A compound microbial feed additive with the functions of enhancing immunity and degrading mycotoxins, and a preparation method and application thereof

Abstract

The present application relates to the technical field of feed additive, in particular to a compound microbial feed additive with the functions of enhancing immunity and degrading mycotoxin, a preparation method and application thereof, which comprises Bacillus subtilis powder, Saccharomyces cerevisiae powder, modified soybean hull pectin, modified gamma-polyglutamic acid, sodium gluconate and a carrier; the present application effectively solves the problem of the decline of storage stability and mycotoxin adsorption function of the compound microbial feed additive containing Bacillus subtilis and Saccharomyces cerevisiae in the application scenario of piglet growth-promoting copper-containing diet by the synergistic effect of modified soybean hull pectin, modified gamma-polyglutamic acid and sodium gluconate, improves the immunity enhancement and toxin control effect in the actual feeding stage of animals, and does not significantly affect the normal existence and utilization of essential trace elements such as zinc, manganese and iron in feed.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, its preparation method, and its application. Background Technology

[0002] Mycotoxin contamination is a long-standing common problem in the feed industry. Zearalenone and aflatoxin B1 are representative toxins with high detection rates in corn-based diets. Long-term intake can cause systemic harm to farmed animals, including decreased immune function, impaired reproductive performance, and liver and kidney damage. In the biocontrol technology of mycotoxins, compound microbial feed additives composed of Bacillus subtilis and Saccharomyces cerevisiae as core components have been widely used due to their complementary functions in mycotoxin degradation and adsorption, as well as enhancing animal immunity. The relevant mechanisms of action have been fully reported in existing literature.

[0003] However, these compound additives exhibit significant functional degradation in practical applications of growth-promoting diets for piglets. The amount of copper sulfate used in growth-promoting diets for piglets can reach 100 mg / kg to 125 mg / kg (based on elemental copper). When this type of compound microbial agent is mixed into the aforementioned copper-containing feed at the conventional recommended dosage (100 g / ton to 500 g / ton), after pelleting, storage, and transportation (usually 2-12 weeks), the copper ions in the feed matrix cause dual interference with the storage stability of Bacillus subtilis spores and the toxin adsorption function of the Saccharomyces cerevisiae cell wall. Ultimately, this leads to a significant decrease in the retention rate of viable bacteria and the adsorption retention rate of typical mycotoxins.

[0004] To address the aforementioned issues, existing technologies primarily employ two approaches: microencapsulation and the addition of chelating agents. Microencapsulation is complex and costly, and the cell wall material is at risk of breakage under the high temperature, high humidity, and mechanical shearing conditions of granulation, allowing copper ions to still penetrate and contact the microbial cells through the damaged pores. Chelating agents, such as citric acid and ethylenediaminetetraacetic acid compounds, lack selectivity for copper ions and simultaneously bind essential micronutrients like zinc, manganese, and iron, disrupting the micronutrient balance in the feed. Therefore, there is an urgent need to develop a novel compound microbial feed additive that can effectively protect the function of the compound microbial agent in copper-containing feed matrices without interfering with essential micronutrients in the feed. Summary of the Invention

[0005] (1) Technical problems to be solved

[0006] The purpose of this invention is to provide a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, as well as its preparation method and application, in order to solve the problem that the compound microbial feed additive containing Bacillus subtilis and Saccharomyces cerevisiae, when used in high-copper diets for promoting growth in piglets, suffers from decreased storage stability and toxin adsorption function due to the dual interference of copper ions in the feed matrix on the stability of the microbial agent spores and the adsorption function of yeast cell wall toxins.

[0007] (2) Technical solution

[0008] To achieve the above objectives, on the one hand, the present invention provides a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, comprising the following parts by weight: 15-30 parts of Bacillus subtilis powder, 10-25 parts of Saccharomyces cerevisiae powder, 8-20 parts of modified soybean hull pectin, 5-15 parts of modified γ-polyglutamic acid, 3-10 parts of sodium gluconate, and 20-50 parts of carrier;

[0009] The viable count of the Bacillus subtilis powder is not less than 2 × 10⁻⁶. 10 CFU / g; the viable count of the brewer's yeast powder is not less than 1×10⁻⁶. 10 CFU / g.

[0010] Furthermore, the carrier is selected from one or more of corn starch, maifanite powder, zeolite powder, and defatted rice bran; the sodium gluconate is food-grade sodium gluconate with a purity of not less than 98%.

[0011] Furthermore, the preparation method of the modified soybean hull pectin includes the following steps:

[0012] S11. In a reaction vessel, soybean hull pectin and 2-(N-morpholino)ethanesulfonic acid buffer were added. The stirring device was turned on and the mixture was stirred and dissolved at 25°C. Nitrogen gas was purged for protection. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially. The mixture was stirred and activated at 25°C. L-cysteine ​​was then added, and the temperature was raised to 35°C. The reaction was continued with stirring. The pH was maintained between 5.5 and 6.0 during the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain intermediate 1.

[0013] S12. Take intermediate 1 obtained in step S11 and add it to the reaction vessel. Add a mixed solution of dimethyl sulfoxide and deionized water and stir to dissolve at 40°C. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in sequence and stir to activate at 25°C. Dissolve L-histidine methyl ester hydrochloride in deionized water and slowly add it dropwise to the reaction system. Maintain the pH between 6.0 and 6.5. Heat to 40°C and continue stirring to react. After the reaction is completed, cool to room temperature, dialyze, and freeze dry to obtain modified soybean skin pectin.

[0014] Furthermore, the preparation method of the modified γ-polyglutamic acid includes the following steps:

[0015] S21. In a reaction vessel, add sodium γ-polyglutamate and deionized water, stir and dissolve at 25°C, adjust the pH to 5.5 with 0.1 mol / L hydrochloric acid, then add ethanol, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, stir and activate at 25°C, dissolve dodecylamine in ethanol and slowly add it dropwise to the reaction system, raise the temperature to 35°C and continue stirring, maintaining the pH between 5.5 and 6.0 during the reaction, dialyze and freeze dry to obtain intermediate 2;

[0016] S22. Take intermediate 2 obtained in step S21 and add it to the reaction vessel. Add 2-(N-morpholino)ethanesulfonic acid buffer and ethanol, stir and dissolve at 30°C. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stir and activate at 25°C. Dissolve 3-amino-1-propanesulfonic acid in 2-(N-morpholino)ethanesulfonic acid buffer, adjust the pH to 5.5 and slowly add it dropwise to the reaction system. Heat to 35°C and continue stirring. Maintain the pH between 5.5 and 6.0 during the reaction. After the reaction is completed, dialyze and freeze dry to obtain modified γ-polyglutamic acid.

[0017] On the other hand, the present invention also provides a method for preparing a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, comprising the following steps:

[0018] S1. Premix: Modified soybean skin pectin, modified γ-polyglutamic acid, and sodium gluconate are pulverized and passed through a 100-mesh sieve. They are then added to a V-type mixer in a clean environment with a relative humidity of no more than 40% and a temperature of no more than 25℃ and mixed for 15-20 minutes to obtain a premixed protective agent.

[0019] S2. Total mixing: Add Bacillus subtilis powder, Saccharomyces cerevisiae powder and carrier to the premixed protective agent obtained in step S1, and continue mixing in a V-type mixer for 30-45 minutes. During the mixing process, the material temperature should not exceed 30°C to obtain a mixture.

[0020] S3. Packaging: The mixture obtained in step S2 is filled with nitrogen and sealed in a package to obtain a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins.

[0021] On the other hand, the present invention also provides an application of a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins. The compound microbial feed additive is used to prepare compound feed for piglets and growing-finishing pigs. The specific form of the application is: the compound microbial feed additive is added to the compound feed at an addition amount of 100g / ton to 500g / ton and mixed evenly.

[0022] Furthermore, the amount of copper sulfate added to the compound feed is 100 mg / kg to 125 mg / kg based on copper element.

[0023] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0024] 1. This invention effectively solves the technical problem of decreased storage stability and mycotoxin adsorption function of compound microbial feed additives containing Bacillus subtilis and Saccharomyces cerevisiae in the application scenario of copper-containing diets for piglet growth promotion by synergistic combination of modified soybean hull pectin, modified γ-polyglutamic acid and sodium gluconate. It enables the compound microbial agent to maintain a high level of viable bacteria and adsorption capacity for zearalenone and aflatoxin B1 even under long-term storage conditions coexisting with copper-containing feed, and significantly improves the immune enhancement and toxin control effect in the actual feeding stage of animals.

[0025] 2. The protection system adopted in this invention has good selectivity for copper ions. While effectively controlling the interference of free copper ions on the microbial agent, it does not significantly affect the normal existence and utilization of essential trace elements such as zinc, manganese, and iron in the feed. This avoids the shortcomings of conventional chelating agents in disrupting the balance of trace elements in the feed, and balances the protection of microbial agent function with the nutritional safety of feed.

[0026] 3. The preparation process of the compound microbial feed additive of the present invention is mild and fully compatible with the premixing, mixing and packaging processes commonly used in the current feed industry. It does not require additional special equipment, the production cost is controllable, and it is suitable for compound feed application for piglets and growing-finishing pigs, which is convenient for large-scale promotion. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the preparation process of a compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions, as described in Example 1 of the present invention.

[0028] Figure 2 This is a physical image of a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, as described in Embodiment 1 of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0030] Example 1: This example discloses a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, comprising the following parts by weight: 20 parts of Bacillus subtilis powder, 18 parts of Saccharomyces cerevisiae powder, 14 parts of modified soybean hull pectin, 10 parts of modified γ-polyglutamic acid, 6 parts of sodium gluconate, and 32 parts of carrier.

[0031] The Bacillus subtilis strain mentioned is the one with preservation number GDMCC 1.372, and the viable count of the powder is 5 × 10⁻⁶. 10 CFU / g; the brewer's yeast is strain with preservation number GDMCC 2.167, and the viable count in the powder is 3×10⁻⁶. 10 CFU / g.

[0032] The carrier is a mixture of corn starch and zeolite powder (mass ratio 1:1); the sodium gluconate is food-grade sodium gluconate with a purity of 99.2%.

[0033] The preparation method of the modified soybean peel pectin includes the following steps:

[0034] S11. In a 2L glass-jacketed reactor, add 10.0g of soybean peel pectin and 500mL of 2-(N-morpholino)ethanesulfonic acid buffer (concentration 0.05mol / L, pH 5.5). Turn on the stirrer and stir to dissolve at 25℃ for 2h. Purge with nitrogen for protection. Then add 3.24g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.94g of N-hydroxysuccinimide sequentially. Activate by stirring at 25℃ for 60min. Add 5.32g of... L-cysteine ​​was pre-dissolved in 20 mL of deionized water and slowly added dropwise to the reaction system. The temperature was raised to 35 °C and the reaction was stirred for 24 h. During the reaction, nitrogen protection was maintained throughout and the pH of the reaction system was maintained between 5.5 and 6.0 with 0.1 mol / L sodium hydroxide solution. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against deionized water for 72 h. The dialysate was changed every 8 h. After dialysis, the solution was freeze-dried to obtain intermediate 1.

[0035] S12. Take 10.0g of intermediate 1 obtained in step S11 and add it to a 2L three-necked flask. Add a mixed solution of 200mL dimethyl sulfoxide and 200mL deionized water, and stir to dissolve at 40℃ for 3h. Then add 3.24g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.94g of N-hydroxysuccinimide, and activate by stirring at 25℃ for 60min. Add 5.61g of... L-histidine methyl ester hydrochloride was pre-dissolved in 50 mL of deionized water and slowly added dropwise to the reaction system. The pH of the reaction system was adjusted and maintained between 6.0 and 6.5 using 0.1 mol / L sodium hydroxide solution. The temperature was raised to 40 °C and stirred for 24 h. After the reaction was completed, the mixture was cooled to room temperature and transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. The mixture was first dialyzed with a 20% (v / v) ethanol aqueous solution for 24 h, and then dialyzed with deionized water for 48 h. The dialysate was changed every 8 h. After the dialysis was completed, the mixture was freeze-dried to obtain modified soybean skin pectin.

[0036] The preparation method of the modified γ-polyglutamic acid includes the following steps:

[0037] S21. In a 2L glass-jacketed reactor, add 10.0g of sodium γ-polyglutamate with a molecular weight of approximately 500,000 Da and 500mL of deionized water. Stir and dissolve at 25°C for 2 hours. Adjust the pH to 5.5 with 0.1mol / L hydrochloric acid, then add 100mL of ethanol. Next, add 3.74g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.24g of N-hydroxysuccinimide. Activate by stirring at 25°C for 60 minutes. Dissolve 3.61g of dodecylamine in 50mL of ethanol and slowly add it dropwise to the reaction system. Heat to 35°C and continue stirring for 24 hours. During the reaction, the pH of the reaction system was maintained between 5.5 and 6.0 using 0.1 mol / L sodium hydroxide solution. After the reaction was completed, the reaction solution was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da at a pressure of 0.2 MPa. The membrane was washed with deionized water for 5 membrane volumes to fully remove free dodecylamine and unreacted small molecules. The ultrafiltration solution was then transferred to a dialysis bag with a molecular weight cutoff of 14,000 Daltons. The solution was dialyzed with 30% ethanol aqueous solution for 24 h, followed by dialyzed with deionized water for 48 h. The dialysate was changed every 12 h. After the dialysis was completed, the solution was freeze-dried to obtain intermediate 2.

[0038] S22. Take 10.0g of intermediate 2 obtained in step S21 and add it to another 2L glass-jacketed reactor. Add 500mL of 2-(N-morpholino)ethanesulfonic acid buffer (concentration 0.05mol / L, pH 5.5) and 50mL of ethanol. Stir and dissolve at 30℃ for 3h. Then add 6.53g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3.91g of N-hydroxysuccinimide. Stir and activate at 25℃ for 60min. Dissolve 4.74g of 3-amino-1-propanesulfonic acid in 50mL of 2-(N-morpholino)ethanesulfonic acid buffer (concentration 0.05mol / L, pH 5.5). In step 5.5), the pH was adjusted to 5.5 with 0.1 mol / L sodium hydroxide solution and then slowly added dropwise to the reaction system. The temperature was raised to 35°C and the reaction was stirred for 24 hours. During the reaction, the pH of the reaction system was maintained between 5.5 and 6.0 with 0.1 mol / L sodium hydroxide solution. After the reaction was completed, the reaction solution was ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da at a pressure of 0.2 MPa. The membrane was washed with deionized water at a constant volume for 5 membrane volumes to fully remove free 3-amino-1-propanesulfonic acid and unreacted small molecules. Then, the ultrafiltration residue was transferred to a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed with deionized water for 72 hours. The dialysate was changed every 8 hours. After dialysis, the solution was freeze-dried to obtain modified γ-polyglutamic acid.

[0039] The preparation method of the compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins includes the following steps:

[0040] S1. Premix: Modified soybean skin pectin, modified γ-polyglutamic acid, and sodium gluconate are pulverized and passed through a 100-mesh sieve, and added to a V-type mixer in a clean environment with a relative humidity of 35% and a temperature of 23°C. The mixture is mixed for 18 minutes to obtain a premixed protective agent.

[0041] S2. Total mixing: Add Bacillus subtilis powder, Saccharomyces cerevisiae powder and carrier to the premixed protective agent obtained in step S1, and continue mixing in a V-type mixer for 40 minutes. During the mixing process, the material temperature is maintained at 28°C to obtain a mixture.

[0042] S3. Packaging: The mixture obtained in step S2 is filled with nitrogen and sealed in a package to obtain a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins. The process is as follows: Figure 1 As shown, the actual object is as follows Figure 2 As shown.

[0043] The application of the compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins is as follows: the compound microbial feed additive is used to prepare compound feed for piglets and growing-finishing pigs; the specific form of application is: the compound microbial feed additive is added to the compound feed at an addition rate of 300g / ton and mixed evenly.

[0044] The amount of copper sulfate added to the compound feed is 112 mg / kg, calculated as copper element.

[0045] Example 2: This example is based on Example 1, but differs from Example 1 in that it includes the following components by weight: 15 parts of Bacillus subtilis powder, 10 parts of Saccharomyces cerevisiae powder, 8 parts of modified soybean hull pectin, 5 parts of modified γ-polyglutamic acid, 3 parts of sodium gluconate, and 20 parts of carrier; other components and preparation methods are the same as in Example 1.

[0046] Example 3: This example is based on Example 1, but differs from Example 1 in that it includes the following components by weight: 30 parts of Bacillus subtilis powder, 25 parts of Saccharomyces cerevisiae powder, 20 parts of modified soybean hull pectin, 15 parts of modified γ-polyglutamic acid, 10 parts of sodium gluconate, and 50 parts of carrier; other components and preparation methods are the same as in Example 1.

[0047] Example 4: This example differs from Example 1 in that, in the application of the compound microbial feed additive, the compound microbial feed additive is added to the compound feed at a dosage of 100g / ton and mixed evenly. The amount of copper sulfate added to the compound feed is 100mg / kg (calculated as copper element). Other components and preparation methods are the same as in Example 1.

[0048] Example 5: This example differs from Example 1 in that, in the application of the compound microbial feed additive, the compound microbial feed additive is added to the compound feed at a dosage of 500g / ton and mixed evenly. The amount of copper sulfate added to the compound feed, calculated as copper element, is 125mg / kg. Other components and preparation methods are the same as in Example 1.

[0049] Comparative Example 1: This comparative example is based on Example 1, except that an equal amount of ordinary soybean skin pectin is used instead of modified soybean skin pectin in this comparative example. Other components and preparation methods are the same as in Example 1.

[0050] Comparative Example 2: This comparative example is based on Example 1, but differs from Example 1 in that the modified soybean peel pectin in this comparative example only completes step S11 and does not perform step S12. Intermediate 1 directly replaces the modified soybean peel pectin, and the other components and preparation methods are the same as in Example 1.

[0051] Comparative Example 3: This comparative example is based on Example 1, but differs from Example 1 in that soybean hull pectin is directly processed in step S12 to replace the modified soybean hull pectin. Other components and preparation methods are the same as in Example 1.

[0052] Comparative Example 4: This comparative example is based on Example 1, except that an equal amount of ordinary sodium γ-polyglutamate is used instead of modified γ-polyglutamic acid in this comparative example. Other components and preparation methods are the same as in Example 1.

[0053] Comparative Example 5: This comparative example is based on Example 1, but differs from Example 1 in that the modified γ-polyglutamic acid only completes step S21 and does not perform step S22. Intermediate 2 directly replaces the modified γ-polyglutamic acid, while other components and preparation methods are the same as in Example 1.

[0054] Comparative Example 6: This comparative example is based on Example 1, but differs from Example 1 in that the sodium γ-polyglutamate is directly processed in step S22 to replace the modified γ-polyglutamate. Other components and preparation methods are the same as in Example 1.

[0055] Comparative Example 7: This comparative example is based on Example 1, but differs from Example 1 in that it does not contain sodium gluconate. Its amount is supplemented by an equal amount of carrier, and the total amount of the formula remains unchanged. Other components and preparation methods are the same as in Example 1.

[0056] Comparative Example 8: This comparative example is based on Example 1, but differs from Example 1 in that it does not contain modified soybean skin pectin, modified γ-polyglutamic acid, and sodium gluconate. The amounts of the three are supplemented by an equal amount of carrier. That is, this comparative example consists only of 20 parts of Bacillus subtilis powder, 18 parts of Saccharomyces cerevisiae powder, and 62 parts of carrier, and is a blank composite microbial agent control without any protective components.

[0057] Comparative Example 9: This comparative example is a comparison with existing microcapsule encapsulation methods. An equal amount of Bacillus subtilis powder (viable count 5 × 10⁻⁶) as in Example 1 was taken. 10 20 portions of CFU / g and brewer's yeast powder (live count 3×10⁻⁶) 1018 portions (CFU / g) of the microbial powder were encapsulated using a sodium alginate-calcium chloride microencapsulation process: the above-mentioned bacterial powder was dispersed in a 2% (w / w) sodium alginate aqueous solution, and the mixture was dripped into a 0.1 mol / L calcium chloride solution using a peristaltic pump. After solidification for 30 min, the mixture was filtered, washed with deionized water, and freeze-dried to obtain microencapsulated composite bacterial powder. The obtained microencapsulated composite bacterial powder was mixed with 32 portions of carrier in a V-type mixer for 40 min, and then sealed with nitrogen gas to obtain a microencapsulated composite microbial feed additive. Copper sulfate was added at a dosage of 300 g / ton to a compound feed with a copper element content of 112 mg / kg and mixed thoroughly.

[0058] Comparative Example 10: This comparative example is a comparison with existing EDTA chelating agent solutions. 20 parts of Bacillus subtilis powder, 18 parts of Saccharomyces cerevisiae powder, and 32 parts of carrier, equal in amount to those in Example 1, were taken. Additionally, 6 parts of disodium ethylenediaminetetraacetate (EDTA-2Na, food grade, purity ≥99%) were added (the amount was equal to that of sodium gluconate in Example 1; 20 parts of modified soybean hull pectin and modified γ-polyglutamic acid were supplemented with an equal amount of carrier). The mixture was added to a V-type mixer and mixed for 40 min in a clean environment with a relative humidity of 35% and a temperature of 23°C. The mixture was then sealed in a nitrogen-filled package to obtain an EDTA chelating agent-type compound microbial feed additive. Copper sulfate was added at an addition rate of 300 g / ton to a compound feed with an addition rate of 112 mg / kg (calculated as copper element) and mixed thoroughly.

[0059] Experimental verification:

[0060] Experiment 1: Verification of the retention rate of viable bacteria in copper-containing compound feed during storage. The results are shown in Table 1.

[0061] Test Samples: Examples 1-5 and Comparative Examples 1-10 were mixed into copper sulfate-containing compound feed at their respective addition amounts (Examples 1-3 and Comparative Examples 1-10 were added at 300 g / ton with a copper concentration of 112 mg / kg; Example 4 was added at 100 g / ton with a copper concentration of 100 mg / kg; and Example 5 was added at 500 g / ton with a copper concentration of 125 mg / kg). After thorough mixing, the samples were placed in sealed aluminum foil sample bags and stored in the dark at 25±1℃ and 60±5% relative humidity. Six parallel samples were prepared for each group.

[0062] Experimental Methods: Samples were collected at the end of weeks 0, 4, and 12. Bacillus subtilis viable counts were performed according to GB / T 23181-2008, using nutrient agar at 37℃ for 48 hours, followed by colony counting. Saccharomyces cerevisiae viable counts were performed according to NY / T2054-2011, using YPD substrate at 30℃ for 72 hours, followed by colony counting. The viable count retention rate (%) at each time point was calculated as follows: (Viable count at each week / Viable count at week 0) × 100%.

[0063] Table 1. Viable cell count retention rate (%) of Bacillus subtilis (Bs) and Saccharomyces cerevisiae (Sc) in each group:

[0064]

[0065] Experiment 2: Verification of the retention rate of mycotoxin adsorption function in copper-containing compound feed during storage. The results are shown in Table 2.

[0066] Test samples: Samples from the same batch as those in Test 1, with consistent storage conditions and time points.

[0067] Experimental Method: Samples from each group were collected at the end of weeks 0, 4, and 12. 1.0 g of each sample was accurately weighed into a 10 mL centrifuge tube. ZEA adsorption rate was determined by adding phosphate buffer (pH 10) containing zearalenone (ZEA). 5.0 mL of aflatoxin B1 (ZEA concentration 1.0 μg / mL) was incubated in a 37℃ water bath with shaking for 2 h, then centrifuged at 8000 r / min for 15 min. The supernatant was filtered through a 0.22 μm filter membrane, and the residual ZEA concentration was determined by high performance liquid chromatography (HPLC, fluorescence detector, excitation wavelength 274 nm, emission wavelength 440 nm, C18 column, methanol:water = 65:35 mobile phase). AFB1 adsorption rate determination: Phosphate buffer containing aflatoxin B1 (AFB1) (pH 6.0, AFB1 concentration 50 ng / mL) was used instead of the ZEA standard solution, and the residual AFB1 concentration was determined by HPLC (fluorescence detector, excitation wavelength 365 nm, emission wavelength 440 nm, C18 column, methanol:water = 45:55 mobile phase). Adsorption rate (%) = (initial toxin concentration - residual toxin concentration) / initial toxin concentration × 100%. The adsorption rate retention rate at subsequent time points was calculated based on the adsorption rate of each group at week 0. Six parallel samples were prepared for each group.

[0068] Table 2. Adsorption rates of ZEA and AFB1 in each group (%):

[0069]

[0070] Experiment 3: Validation of the selectivity of the protective system for essential trace elements in feed. The results are shown in Table 3.

[0071] Test Samples: According to the formulation ratio of Example 1, the three protective components—modified soybean hull pectin, modified γ-polyglutamic acid, and sodium gluconate—were dissolved in simulated feed matrix buffer (50 mmol / L acetate-sodium acetate buffer, pH 6.0) in equal proportions to the actual mass fraction of each component in the total formulation as described in Example 1, to prepare a test solution with a concentration of 3.0 g / L (corresponding to the relative concentration of the three protective components in the actual matrix after adding 300 g / ton of copper-containing feed in Example 1); an equimolar copper capture equivalent of EDTA-2Na (food grade, purity ≥99%) solution was used as a non-selective chelating agent control; and a blank solution with an equal amount of carrier replacing the three protective components was used as a negative control.

[0072] Experimental method: In each test solution, the concentrations of each element were converted according to the typical formulation of growth-promoting compound feed for piglets, and CuSO4·5H2O (Cu) was added simultaneously. 2+ Final concentration 10.0 mg / L), ZnSO4·7H2O (Zn 2+ Final concentration 7.0 mg / L), MnSO4·H2O (Mn 2+ Final concentration 3.5 mg / L) and FeCl3·6H2O (Fe 3+ The final concentration was 7.0 mg / L. After equilibration at 25℃ for 24 h, the mixture was centrifuged at 8000 r / min for 15 min. The supernatant was filtered through a 0.22 μm filter membrane, and the residual concentration of each element was simultaneously determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The capture rate (%) of each element was calculated as (addition amount - residual amount) / addition amount × 100%. Six parallel samples were prepared for each group.

[0073] Table 3. Capture rate (%) of each element in the feed matrix by the protection system:

[0074]

[0075] Experiment 4: Verification of the feeding effect on piglets. The results are shown in Table 4.

[0076] Experimental Samples and Animal Grouping: Forty-eight healthy weaned piglets (28 days old, initial weight 7.2±0.6 kg) were randomly divided into 6 groups of 8 piglets each, housed individually. Feed was premixed and sealed for 8 weeks to simulate actual feed supply chain conditions before being opened and fed for 28 days. The group designs were as follows: Group I (normal control): basal diet, copper supplemented at nutritional requirements (6 mg / kg), no exogenous mycotoxins added, and no product of this invention added; Group II (model control): high copper diet (Cu... Group II (Example 1): The same feed as Group II, with the addition of 300g / ton of the product of Example 1, stored for 8 weeks before feeding; Group III (Comparative Example 8): The same feed as Group II, with the addition of 300g / ton of Comparative Example 8 (blank compound microbial agent), stored for 8 weeks before feeding; Group V (Comparative Example 9): The same feed as Group II, with the addition of 300g / ton of Comparative Example 9 (microencapsulated type), stored for 8 weeks before feeding; Group VI (Comparative Example 10): The same feed as Group II, with the addition of 300g / ton of Comparative Example 10 (EDTA chelated form), stored for 8 weeks before feeding.

[0077] Experimental Methods: Blood was collected from the anterior vena cava on the morning of day 28 before feeding, after fasting. Serum was prepared, and the following indicators were measured: serum immunoglobulin G (IgG) and immunoglobulin A (IgA) levels were determined using ELISA (according to the kit instructions); serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined using the rate method (automated biochemical analyzer); serum ZEA and AFB1 residues were determined using LC-MS / MS (multiple reaction monitoring mode, ZEA quantitative ion pair 319.1→175.1, AFB1 quantitative ion pair 313.0→241.0). Average daily gain (ADG) was calculated as total weight gain over the 28-day feeding period divided by the number of feeding days.

[0078] Table 4. Results of key indicators for each group of piglets after 28 days of feeding:

[0079]

[0080] Based on the results in Tables 1, 2, 3, and 4, it can be concluded that this invention effectively solves the problems of decreased storage stability and mycotoxin adsorption function of compound microbial feed additives containing Bacillus subtilis and Saccharomyces cerevisiae in the application scenario of copper-containing diets for piglet growth promotion by synergistically combining modified soybean hull pectin, modified γ-polyglutamic acid, and sodium gluconate. This allows the compound microbial agent to maintain a high level of viable bacteria and adsorption capacity for zearalenone and aflatoxin B1 even under long-term storage conditions coexisting with copper-containing feed, thereby improving the immune enhancement and toxin control effect in animals during the actual feeding stage, without significantly affecting the normal presence and utilization of essential trace elements such as zinc, manganese, and iron in the feed.

[0081] Experiment 5: Verify the safety of compound microbial feed additives.

[0082] The compound microbial feed additives prepared in Examples 1-5 were mixed into compound feeds according to their respective addition amounts. Acute oral toxicity evaluation was conducted according to GB 15193.3-2014 "National Food Safety Standard - Acute Oral Toxicity Test". The results showed that the acute oral toxicity of the products in each example was practically non-toxic. The levels of harmful substances such as lead, arsenic, mercury, and cadmium, as well as the total mold count, were tested according to GB / T 13078-2017 "Feed Hygiene Standard". All indicators met the limits specified in this standard. The compound microbial feed additives prepared in Examples 1-5 have good feed safety.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins, characterized in that, The product contains the following components by weight: 15-30 parts of Bacillus subtilis powder, 10-25 parts of Saccharomyces cerevisiae powder, 8-20 parts of modified soybean hull pectin, 5-15 parts of modified γ-polyglutamic acid, 3-10 parts of sodium gluconate, and 20-50 parts of carrier. The viable count of the Bacillus subtilis powder is not less than 2 × 10⁻⁶. 10 CFU / g; the viable count of the brewer's yeast powder is not less than 1×10⁻⁶. 10 CFU / g.

2. The compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions according to claim 1, characterized in that, The carrier is selected from one or more of corn starch, maifanite powder, zeolite powder, and defatted rice bran; the sodium gluconate is food-grade sodium gluconate with a purity of not less than 98%.

3. The compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions according to claim 1, characterized in that, The preparation method of the modified soybean peel pectin includes the following steps: S11. In a reaction vessel, soybean hull pectin and 2-(N-morpholino)ethanesulfonic acid buffer were added. The stirring device was turned on and the mixture was stirred and dissolved at 25°C. Nitrogen gas was purged for protection. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially. The mixture was stirred and activated at 25°C. L-cysteine ​​was then added, and the temperature was raised to 35°C. The reaction was continued with stirring. The pH was maintained between 5.5 and 6.0 during the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain intermediate 1. S12. Take intermediate 1 obtained in step S11 and add it to the reaction vessel. Add a mixed solution of dimethyl sulfoxide and deionized water and stir to dissolve at 40°C. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in sequence and stir to activate at 25°C. Dissolve L-histidine methyl ester hydrochloride in deionized water and slowly add it dropwise to the reaction system. Maintain the pH between 6.0 and 6.

5. Heat to 40°C and continue stirring to react. After the reaction is completed, cool to room temperature, dialyze, and freeze dry to obtain modified soybean skin pectin.

4. The compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions according to claim 1, characterized in that, The preparation method of the modified γ-polyglutamic acid includes the following steps: S21. In a reaction vessel, add sodium γ-polyglutamate and deionized water, stir and dissolve at 25°C, adjust the pH to 5.5 with 0.1 mol / L hydrochloric acid, then add ethanol, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, stir and activate at 25°C, dissolve dodecylamine in ethanol and slowly add it dropwise to the reaction system, raise the temperature to 35°C and continue stirring, maintaining the pH between 5.5 and 6.0 during the reaction, dialyze and freeze dry to obtain intermediate 2; S22. Take intermediate 2 obtained in step S21 and add it to the reaction vessel. Add 2-(N-morpholino)ethanesulfonic acid buffer and ethanol, stir and dissolve at 30°C. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stir and activate at 25°C. Dissolve 3-amino-1-propanesulfonic acid in 2-(N-morpholino)ethanesulfonic acid buffer, adjust the pH to 5.5 and slowly add it dropwise to the reaction system. Heat to 35°C and continue stirring. Maintain the pH between 5.5 and 6.0 during the reaction. After the reaction is completed, dialyze and freeze dry to obtain modified γ-polyglutamic acid.

5. A method for preparing a compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Premix: Modified soybean skin pectin, modified γ-polyglutamic acid, and sodium gluconate are pulverized and passed through a 100-mesh sieve. They are then added to a V-type mixer in a clean environment with a relative humidity of no more than 40% and a temperature of no more than 25℃ and mixed for 15-20 minutes to obtain a premixed protective agent. S2. Total mixing: Add Bacillus subtilis powder, Saccharomyces cerevisiae powder and carrier to the premixed protective agent obtained in step S1, and continue mixing in a V-type mixer for 30-45 minutes. During the mixing process, the material temperature should not exceed 30°C to obtain a mixture. S3. Packaging: The mixture obtained in step S2 is filled with nitrogen and sealed in a package to obtain a compound microbial feed additive with functions of enhancing immunity and degrading mycotoxins.

6. The application of a compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions according to any one of claims 1-4, characterized in that, The compound microbial feed additive is used to prepare compound feed for piglets and growing-finishing pigs; the specific form of application is: the compound microbial feed additive is added to the compound feed at an addition amount of 100g / ton to 500g / ton and mixed evenly.

7. The application of the compound microbial feed additive with immune-enhancing and mycotoxin-degrading functions according to claim 6, characterized in that, The amount of copper sulfate added to the compound feed is 100 mg / kg to 125 mg / kg, calculated as copper element.

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