Compound de-mold agent for biological feed and its preparation method

Abstract

This invention relates to the field of feed additive technology, and provides a compound mycotoxin binder for biological feed and its preparation method, comprising: preparing chitosan-intercalated modified montmorillonite by intercalating chitosan molecules into the interlayer of montmorillonite through an intercalation modification reaction; preparing a trehalose-β-cyclodextrin coating solution through an inclusion reaction; coating the surface of a mixed powder particle composed of chitosan-intercalated modified montmorillonite and yeast cell wall with the trehalose-β-cyclodextrin coating solution to obtain coated modified composite particles; and embedding laccase and glucose oxidase into the coated modified composite particles to obtain the compound mycotoxin binder for biological feed. This invention can achieve broad-spectrum and efficient adsorption and biodegradation of aflatoxin B1, zearalenone, and vomitoxin, while reducing the non-specific inhibition of the activity of exogenous enzymes such as phytase in feed by the mycotoxin binder.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, and in particular to a compound mycotoxin binder for biological feed and its preparation method. Background Technology

[0002] Mycotoxin contamination is a long-standing and significant safety hazard for the feed industry and livestock farming. Aflatoxin B1, zearalenone, and vomitoxin are the three most prevalent and harmful mycotoxins in feed ingredients and finished feed products. They cause severe damage to the liver, reproductive system, and immune system of animals, leading to decreased feed intake, stunted growth, reproductive disorders, and even acute poisoning and death in livestock and poultry. Various mycotoxin binders, including aluminosilicate adsorbents, yeast cell wall degrading agents, and microbial enzyme degrading agents, have been widely used in livestock and poultry farming and are of great importance in ensuring feed quality and safety and animal health.

[0003] However, existing mycotoxin binders have significant shortcomings in practical applications. On the one hand, aluminosilicate degraders, such as montmorillonite and zeolite, have good degradation effects on polar toxins like aflatoxin B1, but their interlayer structure carries a negative charge, leading to strong non-specific electrostatic degradation of exogenous enzymes like positively charged phytase under acidic conditions. Studies have shown that ordinary montmorillonite can degrade phytase by up to 94% at pH 2.0, resulting in a significant reduction in the activity of phytase added to feed, leading to decreased phosphorus utilization and increased feed costs. On the other hand, most existing mycotoxin binders are physical adsorption products, with extremely limited degradation capabilities for weakly polar toxins such as vomitoxin. Even under intestinal pH conditions, the degradation rate is less than 80%, making it difficult to meet the needs of controlling complex toxin contamination in actual production. In addition, existing products often use high-temperature treatment or high-speed shearing during processing, which can easily lead to the loss of activity of biological enzymes. Moreover, most products lack specific protective measures for enzymes, so while mycotoxin binders perform their detoxification function, they inevitably sacrifice the biological efficacy of exogenous enzymes in feed.

[0004] Chinese patent CN102028129A discloses a novel mycotoxin detoxifier for feed. This detoxifier, composed of modified montmorillonite, yeast cell walls, and chitosan, can effectively remove contamination from feed ingredients such as corn and grains, as well as various mycotoxins including aflatoxin, zearalenone, T2 toxin, and vomitoxin, preventing various diseases in livestock and poultry caused by consuming mycotoxin-contaminated feed. However, this detoxifier still has shortcomings: the inorganic adsorbent, mainly modified montmorillonite, has a strong non-specific electrostatic adsorption effect on positively charged exogenous enzyme preparations such as feed phytase, easily leading to a significant reduction in the activity of phytase added to the feed; simultaneously, the product lacks targeted protection measures for biological enzyme preparations during processing, inevitably sacrificing the biological efficacy of exogenous enzymes in the feed while performing its detoxification function.

[0005] Therefore, how to provide a compound mycotoxin binder for biological feed that has both broad-spectrum toxin adsorption and degradation capabilities, while minimizing the impact on the activity of exogenous enzymes such as phytase in feed, can efficiently degrade various mycotoxins such as aflatoxin B1, zearalenone, and vomitoxin, significantly reduce non-specific adsorption of phytase, and has good processing and storage stability is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of this, in order to overcome the shortcomings of the prior art, the present invention aims to provide a compound mycotoxin binder for biological feed and its preparation method.

[0007] According to one aspect of the present invention, a method for preparing a composite mycotoxin binder for biological feed is provided. The method includes: preparing chitosan-intercalated modified montmorillonite by inserting chitosan molecules into the interlayer of montmorillonite through an intercalation modification reaction; preparing a trehalose-β-cyclodextrin coating solution through an inclusion reaction; coating the surface of a mixed powder particle composed of chitosan-intercalated modified montmorillonite and yeast cell wall with the trehalose-β-cyclodextrin coating solution to obtain coated modified composite particles; and embedding laccase and glucose oxidase into the coated modified composite particles to obtain a composite mycotoxin binder for biological feed.

[0008] Optionally, the preparation method of the compound mycotoxin binder for biological feed of the present invention involves preparing a sodium-based montmorillonite suspension, using citric acid as a crosslinking aid and protonation promoter, and inserting chitosan macromolecules into the interlayer of montmorillonite through ion exchange between protonated amino cations on the chitosan molecular chain and sodium ions in the interlayer of montmorillonite, thereby obtaining chitosan intercalated modified montmorillonite with interlayer domain expansion and surface charge shielding.

[0009] Optionally, in the preparation method of the composite mycotoxin binder for biological feed of the present invention, chitosan-intercalated modified montmorillonite is prepared in the following manner:

[0010] Add 100 parts by weight of sodium montmorillonite with a particle size of 200 mesh to 500 parts by weight of deionized water and stir for 30 minutes to prepare a sodium montmorillonite suspension.

[0011] Two parts by weight of citric acid were added to a sodium-based montmorillonite suspension under constant temperature of 65℃ and stirring at 300 r / min. After 10 min, eight parts by weight of chitosan with a degree of deacetylation of not less than 90% and one part by weight of citric acid were added. The mixture was stirred at constant temperature for another 150 min. After standing for 12 h, the reactants were washed and dried at 105℃ to constant weight and passed through a 300-mesh sieve to obtain chitosan-intercalated modified montmorillonite.

[0012] Optionally, the preparation method of the compound mycotoxin binder for biological feed of the present invention involves dissolving 3 parts by weight of trehalose and 1 part by weight of β-cyclodextrin in 23 parts by weight of deionized water at 50°C, magnetically stirring until the solution temperature drops to 40°C, maintaining a constant temperature and continuing magnetic stirring for 30 minutes to obtain a trehalose-β-cyclodextrin coated solution.

[0013] Optionally, the preparation method of the compound mycotoxin binder for biological feed of the present invention involves mixing 450-600 parts by weight of chitosan-intercalated modified montmorillonite with 250-400 parts by weight of yeast cell wall using a three-dimensional motion mixer. The spindle speed of the three-dimensional motion mixer is 8 r / min, and the mixing time is 15 min. The yeast cell wall is a Saccharomyces cerevisiae cell wall with a β-glucan content of not less than 20%, thereby obtaining mixed powder particles composed of chitosan-intercalated modified montmorillonite and yeast cell wall.

[0014] Optionally, the preparation method of the compound mycotoxin binder for biological feed of the present invention involves spraying a trehalose-β-cyclodextrin coating solution onto the surface of a mixed powder particle composed of chitosan-intercalated modified montmorillonite and yeast cell walls in the following manner:

[0015] The mixed powder particles are loaded into a fluidized bed material container and preheated to 45°C. The mixed powder particles are fluidized in the hot airflow by controlling the induced draft frequency.

[0016] The fluidized bed inlet air temperature was set to 55℃ and the outlet air temperature to 30℃. Trehalose-β-cyclodextrin coating liquid was injected into the fluidized bed storage tank and atomized and sprayed onto the surface of the mixed powder particles in the fluidized state.

[0017] Optionally, in the preparation method of the compound mycotoxin binder for biological feed of the present invention, the weight ratio of the mixed powder particles in the fluidized bed material container to the trehalose-β-cyclodextrin coating liquid in the fluidized bed storage tank is 90:10, and the trehalose-β-cyclodextrin coating liquid is delivered to the atomizing nozzle at a rate of 10 g / min and sprayed under an atomizing pressure of 0.25 MPa.

[0018] Optionally, in the preparation method of the composite mycotoxin binder for biological feed of the present invention, after spraying, the air temperature is maintained at 55°C for 20 minutes, and then air at 20°C is introduced to cool the coated material to 30°C, thereby obtaining coated modified composite granules.

[0019] Optionally, the preparation method of the compound mycotoxin binder for biological feed of the present invention involves adding 4-7 parts by weight of laccase, 3-5 parts by weight of glucose oxidase, and 100 parts by weight of coated modified composite particles into an anchor mixer. The enzyme activity of laccase is not less than 10,000 U / g, and the enzyme activity of glucose oxidase is not less than 5,000 U / g. The mixture is stirred at a speed of 50 r / min for 10 min, and the uniformly mixed compound mycotoxin binder for biological feed is vacuum sealed and packaged.

[0020] According to another aspect of the present invention, a compound mycotoxin binder for biological feed is provided, which is prepared according to the above-described preparation method.

[0021] The compound mycotoxin binder for biological feed and its preparation method of the present invention have the following beneficial technical effects:

[0022] 1. By modifying sodium-based montmorillonite with chitosan intercalation, chitosan macromolecules are introduced into the interlayer of montmorillonite, effectively shielding the structural negative charge between the montmorillonite layers and reducing the electrostatic adsorption of positively charged phytase by montmorillonite.

[0023] 2. The compound powder is coated with a trehalose-β-cyclodextrin protective solution. The resulting glassy protective film after drying has good moisture and oxygen barrier properties. It physically seals the residual active sites at the edge of montmorillonite and blocks the non-specific contact path between phytase and the adsorbent surface.

[0024] 3. By combining chitosan-intercalated modified montmorillonite with yeast cell walls, the strong degradation ability of modified montmorillonite against polar toxins such as aflatoxin B1 and zearalenone is retained, while the specific degradation effect of esterified mannan in yeast cell walls is utilized to enhance the degradation effect against weakly polar toxins such as vomitoxin.

[0025] 4. Laccase and glucose oxidase are embedded in low-temperature, low-shear, mixed-intercalation modified composite particles to avoid damage to the higher-order structure of enzyme proteins by high temperature and severe mechanical force; at the same time, trehalose, as a natural protective agent, forms a glassy microenvironment in the coating layer, fixes the enzyme molecular conformation, and enhances the thermal stability and anti-inactivation ability of enzyme preparations during processing and storage. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic flowchart of the preparation method of the compound mycotoxin binder for biological feed according to the present invention;

[0028] Figure 2 A schematic diagram showing the degradation rate of different toxins by the compound mycotoxin binder for biological feed prepared according to exemplary embodiments 1-3 of the present invention;

[0029] Figure 3 The above are graphs showing the measured phytase activity data of exemplary embodiments 1-3 according to the present invention.

[0030] Figure 4 This is a graph showing the phytase activity retention rate data of exemplary embodiments 1-3 according to the present invention. Detailed Implementation

[0031] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0032] Figure 1 This is a schematic flowchart of the preparation method of the compound mycotoxin binder for biological feed according to the present invention, as shown below. Figure 1 As shown, the method of the present invention is implemented in the following manner:

[0033] Step S1: Prepare chitosan-intercalated modified montmorillonite;

[0034] Step S2: Prepare trehalose-β-cyclodextrin coating solution;

[0035] Step S3: Prepare mixed powder particles;

[0036] Step S4: Fluidized bed coating;

[0037] Step S5: Low-temperature enzyme embedding and final mixing.

[0038] The method of the present invention will be further described in detail below in specific embodiments.

[0039] Example 1

[0040] Exemplary Example 1 of the present invention provides a compound mycotoxin binder for biological feed. Specifically, the compound mycotoxin binder for biological feed in this embodiment is prepared according to the following steps:

[0041] Step 1: Preparation of chitosan-intercalated modified montmorillonite

[0042] 100g of sodium montmorillonite (200 mesh) was placed in a reaction vessel, and 500g of deionized water was added. The mixture was stirred and dispersed for 30 minutes to form a uniform sodium montmorillonite suspension. The sodium montmorillonite suspension was heated to 65℃ in a water bath and maintained at a constant temperature. 2g of citric acid was added under stirring at 300r / min. After stirring for 10 minutes, 8g of chitosan with a degree of deacetylation of not less than 90% and 1g of citric acid were added, and the mixture was stirred at 300r / min for 150 minutes. After standing for 12 hours, the reaction mixture was washed, dried at 105℃ to constant weight, and passed through a 300-mesh sieve to obtain chitosan-intercalated modified montmorillonite.

[0043] Step 2: Preparation of Trehalose-β-Cyclodextrin Coating Solution

[0044] 3g of trehalose and 1g of β-cyclodextrin were accurately weighed and mixed, then added to 23g of 50℃ deionized water. Magnetic stirring was turned on to fully dissolve the materials. As stirring continued, the solution was allowed to cool naturally to 40℃. After the temperature stabilized, it was maintained at a constant temperature of 40℃ and magnetic stirring was continued for 30 minutes to obtain the trehalose-β-cyclodextrin coated solution.

[0045] Step 3: Preparation of mixed powder particles

[0046] 600g of chitosan-intercalated modified montmorillonite and 250g of yeast cell wall (derived from Saccharomyces cerevisiae, with a β-glucan content of not less than 20%) were put into a three-dimensional motion mixer. The spindle speed was set to 8r / min and the mixture was mixed for 15min to ensure that the two powders were fully dispersed and uniform, thus obtaining mixed powder particles composed of chitosan-intercalated modified montmorillonite and yeast cell wall.

[0047] Step 4: Fluidized bed coating

[0048] The above-mentioned mixed powder particles were loaded into a fluidized bed material container and preheated to a material temperature of 45°C. The induced draft frequency was adjusted to ensure good fluidization of the material in the hot airflow. The inlet air temperature was set to 55°C and the outlet air temperature to 30°C. Trehalose-β-cyclodextrin coating solution was injected into the fluidized bed storage tank, with a weight ratio of 90:10 between the mixed powder particles in the fluidized bed material container and the trehalose-β-cyclodextrin coating solution in the fluidized bed storage tank. A peristaltic pump was turned on to deliver the coating solution to the atomizing nozzle at a rate of 10 g / min. Under an atomization pressure of 0.25 MPa, the coating solution was atomized into fine droplets and uniformly sprayed onto the surface of the fluidized material particles. After spraying, the inlet air temperature was maintained at 55°C for continued fluidization and drying for 20 minutes. Then, 20°C air was introduced to cool the coated material to 30°C, thus obtaining the coated modified composite particles.

[0049] Step 5: Low-temperature enzyme intercalation and final mixing

[0050] The above-mentioned coated and modified composite particles were transferred to an anchor mixer. For every 100g of coated and modified composite particles, 7g of laccase (enzyme activity ≥10000U / g) and 5g of glucose oxidase (enzyme activity ≥5000U / g) were added. The anchor mixer was started and mixed at 50 rpm for 10 minutes. After mixing, the mixture was vacuum-sealed in double-layer aluminum foil bags to obtain a compound mycotoxin binder for biological feed.

[0051] Example 2

[0052] Exemplary Example 2 of the present invention provides a compound mycotoxin binder for biological feed. Specifically, the compound mycotoxin binder for biological feed in this embodiment is prepared according to the following steps:

[0053] Step 1: Preparation of chitosan-intercalated modified montmorillonite

[0054] 100g of sodium montmorillonite (200 mesh) was placed in a reaction vessel, and 500g of deionized water was added. The mixture was stirred and dispersed for 30 minutes to form a uniform sodium montmorillonite suspension. The sodium montmorillonite suspension was heated to 65℃ in a water bath and maintained at a constant temperature. 2g of citric acid was added under stirring at 300r / min. After stirring for 10 minutes, 8g of chitosan with a degree of deacetylation of not less than 90% and 1g of citric acid were added, and the mixture was stirred at 300r / min for 150 minutes. After standing for 12 hours, the reaction mixture was washed, dried at 105℃ to constant weight, and passed through a 300-mesh sieve to obtain chitosan-intercalated modified montmorillonite.

[0055] Step 2: Preparation of Trehalose-β-Cyclodextrin Coating Solution

[0056] 3g of trehalose and 1g of β-cyclodextrin were accurately weighed and mixed, then added to 23g of 50℃ deionized water. Magnetic stirring was turned on to fully dissolve the materials. As stirring continued, the solution was allowed to cool naturally to 40℃. After the temperature stabilized, it was maintained at a constant temperature of 40℃ and magnetic stirring was continued for 30 minutes to obtain the trehalose-β-cyclodextrin coated solution.

[0057] Step 3: Preparation of mixed powder particles

[0058] 450g of chitosan-intercalated modified montmorillonite and 400g of yeast cell wall (derived from Saccharomyces cerevisiae, with a β-glucan content of not less than 20%) were put into a three-dimensional motion mixer. The spindle speed was set to 8r / min and the mixture was mixed for 15min to ensure that the two powders were fully dispersed and uniform, thus obtaining mixed powder particles composed of chitosan-intercalated modified montmorillonite and yeast cell wall.

[0059] Step 4: Fluidized bed coating

[0060] The above-mentioned mixed powder particles were loaded into a fluidized bed material container and preheated to a material temperature of 45°C. The induced draft frequency was adjusted to ensure good fluidization of the material in the hot airflow. The inlet air temperature was set to 55°C and the outlet air temperature to 30°C. Trehalose-β-cyclodextrin coating solution was injected into the fluidized bed storage tank, with a weight ratio of 90:10 between the mixed powder particles in the fluidized bed material container and the trehalose-β-cyclodextrin coating solution in the fluidized bed storage tank. A peristaltic pump was turned on to deliver the coating solution to the atomizing nozzle at a rate of 10 g / min. Under an atomization pressure of 0.25 MPa, the coating solution was atomized into fine droplets and uniformly sprayed onto the surface of the fluidized material particles. After spraying, the inlet air temperature was maintained at 55°C for continued fluidization and drying for 20 minutes. Then, 20°C air was introduced to cool the coated material to 30°C, thus obtaining the coated modified composite particles.

[0061] Step 5: Low-temperature enzyme intercalation and final mixing

[0062] The above-mentioned coated and modified composite granules were transferred to an anchor mixer. For every 100g of coated and modified composite granules, 4g of laccase (enzyme activity ≥10000U / g) and 3g of glucose oxidase (enzyme activity ≥5000U / g) were added. The anchor mixer was started and mixed at 50 rpm for 10 minutes. After mixing, the mixture was vacuum-sealed in double-layer aluminum foil bags to obtain a compound mycotoxin binder for biological feed.

[0063] Example 3

[0064] Exemplary Example 3 of the present invention provides a compound mycotoxin binder for biological feed. Specifically, the compound mycotoxin binder for biological feed in this embodiment is prepared according to the following steps:

[0065] Step 1: Preparation of chitosan-intercalated modified montmorillonite

[0066] 100g of sodium montmorillonite (200 mesh) was placed in a reaction vessel, and 500g of deionized water was added. The mixture was stirred and dispersed for 30 minutes to form a uniform sodium montmorillonite suspension. The sodium montmorillonite suspension was heated to 65℃ in a water bath and maintained at a constant temperature. 2g of citric acid was added under stirring at 300r / min. After stirring for 10 minutes, 8g of chitosan with a degree of deacetylation of not less than 90% and 1g of citric acid were added, and the mixture was stirred at 300r / min for 150 minutes. After standing for 12 hours, the reaction mixture was washed, dried at 105℃ to constant weight, and passed through a 300-mesh sieve to obtain chitosan-intercalated modified montmorillonite.

[0067] Step 2: Preparation of Trehalose-β-Cyclodextrin Coating Solution

[0068] 3g of trehalose and 1g of β-cyclodextrin were accurately weighed and mixed, then added to 23g of 50℃ deionized water. Magnetic stirring was turned on to fully dissolve the materials. As stirring continued, the solution was allowed to cool naturally to 40℃. After the temperature stabilized, it was maintained at a constant temperature of 40℃ and magnetic stirring was continued for 30 minutes to obtain the trehalose-β-cyclodextrin coated solution.

[0069] Step 3: Preparation of mixed powder particles

[0070] 500g of chitosan-intercalated modified montmorillonite and 320g of yeast cell wall (derived from Saccharomyces cerevisiae, with a β-glucan content of not less than 20%) were put into a three-dimensional motion mixer. The spindle speed was set to 8r / min and the mixture was mixed for 15min to ensure that the two powders were fully dispersed and uniform, thus obtaining mixed powder particles composed of chitosan-intercalated modified montmorillonite and yeast cell wall.

[0071] Step 4: Fluidized bed coating

[0072] The above-mentioned mixed powder particles were loaded into a fluidized bed material container and preheated to a material temperature of 45°C. The induced draft frequency was adjusted to ensure good fluidization of the material in the hot airflow. The inlet air temperature was set to 55°C and the outlet air temperature to 30°C. Trehalose-β-cyclodextrin coating solution was injected into the fluidized bed storage tank, with a weight ratio of 90:10 between the mixed powder particles in the fluidized bed material container and the trehalose-β-cyclodextrin coating solution in the fluidized bed storage tank. A peristaltic pump was turned on to deliver the coating solution to the atomizing nozzle at a rate of 10 g / min. Under an atomization pressure of 0.25 MPa, the coating solution was atomized into fine droplets and uniformly sprayed onto the surface of the fluidized material particles. After spraying, the inlet air temperature was maintained at 55°C for continued fluidization and drying for 20 minutes. Then, 20°C air was introduced to cool the coated material to 30°C, thus obtaining the coated modified composite particles.

[0073] Step 5: Low-temperature enzyme intercalation and final mixing

[0074] The above-mentioned coated and modified composite granules were transferred to an anchor mixer. For every 100g of coated and modified composite granules, 5.5g of laccase (enzyme activity ≥10000U / g) and 4g of glucose oxidase (enzyme activity ≥5000U / g) were added. The anchor mixer was started and mixed at 50 rpm for 10 minutes. After mixing, the mixture was vacuum-sealed in double-layer aluminum foil bags to obtain a compound mycotoxin binder for biological feed.

[0075] Example 4

[0076] The mycotoxin degradation rate of the compound mycotoxin binders prepared in Examples 1 to 3 was determined.

[0077] First, prepare the toxin working solution and control solution shown in Table 1.

[0078] Table 1

[0079]

[0080] In this embodiment, when preparing the relevant toxin working solutions and control solutions according to Table 1, the solvent was 50 mL of methanol, and the stock solution concentration was 20000 ppb. The citrate buffer (pH 3.0) was prepared as follows: 15.36 g of anhydrous citric acid and 14.32 g of disodium hydrogen phosphate dodecahydrate were weighed, dissolved in 900 mL of water, and the pH was adjusted to 3.0 with phosphoric acid solution, then brought to a final volume of 1000 mL. The sodium hydrogen phosphate buffer (pH 6.5) was prepared as follows: 5.46 g of sodium dihydrogen phosphate dihydrate and 5.37 g of disodium hydrogen phosphate dodecahydrate were weighed, dissolved in 900 mL of water, and the pH was adjusted to 6.5 with phosphoric acid solution or sodium hydroxide solution, then brought to a final volume of 1000 mL.

[0081] For degradation experiments, 10 mL of the toxin working solution was placed in a 50 mL centrifuge tube, and 0.0100 g of the compound mycotoxin binder for the test organism was added. The mixture was vortexed for 15 seconds and incubated at 37°C and 180 rpm for 4 hours (gastric phase simulation). After incubation, 200 μL of the reaction solution was taken, centrifuged at 5000 rpm for 10 min, and the supernatant was used to determine the residual toxin concentration using an ELISA kit. Subsequently, the pH of the remaining reaction solution was adjusted to 6.5 with 10 mol / L sodium hydroxide solution, and incubated at 37°C and 180 rpm for 240 min (intestinal phase simulation). The supernatant was then centrifuged using the same method to determine the residual toxin concentration.

[0082] A control group was set up for the degradation assay of each toxin. The control group used the corresponding control solution prepared in Table 1. The control group was not added with compound mycotoxin binder for biological feed.

[0083] The degradation rate is calculated using the following formula:

[0084] Y = (C0−C) / C0×100%

[0085] Wherein, C0 is the toxin concentration in the control group, C is the residual toxin concentration in the supernatant after the reaction with the compound mycotoxin binder for biological feed, and Y is the degradation rate.

[0086] The degradation rate of different toxins by the compound mycotoxin binders for biological feed prepared according to exemplary embodiments 1 to 3 of the present invention is shown in Table 2 below; Figure 2 This is a schematic diagram showing the degradation rate of different toxins by the compound mycotoxin binder for biological feed prepared according to exemplary embodiments 1-3 of the present invention.

[0087] Table 2 Degradation rate of mycotoxins

[0088]

[0089] In this embodiment, as Figure 2 As shown in Table 2, chitosan intercalation modification expands the interlayer spacing of montmorillonite, providing ample interlayer capture space for aflatoxin B1 and zearalenone. Simultaneously, esterified mannan in the yeast cell wall binds to vomitoxin through hydrogen bonds and hydrophobic interactions, achieving physical adsorption and detoxification. The bio-feed composite mycotoxin binders prepared in Examples 1 to 3 achieved degradation rates of over 98% for aflatoxin B1 and zearalenone under both pH conditions, indicating that the chitosan-intercalated montmorillonite maintains a stable ability to degrade polar toxins.

[0090] Esterified mannan in yeast cell walls contains numerous hydroxyl groups and hydrophobic regions, which can bind to vomitoxin molecules through hydrogen bonding and hydrophobic interactions. Therefore, adjusting the amount of yeast cell wall increases the number of effective adsorption sites in the system, thereby improving the vomitoxin degradation rate. In all three examples, the degradation rate of vomitoxin was better at pH 6.5 than at pH 3.0. This is related to the dissociation state of the vomitoxin molecules and the pH-dependent changes in the surface charge of the yeast cell wall; hydrophobic interactions and hydrogen bonding are more favorable in the near-neutral intestinal environment.

[0091] The phytase activity retention rate of the compound mycotoxin binders for biological feed prepared in Exemplary Embodiments 1, 2, and 3 of the present invention was determined according to the spectrophotometric method for determination of phytase activity in feed (GB / T 18634-2009). The results are shown in Table 3.

[0092] Table 3

[0093]

[0094] Figure 3 This is a schematic diagram illustrating the measured phytase activity according to exemplary embodiments 1-3 of the present invention. Figure 4 This is a schematic diagram illustrating the phytase activity retention rate according to exemplary embodiments 1-3 of the present invention, as shown below. Figure 3 , Figure 4 As shown in Table 3, chitosan intercalation modification neutralizes the negative charge between montmorillonite layers through chitosan cations, reducing the electrostatic adsorption of positively charged phytase molecules. Trehalose-β-cyclodextrin coating forms a dense glassy protective film on the particle surface, physically shielding residual active adsorption sites at the edges of montmorillonite and blocking the contact path between phytase and the adsorbent surface. Low-temperature enzyme embedding employs low-shear anchoring stirring to avoid mechanical damage to the higher-order structure of the enzyme protein, minimizing the impact on phytase activity while efficiently degrading mycotoxins. The phytase activity retention rates of the compound mycotoxin binders prepared in Examples 1 to 3 were not less than 86.5% after testing. In practical applications, the phytase activity retention rate can be controlled by adjusting the ratio of laccase and glucose oxidase.

[0095] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a compound mycotoxin binder for biological feed, characterized in that, The method includes: preparing chitosan-intercalated modified montmorillonite by inserting chitosan molecules into the interlayer of montmorillonite through an intercalation modification reaction; preparing a trehalose-β-cyclodextrin coating solution through an inclusion reaction; coating the surface of a mixed powder particle composed of chitosan-intercalated modified montmorillonite and yeast cell wall with the trehalose-β-cyclodextrin coating solution to obtain coated modified composite particles; and embedding laccase and glucose oxidase into the coated modified composite particles to obtain a composite mycotoxin binder for biological feed.

2. The preparation method of the compound mycotoxin binder for biological feed according to claim 1, characterized in that, By preparing a sodium-based montmorillonite suspension and using citric acid as a crosslinking aid and protonation promoter, chitosan macromolecules are inserted into the interlayer of montmorillonite through ion exchange between protonated amino cations on the chitosan molecular chain and sodium ions in the interlayer of montmorillonite, resulting in chitosan intercalated modified montmorillonite with interlayer domain expansion and surface charge shielding.

3. The preparation method of the compound mycotoxin binder for biological feed according to claim 1, characterized in that, Chitosan-intercalated modified montmorillonite was prepared in the following manner: Add 100 parts by weight of sodium montmorillonite with a particle size of 200 mesh to 500 parts by weight of deionized water and stir for 30 minutes to prepare a sodium montmorillonite suspension. Two parts by weight of citric acid were added to a sodium-based montmorillonite suspension under constant temperature of 65℃ and stirring at 300 r / min. After 10 min, eight parts by weight of chitosan with a degree of deacetylation of not less than 90% and one part by weight of citric acid were added. The mixture was stirred at constant temperature for another 150 min. After standing for 12 h, the reactants were washed and dried at 105℃ to constant weight and passed through a 300-mesh sieve to obtain chitosan-intercalated modified montmorillonite.

4. The preparation method of the compound mycotoxin binder for biological feed according to claim 1, characterized in that, Dissolve 3 parts by weight of trehalose and 1 part by weight of β-cyclodextrin in 23 parts by weight of 50°C deionized water, and stir magnetically until the solution temperature drops to 40°C. Maintain the constant temperature and continue stirring magnetically for 30 minutes to obtain the trehalose-β-cyclodextrin coated solution.

5. The preparation method of the compound mycotoxin binder for biological feed according to claim 1, characterized in that, 450-600 parts by weight of chitosan-intercalated modified montmorillonite and 250-400 parts by weight of yeast cell wall were mixed using a three-dimensional motion mixer. The spindle speed of the three-dimensional motion mixer was 8 r / min and the mixing time was 15 min. The yeast cell wall was a brewer's yeast cell wall with a β-glucan content of not less than 20%. The result was a mixed powder particle composed of chitosan-intercalated modified montmorillonite and yeast cell wall.

6. The method for preparing the compound mycotoxin binder for biological feed according to claim 1, characterized in that, The trehalose-β-cyclodextrin coating solution was sprayed onto the surface of a mixed powder particle composed of chitosan-intercalated modified montmorillonite and yeast cell walls in the following manner: The mixed powder particles are loaded into a fluidized bed material container and preheated to 45°C. The mixed powder particles are fluidized in the hot airflow by controlling the induced draft frequency. The fluidized bed inlet air temperature was set to 55℃ and the outlet air temperature to 30℃. Trehalose-β-cyclodextrin coating liquid was injected into the fluidized bed storage tank and atomized and sprayed onto the surface of the mixed powder particles in the fluidized state.

7. The preparation method of the compound mycotoxin binder for biological feed according to claim 6, characterized in that, The weight ratio of the mixed powder particles in the fluidized bed material container to the trehalose-β-cyclodextrin coating liquid in the fluidized bed storage tank is 90:

10. The trehalose-β-cyclodextrin coating liquid is delivered to the atomizing nozzle at a rate of 10 g / min and sprayed at an atomization pressure of 0.25 MPa.

8. The method for preparing the compound mycotoxin binder for biological feed according to claim 6, characterized in that, After spraying, maintain an air intake of 55°C for 20 minutes, then introduce 20°C air to cool the coated material to 30°C, thus obtaining coated modified composite particles.

9. The preparation method of the compound mycotoxin binder for biological feed according to claim 1, characterized in that, Add 4-7 parts by weight of laccase, 3-5 parts by weight of glucose oxidase, and 100 parts by weight of coated modified composite particles to an anchor mixer. The enzyme activity of laccase should be no less than 10,000 U / g, and the enzyme activity of glucose oxidase should be no less than 5,000 U / g. Mix at 50 r / min for 10 min. Vacuum seal the uniformly mixed biological feed with a compound mycotoxin binder.

10. A compound mycotoxin binder for biological feed, characterized in that, The compound mycotoxin binder for biological feed is prepared according to the method described in any one of claims 1 to 9.

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