwise adding 15 +/-1 ℃ deionized water into the centrifugal tube, and then carrying out secondary centrifugation for 40 min at 15 +/-1 ℃ by adopting 8000 g of centrifugal force.
d) And finally, carrying out vacuum freeze drying on the centrifuged precipitate to obtain the high-purity alpha-zein, wherein the vacuum freeze drying conditions are as follows: the precooling time is 12h, the precooling temperature is-60 ℃, the freeze-drying time is 72h, and the vacuum degree is 40 pa.
e) Dissolving alpha-zein in glacial acetic acid according to the proportion of 1.0wt%, and heating in water bath at 80 ℃ for 120 min to obtain an alpha-zein acetic acid solution.
f) And (2) dripping deionized water at 15 ℃ into an alpha-zein acetic acid solution at 80 ℃ to obtain the alpha-zein acetic acid aqueous solution, wherein the mixing volume ratio of the deionized water to the alpha-zein acetic acid solution is 10: 1.
g) And f, carrying out vacuum freeze drying on the zein acetic acid aqueous solution obtained in the step f, wherein the vacuum freeze drying conditions are as follows: and (3) precooling for 12h, wherein the precooling temperature is-60 ℃, the freeze-drying time is 72h, and the vacuum degree is 40pa, so that the alpha-zein nanofiber is obtained.
Scanning electron microscope results show that the fiber is in a filament shape, the diameter of the fiber is 50-300 nm, and the fibers are mutually crosslinked to form a three-dimensional network structure.
Example 3
A preparation method of alpha-zein nano fibers comprises the following steps:
a) commercially available zein (3.0 g) is weighed on an analytical balance, dissolved in 20 mL of 90% (v/v) ethanol aqueous solution, vortexed to dissolve completely, and then placed at 4 + -1 deg.C for 10 h.
b) And (3) centrifuging the solution after standing for 10 hours in the step a for 30 minutes by a low-temperature refrigerated centrifuge at the temperature of 4 +/-1 ℃ by using a centrifugal force of 12000 g.
c) And (3) subpackaging the centrifuged supernatant into a centrifugal tube, keeping the temperature of the centrifugal tube at 4 +/-1 ℃, then dropwise adding 15 +/-1 ℃ deionized water into the centrifugal tube, and then carrying out secondary centrifugation for 30 min at 15 +/-1 ℃ by adopting 12000 g of centrifugal force.
d) And finally, carrying out vacuum freeze drying on the centrifuged precipitate to obtain the high-purity alpha-zein, wherein the vacuum freeze drying conditions are as follows: the precooling time is 10h, the precooling temperature is-50 ℃, the freeze-drying time is 48h, and the vacuum degree is 30 pa.
e) Dissolving alpha-zein in glacial acetic acid according to the proportion of 5.0 wt%, and heating in water bath at 60 ℃ for 80 min to obtain an alpha-zein acetic acid solution.
f) And (2) dripping deionized water at 12 ℃ into an alpha-zein acetic acid solution at 60 ℃ to obtain the alpha-zein acetic acid aqueous solution, wherein the mixing volume ratio of the deionized water to the alpha-zein acetic acid solution is 20: 1.
g) And f, carrying out vacuum freeze drying on the zein acetic acid aqueous solution obtained in the step f, wherein the vacuum freeze drying conditions are as follows: and (3) precooling for 10h, wherein the precooling temperature is-50 ℃, the freeze-drying time is 36h, and the vacuum degree is 30pa, so that the alpha-zein nanofiber is obtained.
Scanning electron microscope results show that the fiber is in a filament shape, the diameter of the fiber is 50-300 nm, and the fibers are mutually crosslinked to form a three-dimensional network structure.
Example 4
A preparation method of zein nanofibers comprises the following steps:
a) dissolving zein in glacial acetic acid according to the proportion of 5.0 wt%, and heating in water bath at 60 deg.C for 80 min to obtain zein acetic acid solution.
b) And (2) dropping deionized water at 10 ℃ into a zein acetic acid solution at 50 ℃, wherein the mixing volume ratio of the deionized water to the zein acetic acid solution is 100:1, so as to obtain the zein acetic acid aqueous solution.
c) Carrying out vacuum freeze-drying on the zein acetic acid aqueous solution, wherein the vacuum freeze-drying conditions are as follows: precooling for 10h, wherein the precooling temperature is-50 ℃, the freeze-drying time is 36h, and the vacuum degree is 30pa, so that the zein nanofiber is obtained.
Scanning electron microscope results show that the fiber is in a filament shape, the diameter of the fiber is 50-300 nm, and the fibers are mutually crosslinked to form a three-dimensional network structure.
Example 5
A preparation method of zein nanofibers comprises the following steps:
a) dissolving zein in glacial acetic acid according to the proportion of 1.0wt%, and heating in 80 deg.C water bath for 5min to obtain zein acetic acid solution.
b) And (2) dripping deionized water at 4 ℃ into a zein acetic acid solution at 80 ℃, wherein the mixing volume ratio of the deionized water to the zein acetic acid solution is 1000:1, so as to obtain the zein acetic acid aqueous solution.
c) Carrying out vacuum freeze-drying on the zein acetic acid aqueous solution, wherein the vacuum freeze-drying conditions are as follows: precooling for 4h, wherein the precooling temperature is-60 ℃, the freeze-drying time is 24h, and the vacuum degree is 40pa, so that the zein nanofiber is obtained.
Scanning electron microscope results show that the fiber is in a filament shape, the diameter of the fiber is 50-300 nm, and the fibers are mutually crosslinked to form a three-dimensional network structure.
Example 6
A preparation method of zein nanofibers comprises the following steps:
a) dissolving zein in glacial acetic acid according to the proportion of 10 wt%, and heating in water bath at 60 deg.C for 80 min to obtain zein acetic acid solution.
b) And (2) dropping deionized water at 15 ℃ into a zein acetic acid solution at 50 ℃, wherein the mixing volume ratio of the deionized water to the zein acetic acid solution is 100:1, so as to obtain the zein acetic acid aqueous solution.
c) Carrying out vacuum freeze-drying on the zein acetic acid aqueous solution, wherein the vacuum freeze-drying conditions are as follows: precooling for 12h, wherein the precooling temperature is-40 ℃, the freeze-drying time is 72h, and the vacuum degree is 10pa, so that the zein nanofiber is obtained.
Scanning electron microscope results show that the fiber is in a filament shape, the diameter of the fiber is 50-300 nm, and the fibers are mutually crosslinked to form a three-dimensional network structure.
Claims (8)
1. A preparation method of zein nano fibers is characterized by comprising the following steps: the preparation method of the zein nanofiber comprises the following steps: dissolving zein in glacial acetic acid, and heating in a water bath at 40-80 ℃ for 5-120 min to obtain a zein acetic acid solution; dripping deionized water at 4-15 ℃ into a zein acetic acid solution at 40-80 ℃ to obtain a zein acetic acid aqueous solution; and (3) carrying out vacuum freeze drying on the zein acetic acid aqueous solution to obtain the zein nano fiber.
2. The method of making zein nanofibers according to claim 1, wherein: the concentration of the zein in the glacial acetic acid is 0.1-10 wt%.
3. The method of making zein nanofibers according to claim 1, wherein: the vacuum freeze-drying conditions are as follows: the precooling time is 4-12 h, the precooling temperature is-40 to-60 ℃, the freeze-drying time is 24-72 h, and the vacuum degree is 10-40 pa.
4. The method of making zein nanofibers according to claim 1, wherein: the mixing volume ratio of the deionized water to the zein acetic acid solution is 5: 1-1000: 1.
5. The method of making zein nanofibers according to claim 1, wherein: the zein is alpha-zein.
6. The method of making zein nanofibers according to claim 5, wherein: the extraction method of the alpha-zein comprises the following steps:
dissolving zein in an ethanol water solution with the volume percentage of 85-95%, completely dissolving the zein by vortex, and placing the zein at the temperature of 4 +/-1 ℃; and carrying out low-temperature centrifugal treatment; taking supernatant after low-temperature centrifugation, and keeping the supernatant at 4 +/-1 ℃;
dripping deionized water at 15 +/-1 ℃ into the supernatant after low-temperature centrifugation, completely mixing, and performing secondary centrifugation treatment at 15 +/-1 ℃;
and (4) carrying out vacuum freeze drying on the precipitate after the secondary centrifugation treatment to obtain the alpha-zein.
7. The method of making zein nanofibers according to claim 6, wherein: and the low-temperature centrifugal treatment and the secondary centrifugal treatment are both carried out for 20-40 min by adopting a centrifugal force of 8000-12000 g.
8. The method of making zein nanofibers according to claim 6, wherein: the vacuum freeze-drying conditions are as follows: the precooling time is 4-12 h, the precooling temperature is-40 to-60 ℃, the freeze-drying time is 24-72 h, and the vacuum degree is 10-40 pa.
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| CN109012520A (en) * | 2018-07-18 | 2018-12-18 | 湖北工业大学 | A method of anti-solvent is limited based on gel network and prepares core-shell microcapsule |
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