Composite enzyme preparation for degrading aflatoxin, composite enzyme preparation solution, preparation method and application thereof

Abstract

The present invention belongs to the field of biodegradation of mycotoxins, and specifically relates to a complex enzyme preparation for degrading aflatoxins, a complex enzyme preparation solution, and its preparation method and application. The complex enzyme preparation comprises: 35-45 wt% tyrosinase, 20-30 wt% cellulase, 8-12 wt% pectinase, 8-12 wt% xylanase, and 10-20 wt% mushroom laccase. Compared with existing chemical and biological detoxification technologies, the complex enzyme preparation of the present invention contains safe and non-toxic components, is simple to use, and has low economic costs. After treatment with the enzyme preparation solution, the cellulose and hemicellulose in the peanut meal are initially decomposed, making it easier for animals to digest and absorb, and having higher nutritional value and better economic benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biodegradation of mycotoxins, and more specifically, relates to a composite enzyme preparation for degrading aflatoxin, a composite enzyme preparation solution, and a preparation method and application thereof. Background Art

[0002] Peanut meal, a byproduct of peanut oil extraction, is rich in nutrients such as plant protein, cellulose, polysaccharides, and flavonoids. Its balanced amino acid profile, rich nutrition, palatability, and high digestibility make it an ideal feed raw material. However, due to environmental factors such as moisture, temperature, and ventilation during production, processing, storage, and transportation, peanut meal is susceptible to contamination by molds such as Aspergillus flavus and Aspergillus parasiticus, which in turn produce secondary metabolites—aflatoxins. Aflatoxins are highly toxic substances with carcinogenic, teratogenic, and mutagenic properties. Among them, aflatoxin B1 (AFB1) is the most toxic and poses the most serious threat. Studies have shown that long-term ingestion of trace amounts of aflatoxins in animals can cause chronic poisoning, leading to decreased growth rate, reduced feed utilization, weight loss, and even death.

[0003] Currently reported methods for removing aflatoxins include physical, chemical, and biological methods. Physical and chemical methods are primarily used for removing aflatoxins in simple systems, and most research is limited to the experimental stage. They are ineffective against aflatoxin contamination in complex systems such as peanut meal. Biological methods have been extensively studied in recent years due to their safety, efficiency, and ease of operation. Enzymatic degradation has become a hot topic of research. Enzymatic degradation primarily utilizes specific enzyme preparations to selectively degrade aflatoxins within the system, achieving the goal of detoxification. Although numerous enzymes capable of degrading aflatoxins have been reported, most research remains at the laboratory stage, and commercial enzyme preparations for degrading aflatoxins in peanut meal are scarce. This is due to the complex composition of peanut meal. Pure enzymes are easily inactivated by the various components of peanut meal upon addition, reducing the enzyme's efficiency in degrading aflatoxins and failing to achieve the desired aflatoxin removal goal. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide a composite enzyme preparation for degrading aflatoxin, a composite enzyme preparation solution and its preparation method and application. The composite enzyme preparation overcomes the problem that pure enzyme is inactivated in peanut meal or the degradation efficiency of aflatoxin is reduced, and the removal rate of aflatoxin is as high as 89.82% (such as Figure 13 The residual aflatoxin in the detoxified peanut meal was 9.7 μg / kg, far below the national standard of 50 μg / kg for peanut meal, thus meeting the national standard. This method is environmentally friendly, safe, cost-effective, and the detoxified peanut meal is highly palatable, demonstrating its significant practical value.

[0005] To achieve the above objectives, the first aspect of the present invention provides a composite enzyme preparation for degrading aflatoxin, the composite enzyme preparation comprising:

[0006] 35-45 wt% tyrosinase, 20-30 wt% cellulase, 8-12 wt% pectinase, 8-12 wt% xylanase, 10-20 wt% mushroom laccase.

[0007] A second aspect of the present invention provides a composite enzyme preparation solution for degrading aflatoxin, the composite enzyme preparation solution comprising:

[0008] The above-mentioned complex enzyme preparation, a phosphate buffer capable of dissolving the complex enzyme preparation, and relative to the complex enzyme preparation solution:

[0009] 0.4-0.8wt% NaCl, 0.8-1.2wt% Tween-20, 0.4-0.6wt% guaiacol;

[0010] The concentration of the complex enzyme preparation in the complex enzyme preparation solution is 0.1-0.6 U / mL.

[0011] The mechanism by which the composite enzyme preparation of the present invention can be used to degrade aflatoxins is as follows: a portion of the AFB1 in the moldy peanut meal exists on the surface of the solid powder, while the other portion is wrapped inside the solid by cellulose, polysaccharides, proteins, etc. The traditional enzymatic degradation method is relatively efficient in removing the AFB1 on the surface of the peanut meal solid powder, while the AFB1 embedded in the solid of the peanut meal is basically unable to be degraded because it cannot contact the degrading enzyme. The composite enzyme preparation of the present invention contains cellulase that can destroy the cellulose tissue in the peanut meal, pectinase and xylanase that can destroy the polysaccharide structure, and under the action of mechanical stirring, it fully releases the AFB1 wrapped by cellulose, polysaccharides, proteins, etc. The AFB1 toxin is then degraded under the action of polyphenol oxidase, tyrosinase, and mushroom laccase. Other additives such as Tween-20 and guaiacol are mainly used to improve the degradation efficiency of the enzyme (Tween-20 is mainly used to increase the solubility of AFB1 in water, and guaiacol mainly acts as an electron transfer intermediate). The enzyme preparation catalyzes the degradation process of aflatoxin AFB1, such as Figure 2 and 3 As shown in the figure, the amino acid residues His109, Ser110, His111, Ser113, His452, and Asp456 in the active center of mushroom laccase bind to aflatoxin AFB1 through hydrogen bonds, and bind to Cu 2+ Together with water molecules, they form a hydrogen bond network, destroying the structure of aflatoxin and degrading the highly toxic AFB1 into the less toxic AFQ1 (such as Figure 3The degradation products are basically non-toxic to animals, thus achieving the purpose of removing aflatoxin AFB1 from moldy peanut meal.

[0012] As a preferred solution, the phosphate buffer is a 40-60 mM phosphate buffer with a pH of 4-10.

[0013] The third aspect of the present invention provides a method for preparing the above-mentioned complex enzyme preparation solution, which comprises: dissolving the complex enzyme preparation in a phosphate buffer solution, and then uniformly mixing it with NaCl, Tween-20, and guaiacol.

[0014] A fourth aspect of the present invention provides the use of the above-mentioned composite enzyme preparation solution for degrading aflatoxin in removing aflatoxin from peanut meal.

[0015] As a preferred solution, the application includes:

[0016] The compound enzyme preparation solution is mixed with peanut meal for detoxification, centrifuged, and dried to obtain detoxified peanut meal.

[0017] As a preferred solution, the application further includes: drying the moldy peanut meal, crushing it and passing it through a 100-mesh sieve, and then evenly mixing the complex enzyme preparation solution with the peanut meal.

[0018] As a preferred solution, the above application meets at least one of the following conditions:

[0019] a. The material-liquid ratio of peanut meal to complex enzyme preparation solution is 1g:5-50mL;

[0020] b. The detoxification temperature is 25-55℃;

[0021] c. The speed of detoxification is 100-300rpm;

[0022] d. The detoxification time is 6-72h.

[0023] As a further preferred embodiment, the solid-liquid ratio of peanut meal to complex enzyme preparation solution is 1g:5-20mL.

[0024] As a preferred solution, the above application meets at least one of the following conditions:

[0025] e. The centrifugal speed is 3000-5000rpm;

[0026] f. Drying temperature is 35-55℃;

[0027] g. Drying time is 10-24h;

[0028] h. Dry until the moisture content is less than 10%.

[0029] In the present invention, after the detoxified peanut meal is obtained, it is crushed to obtain the detoxified peanut meal used as feed.

[0030] Beneficial effects of the present invention:

[0031] The present invention can complete the detoxification treatment of peanut meal by mixing, oscillating, centrifuging, drying and other operations with a composite enzyme preparation solution and peanut meal with excessive aflatoxin. The detoxification rate is as high as 89.82%, and the aflatoxin residue is 9.7 μg / kg peanut meal, which is far less than the aflatoxin content of less than 50 μg / kg in feed peanut meal stipulated in the national standard.

[0032] Compared with existing chemical and biological detoxification technologies, the components contained in the composite enzyme preparation of the present invention are safe and non-toxic, the method of use is simple, and the economic cost is low. After being treated with the enzyme preparation solution, the cellulose, hemicellulose, etc. in the peanut meal are preliminarily decomposed, making it easier for animals to digest and absorb, with higher nutritional value and better economic benefits.

[0033] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of aflatoxin content determination in peanut meal before and after detoxification in Example 27 are shown. (Wherein, "LU" represents fluorescence intensity; time represents time)

[0035] Figure 2 The mechanism of aflatoxin degradation by enzyme preparations is shown.

[0036] Figure 3 The products after aflatoxin degradation are shown. (“Laccase” refers to mushroom laccase)

[0037] Figure 4 The effect of the enzyme amount in Examples 1 to 6 on the aflatoxin residue in peanut meal is shown.

[0038] Figure 5 The effect of enzyme amount on the degradation of aflatoxin in peanut meal in Examples 1 to 6 is shown.

[0039] Figure 6 The effect of the pH of the systems of Examples 7 to 13 on the residual amount of aflatoxin in peanut meal is shown.

[0040] Figure 7 The effect of pH of the systems of Examples 7 to 13 on the degradation of aflatoxin in peanut meal is shown.

[0041] Figure 8 The effect of reaction temperature on the residual amount of aflatoxin in peanut meal in Examples 14 to 17 is shown.

[0042] Figure 9 The effect of reaction temperature on the degradation of aflatoxin in peanut meal in Examples 14 to 17 is shown.

[0043] Figure 10 The effect of detoxification time on the residual amount of aflatoxin in peanut meal in Examples 18 to 22 is shown.

[0044] Figure 11 The effect of detoxification time on the degradation of aflatoxin in peanut meal in Examples 18 to 22 is shown.

[0045] Figure 12 The effect of the material-liquid ratio of Examples 23 to 27 on the residual amount of aflatoxin is shown.

[0046] Figure 13 The effect of the material-liquid ratio of Examples 23 to 27 on the degradation of aflatoxin in peanut meal is shown. DETAILED DESCRIPTION

[0047] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Instead, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0048] In the embodiment of the present invention, the composition of the composite enzyme preparation adopted is:

[0049] 40wt% tyrosinase, 25wt% cellulase, 10wt% pectinase, 10wt% xylanase, 15wt% mushroom laccase.

[0050] In the present invention, U is the activity unit of the complex enzyme preparation, and U is defined as the amount of the complex enzyme preparation required to degrade 1 mg of aflatoxin.

[0051] Examples 1 to 6: Optimal enzyme amounts for complex enzyme preparations

[0052] The complex enzyme preparation was weighed and dissolved in 50 mM phosphate buffer at pH 6.0 to prepare complex enzyme preparation solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 U / mL, respectively, as Examples 1 to 6. Each complex enzyme preparation solution also included 0.6 wt% NaCl, 1 wt% Tween-20, and 0.5 wt% guaiacol.

[0053] A certain amount of moldy peanut meal was weighed, pulverized at high speed, and passed through a 100-mesh sieve. 20 mL of a complex enzyme solution with varying concentrations was added to 2 g of the sieve residue. The mixture was shaken and reacted at 37°C for 24 hours. After the reaction, it was centrifuged at 5000 rpm for 10 minutes. 10 mL of a 70% methanol aqueous solution was added to the peanut meal precipitate to extract aflatoxin B1 for detection of aflatoxin B1 content.

[0054] The results are as attached Figure 4 and 5 As shown, the complex enzyme preparation can effectively degrade aflatoxins in peanut meal. With increasing concentrations (0.1-0.6 U / mL), the aflatoxin removal rate of the complex enzyme preparation increased significantly. At a concentration of 0.5 U / mL, the aflatoxin removal rate reached 69.2%, with a residual aflatoxin content of 29.4 μg / kg of peanut meal.

[0055] Examples 7-13: Optimal pH of complex enzyme preparations

[0056] Phosphate buffer solutions of pH 4, 5, 6, 7, 8, 9, and 10 were prepared, and appropriate amounts of the complex enzyme preparation were weighed to prepare complex enzyme preparation solutions with a concentration of 0.5 U / mL. The complex enzyme preparation solutions were stirred and mixed to prepare complex enzyme preparation solutions with different pH values, respectively serving as Examples 7 to 13. Each complex enzyme preparation solution also contained 0.6 wt% NaCl, 1 wt% Tween-20, and 0.5 wt% guaiacol.

[0057] A certain amount of moldy peanut meal was weighed, crushed at high speed and passed through a 100-mesh sieve. 20 mL of complex enzyme preparation solution with different pH values ​​was added to 2 g of the sieve material respectively. The above mixture was shaken and reacted at 37°C for 24 h. After the reaction was completed, it was centrifuged at 5000 rpm for 10 min. 10 mL of 70% methanol aqueous solution was added to the peanut meal precipitate to extract aflatoxin B1, which was used to detect the aflatoxin B1 content.

[0058] The results are as attached Figure 6-7 As shown, the pH of the complex enzyme preparation solution has a significant effect on the detoxification efficiency. When the pH of the complex enzyme solution is 6.0, the detoxification rate is 73.99%, and the residual aflatoxin content is 24.8μg / Kg peanut meal. It can be seen that the optimal pH of the enzyme complex preparation is 6.0.

[0059] Examples 14-17: Optimum temperature of complex enzyme preparation

[0060] A composite enzyme preparation was weighed to prepare a composite enzyme preparation solution with a concentration of 0.5 U / mL. A certain amount of moldy peanut meal was then weighed, crushed at high speed, and passed through a 100-mesh sieve. 20 mL of a composite enzyme preparation solution with a pH of 6.0 was added to 2 g of the sieve residue, and the mixture was placed in an oscillation reaction at 25°C, 35°C, 45°C, and 55°C for 24 hours, respectively, as Examples 14 to 17. After the reaction was completed, the mixture was centrifuged at 5000 rpm for 10 minutes, the supernatant was discarded, and 10 mL of a 70% methanol aqueous solution was added to the precipitate to extract aflatoxin B1 for the detection of aflatoxin B1 content.

[0061] The results are as attached Figure 8 and 9 As shown, reaction temperature has a significant effect on detoxification efficiency. As the reaction temperature increases (25-55°C), the detoxification rate of the complex enzyme preparation increases significantly (57.0-85.2%). When the reaction temperature is 55°C, the detoxification rate is the highest at 85.2%, and the residual aflatoxin is 14.1μg / kg peanut meal. It can be seen that the optimal reaction temperature is 45°C, at which time the detoxification rate is 80.8%, and the residual aflatoxin is 18.4μg / kg peanut meal.

[0062] Examples 18-22: Optimal detoxification time of complex enzyme preparations

[0063] The complex enzyme preparation was weighed to prepare a complex enzyme preparation solution with a concentration of 0.5U / mL. Then, the moldy peanut meal was crushed at high speed and passed through a 100-mesh sieve. 20mL of the complex enzyme preparation solution with a pH of 6.0 was added to 2g of the sieve undersize. The above mixture was placed at 45°C and oscillated for 6, 12, 24, 48, and 72h, respectively, as Examples 18 to 22. After the reaction was completed, the mixture was centrifuged at 5000rpm for 10min, the supernatant was discarded, and 10mL of 70% methanol aqueous solution was added to the precipitate to extract aflatoxin B1 for the detection of aflatoxin B1 content.

[0064] The results are as attached Figure 10 and 11 As shown in the results, the detoxification time had a significant effect on the detoxification efficiency of the complex enzyme preparation. As the detoxification time increased (6-72h), the detoxification rate of the complex enzyme preparation increased from 65.6% to 85.7%, and the toxin residue decreased from 95.4μg / Kg to 13.6μg / Kg peanut meal.

[0065] When the detoxification time is 72h, the detoxification rate is 85.7%, and the toxin residue is 13.6μg / Kg peanut meal. It can be seen that the optimal detoxification time is 48h, at which time the detoxification rate is 84.5%, and the aflatoxin residue is 14.6μg / Kg peanut meal.

[0066] Examples 23-27: Optimal material-liquid ratio of complex enzyme preparation

[0067] First, weigh an appropriate amount of complex enzyme preparation to prepare a complex enzyme preparation solution with a concentration of 0.5U / mL. The moldy peanut meal was crushed at high speed and passed through a 100-mesh sieve. 2mL, 10mL, 20mL, 30mL, and 40mL of the complex enzyme preparation solution with a pH of 6.0 were added to 2g of the sieve material, respectively. The corresponding material-liquid ratios were 1:1, 1:5, 1:10, 1:15, and 1:20, respectively, as Examples 23 to 27. The mixture was placed at 45°C and shaken for 48h. After the reaction was complete, it was centrifuged at 5000rpm for 10min, the supernatant was discarded, and 10mL of 70% methanol aqueous solution was added to the precipitate to extract aflatoxin B1 for the detection of aflatoxin B1 content.

[0068] The results are as attached Figure 12-13 The results showed that the material-liquid ratio significantly affected the detoxification efficiency of the compound enzyme preparation. As the material-liquid ratio decreased (1:1-1:20), the detoxification rate of the compound enzyme preparation increased from 23.5% to 89.8%, and the aflatoxin residue decreased from 95.4μg / kg to 9.7μg / kg of peanut meal. When the material-liquid ratio was 1:20, the detoxification rate was 89.8%, and the toxin residue was 9.7μg / kg of peanut meal.

[0069] like Figure 1 The following table shows the results of aflatoxin content determination in peanut meal before and after detoxification in Example 27. The vertical axis "LU" represents fluorescence intensity. Without the enzyme preparation, the peanut meal contained a very high AFB1 toxin content, as indicated by high fluorescence intensity. After the enzyme preparation was added for degradation, the residual AFB1 in the peanut meal was very low, as indicated by low fluorescence intensity. This demonstrates that the enzyme preparation is highly effective in removing AFB1 from peanut meal.

[0070] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Application of a composite enzyme preparation solution for degrading aflatoxin in removing aflatoxin from peanut meal, characterized in that: The complex enzyme preparation solution includes: A complex enzyme preparation, a phosphate buffer capable of dissolving the complex enzyme preparation, and relative to the complex enzyme preparation solution: 0.4~0.8wt% NaCl, 0.8~1.2wt% Tween-20, 0.4~0.6wt% guaiacol; Wherein, the concentration of the complex enzyme preparation in the complex enzyme preparation solution is 0.5-0.6U / mL; The complex enzyme preparation includes: 35-45wt% tyrosinase, 20-30wt% cellulase, 8-12wt% pectinase, 8-12wt% xylanase, 10-20wt% mushroom laccase; The phosphate buffer is a 40-60 mM phosphate buffer with a pH of 6; The moldy peanut meal is dried, crushed, and passed through a 100-mesh sieve, and then the complex enzyme preparation solution is mixed evenly with the peanut meal, detoxified, centrifuged, and dried to obtain detoxified peanut meal; The material-liquid ratio of peanut meal to complex enzyme preparation solution is 1g:10-20mL; The temperature for detoxification is 45-55℃; The detoxification time is 48-72h.

2. The use according to claim 1, characterized in that The preparation method of the complex enzyme preparation solution comprises: The complex enzyme preparation is dissolved in phosphate buffer, and then mixed evenly with NaCl, Tween-20 and guaiacol.

3. The use according to claim 1, wherein: The detoxification speed is 100-300rpm.

4. The use according to claim 1, wherein Meet at least one of the following conditions: The centrifugal speed is 3000-5000rpm; The drying temperature is 35-55℃; The drying time is 10-24h; Dry to a moisture content of less than 10%.

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