Modified biochar immobilized zebrafish nitrifying bacteria microspheres, preparation method and application thereof

Abstract

The present application relates to feed additives and organic pollutant degradation technical field, specifically relates to a kind of modified biochar immobilized ZEN degradation bacteria microspheres and preparation method and application.The microsphere is by core material, inner layer wall material and outer layer wall material composition;Core material is by carrier, soluble starch and corn scab ketone degrading bacteria preparation;Carrier is by citric acid and octadecyl trimethylammonium chloride to biochar combined modification preparation;Corn scab ketone degrading bacteria is Bacillus Subtilis 168;Inner layer wall material is by sodium alginate and calcium chloride crosslinking preparation;Outer layer wall material is chitosan.The degradation bacteria microsphere of the present application can effectively maintain the activity of corn scab ketone degrading bacteria, combined with the adsorption of biochar and the biological detoxification of degradation bacteria, can realize the efficient degradation of corn scab ketone.

Description

Technical Field

[0001] This invention relates to the field of feed additives and organic pollutant degradation technology, specifically to a modified biochar-immobilized ZEN-degrading microsphere, its preparation method, and its application. Background Technology

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a secondary metabolite mainly produced by fungi such as Fusarium, Aspergillus, and Penicillium. Crops such as corn, soybeans, and wheat, as well as their byproducts, are highly susceptible to ZEN contamination under high temperature and humidity conditions. ZEN has a strong estrogenic effect, primarily harming the reproductive function of livestock and poultry in animal husbandry, causing infertility, abortion, birth defects, and even death. Furthermore, ZEN can cause liver toxicity, immunotoxicity, and carcinogenic damage in livestock and poultry, severely reducing the economic benefits of animal husbandry. Therefore, detoxification treatment of ZEN-contaminated feed is of great significance.

[0003] Currently, methods for removing mycotoxins from feed include physical detoxification, chemical detoxification, and biological detoxification. Among these, biological detoxification utilizes the biotransformation and adsorption activities of specific microorganisms to remove mycotoxins. This method has become an important development direction for mycotoxin removal in feed due to its high efficiency, low toxicity, high specificity, environmental friendliness, low cost, and ease of industrial production. However, in actual production, the direct application of free microorganisms is often limited by environmental factors leading to decreased microbial activity and consequently reduced remediation efficiency of biological detoxification methods.

[0004] Microbial immobilization technology, which confines microorganisms within a suitable carrier space through adsorption, encapsulation, and cross-linking, can effectively enhance their environmental resistance and metabolic activity, attracting widespread attention in the removal of environmental pollutants. Among these factors, the properties of the carrier are a key influence on the activity and biomass of immobilized microorganisms, thus limiting the successful implementation of microbial immobilization technology. Biochar, a carbon-rich material produced by the pyrolysis of agricultural waste under anaerobic conditions, possesses abundant microporous structures and functional groups, a large specific surface area, low cytotoxicity and cost, rich nutrients, and environmentally friendly properties, effectively providing microorganisms with a suitable and unfavorable environment. Optimizing biochar from rice straw as a microbial immobilization carrier can provide new ideas for the resource development and utilization of rice straw, becoming an important pathway for circular agriculture and a low-carbon economy. However, our experiments revealed that directly using biochar as a carrier to immobilize microorganisms not only makes it difficult to maintain their activity in the gastrointestinal environment but also results in poor selective adsorption of zearalenone.

[0005] Based on existing literature and patent achievements, current technologies for detoxifying mycotoxins in feed can be categorized into four types: Type 1 involves solely utilizing microorganisms with mycotoxin-degrading properties to degrade the toxins; Type 2 combines mycotoxin-degrading microorganisms with nutrients to degrade mycotoxins; Type 3 involves directly adding adsorbents such as zeolite, aluminosilicates, and yeast cell walls to the feed to adsorb mycotoxins; and Type 4 involves using bentonite, montmorillonite, etc., in combination with detoxifying microorganisms to remove mycotoxins. Of these technologies for addressing mycotoxins in feed, Types 1 and 2 are more frequently reported. However, the microbial activity and quantity of these products decline rapidly during shelf life, and microbial activity is greatly affected by gastrointestinal factors (pH and digestive enzymes), making it difficult to maintain detoxification effectiveness. Type 3, using adsorbents alone, has significant limitations when dealing with structurally specific mycotoxins, and the physical adsorption of mycotoxins by adsorbents is easily desorbed and re-released under the influence of gastrointestinal factors. Type 4 has the fewest reports, and although existing results involve both adsorbents and detoxifying microorganisms, the preparation process is simply a mixture of these raw materials, which cannot achieve stable solidification of microorganisms. This will lead to a decrease in microbial activity and low remediation efficiency.

[0006] Currently, biochar-based microbial immobilization technology mainly focuses on the immobilization technique of directly adsorbing microorganisms using biochar; there are few reports on the detoxification of fungi and toxins using biochar as an adsorbent. For example, Chinese patent (CN114768758A) focuses on using the activator K2CO3 to optimize the carbonization and activation conditions such as temperature and time for the adsorption and removal of aflatoxin by biochar made from peanut shells, without involving microbial immobilization technology; while Chinese patent (CN110683911A) mainly targets the control of aflatoxin in the planting field. This technology uses corn straw biochar to adsorb aflatoxin-controlling bacteria and NPK compound fertilizer. One of the purposes of using biochar is to slow-release NPK fertilizer, and Bacillus subtilis and chitosan are used to improve plant immunity; therefore, the above-mentioned disclosed technical solutions are not inspiring for this invention in terms of preparation process and the purpose of raw material use. Summary of the Invention

[0007] To address the problem that the direct application of free microorganisms in actual production is often limited by environmental factors leading to a decline in microbial activity, and the problem that directly using biochar as a carrier to immobilize microorganisms not only makes it difficult to maintain the activity of microorganisms in the gastrointestinal environment, but also results in poor selective adsorption capacity for zearalenone, the present invention aims to provide a modified biochar-immobilized ZEN-degrading microsphere, its preparation method, and its application.

[0008] Our previous research has shown that biochar prepared from rice straw is safe and controllable within limited dosage ranges and can be used as a feed additive in animal production to reduce the emission of harmful gases from animal feces and regulate the structure and metabolites of the animal's intestinal microbiota. This invention utilizes the characteristics of modified biochar to effectively improve its microbial carrying capacity and pollutant adsorption capacity. It employs two immobilization technologies: adsorption and encapsulation. Modified biochar adsorbs degrading bacteria to form a core material, which is then encapsulated within a double-layer wall material to form microspheres. Chitosan, as the outermost wall material, inhibits the growth of harmful bacteria while promoting the growth of degrading bacteria within. These microspheres effectively enhance the degradation of zearalenone while maintaining the activity of the microbial agent for a long time. Therefore, using modified biochar as an immobilization carrier for the detoxification and remediation of zearalenone in feed is a safe, environmentally friendly, and highly efficient remediation technology.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows.

[0010] A modified biochar-immobilized ZEN-degrading microsphere comprises a core material, an inner wall material, and an outer wall material. The core material is prepared from a carrier, soluble starch, and zearalenone-degrading bacteria. The carrier is prepared by co-modifying biochar with citric acid and octadecyltrimethylammonium chloride. The zearalenone-degrading bacteria is Bacillus subtilis 168. The inner wall material is prepared by cross-linking sodium alginate and calcium chloride. The outer wall material is chitosan.

[0011] Furthermore, the carrier is prepared by the following method: a citric acid solution and an octadecyltrimethylammonium chloride solution are mixed to prepare a mixed modifier; biochar is mixed evenly with the mixed modifier, subjected to constant temperature oscillation, then washed until the pH is stable, and filtered to obtain modified biochar.

[0012] Furthermore, the concentration of the citric acid solution is 0.55–0.65 mol / L; the concentration of the octadecyltrimethylammonium chloride solution is 35–45 g / L; the volume ratio of the citric acid solution to the octadecyltrimethylammonium chloride solution is 1:1; and the dosage ratio of the biochar to the mixed modifier is 1 g:10 mL.

[0013] This invention also provides a method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres, comprising the following steps:

[0014] S1. A mixed modifier is prepared by mixing citric acid solution and octadecyltrimethylammonium chloride solution; after the biochar is mixed evenly with the mixed modifier, it is subjected to constant temperature shaking, then washed until the pH is stable, and filtered to obtain modified biochar;

[0015] S2. Prepare a zearalenone-degrading bacteria suspension from the zearalenone-degrading bacteria; mix the modified biochar from S1 with soluble starch evenly, and then add it to the zearalenone-degrading bacteria suspension under stirring to obtain the core material of the modified biochar-immobilized ZEN-degrading bacteria microspheres.

[0016] S3. After mixing sodium alginate solution with the core material of S2 evenly, calcium chloride solution is added to carry out cross-linking reaction to obtain cross-linked microspheres.

[0017] S4. Add the cross-linked microspheres from S3 to the chitosan solution and oscillate at a constant temperature to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0018] Further, in S1, the concentration of the citric acid solution is 0.55–0.65 mol / L; the concentration of the octadecyltrimethylammonium chloride solution is 35–45 g / L; the volume ratio of the citric acid solution to the octadecyltrimethylammonium chloride solution is 1:1; and the dosage ratio of the biochar to the mixed modifier is 1 g:10 mL.

[0019] Furthermore, in S2, the mass ratio of modified biochar to soluble starch is 1:2–3; the concentration of the zearalenone-degrading bacterial suspension is 10. 8 ~10 9 cfu / mL; the ratio of modified biochar to zearalenone-degrading bacterial suspension is 1g:13-18mL.

[0020] Furthermore, in S3, the concentration of the sodium alginate solution is 43–150 g / L; the calcium chloride solution is a 2% (w / v) calcium chloride solution. The volume ratio of the sodium alginate solution to the calcium chloride solution is 40–140:100.

[0021] Furthermore, in S4, the chitosan solution is a chitosan acetate solution with a mass-volume ratio of 2% and a pH of 5.7–6.0.

[0022] Furthermore, in S1, the conditions for isothermal oscillation are: temperature 30℃, oscillation frequency 150~200r / min, and oscillation time 24~36h. In S4, the conditions for isothermal oscillation are: temperature 30℃, oscillation frequency 160~180r / min, and oscillation time 1~2h.

[0023] Furthermore, in S2, the zearalenone-degrading bacterial suspension was prepared by the following method: single colonies of zearalenone-degrading bacteria were cultured in LB liquid medium under constant temperature shaking for 18-24 hours until the logarithmic growth phase, centrifuged, washed and the precipitate was resuspended in physiological saline to obtain the zearalenone-degrading bacterial suspension.

[0024] Furthermore, the LB liquid culture medium is formulated as follows: 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium chloride, and pH adjusted to 7.0-7.2.

[0025] This invention also provides the application of modified biochar-immobilized ZEN-degrading microspheres in the degradation of zearalenone.

[0026] Furthermore, during the degradation or removal of zearalenone, the modified biochar-immobilized ZEN-degrading microspheres are added to the feed at a rate of 3% to 6% based on the mass of the modified biochar.

[0027] The beneficial effects of this invention are:

[0028] 1. This invention uses modified biochar prepared by co-modification of biochar with citric acid and octadecyltrimethylammonium chloride as a carrier. Zearalenone-degrading bacteria are adsorbed onto the carrier as the core material, and the outer layer is coated with a double-layer wall material of sodium alginate and chitosan to obtain modified biochar-immobilized ZEN-degrading bacteria microspheres. Soluble starch provides nutrients for the degrading bacteria and, in combination with sodium alginate, improves the stability of the microspheres in gastrointestinal fluids. Chitosan, as the outermost wall material, inhibits the growth of harmful bacteria while promoting the growth of the internal degrading bacteria. The degrading bacteria microspheres of this invention effectively maintain the activity of zearalenone-degrading bacteria. Combining the adsorption effect of biochar and the biodetoxification effect of the degrading bacteria, highly efficient degradation of zearalenone can be achieved.

[0029] 2. The microspheres of the present invention have a double-walled embedded immobilized core structure, which can target the characteristics of the gastrointestinal environment, so that the degrading bacteria in the core are not affected by gastrointestinal factors (such as pH and digestive enzymes), which can significantly increase the number of live bacteria loaded and the preservation time, and the product is non-toxic and environmentally friendly.

[0030] 3. This invention uses citric acid and octadecyltrimethylammonium chloride to jointly modify biochar to obtain modified biochar. Octadecyltrimethylammonium chloride acts as a cationic surfactant, significantly improving the selective adsorption capacity of biochar for zearalenone; while citric acid can adjust the pH value of the biochar, increasing the content of elements such as carbon, nitrogen, and sulfur, creating a suitable habitat for degrading bacteria.

[0031] 4. The microspheres prepared by the method of this invention have a very significant degradation effect on zearalenone (ZEN). Even 60 days after preparation, the detoxification rate of ZEN by these microspheres can still reach 91.52%, and the resolution rate of ZEN in the gastrointestinal environment is no higher than 9.5%. Attached Figure Description

[0032] Figure 1 This is a flowchart of a method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres provided in Example 1.

[0033] Figure 2 This is a product image of the modified biochar-immobilized ZEN-degrading microspheres prepared in Example 1.

[0034] Figure 3 The microstructures of the modified biochar (A) and the modified biochar-immobilized ZEN-degrading bacteria (B) in Example 1 are shown.

[0035] Figure 4 Test data on the removal performance of zearalenone by free bacteria and modified biochar-immobilized ZEN-degrading bacterial microspheres.

[0036] Figure 5 This is the analytical data of modified biochar in artificial gastrointestinal fluid.

[0037] Figure 6 This is experimental data on testosterone levels in mice exposed to zearalenone.

[0038] Figure 7 This is experimental data on the SOD level in the liver of mice exposed to zearalenone. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Bacillus subtilis 168 was purchased from the China Microbial Culture Collection Network.

[0042] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] The following embodiments of the present invention provide modified biochar-immobilized ZEN-degrading microspheres, which are composed of a core material, an inner wall material, and an outer wall material; the core material is prepared from a carrier, soluble starch, and zearalenone-degrading bacteria; the carrier is prepared by co-modifying biochar with citric acid and octadecyltrimethylammonium chloride; the zearalenone-degrading bacteria is Bacillus subtilis 168; the inner wall material is prepared by cross-linking sodium alginate and calcium chloride; and the outer wall material is chitosan.

[0044] Specifically, the carrier is prepared by the following method: citric acid and octadecyltrimethylammonium chloride are mixed to form a mixed modifier; biochar is mixed evenly with the mixed modifier, subjected to constant temperature shaking, then washed until pH stabilized, and filtered to obtain modified biochar. The concentration of citric acid is 0.55–0.65 mol / L; the concentration of octadecyltrimethylammonium chloride is 35–45 g / L; the volume ratio of citric acid to octadecyltrimethylammonium chloride is 1:1; and the dosage ratio of biochar to the mixed modifier is 1 g:10 mL.

[0045] The preparation method of the modified biochar-immobilized ZEN-degrading bacterial microspheres described above will be explained in detail below.

[0046] Example 1

[0047] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres (the specific preparation process is as follows) Figure 1 (As shown), including the following steps:

[0048] S1. Preparation of modified biochar

[0049] Take 5 mL each of 0.6 mol / L citric acid and 40 g / L octadecyltrimethylammonium chloride, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0050] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0051] Prepare LB liquid culture medium: Weigh 10g tryptone, 5g yeast extract, and 10g sodium chloride, dissolve them in 1L deionized water, adjust the pH to 7.1, and autoclave at 121℃ for 20min.

[0052] Preparation of Bacillus subtilis 168 bacterial suspension: Pick a single colony of Bacillus subtilis 168 and add it to 200 mL of LB liquid medium. Incubate at 30°C with shaking at 180 rpm for 20 h until the logarithmic growth phase. Centrifuge the bacterial suspension at 4000 rpm for 5 min, discard the supernatant, wash the precipitate with sterile physiological saline, collect the bacterial sludge, and resuspend it in 200 mL of sterile physiological saline to prepare a bacterial suspension with a concentration of 1.0 × 10⁻⁶. 8cfu / mL.

[0053] Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 360mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:18. Gradually add 70g of the mixture of modified biochar and soluble starch to the 360mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0054] S3. Preparation of the inner wall material of ZEN-degrading microspheres immobilized with modified biochar

[0055] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a 2% (w / v) calcium chloride solution. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 360mL of the core material suspension of modified biochar immobilized ZEN-degrading microspheres prepared by S2, stir until homogeneous, and then place the mixture into a 2mm diameter syringe. Squeeze the syringe from a height of 25cm to drip the solution into 100mL of the 2% (w / v) calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres.

[0056] S4. Preparation of the outer wall material of ZEN-degrading bacterial microspheres immobilized with modified biochar

[0057] 2g of chitosan was dissolved in 100mL of 1% glacial acetic acid solution to prepare a 2% (w / v) chitosan-glacial acetic acid solution. The pH was adjusted to 6.0 using 1mol / L NaOH solution, and the solution was autoclaved at 121℃ for 20min and cooled for later use. The cross-linked microspheres prepared in S3 were added to 100mL of the 2% (w / v) chitosan-glacial acetic acid solution and coated by shaking at 180r / min for 1h at 30℃. The microspheres were washed three times with sterile deionized water and then dried at 35℃ for 24h to obtain modified biochar-immobilized ZEN-degrading microspheres. The product image and microstructure of the microspheres are shown below. Figure 2-3 As shown.

[0058] Example 2

[0059] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0060] S1. Preparation of modified biochar

[0061] Take 5 mL each of 0.55 mol / L citric acid and 35 g / L octadecyltrimethylammonium chloride, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through an 80-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 200 r / min for 30 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 70℃ for 8 h to obtain modified biochar.

[0062] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0063] Prepare LB liquid culture medium: Weigh 12g tryptone, 6g yeast extract, and 12g sodium chloride, dissolve them in 1L deionized water, adjust the pH to 7.2, and autoclave at 121℃ for 20min.

[0064] Preparation of Bacillus subtilis 168 bacterial suspension: Pick a single colony of Bacillus subtilis 168 and add it to 200 mL of LB liquid medium. Incubate at 30°C with shaking at 180 rpm for 24 h until the logarithmic growth phase. Centrifuge the bacterial suspension at 4000 rpm for 5 min, discard the supernatant, wash the precipitate with sterile physiological saline, collect the bacterial sludge, and resuspend it in 200 mL of sterile physiological saline to prepare a bacterial suspension with a concentration of 1.0 × 10⁻⁶. 9 cfu / mL.

[0065] Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 320mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:16. Gradually add 70g of the mixture of modified biochar and soluble starch to the 320mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0066] S3. Preparation of the inner wall material of ZEN-degrading microspheres immobilized with modified biochar

[0067] Add 6g of sodium alginate to 80mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 75g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a 2% calcium chloride solution. Autoclave at 121℃ for 20min and cool for later use. Add 80mL of the 75g / L sodium alginate solution to 320mL of the core material suspension of modified biochar immobilized ZEN-degrading microspheres prepared by S2 and stir until homogeneous. Then, place the above mixture into a 2mm diameter syringe and squeeze the syringe from a height of 25cm to drip the solution into 100mL of a 2% calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres.

[0068] S4. Preparation of the outer wall material of ZEN-degrading bacterial microspheres immobilized with modified biochar

[0069] 2g of chitosan was dissolved in a 1% glacial acetic acid solution to prepare a 2% (w / v) chitosan-glacial acetic acid solution. The pH was adjusted to 5.9 using 1mol / L NaOH solution, and the solution was autoclaved at 121℃ for 20 min and then cooled for later use. The cross-linked microspheres prepared in S3 were added to 100mL of the 2% (w / v) chitosan-glacial acetic acid solution and coated by shaking at 170r / min for 1.5h at 30℃. The microspheres were washed three times with sterile deionized water and then dried at 32℃ for 30h to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0070] Example 3

[0071] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0072] S1. Preparation of modified biochar

[0073] Take 5 mL each of 0.65 mol / L citric acid and 45 g / L octadecyltrimethylammonium chloride, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 90-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 150 r / min for 36 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 60℃ for 9 h to obtain modified biochar.

[0074] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0075] Prepare LB liquid culture medium: Weigh 8g tryptone, 4g yeast extract, and 8g sodium chloride, dissolve them in 1L deionized water, adjust the pH to 7.0, and autoclave at 121℃ for 20min.

[0076] Preparation of Bacillus subtilis 168 bacterial suspension: Pick a single colony of Bacillus subtilis 168 and add it to 200 mL of LB liquid medium. Incubate at 30°C with shaking at 180 rpm for 18 h until the logarithmic growth phase. Centrifuge the bacterial suspension at 4000 rpm for 5 min, discard the supernatant, wash the precipitate with sterile physiological saline, collect the bacterial sludge, and resuspend it in 200 mL of sterile physiological saline to prepare a bacterial suspension with a concentration of 1.0 × 10⁻⁶. 9 cfu / mL.

[0077] Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0078] S3. Preparation of the inner wall material of ZEN-degrading microspheres immobilized with modified biochar

[0079] Add 6g of sodium alginate to 140mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a concentration of 43g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a 2% calcium chloride solution. Autoclave at 121℃ for 20min and cool for later use. Add 140mL of the 43g / L sodium alginate solution to 260mL of the core material suspension of modified biochar immobilized ZEN-degrading microspheres prepared by S2 and stir until homogeneous. Then, place the mixture into a 2mm diameter syringe and squeeze the syringe from a height of 25cm to drip the solution into 100mL of a 2% (w / v) calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres.

[0080] S4. Preparation of the outer wall material of ZEN-degrading bacterial microspheres immobilized with modified biochar

[0081] 2g of chitosan was dissolved in a 1% glacial acetic acid solution to prepare a 2% (w / v) chitosan-glacial acetic acid solution. The pH was adjusted to 5.7 using 1mol / L NaOH solution, and the solution was autoclaved at 121℃ for 20 min and then cooled for later use. The cross-linked microspheres prepared in S3 were added to 100mL of the 2% (w / v) chitosan-glacial acetic acid solution and coated by shaking at 160r / min for 2h at 30℃. The microspheres were washed three times with sterile deionized water and then dried at 30℃ for 48h to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0082] The modified biochar-immobilized ZEN-degrading bacterial microspheres prepared in Example 1 are used as an example to illustrate the performance of the modified biochar-immobilized ZEN-degrading bacterial microspheres prepared in Example 1 of the present invention in removing zearalenone.

[0083] 1. De-ZEN removal experiment by modified biochar-immobilized ZEN-degrading bacterial microspheres.

[0084] Take 50 μL of 2 mg / mL zearalenone solution and add it to 9.95 mL of LB liquid medium to form a 10 mL system. Add 3 g of modified biochar-immobilized ZEN-degrading bacterial microspheres (treatment group) or an equal amount of free bacteria (Bacillus subtilis 168, control group) to this system, with three replicates for both groups. Then, after incubation at 30℃ with shaking at 180 rpm for 12 h, centrifuge at 5000 rpm for 10 min, collect the supernatant, add an equal volume of methanol, filter through a 0.22 nm filter membrane, and determine the ZEN content in the supernatant by high performance liquid chromatography.

[0085] Preparation of 2 mg / mL zearalenone (ZEN) standard solution: Dissolve 20 mg of zearalenone standard in 10 mL of methanol solution. High-performance liquid chromatography (HPLC) conditions: C18 column, column temperature 30℃, wavelength 274 nm, flow rate 1 mL / min, mobile phase methanol:water = 8:2. The removal performance of ZEN-degrading bacteria microspheres immobilized with modified biochar was tested at 0, 7, 15, 30, and 60 days after preparation. The experimental results are as follows: Figure 4 As shown.

[0086] from Figure 4The detoxification test results showed that the detoxification rate of zearalenone by the modified biochar-immobilized ZEN-degrading bacteria microspheres decreased from 96.96% at 0 days to 91.52% at 60 days, a decrease of only 5.61%. In contrast, the detoxification rate of zearalenone by free bacteria decreased from 78.71% at 0 days to 46.74% at 60 days, a decrease of 40.62%. Furthermore, the detoxification rate of zearalenone by the modified biochar-immobilized ZEN-degrading bacteria microspheres was 23.19%, 52.26%, 56.32%, 55.40%, and 95.81% higher than that of free bacteria at 0 days, 7 days, 15 days, 30 days, and 60 days, respectively. This indicates that, compared to non-immobilized free bacteria, the modified biochar-immobilized ZEN-degrading bacteria microspheres can effectively maintain the activity of zearalenone-degrading bacteria and have a significant removal effect on zearalenone.

[0087] 2. Desorption experiment of ZEN by modified biochar-immobilized ZEN-degrading bacterial microspheres in gastrointestinal fluid.

[0088] Preparation of artificial gastric juice: Add 1g of pepsin and 80mL of deionized water to 1.64mL of 9.5% dilute hydrochloric acid, shake well, and then dilute with distilled water to 100mL, with a pH of 1.8.

[0089] Preparation of artificial intestinal fluid: Dissolve 0.68g of potassium dihydrogen phosphate in 50mL of deionized water, and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution; dissolve 1g of pancreatic enzyme in an appropriate amount of water, mix the two solutions, and dilute with water to 100mL.

[0090] Take 50 μL of 2 mg / mL zearalenone solution and add it to 9.95 mL of LB liquid medium to form a 10 mL system. Add 3 g of modified biochar-immobilized ZEN-degrading microspheres to this system, setting up 3 replicates. Then, incubate at 30℃ with shaking at 180 r / min for 12 h, centrifuge at 5000 r / min for 10 min, take the supernatant, add an equal volume of methanol, filter through a 0.22 nm filter membrane, and determine the zearalenone concentration in the supernatant (X1, zearalenone concentration in the supernatant after adsorption) by high performance liquid chromatography.

[0091] The precipitate after adsorption and centrifugation was collected, and 10 mL of artificial gastric fluid and artificial intestinal fluid were added. The mixture was incubated at 37℃ for 2 hours in a constant-temperature shaking water bath at 120 rpm until the adsorption was complete. At 30, 60, and 120 min of the adsorption process, the mixture was centrifuged at 5000 rpm for 10 min. An equal volume of methanol was added to the supernatant, and the solution was filtered through a 0.22 nm filter. The zearalenone content in the supernatant (X2, zearalenone concentration in the supernatant after adsorption) was determined by high-performance liquid chromatography. A blank control group (X0, zearalenone concentration in the blank group) was set up. The experimental results are shown in […]. Figure 5Zearalenone resolution rate / % = [X2 / (X0-X1)] × 100%.

[0092] from Figure 5 The results of the gastrointestinal fluid analysis test showed that the resolution rate of zearalenone adsorbed by the microspheres in the artificial gastric fluid decreased from 8.45% at 30 min to 7.46% at 120 min; the resolution rate of zearalenone adsorbed by the microspheres in the artificial intestinal fluid decreased from 9.5% at 30 min to 8.86% at 120 min. Overall, the resolution rate of zearalenone adsorbed by the microspheres in both the artificial gastric fluid and artificial intestinal fluid gradually decreased with the resolution time, and the overall resolution rate was not higher than 9.5%. It can be seen that the modified biochar immobilized ZEN-degrading bacterial microspheres prepared in Example 1 of this invention have a good ability to remove zearalenone in the gastrointestinal environment.

[0093] 3. The protective effect of modified biochar-immobilized ZEN-degrading bacterial microspheres on poisoned mice.

[0094] Twenty-four 21-day-old male Kunming mice were randomly divided into four treatment groups: a control group (treatment 1), a zearalenone-treated group (ZEN group, treatment 2), a free bacteria + zearalenone-treated group (ZEN + free bacteria group, treatment 3), and a modified biochar-immobilized ZEN-degrading microspheres + zearalenone-treated group (ZEN + immobilized microspheres group, treatment 4). Each treatment group contained six male mice.

[0095] Mice had free access to food and water during the experiment. They were administered zearalenone via gavage daily for 35 consecutive days. Mice in treatment groups 2, 3, and 4 were administered 60 mg / mL zearalenone daily (0.2 mL per mouse), while the control group received an equal volume of physiological saline. Treatment groups 1 and 2 were fed a normal diet, while treatment groups 3 and 4 were fed diets containing free bacteria and diets containing 3% modified biochar-immobilized ZEN-degrading microspheres, respectively. The experiment was approved by the university's animal management committee, and the procedures were strictly implemented in accordance with the university's experimental animal management regulations.

[0096] On day 35 of the experiment, liver samples and blood were collected from mice and centrifuged to prepare serum. The procedures for mouse testosterone and superoxide dismutase (SOD) assays were performed according to the manufacturer's instructions. Liver SOD levels were measured using the hydroxylamine method, and serum testosterone levels were measured using an ELISA method. Absorbance values ​​at 550 nm and 450 nm wavelengths were detected using a microplate reader.

[0097] from Figure 6The testosterone levels of mice in the ZEN group and the ZEN+ free bacteria group were significantly lower than those in the control group and the ZEN+ immobilized microsphere group (P<0.05), while there was no significant difference between the ZEN+ immobilized microsphere group and the control group (P>0.05). The testosterone levels of mice in the ZEN+ free bacteria group were 11.32% (P<0.05) and 9.22% (P<0.05) lower than those in the control group and the ZEN+ immobilized microsphere group, respectively.

[0098] from Figure 7 The results of SOD detection showed that the liver SOD levels of mice in the ZEN group and the ZEN+ free bacteria group were significantly lower than those in the control group and the ZEN+ immobilized microsphere group (P<0.05), while there was no significant difference between the ZEN+ immobilized microsphere group and the control group (P>0.05). The liver SOD levels of mice in the ZEN+ free bacteria group were 11.09% (P<0.05) and 7.84% (P<0.05) lower than those in the control group and the ZEN+ immobilized microsphere group, respectively.

[0099] Therefore, it can be seen that the modified biochar immobilized ZEN-degrading microspheres prepared in Example 1 of this invention, when added to the diet of mice as a feed additive, can effectively target the ability of mice to remove zearalenone in the gastrointestinal environment and play a good protective role for mice exposed to the toxin.

[0100] Example 4

[0101] A method for preparing ZEN-degrading microspheres immobilized with modified biochar is basically the same as the method in Example 1, except that 50g of the modified biochar prepared in S1 is added to a beaker, and 50g of soluble starch is added to the beaker at a mass ratio of 1:1, and mixed well to obtain a mixture of modified biochar and soluble starch.

[0102] Example 5

[0103] A method for preparing ZEN-degrading microspheres immobilized with modified biochar is basically the same as the method in Example 1, except that 33.3g of the modified biochar prepared in S1 is added to a beaker, and 50g of soluble starch is added to the beaker at a mass ratio of 1:1.5, and mixed well to obtain a mixture of modified biochar and soluble starch.

[0104] Example 6

[0105] A method for preparing ZEN-degrading microspheres immobilized with modified biochar is basically the same as the method in Example 1, except that 25g of the modified biochar prepared in S1 is added to a beaker, and 50g of soluble starch is added to the beaker at a mass ratio of 1:2, and mixed well to obtain a mixture of modified biochar and soluble starch.

[0106] Example 7

[0107] A method for preparing ZEN-degrading microspheres immobilized with modified biochar is basically the same as the method in Example 1, except that 16.7g of the modified biochar prepared in S1 is added to a beaker, and 50g of soluble starch is added to the beaker at a mass ratio of 1:3, and mixed well to obtain a mixture of modified biochar and soluble starch.

[0108] We first optimized the addition ratio of modified biochar obtained by co-modification with citric acid and octadecyltrimethylammonium chloride to soluble starch, with the target being the carrying capacity of degrading bacteria. The results are shown in Table 1.

[0109] Table 1. Effect of the ratio of modified biochar to soluble starch on the number of bacteria carrying the degradation process.

[0110]

[0111] As shown in Table 1, the carrying capacity of the core material for degrading bacteria gradually increases with decreasing modified biochar content. The highest carrying capacity is achieved when the ratio of modified biochar to soluble starch is 1:2.5. Subsequently, the carrying capacity decreases with further reduction in the amount of modified biochar. Therefore, the optimal ratio of modified biochar obtained from the co-modification with citric acid-octadecyltrimethylammonium chloride to soluble starch is 1:2.5, at which point the core material exhibits the highest carrying capacity for degrading bacteria.

[0112] Example 8

[0113] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0114] S1. Preparation of modified biochar

[0115] Take 10 mL of 0.6 mol / L citric acid as a modifier. Grind and sieve rice straw biochar to a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to a beaker containing 10 mL of modifier at a mass-to-volume ratio of biochar to modifier of 1:10. Then, place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min for 24 h. Afterward, wash with distilled water until the pH stabilizes, filter, and dry the resulting modified biochar powder at 80℃ for 10 h to obtain the modified biochar.

[0116] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0117] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of the bacterial suspension to be 360mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:18. Gradually add 70g of the mixture of modified biochar and soluble starch to the 360mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0118] S3. Preparation of ZEN-degrading microspheres immobilized with modified biochar

[0119] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a calcium chloride solution with a mass-to-volume ratio of 2%. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 360mL of the core material suspension of the modified biochar immobilized ZEN-degrading microspheres prepared by S2 and stir until homogeneous. Then, place the mixture into a 2mm diameter syringe and squeeze the syringe from a height of 25cm to drip the solution into 100mL of the 2% mass-to-volume ratio calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres. Then, dry the crosslinked microspheres at 35℃ for 24h to obtain modified biochar immobilized ZEN-degrading microspheres.

[0120] Example 9

[0121] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres is basically the same as the method in Example 1, except that...

[0122] S1. Preparation of modified biochar: 10 mL of 0.6 mol / L citric acid was used as a modifier. Rice straw biochar was ground and sieved through a 100-mesh sieve to obtain the biochar to be modified. At a mass-to-volume ratio of biochar to modifier of 1:10, 1 g of biochar was added to a beaker containing 10 mL of modifier. The beaker was then placed on a magnetic stirrer and stirred until a uniform suspension of biochar was formed. The beaker was then placed on a shaker at 30℃ and 180 r / min for 24 h. Afterward, the mixture was washed with distilled water until the pH stabilized, then filtered. The resulting modified biochar powder was dried at 80℃ for 10 h to obtain the modified biochar.

[0123] Example 10

[0124] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0125] S1. Preparation of modified biochar

[0126] Take 5 mL each of 0.6 mol / L citric acid and 1.5 mol / L sulfuric acid, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0127] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0128] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of the bacterial suspension to be 360mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:18. Gradually add 70g of the mixture of modified biochar and soluble starch to the 360mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0129] S3. Preparation of ZEN-degrading microspheres immobilized with modified biochar

[0130] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a calcium chloride solution with a mass-to-volume ratio of 2%. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 360mL of the core material suspension of the modified biochar immobilized ZEN-degrading microspheres prepared by S2 and stir until homogeneous. Then, place the mixture into a 2mm diameter syringe and squeeze the syringe from a height of 25cm to drip the solution into 100mL of the 2% mass-to-volume ratio calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres. Then, dry the crosslinked microspheres at 35℃ for 24h to obtain modified biochar immobilized ZEN-degrading microspheres.

[0131] Example 11

[0132] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres is basically the same as the method in Example 1, except that...

[0133] S1. Preparation of modified biochar

[0134] Take 5 mL each of 0.6 mol / L citric acid and 1.5 mol / L sulfuric acid, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0135] Example 12

[0136] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0137] S1. Preparation of modified biochar

[0138] Take 5 mL each of 0.6 mol / L citric acid and 40 g / L octadecyltrimethylammonium chloride, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0139] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0140] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of the bacterial suspension to be 360mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:18. Gradually add 70g of the mixture of modified biochar and soluble starch to the 360mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0141] S3. Preparation of ZEN-degrading microspheres immobilized with modified biochar

[0142] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a 2% (w / v) calcium chloride solution. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 360mL of the core material suspension of the modified biochar-immobilized ZEN-degrading microspheres prepared in S2, stir until homogeneous, and then place the mixture into a 2mm diameter syringe. Squeeze the syringe from a height of 25cm to drip the solution into 100mL of a 2% (w / v) calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres. Then dry the crosslinked microspheres at 35℃ for 24h to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0143] Example 13

[0144] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0145] S1. Preparation of modified biochar

[0146] Take 10 mL of 0.6 mol / L citric acid as a modifier. Grind and sieve rice straw biochar to a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to a beaker containing 10 mL of modifier at a mass-to-volume ratio of biochar to modifier of 1:10. Then, place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min for 24 h. Afterward, wash with distilled water until the pH stabilizes, filter, and dry the resulting modified biochar powder at 80℃ for 10 h to obtain the modified biochar.

[0147] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0148] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0149] S3. Preparation of ZEN-degrading microspheres immobilized with modified biochar

[0150] 6g of sodium alginate was added to 40mL of deionized water and stirred until homogeneous, yielding a sodium alginate solution with a mass-to-volume ratio of 150g / L. 2g of calcium chloride was dissolved in 100mL of deionized water to prepare a 2% (w / v) calcium chloride solution. The solution was then autoclaved at 121℃ for 20 minutes and cooled for later use. 40mL of the 150g / L sodium alginate solution was added to 260mL of the core material suspension of the modified biochar-immobilized ZEN-degrading microspheres prepared in S2, and stirred until homogeneous. The mixture was then placed into a 2mm diameter syringe and squeezed from a height of 25cm, causing the solution inside to drip dropwise into 100mL of a 2% (w / v) calcium chloride solution for cross-linking for 1 hour. The microspheres were washed three times with sterile deionized water to obtain cross-linked microspheres. The cross-linked microspheres were then dried at 35℃ for 24 hours to obtain the modified biochar-immobilized ZEN-degrading microspheres.

[0151] Example 14

[0152] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0153] S1. Preparation of modified biochar

[0154] Take 10 mL of 0.6 mol / L citric acid as a modifier. Grind and sieve rice straw biochar to a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to a beaker containing 10 mL of modifier at a mass-to-volume ratio of biochar to modifier of 1:10. Then, place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min for 24 h. Afterward, wash with distilled water until the pH stabilizes, filter, and dry the resulting modified biochar powder at 80℃ for 10 h to obtain the modified biochar.

[0155] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0156] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0157] S3. Preparation of the inner wall material of ZEN-degrading microspheres immobilized with modified biochar

[0158] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a calcium chloride solution with a mass-to-volume ratio of 2%. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 260mL of the core material suspension of modified biochar immobilized ZEN-degrading microspheres prepared by S2, stir until homogeneous, and then place the mixture into a 2mm diameter syringe. Squeeze the syringe from a height of 25cm to drip the solution into 100mL of a 2% calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres.

[0159] S4. Preparation of the outer wall material of ZEN-degrading bacterial microspheres immobilized with modified biochar

[0160] 2 g of chitosan was dissolved in 100 mL of 1% glacial acetic acid solution to prepare a 2% (w / v) chitosan-glacial acetic acid solution. The pH was adjusted to 6.0 using 1 mol / L NaOH solution, and the solution was autoclaved at 121 °C for 20 min and then cooled for later use. The cross-linked microspheres prepared in S3 were added to 100 mL of the 2% (w / v) chitosan-glacial acetic acid solution and coated by shaking at 180 r / min for 1 h at 30 °C. The microspheres were washed three times with sterile deionized water and then dried at 35 °C for 24 h to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0161] Example 15

[0162] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0163] S1. Preparation of modified biochar

[0164] Take 5 mL each of 0.6 mol / L citric acid and 1.5 mol / L sulfuric acid, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0165] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0166] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0167] S3. Preparation of ZEN-degrading microspheres immobilized with modified biochar

[0168] 6g of sodium alginate was added to 40mL of deionized water and stirred until homogeneous, yielding a sodium alginate solution with a mass-to-volume ratio of 150g / L. 2g of calcium chloride was dissolved in 100mL of deionized water to prepare a 2% (w / v) calcium chloride solution. The solution was then autoclaved at 121℃ for 20 minutes and cooled for later use. 40mL of the 150g / L sodium alginate solution was added to 260mL of the core material suspension of the modified biochar-immobilized ZEN-degrading microspheres prepared in S2, and stirred until homogeneous. The mixture was then placed into a 2mm diameter syringe and squeezed from a height of 25cm, causing the solution inside to drip dropwise into 100mL of a 2% (w / v) calcium chloride solution for cross-linking for 1 hour. The microspheres were washed three times with sterile deionized water to obtain cross-linked microspheres. The cross-linked microspheres were then dried at 35℃ for 24 hours to obtain the modified biochar-immobilized ZEN-degrading microspheres.

[0169] Example 16

[0170] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0171] S1. Preparation of modified biochar

[0172] Take 5 mL each of 0.6 mol / L citric acid and 1.5 mol / L sulfuric acid, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0173] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0174] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0175] S3. Preparation of the inner wall material of ZEN-degrading microspheres immobilized with modified biochar

[0176] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a calcium chloride solution with a mass-to-volume ratio of 2%. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 260mL of the core material suspension of modified biochar immobilized ZEN-degrading microspheres prepared by S2, stir until homogeneous, and then place the mixture into a 2mm diameter syringe. Squeeze the syringe from a height of 25cm to drip the solution into 100mL of a 2% calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres.

[0177] S4. Preparation of the outer wall material of ZEN-degrading bacterial microspheres immobilized with modified biochar

[0178] 2 g of chitosan was dissolved in 100 mL of 1% glacial acetic acid solution to prepare a 2% (w / v) chitosan-glacial acetic acid solution. The pH was adjusted to 6.0 using 1 mol / L NaOH solution, and the solution was autoclaved at 121 °C for 20 min and then cooled for later use. The cross-linked microspheres prepared in S3 were added to 100 mL of the 2% (w / v) chitosan-glacial acetic acid solution and coated by shaking at 180 r / min for 1 h at 30 °C. The microspheres were washed three times with sterile deionized water and then dried at 35 °C for 24 h to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0179] Example 17

[0180] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0181] S1. Preparation of modified biochar

[0182] Take 5 mL each of 0.6 mol / L citric acid and 40 g / L octadecyltrimethylammonium chloride, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0183] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0184] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0185] S3. Preparation of ZEN-degrading microspheres immobilized with modified biochar

[0186] 6g of sodium alginate was added to 40mL of deionized water and stirred until homogeneous, yielding a sodium alginate solution with a mass-to-volume ratio of 150g / L. 2g of calcium chloride was dissolved in 100mL of deionized water to prepare a 2% (w / v) calcium chloride solution. The solution was then autoclaved at 121℃ for 20 minutes and cooled for later use. 40mL of the 150g / L sodium alginate solution was added to 260mL of the core material suspension of the modified biochar-immobilized ZEN-degrading microspheres prepared in S2, and stirred until homogeneous. The mixture was then placed into a 2mm diameter syringe and squeezed from a height of 25cm, causing the solution inside to drip dropwise into 100mL of a 2% (w / v) calcium chloride solution for cross-linking for 1 hour. The microspheres were washed three times with sterile deionized water to obtain cross-linked microspheres. The cross-linked microspheres were then dried at 35℃ for 24 hours to obtain the modified biochar-immobilized ZEN-degrading microspheres.

[0187] Example 18

[0188] A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres includes the following steps:

[0189] S1. Preparation of modified biochar

[0190] Take 5 mL each of 0.6 mol / L citric acid and 40 g / L octadecyltrimethylammonium chloride, and mix them evenly in a beaker to obtain 10 mL of mixed modifier. Take rice straw biochar, grind and sieve it through a 100-mesh sieve to obtain the biochar to be modified. Add 1 g of biochar to the beaker containing 10 mL of mixed modifier at a mass-to-volume ratio of biochar to mixed modifier of 1:10. Then place the beaker on a magnetic stirrer and stir until a uniform suspension of biochar is formed. Place the beaker on a shaker at 30℃ and 180 r / min and shake for 24 h. After washing with distilled water until the pH stabilizes, filter and dry the obtained modified biochar powder at 80℃ for 10 h to obtain modified biochar.

[0191] S2. Preparation of modified biochar-immobilized ZEN-degrading bacterial microsphere core material

[0192] A Bacillus subtilis 168 bacterial suspension was prepared according to the method in Example 1, with a suspension concentration of 1.0 × 10⁻⁶. 8 CFU / mL. Add 20g of the modified biochar prepared by S1 to a beaker, and add 50g of soluble starch at a mass ratio of 1:2.5. Mix well to obtain a mixture of modified biochar and soluble starch. Then, determine the volume of bacterial suspension to be 260mL according to a mass-to-volume ratio of modified biochar to bacterial suspension of 1:13. Gradually add 70g of the mixture of modified biochar and soluble starch to the 260mL bacterial suspension under stirring to obtain a core material suspension of modified biochar immobilized ZEN-degrading bacterial microspheres.

[0193] S3. Preparation of the inner wall material of ZEN-degrading microspheres immobilized with modified biochar

[0194] Add 6g of sodium alginate to 40mL of deionized water and stir until homogeneous to obtain a sodium alginate solution with a mass-to-volume ratio of 150g / L. Dissolve 2g of calcium chloride in 100mL of deionized water to prepare a calcium chloride solution with a mass-to-volume ratio of 2%. Autoclave at 121℃ for 20min and cool for later use. Add 40mL of the 150g / L sodium alginate solution to 260mL of the core material suspension of modified biochar immobilized ZEN-degrading microspheres prepared by S2, stir until homogeneous, and then place the mixture into a 2mm diameter syringe. Squeeze the syringe from a height of 25cm to drip the solution into 100mL of a 2% calcium chloride solution for crosslinking for 1h. Wash three times with sterile deionized water to obtain crosslinked microspheres.

[0195] S4. Preparation of the outer wall material of ZEN-degrading bacterial microspheres immobilized with modified biochar

[0196] 2 g of chitosan was dissolved in 100 mL of 1% glacial acetic acid solution to prepare a 2% (w / v) chitosan-glacial acetic acid solution. The pH was adjusted to 6.0 using 1 mol / L NaOH solution, and the solution was autoclaved at 121 °C for 20 min and then cooled for later use. The cross-linked microspheres prepared in S3 were added to 100 mL of the 2% (w / v) chitosan-glacial acetic acid solution and coated by shaking at 180 r / min for 1 h at 30 °C. The microspheres were washed three times with sterile deionized water and then dried at 35 °C for 24 h to obtain modified biochar-immobilized ZEN-degrading microspheres.

[0197] We optimized the degradation conditions of ZEN by ZEN-degrading microspheres immobilized with modified biochar, taking into account factors such as the type of modifier, the mass-to-volume ratio of modified biochar to bacterial suspension, and the presence or absence of chitosan. The effects of different factors on the ZEN degradation rate are shown in Table 2.

[0198] Table 2. Effects of different factors on ZEN degradation rate

[0199]

[0200]

[0201] Note: "-" indicates that no chitosan was added, and "+" indicates that chitosan was added.

[0202] As shown in Table 2, when citric acid is used as the modifier, the ratio of modified biochar to bacterial suspension is 1:18, and chitosan is used as the outer wall material, resulting in the highest ZEN degradation rate (88.95%). When citric acid and sulfuric acid are used as modifiers, the ratio of modified biochar to bacterial suspension is 1:18, and chitosan is not added, resulting in the highest ZEN degradation rate (94.63%). When citric acid and octadecyltrimethylammonium chloride are used as modifiers, the ratio of modified biochar to bacterial suspension is 1:18, and chitosan is used as the outer wall material, resulting in the highest ZEN degradation rate (96.96%). Considering all factors, the optimal conditions are selected: citric acid and octadecyltrimethylammonium chloride as the modifier, a modified biochar to bacterial suspension ratio of 1:18, and chitosan as the outer wall material. Under these conditions, the ZEN degradation rate is the highest (96.96%).

[0203] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified biochar-immobilized ZEN-degrading bacterial microsphere, comprising a core material, an inner wall material, and an outer wall material; characterized in that, The core material is prepared from a carrier, soluble starch, and zearalenone-degrading bacteria; the carrier is prepared by combined modification of biochar with citric acid and octadecyltrimethylammonium chloride; the zearalenone-degrading bacteria is Bacillus subtilis (…). Bacillus subtilis 168; The inner wall material is prepared by cross-linking sodium alginate and calcium chloride; The outer wall material is chitosan.

2. The modified biochar-immobilized ZEN-degrading bacterial microspheres according to claim 1, characterized in that, The carrier is prepared by the following method: A mixed modifier is prepared by mixing citric acid solution and octadecyltrimethylammonium chloride solution; After the biochar and the mixed modifier are mixed evenly, the mixture is subjected to constant temperature shaking, then washed until the pH is stable, and filtered to obtain modified biochar.

3. The modified biochar-immobilized ZEN-degrading microspheres according to claim 2, characterized in that, The concentration of the citric acid solution is 0.55–0.65 mol / L; the concentration of the octadecyltrimethylammonium chloride solution is 35–45 g / L; and the volume ratio of the citric acid solution to the octadecyltrimethylammonium chloride solution is 1:

1. The ratio of biochar to the mixed modifier is 1g:10mL.

4. A method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. A mixed modifier is prepared by mixing citric acid solution and octadecyltrimethylammonium chloride solution; After the biochar and the mixed modifier are mixed evenly, they are subjected to constant temperature oscillation, then washed and filtered to obtain modified biochar. S2. Prepare a suspension of zearalenone-degrading bacteria from zearalenone-degrading bacteria; The modified biochar of S1 was mixed evenly with soluble starch, and then added to the suspension of zearalenone-degrading bacteria under stirring to obtain the core material of ZEN-degrading bacteria microspheres immobilized with modified biochar. S3. After mixing sodium alginate solution with the core material of S2 evenly, calcium chloride solution is added to carry out cross-linking reaction to obtain cross-linked microspheres. S4. Add the cross-linked microspheres from S3 to the chitosan solution and oscillate at a constant temperature to obtain modified biochar-immobilized ZEN-degrading microspheres.

5. The method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres according to claim 4, characterized in that, In S1, the concentration of the citric acid solution is 0.55–0.65 mol / L; the concentration of the octadecyltrimethylammonium chloride solution is 35–45 g / L; and the volume ratio of the citric acid solution to the octadecyltrimethylammonium chloride solution is 1:

1. The ratio of biochar to the mixed modifier is 1g:10mL.

6. The method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres according to claim 4, characterized in that, In S2, the mass ratio of modified biochar to soluble starch is 1:2 to 3; The concentration of the zearalenone-degrading bacteria suspension was 10. 8 ~10 9 cfu / mL; The ratio of modified biochar to zearalenone-degrading bacterial suspension is 1g:13-18mL.

7. The method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres according to claim 4, characterized in that, In S3, the sodium alginate solution concentration is 43–150 g / L; the calcium chloride solution is a 2% (w / v) calcium chloride solution.

8. The method for preparing modified biochar-immobilized ZEN-degrading bacterial microspheres according to claim 4, characterized in that, In S4, the chitosan solution is a chitosan acetate solution with a mass-volume ratio of 2% and a pH of 5.7–6.

0.

9. The application of the modified biochar immobilized ZEN-degrading microspheres according to any one of claims 1 to 3 in the degradation or removal of zearalenone; the application is for non-therapeutic purposes.

10. The application according to claim 9, characterized in that, When degrading or removing zearalenone, 3% to 6% of the modified biochar-immobilized ZEN-degrading microspheres are added to the feed, based on the mass of the modified biochar.

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