Method for degrading aflatoxin through magnetic nanoparticle immobilized biological enzyme

Abstract

The invention discloses a method for degrading aflatoxin by magnetic nanoparticle immobilized biological enzyme, and belongs to the technical field of immobilized biological enzyme. The method comprises the following steps: preparing aminated ferroferric oxide magnetic nanoparticles (Fe3O4 (at) NH2) through a chemical coprecipitation method, and carrying out functional modification on the surfaces of the magnetic nanoparticles by adopting 3-aminopropyltriethoxysilane to obtain a magnetic carrier; the aflatoxin degrading enzyme is covalently bound to the surface of the magnetic carrier through a glutaraldehyde cross-linking method, and finally the magnetic nanoparticle immobilized biological enzyme with high stability and recoverability is obtained. The method solves the problems that the enzyme is easy to fall off and difficult to recover in a traditional enzyme immobilization method, the stability and reuse efficiency of the enzyme in an organic solvent are remarkably improved, and the method is suitable for efficient degradation of aflatoxin in food and feed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immobilized biological enzymes, and particularly relates to a method for degrading aflatoxins by using magnetic nanoparticles to immobilize biological enzymes. BACKGROUND

[0002] Aflatoxins are strong carcinogenic metabolites produced by Aspergillus fungi, which widely pollute grains and feed and pose a serious threat to human health. The biological enzyme degradation method has become a research hotspot due to its high efficiency and environmental friendliness, but free enzymes have the defects of poor stability and difficulty in recycling.

[0003] Although the existing immobilization technologies (such as carrier adsorption and embedding method) can solve the above problems to some extent, there are still the following shortcomings:

[0004] (1) Low specific surface area of the carrier: the enzyme loading capacity of traditional carriers (such as chitosan and silicon dioxide) is limited;

[0005] (2) Poor separation efficiency: non-magnetic carriers need to be recovered by centrifugation or filtration, which is complicated;

[0006] (3) Inactivation of organic solvents: enzymes in oil phase or organic solvents are easily inactivated due to desolvation.

[0007] Magnetic nanoparticles (MNPs) have become an ideal carrier for enzyme immobilization due to their superparamagnetism, high specific surface area and easy separation characteristics. The existing technology (such as CN 119020345 A) has tried to use magnetic carriers for glutamate decarboxylase immobilization, but there is no systematic report on the magnetic immobilization method for aflatoxin-degrading enzymes. SUMMARY

[0008] To solve the above technical problems, the application provides a method for degrading aflatoxins by using magnetic nanoparticles to immobilize biological enzymes.

[0009] To achieve the above purpose, the application provides a method for degrading aflatoxins by using magnetic nanoparticles to immobilize biological enzymes, which comprises the following steps:

[0010] Fe3O4 nanoparticles are dispersed in ethanol, 3-aminopropyltriethoxysilane (APTES) is added, and reflux reaction is performed, and then amino-functionalized magnetic nanoparticles (Fe3O4@NH2) are obtained after magnetic separation;

[0011] The Fe3O4@NH2 is dispersed in a phosphate buffer, glutaraldehyde is added, and oscillation is performed at room temperature, and then an aldehyde-functionalized carrier (Fe3O4@CHO) is obtained after magnetic separation;

[0012] The aflatoxin detoxification enzyme solution is mixed with the Fe3O4@CHO, oscillation treated, magnetically separated, washed with a phosphate buffer solution, and then the magnetic nanoparticle immobilized biological enzyme is obtained;

[0013] The magnetic nanoparticle immobilized biological enzyme is added into the aflatoxin containing liquid to be treated.

[0014] In the present application, 3-aminopropyltriethoxysilane (APTES) forms siloxane bonds (-Si-O-) on the surface of Fe3O4 through a hydrolysis reaction, and introduces amino groups (-NH2). This process not only enhances the dispersibility of the nanoparticles, but also provides active sites for subsequent aldehyde group modification. As a bifunctional crosslinking agent, glutaraldehyde (GA) has aldehyde groups (-CHO) at both ends, which respectively react with the amino groups (-NH2) on the surface of the carrier and the amino groups (-NH2) on the surface of the enzyme molecules to form covalent bonds. The aflatoxin detoxification enzyme reacts with the aldehyde groups of Fe3O4@CHO through the ε-amino groups of the surface lysine residues to form stable covalent bonds.

[0015] Further, the preparation method of the Fe3O4 nanoparticles includes the following steps: dissolving FeCl3·6H2O and FeSO4·7H2O in deionized water, purging with nitrogen, heating to 80℃, adding ammonia water to pH=10, magnetically separating after 1h of reaction, washing with ethanol, and drying to obtain the Fe3O4 nanoparticles. The superparamagnetic property (saturation magnetization about 60emu / g) of Fe3O4 enables the immobilized enzyme to rapidly aggregate under an external magnetic field, with a separation efficiency >95% within 10 minutes, significantly reducing the risk of enzyme loss.

[0016] Further, the molar ratio of FeCl3·6H2O to FeSO4·7H2O is 2:1.

[0017] Further, the molar ratio of the Fe3O4 nanoparticles to 3-aminopropyltriethoxysilane is (2-3):1. In the present application, the molar ratio of Fe3O4 to APTES (2-3:1) ensures a moderate amino group density and avoids steric hindrance.

[0018] Further, the temperature of the reflux reaction is 70-80℃, and the time is 5-6h.

[0019] Further, the pH of the phosphate buffer solution is 7.4.

[0020] Further, the mass concentration of glutaraldehyde (GA) is 2-3%.

[0021] A GA concentration of 2-3wt% can balance the crosslinking density and enzyme activity retention, and a phosphate buffer solution with pH=7.4 can make the tertiary structure of the enzyme more stable.

[0022] Further, the molar ratio of the aflatoxin detoxifying enzyme in the solution to the aldehyde- functionalized carrier is (1-2):1; preferably 2:1. The molar ratio of the enzyme to the carrier in the present application can ensure that the enzyme is fully exposed to the active site, while avoiding conformational rigidity caused by excessive cross-linking.

[0023] Further, the magnetic nanoparticles immobilized biological enzyme is added in an amount of 5-10 mg / mL in the aflatoxin-containing liquid to be treated.

[0024] Compared with the prior art, the present application has the following advantages and technical effects:

[0025] The Fe3O4 core endows the magnetic nanoparticle immobilized biological enzyme with superparamagnetism. After the degradation reaction is completed or when it is necessary to recover, an external magnetic field can be applied to quickly and simply achieve magnetic separation of the magnetic nanoparticle immobilized biological enzyme from the reaction system (containing degradation products and the substrate to be treated), which is the most prominent advantage of the present application, solving the problem of difficult recovery and non-reusable of free enzymes.

[0026] Because the enzyme is firmly immobilized and the carrier is easy to recover by magnetic separation, the magnetic nanoparticle immobilized biological enzyme can be reused in the next degradation reaction after removing the bound products or simple washing (such as with a phosphate buffer), significantly reducing the use cost.

[0027] The magnetic separation process of the present application is simple and fast, without the need for complex centrifugation or filtration steps.

[0028] Immobilization generally improves the tolerance of enzymes to temperature, pH changes, organic solvents or protease degradation, prolonging the service life.

[0029] The magnetic nanoparticles have good dispersibility in solution, which is conducive to the full contact of the enzyme with the substrate (toxin). Through an external magnetic field, directional movement or local enrichment of the magnetic nanoparticle immobilized biological enzyme in the reaction system can also be achieved. DETAILED DESCRIPTION

[0030] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0031] It should be understood that the terms described in the present application are only for describing the particular embodiments, and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0033] Many modifications and variations of this application of the application described herein will be apparent to those of ordinary skill in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0034] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0035] The embodiment of the present application provides a method for degrading aflatoxin by magnetic nanoparticle immobilized biological enzyme, comprising the following steps:

[0036] Fe3O4 nanoparticles are dispersed in ethanol, 3-aminopropyltriethoxysilane (APTES) is added, and reflux reaction is carried out, and then amino-functionalized magnetic nanoparticles (Fe3O4@NH2) are obtained after magnetic separation;

[0037] Fe3O4@NH2 is dispersed in a phosphate buffer, glutaraldehyde is added, and oscillation is carried out at room temperature, and then aldehyde-functionalized carriers (Fe3O4@CHO) are obtained after magnetic separation;

[0038] Aflatoxin detoxification enzyme solution is mixed with Fe3O4@CHO, oscillation treatment is carried out, and then magnetic nanoparticle immobilized biological enzyme is obtained after magnetic separation and washing with a phosphate buffer;

[0039] The magnetic nanoparticle immobilized biological enzyme is added into a to-be-treated solution containing aflatoxin.

[0040] The application prepares amino-modified ferroferric oxide magnetic nanoparticles (Fe3O4@NH2) by a chemical co-precipitation method, modifies the surface of the magnetic nanoparticles by 3-aminopropyltriethoxysilane to obtain a magnetic carrier, and covalently combines aflatoxin-degrading enzyme to the surface of the magnetic carrier by a glutaraldehyde cross-linking method, so as to finally obtain a magnetic nanoparticle immobilized biological enzyme with high stability and recyclability. The application solves the problems of enzyme easy to fall off and difficult to recycle in the traditional enzyme immobilization method, significantly improves the stability and repeated use efficiency of the enzyme in an organic solvent, and is suitable for efficient degradation of aflatoxins in food and feed.

[0041] In the embodiment of the application, the preparation method of the Fe3O4 nanoparticles is as follows: FeCl3·6H2O and FeSO4·7H2O are dissolved in deionized water, nitrogen is introduced for protection, the temperature is raised to 80 DEG C, ammonia water is added dropwise until the pH is 10, magnetic separation is performed after 1 h of reaction, ethanol washing is performed, and drying is performed to obtain Fe3O4 nanoparticles.

[0042] In the embodiment of the application, the molar ratio of FeCl3·6H2O to FeSO4·7H2O is 2:1.

[0043] In the embodiment of the application, the molar ratio of Fe3O4 nanoparticles to APTES is (2-3):1. The molar ratio of Fe3O4 to APTES in the application (2-3:1) ensures moderate amino density and avoids steric hindrance.

[0044] In the embodiment of the application, the temperature of the reflux reaction is 70-80 DEG C, and the time is 5-6 h.

[0045] In the embodiment of the application, the pH of the phosphate buffer is 7.4.

[0046] In the embodiment of the application, the mass concentration of glutaraldehyde is 2-3 wt%; preferably, the concentration is 2.5 wt%.

[0047] In the embodiment of the application, the molar ratio of aflatoxin detoxification enzyme in the aflatoxin detoxification enzyme solution to the aldehyde-modified carrier is (1-2):1; preferably, the molar ratio is 2:1. The molar ratio of enzyme to carrier in the application can ensure that the enzyme fully exposes the active site, while avoiding conformational rigidity caused by excessive cross-linking.

[0048] In the embodiment of the application, the addition amount of the magnetic nanoparticle immobilized biological enzyme in the aflatoxin-containing liquid to be treated is 5-10 mg / mL.

[0049] In the embodiment of the application, the aflatoxin detoxification enzyme is selected from aflatoxin B1-degrading enzyme, which is purchased from Shaanxi Chenming Biological Technology Co., Ltd., and the enzyme activity retention rate is 99%.

[0050] In the present application, 3-aminopropyltriethoxysilane (APTES) forms siloxane bonds (-Si-O-) on the surface of Fe3O4 through a hydrolysis reaction, and introduces amino groups (-NH2). This process not only enhances the dispersibility of the nanoparticles, but also provides active sites for subsequent aldehyde group modification. As a bifunctional crosslinking agent, glutaraldehyde (GA) has two aldehyde groups (-CHO) at its two ends, which respectively react with the amino groups (-NH2) on the surface of the carrier and the amino groups (-NH2) on the surface of the enzyme molecules to form covalent bonds. The aflatoxin detoxification enzyme reacts with the aldehyde groups of Fe3O4@CHO through the ε-amino groups of the surface lysine residues to form stable covalent bonds. The magnetic nanoparticle immobilized biological enzyme of the present application retains the catalytic center of the natural enzyme and can specifically recognize the bifuran ring structure of aflatoxin B1 (AFB1). In the catalytic degradation process, the magnetic nanoparticle immobilized biological enzyme hydrolyzes the lactone ring of AFB1 to generate a nontoxic coumarin derivative (AFB1-8,9-epoxide hydrolysis product), which destroys its carcinogenicity.

[0051] Unless otherwise specified, the room temperature in the present application is 25±2℃.

[0052] It should be noted that the parts of the present application not described in detail are conventional operating means in the art and are not the focus of the present application.

[0053] The technical solutions of the present application are further illustrated by the following examples.

[0054] Example 1

[0055] A method for degrading aflatoxin by magnetic nanoparticle immobilized biological enzyme, comprising the following steps:

[0056] FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water (Fe 3+ with a molar ratio of 2:1, and the solution was protected by nitrogen. The temperature was raised to 80℃, and ammonia water was added dropwise until the pH reached 10. After 1h of reaction, the magnetic nanoparticles were separated by magnetic separation, washed with ethanol, and dried to obtain Fe3O4 nanoparticles. The particle size distribution of the Fe3O4 nanoparticles obtained in this example was 12-16nm, and the dispersion was uniform;

[0057] The above Fe3O4 nanoparticles were dispersed in ethanol, and 3-aminopropyltriethoxysilane (3-aminopropyltriethoxysilane concentration was 2M) was added. The mixture was refluxed at 80℃ for 5h, and the amino-modified magnetic nanoparticles were obtained after magnetic separation. The molar ratio of Fe3O4 nanoparticles to 3-aminopropyltriethoxysilane was 2:1;

[0058] The amino-functionalized magnetic nanoparticles were dispersed in a phosphate buffer solution with pH = 7.4 (the mass concentration of the amino-functionalized magnetic nanoparticles was 2%), 2.5 wt% glutaraldehyde was added (i.e. the concentration of the glutaraldehyde in the phosphate buffer solution), and the mixture was oscillated at room temperature for 4 hours. After magnetic separation, the aldehyde-functionalized carrier was obtained;

[0059] The aflatoxin detoxification enzyme solution (1.5 wt%) was mixed with the aldehyde-functionalized carrier at a molar ratio of 2:1, and the mixture was oscillated at room temperature for 4 hours. After magnetic separation and washing with a phosphate buffer solution, the magnetic nanoparticle immobilized biological enzyme was obtained;

[0060] The magnetic nanoparticle immobilized biological enzyme (5 mg / mL) was added to corn oil containing 10 μg / kg AFB1, and the mixture was oscillated at 37°C for 24 hours. After magnetic separation, the AFB1 content was detected by HPLC.

[0061] In this embodiment, the AFB1 degradation rate was 95%, and the degradation product was non-toxic AFB1-8,9-epoxide.

[0062] Example 2

[0063] A method for degrading aflatoxin by using a magnetic nanoparticle immobilized biological enzyme, comprising the following steps:

[0064] FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water (Fe 3+ The mixture was protected by nitrogen, and the temperature was raised to 80°C. Ammonia was added dropwise until the pH reached 10. After 1 hour of reaction, the mixture was subjected to magnetic separation, ethanol washing, and drying to obtain Fe3O4 nanoparticles. In this embodiment, the particle size distribution of the obtained Fe3O4 nanoparticles was 15-20 nm, and the nanoparticles were uniformly dispersed;

[0065] The Fe3O4 nanoparticles were dispersed in ethanol, and 3-aminopropyl triethoxysilane (the concentration of the 3-aminopropyl triethoxysilane was 2M) was added. The mixture was refluxed at 70°C for 6 hours. After magnetic separation, the amino-functionalized magnetic nanoparticles were obtained. The molar ratio of the Fe3O4 nanoparticles to the 3-aminopropyl triethoxysilane was 3:1.

[0066] The amino-functionalized magnetic nanoparticles were dispersed in a phosphate buffer solution with pH = 7.4 (the mass concentration of the amino-functionalized magnetic nanoparticles was 2%), 3 wt% glutaraldehyde was added (i.e. the concentration of the glutaraldehyde in the phosphate buffer solution), and the mixture was oscillated at room temperature for 4 hours. After magnetic separation, the aldehyde-functionalized carrier was obtained;

[0067] The aflatoxin detoxification enzyme solution was mixed with the aldehyde-functionalized carrier at a molar ratio of 1:1, and the mixture was oscillated at room temperature for 4 hours. After magnetic separation and washing with a phosphate buffer solution, the magnetic nanoparticle immobilized biological enzyme was obtained;

[0068] The magnetic nanoparticle immobilized biological enzyme (10 mg / mL) was added to the corn oil containing 10 μg / kg AFB1, and the mixture was treated at 37°C for 24 h. After magnetic separation, the AFB1 content was detected by HPLC.

[0069] In this embodiment, the AFB1 degradation rate reached 92%, and the degradation product was nontoxic AFB1-8,9-epoxide.

[0070] Example 3

[0071] A method for degrading aflatoxin by using a magnetic nanoparticle immobilized biological enzyme, comprising the following steps:

[0072] FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water (Fe 3+ with a concentration of 1 M) in a molar ratio of 2:1. Nitrogen was introduced for protection, and the temperature was raised to 80°C. Ammonia was added dropwise until the pH reached 10. After 1 h of reaction, magnetic separation was performed, and the product was washed with ethanol and dried to obtain Fe3O4 nanoparticles. In this embodiment, the particle size distribution of the obtained Fe3O4 nanoparticles was 15-20 nm, and the nanoparticles were uniformly dispersed.

[0073] The above Fe3O4 nanoparticles were dispersed in ethanol, and 3-aminopropyl triethoxysilane was added. Refluxing reaction was performed at 70°C for 6 h. After magnetic separation, amino-functionalized magnetic nanoparticles were obtained. The molar ratio of Fe3O4 nanoparticles to 3-aminopropyl triethoxysilane was 2.5:1.

[0074] The above amino-functionalized magnetic nanoparticles were dispersed in a phosphate buffer solution with a pH of 7.4 (i.e., the concentration of glutaraldehyde in the phosphate buffer system). Glutaraldehyde with a concentration of 2 wt% was added. Oscillation was performed at room temperature for 4 h. After magnetic separation, an aldehyde-functionalized carrier was obtained.

[0075] Aflatoxin detoxification enzyme solution (concentration 1.5 wt%) was mixed with the aldehyde-functionalized carrier at a molar ratio of 2:1. Oscillation was performed at room temperature for 4 h. After magnetic separation and washing with a phosphate buffer solution, a magnetic nanoparticle immobilized biological enzyme was obtained.

[0076] The magnetic nanoparticle immobilized biological enzyme (8 mg / mL) was added to the corn oil containing 10 μg / kg AFB1, and the mixture was treated at 37°C for 24 h. After magnetic separation, the AFB1 content was detected by HPLC.

[0077] In this embodiment, the AFB1 degradation rate reached 93%, and the degradation product was nontoxic AFB1-8,9-epoxide.

[0078] The stability and reusability of the magnetic nanoparticle immobilized biological enzyme prepared in Example 1 were tested. The test method and results are as follows:

[0079] a. Temperature stability: The magnetic nanoparticle immobilized bioenzyme and free enzyme (i.e. aflatoxin B1 degrading enzyme directly purchased) were placed in a 40°C water bath, respectively, and the activity was measured every 2 hours. The results showed that the activity of free enzyme decreased to 50% after 12 hours, while the activity of magnetic nanoparticle immobilized bioenzyme remained at 75%.

[0080] b. Organic solvent resistance: After reacting in 50% (volume fraction) ethanol for 24 hours, the activity of magnetic nanoparticle immobilized bioenzyme remained at 85%, while the activity of free enzyme remained at only 30%.

[0081] c. Recycling performance: After being used repeatedly for 5 times, the degradation rate of magnetic nanoparticle immobilized bioenzyme remained > 70%.

[0082] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for degrading aflatoxins by immobilized biological enzymes using magnetic nanoparticles, characterized in that, The method comprises the following steps: Fe3O4 nanoparticles are dispersed in ethanol, 3-aminopropyl triethoxysilane is added, and reflux reaction is performed, and after magnetic separation, amino-functionalized magnetic nanoparticles are obtained; The amino-functionalized magnetic nanoparticles are dispersed in a phosphate buffer, glutaraldehyde is added, and oscillation is performed at room temperature, and after magnetic separation, aldehyde-functionalized carriers are obtained; A solution of aflatoxin detoxification enzyme is mixed with the aldehyde-functionalized carriers, oscillation is performed, magnetic separation is performed, and after washing with a phosphate buffer, magnetic nanoparticle immobilized biological enzymes are obtained; The magnetic nanoparticle immobilized biological enzymes are added into a solution containing aflatoxin to be treated.

2. The method of claim 1, wherein the magnetic nanoparticle-immobilized biological enzyme degrades aflatoxins. The Fe3O4 nanoparticles are prepared by the following steps: FeCl3·6H2O and FeSO4·7H2O are dissolved in water, nitrogen is introduced for protection, the temperature is raised to 80℃, ammonia water is added dropwise until pH=10, reaction is performed for 1h, magnetic separation is performed, ethanol washing is performed, and drying is performed to obtain the Fe3O4 nanoparticles. ​ 3. The method of claim 2, wherein the magnetic nanoparticle-immobilized biological enzyme is a mycotoxin-degrading enzyme. The molar ratio of the FeCl3·6H2O and the FeSO4·7H2O is 2:

1.

4. The method for immobilizing biological enzymes to degrade aflatoxin using magnetic nanoparticles according to claim 1, characterized in that, The molar ratio of the Fe3O4 nanoparticles and the 3-aminopropyl triethoxysilane is (2-3):

1.

5. The method for immobilizing biological enzymes to degrade aflatoxin using magnetic nanoparticles according to claim 1, characterized in that, The reflux reaction is performed at a temperature of 70-80℃ for 5-6h.

6. The method for immobilizing biological enzymes to degrade aflatoxin using magnetic nanoparticles according to claim 1, characterized in that, The pH of the phosphate buffer is 7.

4.

7. The method of claim 1, wherein the magnetic nanoparticle-immobilized biological enzyme is an aflatoxin-degrading enzyme. The mass concentration of the glutaraldehyde is 2-3%.

8. The method of claim 1, wherein the magnetic nanoparticle-immobilized biological enzyme degrades aflatoxins. The molar ratio of the aflatoxin detoxification enzyme in the solution of the aflatoxin detoxification enzyme to the aldehyde-functionalized carriers is (1-2):

1.

9. The method of claim 8, wherein the magnetic nanoparticle-immobilized biological enzyme is an aflatoxin-degrading enzyme. The molar ratio of the aflatoxin detoxification enzyme in the solution of the aflatoxin detoxification enzyme to the aldehyde-functionalized carriers is 2:

1.

10. The method of claim 1, wherein the magnetic nanoparticle-immobilized biological enzyme degrades aflatoxins. The addition amount of the magnetic nanoparticle immobilized biological enzymes in the solution containing aflatoxin to be treated is 5-10mg / mL.