Preparation method of magnetic cottonseed protein nanospheres with high lipase loading

Abstract

The invention discloses a preparation method of magnetic cottonseed protein nanospheres with high lipase loading, which relates to the technical fields of enzyme engineering and lipase loading in the biological industry, and includes: using the antisolvent method to coat nano-Fe3O4 particles with plant polyphenols and cottonseed protein to obtain magnetic cottonseed protein nanospheres; adding the magnetic nanoprotein spheres into a lipase solution, and using the covalent and secondary interactions between the plant polyphenols and the cottonseed protein to load the lipase on the magnetic cottonseed protein nanospheres; separating, washing, and drying to obtain magnetic cottonseed protein nanospheres with high lipase loading. The invention uses the antisolvent method to coat the nano-particle Fe3O4 to prepare magnetic cottonseed protein nanospheres, fixes the lipase on the surface of the nanospheres through the covalent and secondary interactions between the plant polyphenols and the protein, optimizes the parameters, and obtains magnetic cottonseed protein nanospheres with high lipase loading. The obtained magnetic cottonseed protein nanospheres of the invention can realize the efficient catalysis and recycling of lipase.

Description

Technical Field

[0001] The present invention belongs to the technical fields of enzyme engineering and lipase loading in the biological industry. More specifically, the present invention relates to a method for preparing magnetic cottonseed protein nanospheres with high lipase loading. Background Art

[0002] With the rapid development of biotechnology, enzymes, as biocatalysts, have shown great application potential in fields such as the food industry, medicine, environmental protection, and chemical synthesis. Among them, lipase, as an important hydrolase, scientifically named triacylglycerol acylhydrolase, is an enzyme that can catalyze the hydrolysis of oils and fats and is widely present in tissues of animals, plants, and microorganisms (such as molds, bacteria, etc.) containing fats; lipase can catalyze the hydrolysis of triglycerides into fatty acids and glycerol and is widely used in fields such as food oil processing, detergent production, medicine, and biochemistry preparation. However, traditional lipases have problems such as being easily inactivated and difficult to recycle and reuse during use, which severely limits their industrial applications.

[0003] To overcome these challenges, researchers have been committed to developing new enzyme immobilization technologies to improve the stability, activity, and recyclability of enzymes. Among them, the nanocarrier technology has become a popular research direction for enzyme immobilization due to its unique physicochemical properties, such as high specific surface area, good biocompatibility, and easy surface modification. In particular, magnetic nanoparticles, such as Fe3O4, provide a new solution for the recycling of enzymes due to their characteristics of being easily separated and recovered under an external magnetic field.

[0004] Cottonseed protein, as a natural and renewable protein resource, has excellent biodegradability and biocompatibility, and is widely sourced and low-cost. In recent years, research on using cottonseed protein as a carrier material to prepare functionalized nanomaterials by combining with nanotechnology has gradually increased. However, directly immobilizing lipase on cottonseed protein nanospheres often has problems such as low loading efficiency and large loss of enzyme activity during the immobilization process. Summary of the Invention

[0005] An object of the present invention is to solve at least the above problems and / or defects and provide at least the advantages described hereinafter.

[0006] To achieve these objects and other advantages according to the present invention, there is provided a method for preparing magnetic cottonseed protein nanospheres with high lipase loading, including: using the antisolvent method to coat nanosized Fe3O4 particles with cottonseed protein modified by plant polyphenols to obtain magnetic cottonseed protein nanospheres, adding the magnetic nanoprotein spheres into a lipase solution, and using the covalent and secondary interactions between the plant polyphenols and the cottonseed protein to load the lipase on the magnetic cottonseed protein nanospheres, followed by separation, washing, and drying to obtain the magnetic cottonseed protein nanospheres with high lipase loading.

[0007] Preferably, it includes the following steps:

[0008] Step 1: Disperse nano-Fe3O4 in deionized water to obtain a nano-Fe3O4 particle suspension; dissolve plant polyphenols and cottonseed protein powder in deionized water respectively to obtain a plant polyphenol solution and a cottonseed protein solution.

[0009] Step 2: Use the plant polyphenol solution and the cottonseed protein solution to prepare plant polyphenol-modified cottonseed protein; add the nano-Fe3O4 particle suspension to the plant polyphenol-modified cottonseed protein, and disperse it evenly by ultrasonic treatment to obtain a mixed system.

[0010] Step 3: Stir the mixed system, and at the same time dropwise add an anti-solvent to the mixed system. As the anti-solvent is added dropwise, the solubility of the plant polyphenol-modified cottonseed protein decreases, and it gradually precipitates on the surface of the nano-Fe3O4 particles, coating the surface of the nano-Fe3O4 particles. After standing for a certain period of time at a certain temperature, solid-liquid separation is carried out, and after washing and drying, magnetic cottonseed protein nanospheres are obtained.

[0011] Step 4: Add the magnetic cottonseed protein nanospheres to the lipase solution, and use the covalent and secondary interactions between plant polyphenols and cottonseed protein to load lipase on the magnetic cottonseed protein nanospheres. After solid-liquid separation, washing, and drying, magnetic cottonseed protein nanospheres with high lipase loading are obtained.

[0012] Preferably, in Step 1, the mass-volume ratio of nano-Fe3O4 to deionized water is 1-10 g:20-50 mL; the mass-volume ratio of plant polyphenols to deionized water is 1-2 g:20 mL; the mass-volume ratio of cottonseed protein powder to deionized water is 3-5 g:50-100 mL.

[0013] Preferably, in Step 2, the specific method for preparing the plant polyphenol-modified cottonseed protein includes: stirring the cottonseed protein powder solution at a speed of 200-800 rpm, then dropwise adding glutaraldehyde, continuously stirring and then standing to obtain a cross-linked system; stirring the cross-linked system at a speed of 600-1200 rpm, dropwise adding the plant polyphenol solution to the cross-linked system, continuously stirring, heating to 60-80 °C after dropping and holding for 1-2 h, stopping stirring, and cooling to obtain the plant polyphenol-modified cottonseed protein; among them, the volume ratio of the plant polyphenol solution to the cottonseed protein solution is 1:1, and the mass ratio of glutaraldehyde to plant polyphenols is 1-3:5-10.

[0014] Preferably, in Step 1, the plant polyphenol is one of tea polyphenols, apple polyphenols, grape polyphenols, resveratrol, and myricetin.

[0015] Preferably, in the second step, the volume ratio of the plant polyphenol solution, the cottonseed protein solution and the nano-Fe3O4 particle suspension is 1:1:2-4; the ultrasonic dispersion frequency is 40-60 kHz, and the dispersion time is 1-3 h.

[0016] Preferably, in the third step, the anti-solvent is one of petroleum ether, chloroform or benzene; the volume ratio of the nano-Fe3O4 particle suspension to the anti-solvent is 2-4:5-8.

[0017] Preferably, in the third step, the standing temperature is 30-60 °C, and the standing time is 12-24 h.

[0018] Preferably, in the fourth step, the concentration of the lipase solution is 1-1.6 g / mL, and the mass-volume ratio of the magnetic cottonseed protein nanospheres to the lipase solution is 5-10 mg:1 mL.

[0019] Preferably, the nano-Fe3O4 in the first step is replaced by modified nano-Fe3O4. The preparation method of the modified nano-Fe3O4 includes: selecting nano-Fe3O4 with a particle size distribution of 20-500 nm, ultrasonically dispersing it in absolute ethanol, with an ultrasonic frequency of 30-60 kHz and an ultrasonic time of 20-40 min to obtain a nano-Fe3O4 suspension; adding polyvinylpyrrolidone and γ-aminopropyltriethoxysilane to the nano-Fe3O4 suspension, magnetically stirring at a speed of 1200-2400 rpm for 1-2 h, heating to 80-90 °C within 5-10 min, keeping warm for 15-60 min, and then cooling to room temperature; adding sodium dodecylbenzenesulfonate, ultrasonically dispersing, with an ultrasonic frequency of 40-80 kHz and an ultrasonic time of 20-60 min, heating to 60-80 °C and keeping warm for 1-2 h, performing solid-liquid separation, washing and drying to obtain modified nano-Fe3O4, wherein the mass-volume ratio of nano-Fe3O4, absolute ethanol, polyvinylpyrrolidone, sodium dodecylbenzenesulfonate and γ-aminopropyltriethoxysilane is 0.5-1 g:10-30 mL:0.2-0.5 g:0.05-0.2 g:0.1-0.2 g.

[0020] The present invention has at least the following beneficial effects: The present invention discloses a preparation method of magnetic cottonseed protein nanospheres with high lipase loading. Magnetic cottonseed protein nanospheres are prepared by using the anti-solvent method to coat nano-particles Fe3O4, and lipase is immobilized on the surface of the nanospheres through covalent and secondary interactions between plant polyphenols and proteins. By optimizing the parameters, magnetic cottonseed protein nanospheres with high lipase loading are obtained. The magnetic cottonseed protein nanospheres obtained by the present invention can achieve efficient catalysis and recycling of lipase.

[0021] The present invention also uses the organic polymer polyvinylpyrrolidone to coat nano-Fe3O4, which increases the surface area of nano-Fe3O4. Meanwhile, γ-aminopropyltriethoxysilane is used to improve the tightness of the binding between polyvinylpyrrolidone and nano-Fe3O4. At the same time, the introduction of amino groups increases the active sites on the surface of polyvinylpyrrolidone-coated nano-Fe3O4, promoting the binding of cottonseed protein to polyvinylpyrrolidone-coated nano-Fe3O4. Subsequently, sodium dodecylbenzenesulfonate is used as a dispersant to disperse the polyvinylpyrrolidone-coated nano-Fe3O4, reducing the agglomeration between the polyvinylpyrrolidone-coated nano-Fe3O4, and modified nano-Fe3O4 with good dispersibility, small agglomeration and large surface area is prepared. The modified nano-Fe3O4 with good dispersibility, small agglomeration and large surface area can coat more plant polyphenol-modified cottonseed protein, thereby further increasing the loading capacity of the magnetic cottonseed protein nanospheres for lipase.

[0022] Other advantages, objects and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Detailed implementation mode

[0023] The following provides a further detailed description of the present invention so that those skilled in the art can implement it with reference to the text of the specification.

[0024] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0025] Example 1

[0026] This example provides a method for preparing magnetic cottonseed protein nanospheres with high lipase loading, including the following steps:

[0027] Step 1: Disperse 1 g of nano-Fe3O4 in 20 mL of deionized water to obtain a nano-Fe3O4 particle suspension; dissolve 1 g of myricetin tannin in 20 mL of deionized water and dissolve 3 g of cottonseed protein powder in 50 mL of deionized water to obtain a myricetin tannin solution and a cottonseed protein solution respectively;

[0028] Step 2: Stir the cottonseed protein powder solution at a speed of 600 rpm, then dropwise add 0.2 g of glutaraldehyde, continuously stir and then let it stand to obtain a crosslinked system; stir the crosslinked system at a speed of 600 rpm, dropwise add 20 mL of myricetin tannin solution (the volume of the myricetin tannin solution is the same as that of the cottonseed protein solution) to the crosslinked system, continuously stir, after the addition is completed, raise the temperature to 80 °C and keep it warm for 2 h, stop stirring, and obtain myricetin tannin-modified cottonseed protein after cooling; add 40 mL of nano-Fe3O4 particle suspension to the myricetin tannin-modified cottonseed protein, ultrasonically disperse it evenly, the ultrasonic dispersion frequency is 60 kHz, and the dispersion time is 3 h to obtain a mixed system;

[0029] Step 3: Stir the mixed system, and at the same time dropwise add 100 mL of petroleum ether as an anti-solvent to the mixed system. As the anti-solvent is added dropwise, the solubility of the myricetin tannin-modified cottonseed protein decreases, and it gradually precipitates on the surface of the nano-Fe3O4 particles, coating the surface of the nano-Fe3O4 particles. After standing at 30-60 °C for 12-24 h, perform solid-liquid separation, wash and dry to obtain magnetic cottonseed protein nanospheres;

[0030] Step 4: Add 10 mg of magnetic cottonseed protein nanospheres to 1 mL of lipase solution with a concentration of 1.2 g / mL, and use the covalent interaction between myricetin tannin and cottonseed protein to load lipase on the magnetic cottonseed protein nanospheres, perform solid-liquid separation, washing, and drying to obtain magnetic cottonseed protein nanospheres with high lipase loading.

[0031] Example 2

[0032] This example provides a preparation method of magnetic cottonseed protein nanospheres with high lipase loading, including the following steps:

[0033] Step 1: Disperse 4 g of nano-Fe3O4 in 40 mL of deionized water to obtain a nano-Fe3O4 particle suspension; dissolve 2 g of apple polyphenol in 20 mL of deionized water, and dissolve 4 g of cottonseed protein powder in 60 mL of deionized water to obtain an apple polyphenol solution and a cottonseed protein solution respectively;

[0034] Step 2: Stir the cottonseed protein powder solution at a speed of 800 rpm, then dropwise add 0.4 g of glutaraldehyde, continuously stir and then let it stand to obtain a crosslinked system; stir the crosslinked system at a speed of 1200 rpm, dropwise add 60 mL of apple polyphenol solution (the volume of the apple polyphenol solution is the same as that of the cottonseed protein solution) to the crosslinked system, continuously stir, after the addition is completed, raise the temperature to 80 °C and keep it warm for 2 h, stop stirring, and obtain apple polyphenol-modified cottonseed protein after cooling; add 60 mL of nano-Fe3O4 particle suspension to the apple polyphenol-modified cottonseed protein, ultrasonically disperse it evenly, the ultrasonic dispersion frequency is 60 kHz, and the dispersion time is 2 h to obtain a mixed system;

[0035] Step 3: Stir the mixed system, and simultaneously add 120 mL of chloroform dropwise to the mixed system as an anti-solvent. As the anti-solvent is added dropwise, the solubility of the apple polyphenol-modified cottonseed protein decreases, and it gradually precipitates on the surface of the nano-Fe3O4 particles, coating the surface of the nano-Fe3O4 particles. After standing at 60 °C for 12 h, solid-liquid separation is carried out, and after washing and drying, magnetic cottonseed protein nanospheres are obtained;

[0036] Step 4: Add 10 mg of magnetic cottonseed protein nanospheres to 1 mL of lipase solution with a concentration of 1.2 g / mL. Utilize the covalent and secondary interactions between apple polyphenols and cottonseed protein to load lipase on the magnetic cottonseed protein nanospheres. After solid-liquid separation, washing, and drying, magnetic cottonseed protein nanospheres with high lipase loading are obtained.

[0037] Example 3

[0038] This example provides a method for preparing magnetic cottonseed protein nanospheres with high lipase loading, including the following steps:

[0039] Step 1: Disperse 5 g of nano-Fe3O4 in 50 mL of deionized water to obtain a nano-Fe3O4 particle suspension; dissolve 2 g of grape polyphenols in 20 mL of deionized water, and dissolve 5 g of cottonseed protein powder in 100 mL of deionized water to obtain a grape polyphenol solution and a cottonseed protein solution respectively;

[0040] Step 2: Stir the cottonseed protein powder solution at a speed of 800 rpm, and then dropwise add 0.6 g of glutaraldehyde. After continuous stirring and standing, a cross-linked system is obtained; stir the cross-linked system at a speed of 1200 rpm, and dropwise add 100 mL of grape polyphenol solution (the volume of the grape polyphenol solution is the same as that of the cottonseed protein powder solution) to the cross-linked system. After continuous stirring, the temperature is raised to 80 °C and kept warm for 2 h, then stop stirring. After cooling, grape polyphenol-modified cottonseed protein is obtained; add 80 mL of nano-Fe3O4 particle suspension to the grape polyphenol-modified cottonseed protein, and disperse it evenly by ultrasonic wave. The ultrasonic dispersion frequency is 60 kHz, and the dispersion time is 3 h to obtain a mixed system;

[0041] Step 3: Stir the mixed system, and simultaneously add 160 mL of benzene dropwise to the mixed system as an anti-solvent. As the anti-solvent is added dropwise, the solubility of the grape polyphenol-modified cottonseed protein decreases, and it gradually precipitates on the surface of the nano-Fe3O4 particles, coating the surface of the nano-Fe3O4 particles. After standing at 60 °C for 12 h, solid-liquid separation is carried out, and after washing and drying, magnetic cottonseed protein nanospheres are obtained;

[0042] Step 4: Add 10 mg of magnetic cottonseed protein nanoparticles into 1 mL of lipase solution with a concentration of 1.2 g / mL. Utilize the covalent interaction between grape polyphenols and cottonseed protein to load lipase onto the magnetic cottonseed protein nanoparticles. Perform solid-liquid separation, washing, and drying to obtain magnetic cottonseed protein nanoparticles with high lipase loading.

[0043] Example 4

[0044] This example provides a method for preparing magnetic cottonseed protein nanoparticles with high lipase loading. Compared with Example 1, the nano-Fe3O4 in Step 1 is replaced with modified nano-Fe3O4, and the process parameters of the remaining steps are the same as those in Example 1. Among them, the preparation method of the modified nano-Fe3O4 includes:

[0045] Select 10 g of nano-Fe3O4 with a particle size distribution in the range of 20 - 500 nm, ultrasonically disperse it in 250 mL of absolute ethanol, with an ultrasonic frequency of 60 kHz and an ultrasonic time of 40 min to obtain a nano-Fe3O4 suspension.

[0046] Add 2 g of polyvinylpyrrolidone and 1 g of γ-aminopropyltriethoxysilane to the nano-Fe3O4 suspension, magnetically stir at a speed of 1200 rpm for 2 h, heat to 80 °C within 5 min, keep warm for 30 min, and then cool to room temperature; add 0.5 g of sodium dodecylbenzenesulfonate, ultrasonically disperse it, with an ultrasonic frequency of 60 kHz and an ultrasonic time of 30 min, heat to 80 °C and keep warm for 2 h, perform solid-liquid separation, washing, and drying to obtain the modified nano-Fe3O4.

[0047] Example 5

[0048] This example provides a method for preparing magnetic cottonseed protein nanoparticles with high lipase loading. Compared with Example 1, the nano-Fe3O4 in Step 1 is replaced with modified nano-Fe3O4, and the process parameters of the remaining steps are the same as those in Example 1. Among them, the preparation method of the modified nano-Fe3O4 includes:

[0049] Select 10 g of nano-Fe3O4 with a particle size distribution in the range of 20 - 500 nm, ultrasonically disperse it in 200 mL of absolute ethanol, with an ultrasonic frequency of 60 kHz and an ultrasonic time of 40 min to obtain a nano-Fe3O4 suspension.

[0050] Add 3 g of polyvinylpyrrolidone and 2 g of γ-aminopropyltriethoxysilane to the nano-Fe3O4 suspension, magnetically stir at a speed of 2400 rpm for 2 h, heat to 90 °C within 10 min, keep warm for 60 min, and then cool to room temperature; add 1 g of sodium dodecylbenzenesulfonate, ultrasonically disperse it, with an ultrasonic frequency of 80 kHz and an ultrasonic time of 60 min, heat to 80 °C and keep warm for 1 h, perform solid-liquid separation, washing, and drying to obtain the modified nano-Fe3O4.

[0051] Comparative Example 1

[0052] This comparative example provides a method for preparing magnetic cottonseed protein nanospheres with high-loaded lipase. Compared with Example 1, the operation of modifying cottonseed protein with myricetin tannin in Step 2 was not carried out, and in Step 3, the modified cottonseed protein with myricetin tannin was not used to coat nano-Fe3O4. Instead, 20 mL of myricetin tannin solution and 20 mL of cottonseed protein solution were directly mixed with 40 mL of nano-Fe3O4 particle suspension, and ultrasonic dispersion was carried out evenly. The ultrasonic dispersion frequency was 60 kHz, and the dispersion time was 3 h to obtain a mixed system. Subsequently, the mixed system was stirred, and at the same time, 120 mL of chloroform was added dropwise to the mixed system as an antisolvent. As the antisolvent was added dropwise, the solubility of the apple polyphenol-modified cottonseed protein decreased and gradually precipitated on the surface of the nano-Fe3O4 particles to coat the surface of the nano-Fe3O4 particles. After standing at 60 °C for 12 h, solid-liquid separation was carried out, and after washing and drying, magnetic cottonseed protein nanospheres were obtained;

[0053] 10 mg of magnetic cottonseed protein nanospheres were added to 1 mL of lipase solution with a concentration of 1.2 g / mL. Using the covalent and secondary interactions between myricetin tannin and cottonseed protein, the lipase was loaded on the magnetic cottonseed protein nanospheres, and after solid-liquid separation, washing, and drying, magnetic cottonseed protein nanospheres with high-loaded lipase were obtained.

[0054] Comparative Example 2

[0055] This comparative example provides a method for preparing magnetic nanospheres loaded with lipase. Compared with Example 1, 10 mg of nano-Fe3O4 particle suspension was directly added to 1 mL of lipase solution with a concentration of 1.2 g / mL, and after solid-liquid separation, washing, and drying, magnetic nanospheres loaded with lipase were obtained.

[0056] The lipase loading amounts of the magnetic cottonseed protein nanospheres with high-loaded lipase prepared in Examples 1-5, the magnetic cottonseed protein nanospheres with high-loaded lipase prepared in Comparative Example 1, and the magnetic nanospheres loaded with lipase prepared in Comparative Example 2 were measured respectively, and the following table was obtained:

[0057] Lipase loading amount (mg / g) Example 1 205.2 Example 2 194.0 Example 3 188.5 Example 4 252.6 Example 5 248.9 Comparative Example 1 155.7 Comparative Example 2 124.6

[0058] As can be seen from the above table, the samples of magnetic cottonseed protein nanospheres with high-loaded lipase prepared in Examples 1-5 all achieved high lipase loading amounts and were significantly better than Comparative Example 1 and Comparative Example 2; among them, compared with Examples 1-3, the lipase loading amounts of the magnetic cottonseed protein nanosphere samples in Examples 4 and 5 were further improved.

[0059] The number of devices and the processing scale described herein are used to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be apparent to those skilled in the art.

[0060] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A preparation method of a high-load lipase magnetic cottonseed protein nanosphere, characterized in that, It includes the following steps: Step 1: Disperse nano-Fe3O4 in deionized water to obtain a nano-Fe3O4 particle suspension; dissolve plant polyphenols and cottonseed protein powder in deionized water respectively to obtain a plant polyphenol solution and a cottonseed protein solution; Step 2: Stir the cottonseed protein powder solution at a speed of 200-800 rpm, then dropwise add glutaraldehyde, continue stirring and then let it stand to obtain a crosslinked system; stir the crosslinked system at a speed of 600-1200 rpm, dropwise add the plant polyphenol solution to the crosslinked system, continue stirring, after the addition is completed, heat up to 60-80 °C and keep it warm for 1-2 h, stop stirring, and cool to obtain plant polyphenol-modified cottonseed protein; wherein, the volume ratio of the plant polyphenol solution to the cottonseed protein solution is 1:1, and the mass ratio of glutaraldehyde to plant polyphenols is 1-3:5-10; add the nano-Fe3O4 particle suspension to the plant polyphenol-modified cottonseed protein, and disperse it evenly by ultrasonic waves to obtain a mixed system; Step 3: Stir the mixed system, and at the same time dropwise add an anti-solvent to the mixed system. As the anti-solvent is added dropwise, the solubility of the plant polyphenol-modified cottonseed protein decreases and gradually precipitates on the surface of the nano-Fe3O4 particles, coating the surface of the nano-Fe3O4 particles. After standing at a certain temperature for a period of time, solid-liquid separation is carried out, and after washing and drying, magnetic cottonseed protein nanospheres are obtained; the anti-solvent is one of petroleum ether, chloroform or benzene; the volume ratio of the nano-Fe3O4 particle suspension to the anti-solvent is 2-4:5-8; Step 4: Add the magnetic cottonseed protein nanospheres to the lipase solution, and utilize the covalent and secondary interactions between plant polyphenols and cottonseed protein to load the lipase on the magnetic cottonseed protein nanospheres, carry out solid-liquid separation, washing and drying to obtain magnetic cottonseed protein nanospheres with high lipase loading.

2. The preparation method of the magnetic cottonseed protein nanospheres with high-loading lipase according to claim 1, characterized in that, In the said Step 1, the mass-volume ratio of nano-Fe3O4 to deionized water is 1-10 g:20-50 mL; the mass-volume ratio of plant polyphenols to deionized water is 1-2 g:20 mL; the mass-volume ratio of cottonseed protein powder to deionized water is 3-5 g:50-100 mL.

3. The preparation method of the magnetic cottonseed protein nanospheres with high-load lipase according to claim 1, characterized in that, In the said Step 1, the plant polyphenols are one of tea polyphenols, apple polyphenols, grape polyphenols, resveratrol, myricetin tannin.

4. The preparation method of the magnetic cottonseed protein nanospheres with high-loading lipase as claimed in claim 1, characterized in that, In the said Step 2, the volume ratio of the plant polyphenol solution, the cottonseed protein solution to the nano-Fe3O4 particle suspension is 1:1:2-4; the ultrasonic dispersion frequency is 40-60 kHz, and the dispersion time is 1-3 h.

5. The preparation method of the magnetic cottonseed protein nanospheres with high-loading lipase according to claim 1, characterized in that In the said Step 3, the standing temperature is 30-60 °C, and the standing time is 12-24 h.

6. The preparation method of the magnetic cottonseed protein nanospheres with high-loading lipase according to claim 1, characterized in that, In the said Step 4, the concentration of the lipase solution is 1-1.6 g / mL, and the mass-volume ratio of the magnetic cottonseed protein nanospheres to the lipase solution is 5-10 mg:1 mL.

7. The preparation method of the magnetic cottonseed protein nanospheres with high-loading lipase according to claim 1, characterized in that, In step one, the nano-Fe3O4 is replaced with modified nano-Fe3O4, and the preparation method of the modified nano-Fe3O4 includes: selecting nano-Fe3O4 with a particle size distribution of 20 - 500 nm, ultrasonically dispersing it in absolute ethanol, with an ultrasonic frequency of 30 - 60 kHz and an ultrasonic time of 20 - 40 min to obtain a nano-Fe3O4 suspension; adding polyvinylpyrrolidone and γ-aminopropyltriethoxysilane to the nano-Fe3O4 suspension, magnetically stirring at a speed of 1200 - 2400 rpm for 1 - 2 h, heating to 80 - 90 °C within 5 - 10 min, keeping warm for 15 - 60 min, and cooling to room temperature; adding sodium dodecylbenzenesulfonate, ultrasonically dispersing it, with an ultrasonic frequency of 40 - 80 kHz and an ultrasonic time of 20 - 60 min, heating to 60 - 80 °C and then keeping warm for 1 - 2 h, performing solid-liquid separation, washing and drying to obtain the modified nano-Fe3O4, where the mass-volume ratio of nano-Fe3O4, absolute ethanol, polyvinylpyrrolidone, sodium dodecylbenzenesulfonate, and γ-aminopropyltriethoxysilane is 0.5 - 1 g: 10 - 30 mL: 0.2 - 0.5 g: 0.05 - 0.2 g: 0.1 - 0.2 g.