A breeding method for preventing and controlling african swine fever

Abstract

The application discloses a breeding method for preventing and controlling African swine fever, and belongs to the technical field of livestock breeding. The method comprises the following steps: subjecting a yucca extract with steroidal saponin ≥ 85% to enzymolysis by using beta-glucosidase to obtain an enzymolysis yucca product with the sum of secondary saponins and aglycone ≥ 40% and complete steroidal saponin ≤ 30%; adding chitosan modified montmorillonite microparticles and the enzymolysis product to a basic daily ration to obtain a prevention and control feed; and feeding pigs with the feed, and combining with timed and quantitative feeding and water management. The application realizes debittering and enhanced anti-inflammatory activity simultaneously through enzymolysis, and solves the palatability obstacle; the modified montmorillonite forms a physical barrier in the intestinal tract, and cooperates with the immune regulation of the enzymolysis product to block the invasion of viruses. The application realizes zero incidence of pigs in a virus positive environment, simultaneously improves daily weight gain, and reduces the feed to weight ratio, and is suitable for large-scale promotion.

Description

[Technical Field] This invention relates to the field of livestock breeding technology, and in particular to a breeding method for preventing and controlling African swine fever. [Background Technology] African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease of pigs caused by the African swine fever virus (ASFV), with a morbidity and mortality rate that can reach 100%.

[0003] Currently, the prevention and control of African swine fever (ASF) mainly relies on the construction of a biosecurity system, including measures such as farm isolation and disinfection, control of personnel and vehicle access, harmless disposal of dead pigs, and epidemic monitoring and early warning. However, regional prevention and control systems primarily focus on monitoring and regulating the external environment of farms. Existing technologies still have significant shortcomings in improving the overall health and disease resistance of pig herds through feed optimization and feeding management within farms. While some existing technologies control ASF through feed, such as using plant extracts to prepare feed additives, adding organic trace elements and probiotics, these approaches mostly focus on the antiviral or immunomodulatory functions of single additives, failing to systematically integrate the safe handling of feed ingredients, improvement of the palatability of active ingredients, and intestinal barrier protection.

[0004] Yucca extract, as a functional feed additive, primarily contains steroidal saponins, which possess various biological functions such as antioxidation, anti-inflammation, antibacterial activity, and immunomodulation, positively impacting pig growth performance and gut health. However, current steroidal saponin content in yucca extracts is typically 5%-30%, resulting in low purity. High-purity saponins often exhibit a strong bitter taste and pungent odor, affecting feed palatability. While chemical hydrolysis can break down saponins into secondary saponins and aglycones, strong acid conditions can easily produce undesirable byproducts. Therefore, improving the application performance of yucca extract while maintaining its beneficial effects remains a key concern in this field.

[0005] In summary, the existing technology lacks a systematic breeding method for the prevention and control of African swine fever that can organically combine the improvement of the application performance of feed active ingredients with intestinal protection. [Summary of the Invention] In view of the above, it is necessary to provide a breeding method for preventing and controlling African swine fever, which can simultaneously achieve debittering and anti-inflammatory activity through enzymatic hydrolysis to solve palatability problems; modified montmorillonite forms a physical barrier in the intestine and works synergistically with the immune regulation of the enzymatic hydrolysis products to block viral invasion.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preventing and controlling African swine fever in pig farming includes the following steps: (I) Feed formulation: (1) Enzymatic hydrolysis pretreatment: Yucca extract with a steroidal saponin weight percentage of ≥85% is mixed with water to prepare an extract, and β-glucosidase is added for enzymatic hydrolysis to obtain enzymatic hydrolyzed yucca product; in the enzymatic hydrolyzed yucca product, the weight percentage of secondary saponins and aglycones is ≥40%, and the weight percentage of intact steroidal saponins is ≤30%; (2) Modified montmorillonite microparticles and the enzymatic hydrolyzed yucca product are added to the basic diet and mixed to obtain the control feed; the particle size of the modified montmorillonite microparticles is 50-200μm, and the zeta potential after surface modification with a cationic modifier is +15mV to +35mV; (ii) Feeding management: The pigs shall be fed the aforementioned control feed.

[0008] In this invention, the enzymatic hydrolysis treatment is further performed at a temperature of 40-60°C, a pH of 4.0-6.0, and a time of 2-8 hours.

[0009] In this invention, the β-glucosidase has an enzyme activity of 10,000-50,000 U / g and is added at a rate of 0.1%-1.0% of the dry weight of the yucca extract.

[0010] In this invention, the enzymatic hydrolysis pretreatment further includes an enzyme inactivation step: heating the enzymatic hydrolysis product to 80-100℃ and holding for 5-15 minutes, and then drying to obtain enzymatic hydrolyzed yucca product powder.

[0011] In this invention, the cationic modifier is chitosan, wherein the degree of deacetylation of the chitosan is 80%-95% and the weight-average molecular weight is 50kDa-200kDa.

[0012] In this invention, the modified montmorillonite microparticles are added to the feed at a rate of 0.5-2.0 parts by weight, and the enzymatically hydrolyzed yucca product is added to the feed at a rate of 0.05-0.5 parts by weight.

[0013] In this invention, further, step (ii) also includes: adjusting the addition ratio of modified montmorillonite microparticles and enzymatically hydrolyzed yucca products in the prevention and control feed based on the monitoring results of African swine fever virus in the pig farm environment.

[0014] In this invention, the environmental monitoring of African swine fever virus further includes: regularly collecting cotton swab samples from the pigsty environment, using quantitative real-time PCR to detect African swine fever virus nucleic acid, and determining the environmental risk level based on the obtained Ct value; when the Ct value is ≥37, it is a low-risk state, and when the Ct value is <37, it is a high-risk state; under the high-risk state, the addition amount of modified montmorillonite microparticles and enzymatic hydrolysis of yucca products is increased to 1.5-2.0 times the basic addition amount.

[0015] In this invention, further, in step (ii), a three-fixed feeding system of fixed time, fixed quantity, and fixed quality is adopted, feeding 2-4 times a day, with an interval of 4-6 hours between two consecutive feedings; when changing feed, a transition period of 3-5 days is set, during which the old and new feeds are replaced in a gradient of 20%-30% per day.

[0016] Furthermore, this invention also includes a drinking water management step: using purified water for pigs to drink, and adding organic acid to adjust the pH of the drinking water to 3.5-4.5.

[0017] The present invention has at least the following beneficial effects: 1. Currently, there is still a lack of effective drugs and vaccines for the prevention and control of African swine fever. While existing technologies have attempted to improve pigs' disease resistance through anti-inflammatory drugs or immunomodulators, long-term use of chemically synthesized drugs easily leads to drug resistance and drug residue risks, which is inconsistent with the development direction of green farming. The inventors chose yucca extract as a natural anti-inflammatory active ingredient, but unexpectedly discovered during the research process that while high-purity yucca extract is rich in steroidal saponins, it is accompanied by a strong bitter taste and pungent odor, resulting in a significant decrease in pig feed intake and ineffective ingestion of the active ingredients—a palatability barrier that is not significant in conventionally pure yucca extract, representing a previously unaddressed technical blind spot in the field. To address this newly discovered problem, the inventors tried various conventional methods such as sweetener masking, flavoring agent blending, and coating isolation, but none of these methods fundamentally solved the problem. Ultimately, the study found that enzymatic hydrolysis of high-purity yucca extract using β-glucosidase, by hydrolyzing the glycosyl groups of steroidal saponins, reduced the bitterness score from 8.5 to 2.3, restoring feed intake to normal levels and fundamentally eliminating palatability barriers. Furthermore, enzymatic hydrolysis not only removed bitterness but also significantly enhanced anti-inflammatory activity and substantially improved intestinal transmembrane transport.

[0018] 2. Montmorillonite is mainly used in feed to adsorb mycotoxins and prevent diarrhea in piglets. There are also reports of using montmorillonite to adsorb viruses. However, the inventors have modified this adsorption effect into a physical barrier—using chitosan to modify montmorillonite microparticles, allowing them to form an electrostatic complex network with mucus proteins in the intestine, increasing the thickness of the intestinal mucus layer and physically blocking the invasion of ASFV virus particles. Simultaneously, the secondary saponins and aglycones in the enzymatically hydrolyzed yucca products can cross the intestinal epithelium to reach the target sites of ASFV replication (ileum and colonic lymphoid tissue), regulating the local immune microenvironment. The two work together: the physical barrier formed by the modified montmorillonite in the intestinal mucus layer directly blocks the contact between ASFV virus particles and intestinal epithelial cells, delaying the viral invasion process and allowing time for the full release and absorption of the active ingredients in the enzymatically hydrolyzed yucca products; the secondary saponins and aglycones in the enzymatically hydrolyzed yucca products, after crossing the intestinal epithelium, reach the gut-associated lymphoid tissue, regulating the local immune microenvironment and further strengthening the barrier function of the intestinal mucosa. The two work together to form a spatiotemporal synergistic defense line of "physical blocking + penetration regulation". Experiments have shown that the incidence rate of the modified montmorillonite group alone was 35%, the incidence rate of the enzymatically hydrolyzed yucca group alone was 25%, while the incidence rate of the complete scheme of this invention was only 0%. The actual effect is significantly better than the expected value of independent superposition, which confirms the synergistic effect.

[0019] 3. This invention, through a comprehensive solution encompassing feed formulation, feeding management, and water management, achieves excellent disease control results of zero morbidity and zero mortality in large-scale pig farms with persistently positive ASFV nucleic acid tests. Simultaneously, it increases the average daily weight gain of pigs and reduces the feed conversion ratio, thus possessing dual economic value in both disease prevention and efficiency improvement. The operation is simple, requires no large-scale modification of existing breeding facilities, is suitable for large-scale promotion, and has good prospects for widespread application.

Detailed Implementation Methods

[0021] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0022] In this invention, the basal diet is a complete compound feed for pigs that meets national standards, with the following nutritional levels: digestible energy 13.5-14.5 MJ / kg, crude protein 16%-18%, and lysine 0.8%-1.0%.

[0023] Experiments have shown that when the degree of deacetylation of chitosan is 80%-95% and the weight-average molecular weight is 50-200kDa, the zeta potential of modified montmorillonite microparticles is +15mV to +35mV. This positive charge range can ensure that it can effectively bind to negatively charged mucins.

[0024] Example 1 This embodiment provides a method for preventing and controlling African swine fever in livestock farming, which specifically includes the following steps: (a) Feed formulation 1. Preparation of high-purity yucca extract Dried yucca leaves were collected and pulverized through a 40-mesh sieve. The yucca leaf powder was placed in a supercritical fluid extraction vessel, and extraction was carried out for 2 hours at an extraction temperature of 35℃ and an extraction pressure of 25MPa using CO2 as the extraction medium and anhydrous ethanol as the entrainer (10% of the raw material mass). After extraction, the extract was collected, and the ethanol was recovered by concentration under reduced pressure to obtain crude yucca extract.

[0025] The crude yucca extract was redissolved in a 30% ethanol aqueous solution and loaded onto a macroporous adsorption resin column (D101 type). Gradient elution was performed sequentially with 30%, 50%, and 70% (v / v) ethanol aqueous solutions. The eluent fraction of the 70% ethanol aqueous solution was collected, concentrated under reduced pressure, and freeze-dried to obtain a high-purity yucca extract powder.

[0026] HPLC analysis showed that the high-purity yucca extract prepared in this example contained 86.3% steroidal saponins by weight.

[0027] 2. Preparation of enzymatic hydrolysis products of yucca The high-purity yucca extract powder was dissolved in deionized water to prepare an extract with a mass concentration of 5%. The pH was adjusted to 4.0, and the solution was heated to 40°C. β-glucosidase was added for enzymatic hydrolysis. The β-glucosidase was derived from Aspergillus niger, with an enzyme activity of 10000 U / g, and the amount added was 0.1% of the dry weight of the high-purity yucca extract. The hydrolysis time was 2 hours, and the stirring speed was 100 rpm.

[0028] After enzymatic hydrolysis, the hydrolysate was heated to 80°C and held for 5 minutes to inactivate β-glucosidase and terminate the hydrolysis reaction. Then, it was spray-dried to obtain the hydrolyzed yucca product powder.

[0029] HPLC-MS analysis showed that the total weight percentage of secondary saponins and aglycones in the enzymatic hydrolysis of yucca prepared in this example was 43.1%, and the weight percentage of intact steroidal saponins was 28.3%.

[0030] 3. Preparation of modified montmorillonite microparticles Natural montmorillonite (montmorillonite content ≥95%) was crushed and passed through a 200-mesh sieve. It was then dispersed in deionized water to prepare a 5% (w / w) suspension, which was stirred for 2 hours to allow it to fully hydrate and swell. After standing and separating into layers, the upper suspension was collected and centrifuged to obtain purified montmorillonite.

[0031] Chitosan (80% degree of deacetylation, 50 kDa weight average molecular weight) was dissolved in a 1% aqueous acetic acid solution to prepare a 0.5% (w / w) chitosan-acetic acid solution. Purified montmorillonite was dispersed in the chitosan-acetic acid solution (montmorillonite to chitosan mass ratio 20:1), and the mixture was stirred at 40°C for 4 hours. After the reaction, the precipitate was separated by centrifugation, washed with deionized water until neutral, vacuum dried at 60°C, and pulverized through a 200-mesh sieve to obtain chitosan-modified montmorillonite microparticles.

[0032] Tests showed that the modified montmorillonite microparticles had an average particle size of 50 μm and a zeta potential of +15 mV.

[0033] 4. Preparation of feed for disease prevention and control The modified montmorillonite microparticles and enzymatically hydrolyzed yucca products were added to the basal diet and mixed thoroughly to obtain the control feed. The amount of modified montmorillonite microparticles added was 0.5 parts by weight, and the amount of enzymatically hydrolyzed yucca products added was 0.05 parts by weight.

[0034] (ii) Feeding Management The pigs were fed the aforementioned feed for disease control. They were fed three times daily (6:00-7:00, 12:00-13:00, and 18:00-19:00), with a 5-6 hour interval between feedings. The amount of feed given each time was dynamically adjusted according to the pigs' age and weight to ensure no leftover feed remained in the trough. A 3-day transition period was implemented when changing feed, during which the old and new feeds were gradually replaced at a rate of 20% per day.

[0035] (III) Drinking Water Management Purified water is used for the pigs to drink, and citric acid is added to adjust the pH of the drinking water to 3.5.

[0036] Example 2 This embodiment provides a method for preventing and controlling African swine fever in livestock farming, which specifically includes the following steps: (a) Feed formulation 1. Preparation of high-purity yucca extract Dried yucca leaves were collected and pulverized through a 40-mesh sieve. The yucca leaf powder was then placed in a supercritical fluid extraction vessel. Using CO2 as the extraction medium and anhydrous ethanol as the entrainer (15% of the raw material mass), extraction was performed for 3 hours at an extraction temperature of 42.5℃ and an extraction pressure of 32.5 MPa. After extraction, the extract was collected, and the ethanol was recovered by concentration under reduced pressure to obtain the crude yucca extract.

[0037] The crude yucca extract was redissolved in a 30% ethanol aqueous solution and loaded onto a macroporous adsorption resin column (D101 type). Gradient elution was performed sequentially with 30%, 50%, and 70% (v / v) ethanol aqueous solutions. The eluent fraction of the 70% ethanol aqueous solution was collected, concentrated under reduced pressure, and freeze-dried to obtain a high-purity yucca extract powder.

[0038] HPLC analysis showed that the high-purity yucca extract prepared in this example contained 88.7% steroidal saponins by weight.

[0039] 2. Preparation of enzymatic hydrolysis products of yucca The high-purity yucca extract powder was dissolved in deionized water to prepare a 10% (w / w) extract solution. The pH was adjusted to 5.0, and the solution was heated to 50°C. β-glucosidase was added for enzymatic hydrolysis. The β-glucosidase was derived from *Aspergillus niger*, with an enzyme activity of 30,000 U / g, and was added at 0.5% of the dry weight of the high-purity yucca extract. The hydrolysis time was 5 hours, with a stirring speed of 200 rpm.

[0040] After enzymatic hydrolysis, the hydrolysate was heated to 90°C and held for 10 minutes to inactivate β-glucosidase and terminate the hydrolysis reaction. Then, it was spray-dried to obtain the hydrolyzed yucca product powder.

[0041] HPLC-MS analysis showed that the total weight percentage of secondary saponins and aglycones in the enzymatic hydrolysed yucca product prepared in this example was 48.7%, and the weight percentage of intact steroidal saponins was 25.6%.

[0042] 3. Preparation of modified montmorillonite microparticles Natural montmorillonite (montmorillonite content ≥95%) was crushed and passed through a 200-mesh sieve. It was then dispersed in deionized water to prepare a suspension with a mass concentration of 7.5%, and stirred for 3 hours to allow it to fully hydrate and swell. After standing and separating into layers, the upper suspension was collected, centrifuged, and purified montmorillonite was obtained.

[0043] Chitosan (88% deacetylation, weight-average molecular weight 125 kDa) was dissolved in a 1.5% aqueous acetic acid solution to prepare a 1.25% (w / w) chitosan-acetic acid solution. Purified montmorillonite was dispersed in the chitosan-acetic acid solution (montmorillonite to chitosan mass ratio 12.5:1), and the mixture was stirred at 50°C for 6 hours. After the reaction, the precipitate was separated by centrifugation, washed with deionized water until neutral, vacuum dried at 70°C, and pulverized through a 200-mesh sieve to obtain chitosan-modified montmorillonite microparticles.

[0044] The modified montmorillonite microparticles were tested and found to have an average particle size of 125 μm and a zeta potential of +25 mV.

[0045] 4. Preparation of feed for disease prevention and control The modified montmorillonite microparticles and enzymatically hydrolyzed yucca products were added to the basal diet and mixed thoroughly to obtain the control feed. The amount of modified montmorillonite microparticles added was 1.25 parts by weight, and the amount of enzymatically hydrolyzed yucca products added was 0.25 parts by weight.

[0046] (ii) Feeding Management The pigs were fed the aforementioned feed for disease control. They were fed three times daily (6:00-7:00, 12:00-13:00, and 18:00-19:00), with a 5-6 hour interval between feedings. The amount of feed given each time was dynamically adjusted according to the pigs' age and weight to ensure no leftover feed remained in the trough. A four-day transition period was implemented when changing feed, during which the old and new feeds were gradually replaced at a rate of 25% per day.

[0047] (III) Drinking Water Management Purified water is used for the pigs to drink, and citric acid is added to adjust the pH of the drinking water to 4.0.

[0048] Example 3 This embodiment provides a method for preventing and controlling African swine fever in livestock farming, which specifically includes the following steps: (a) Feed formulation 1. Preparation of high-purity yucca extract Dried yucca leaves were collected and pulverized through a 40-mesh sieve. The yucca leaf powder was placed in a supercritical fluid extraction vessel, and extraction was carried out for 4 hours at an extraction temperature of 50℃ and an extraction pressure of 40 MPa using CO2 as the extraction medium and anhydrous ethanol as the entrainer (20% of the raw material mass). After extraction, the extract was collected, and the ethanol was recovered by concentration under reduced pressure to obtain crude yucca extract.

[0049] The crude yucca extract was redissolved in a 30% ethanol aqueous solution and loaded onto a macroporous adsorption resin column (D101 type). Gradient elution was performed sequentially with 30%, 50%, and 70% (v / v) ethanol aqueous solutions. The eluent fraction of the 70% ethanol aqueous solution was collected, concentrated under reduced pressure, and freeze-dried to obtain a high-purity yucca extract powder.

[0050] HPLC analysis showed that the high-purity yucca extract prepared in this example contained 91.7% steroidal saponins by weight.

[0051] 2. Preparation of enzymatic hydrolysis products of yucca The high-purity yucca extract powder was dissolved in deionized water to prepare an extract with a mass concentration of 15%. The pH was adjusted to 6.0, and the solution was heated to 60°C. β-glucosidase was added for enzymatic hydrolysis. The β-glucosidase was derived from Aspergillus niger, with an enzyme activity of 50,000 U / g, and the amount added was 1.0% of the dry weight of the high-purity yucca extract. The hydrolysis time was 8 hours, and the stirring speed was 300 rpm.

[0052] After enzymatic hydrolysis, the hydrolysate was heated to 95°C and held for 15 minutes to inactivate β-glucosidase and terminate the hydrolysis reaction. Then, it was spray-dried to obtain the hydrolyzed yucca product powder.

[0053] HPLC-MS analysis showed that the total weight percentage of secondary saponins and aglycones in the enzymatic hydrolysis of yucca prepared in this example was 56.3%, and the weight percentage of intact steroidal saponins was 22.5%.

[0054] 3. Preparation of modified montmorillonite microparticles Natural montmorillonite (montmorillonite content ≥95%) was crushed and passed through a 200-mesh sieve. It was then dispersed in deionized water to prepare a 10% (w / w) suspension, which was stirred for 4 hours to allow it to fully hydrate and swell. After standing and separating into layers, the upper suspension was collected and centrifuged to obtain purified montmorillonite.

[0055] Chitosan (95% degree of deacetylation, 200 kDa weight average molecular weight) was dissolved in a 2% aqueous acetic acid solution to prepare a 2.0% (w / w) chitosan-acetic acid solution. Purified montmorillonite was dispersed in the chitosan-acetic acid solution (montmorillonite to chitosan mass ratio 5:1), and the mixture was stirred at 60°C for 8 hours. After the reaction, the precipitate was separated by centrifugation, washed with deionized water until neutral, vacuum dried at 80°C, and pulverized through a 200-mesh sieve to obtain chitosan-modified montmorillonite microparticles.

[0056] Tests showed that the modified montmorillonite microparticles had an average particle size of 200 μm and a zeta potential of +35 mV.

[0057] 4. Preparation of feed for disease prevention and control The modified montmorillonite microparticles and enzymatically hydrolyzed yucca products were added to the basal diet and mixed thoroughly to obtain the control feed. The amount of modified montmorillonite microparticles added was 2.0 parts by weight, and the amount of enzymatically hydrolyzed yucca products added was 0.5 parts by weight.

[0058] (ii) Feeding Management The pigs were fed the aforementioned feed for disease control. They were fed four times daily (6:00-7:00, 10:00-11:00, 14:00-15:00, and 18:00-19:00), with a four-hour interval between feedings. The amount of feed given each time was dynamically adjusted according to the pigs' age and weight to ensure no leftover feed remained in the troughs. A five-day transition period was implemented when changing feed, during which the old and new feeds were gradually replaced at a rate of 30% per day.

[0059] (III) Drinking Water Management Purified water is used for the pigs to drink, and citric acid is added to adjust the pH of the drinking water to 4.5.

[0060] Experiment 1: Effect of enzymatic hydrolysis time on the palatability and activity of yucca products Bitterness was determined using an electronic tongue, calibrated with 10mM quinine solution as the reference. Each group of samples was measured in parallel three times and the average value was taken.

[0061] The high-purity yucca extract (steroidal saponin content 86.3%) prepared in Example 1 was used to prepare yucca products with different enzymatic hydrolysis time gradients according to the enzymatic hydrolysis method in Example 2: 0 hours (untreated), 2 hours, 4 hours, 6 hours, and 8 hours.

[0062] A dual-feeder preference test was used. Thirty healthy growing pigs (approximately 30 kg) were selected, with three pigs per pen, for a total of 10 pens. Each pen had two feeders: one containing the test feed (added amount uniformly calculated based on effective saponin equivalents), and the other containing the control feed without the test sample. Feed intake by the pigs for both feeds was recorded over 24 hours. Simultaneously, an LPS-induced porcine alveolar macrophage inflammation model was used to determine the anti-inflammatory activity (TNF-α IC50) of each group of samples. 50 ).

[0063] Table 1. Comparison of composition, bitterness, and activity of yucca products at different enzymatic hydrolysis times. According to the results in Table 1, during enzymatic hydrolysis of 0-4 hours, the content of intact saponins gradually decreased, while the content of secondary saponins and aglycones gradually increased. The bitterness score rapidly decreased from 8.5 to 2.3, feed intake recovered from 1620g / head to 2100g / head, and anti-inflammatory activity gradually increased. At 4 hours of enzymatic hydrolysis, the bitterness was lowest and the anti-inflammatory activity was strongest. Further extending the hydrolysis time to 6 hours and 8 hours resulted in a slight increase in the bitterness score, but a decrease in anti-inflammatory activity. This indicates that excessive hydrolysis caused some of the highly active secondary saponins to be further hydrolyzed into free aglycones with relatively lower activity, and these free aglycones themselves have a certain bitterness. These results show that when the hydrolysis time is controlled within the range of 2-8 hours, the technical effect of intact saponin content ≤30% and the sum of secondary saponin and aglycone content ≥40% can be achieved. Among these, 4 hours of hydrolysis is the preferred option, at which point the bitterness and activity reach the optimal balance.

[0064] Experiment 2: Evaluation of the anti-inflammatory activity and intestinal permeability of the enzymatic hydrolysates Take the untreated high-purity yucca extract (0h) from Experiment 1, the product of enzymatic hydrolysis for 4h, and the chemically hydrolyzed product prepared by the following method: Take the high-purity yucca extract powder from Example 1, add 1.0 mol / L hydrochloric acid solution, hydrolyze at 90°C for 3 hours, neutralize, wash, and dry to obtain the chemically hydrolyzed yucca product.

[0065] Compare the following indicators among the three groups of samples: (1) In vitro anti-inflammatory activity: The IC50 values ​​of TNF-α and IL-6 were measured using an LPS-induced porcine alveolar macrophage inflammation model. 50 .

[0066] (2) Intestinal permeability: Using the Caco-2 cell monolayer model, each sample was added to the top side of the Transwell chamber (simulating the intestinal lumen side), and after incubation for 2 hours, the cumulative transport of the main components on the basal side (simulating the blood side) was measured, and the transmembrane transport rate was calculated.

[0067] Table 2. Comparison of anti-inflammatory activity and permeability of yucca products treated with different methods According to the results in Table 2, although the untreated high-purity yucca extract had a high content of intact steroidal saponins (86.3%), its large molecular weight and strong polarity resulted in a transmembrane transport rate of only 8.5%, and its anti-inflammatory activity was generally weak. While chemical hydrolysis can break down steroidal saponins into secondary saponins and aglycones, the strong acid conditions caused side reactions such as dehydration and cyclization of the aglycones, producing inactive or even antagonistic byproducts. HPLC-MS analysis revealed more than five abnormal degradation products in the chemical hydrolysis products, while the enzymatic hydrolysis products were mainly a mixture of secondary saponins produced by the specific cleavage of β-1,4-glycosidic bonds by β-glucosidase. The two products have fundamentally different chemical compositions. Therefore, the anti-inflammatory activity of the chemically hydrolyzed products decreased rather than increased, with the transmembrane transport rate only increasing to 12.3%. Enzymatic hydrolysis for 4 hours, under mild conditions, converted intact saponins into secondary saponins and aglycones, preserving their natural configuration. The transmembrane transport rate significantly increased to 35.8%, and the anti-inflammatory activity was also significantly enhanced. The above results show that enzymatic hydrolysis not only solves the palatability problem, but also unexpectedly enhances the intestinal permeability and anti-inflammatory activity of the active ingredients. Those skilled in the art cannot infer the effect of the enzymatic hydrolysis method of the present invention from the known effects of chemical hydrolysis.

[0068] Experiment 3: Verification of the control effect under positive environmental conditions in large-scale pig farms A demonstration pig farm (with an annual output of over 1000 pigs) in Longzhou County, Guangxi Province, was selected for verification. Fluorescent quantitative PCR testing of environmental swabs from the pigsty yielded Ct values ​​of 35.55 and 36.41, respectively, indicating that the pig farm was in an environment with ASFV nucleic acid positivity and posed a continuous risk of viral contamination.

[0069] One hundred healthy growing pigs (approximately 60 days old and weighing approximately 25 kg) of similar weight were selected and randomly divided into five groups of 20 pigs each. Each group was fed the following feed: Group A (blank control group): Standard basal diet and standard feeding management.

[0070] Group B (modified montmorillonite group): basal diet + modified montmorillonite (1.25 parts by weight) from Example 2, with routine feeding management.

[0071] Group C (enzymatic hydrolyzed yucca alone): basal diet + 0.25 parts by weight of enzymatic hydrolyzed yucca product from Example 2, with routine feeding management.

[0072] Group D (Untreated Yucca + Montmorillonite Group): Basal diet + Untreated high-purity yucca extract (0.25 parts by weight) + Modified montmorillonite (1.25 parts by weight), with routine feeding management.

[0073] Group E (Complete Scheme of the Invention): Basal diet + Modified montmorillonite (1.25 parts by weight) from Example 2 + Enzymatic hydrolysis of yucca product (0.25 parts by weight), and the feeding and water management of Example 2 were adopted.

[0074] The experiment lasted for 12 weeks. During the experiment, environmental swabs from the pigsty, oral swabs from the pigs, and anticoagulated blood samples were collected monthly. ASFV nucleic acid was detected using quantitative real-time PCR. The ASFV nucleic acid positivity rate, morbidity rate, and growth performance of each group were also recorded. All experimental groups were housed in different pigsties within the same pig farm, with consistent stocking density, ventilation, temperature, humidity, and other environmental conditions.

[0075] During the experiment, cotton swabs were collected from the pig farm environment every month. The results showed that ASFV nucleic acid was consistently positive (Ct value range 33.2-37.8), indicating that the pig farm environment was under continuous risk of virus exposure.

[0076] Table 3. Control efficacy of each group under persistently positive environmental conditions. According to the results in Table 3, under the condition of persistent ASFV nucleic acid positivity in the pig farm environment, the morbidity rate of group A (blank control) was 65%, and the mortality rate was 60%. Group B (montmorillonite alone) had a morbidity rate of 35% and a mortality rate of 30%; Group C (enzymatically hydrolyzed yucca alone) had a morbidity rate of 25% and a mortality rate of 20%. If the effects of the two are simply superimposed, the expected morbidity rate is 1 - (1 - 0.35) × (1 - 0.25) = 51.25%. Group E (the complete scheme of this invention) had an actual morbidity rate of 0% and a mortality rate of 0%, which is far lower than the expected value of independent superposition, and the difference is statistically significant (P < 0.05). Analysis suggests that the physical barrier formed by modified montmorillonite in the intestinal mucus layer directly blocks the contact between ASFV viral particles and intestinal epithelial cells, delaying the viral invasion process and buying time for the full release and absorption of active ingredients in the enzymatically hydrolyzed yucca products. The secondary saponins and aglycones in the enzymatically hydrolyzed yucca products reach the gut-associated lymphoid tissue after passing through the intestinal epithelium, regulating the local immune microenvironment and further strengthening the barrier function of the intestinal mucosa, thus forming a spatiotemporally synergistic defense line of "physical barrier + penetration regulation".

[0077] The morbidity rate in group D (untreated yucca + montmorillonite) was 30% (6 / 20), and the mortality rate was 25% (5 / 20), lower than group A but higher than group C (25%, 5 / 20) and group E (0%). The amount of untreated yucca extract added to group D was 0.25 parts by weight, consistent with the amount added to the enzymatically hydrolyzed yucca product in group C. According to Table 1, the feed intake of untreated yucca extract was 1620 g / head·24h, and the feed intake of the enzymatically hydrolyzed product (4h) was 2100 g / head·24h. The actual feed intake of the untreated group was approximately 77% of that of the enzymatically hydrolyzed group. Although the untreated yucca had a higher content of steroidal saponins, the actual feed intake was insufficient due to palatability issues, resulting in a significantly lower intake of effective components compared to group C. Therefore, the control effect was inferior to group C and even less effective than group E. This result validates the necessity of enzymatic hydrolysis in improving palatability and ensuring the intake of effective components.

[0078] The average daily weight gain of group E (the complete scheme of this invention) was significantly higher than that of group A, increasing by 13.9%, and the feed conversion ratio decreased from 3.42 to 3.05, indicating that this invention can improve the growth performance of pigs while controlling ASFV. The morbidity and mortality rates of group E were both 0%, further confirming the excellent control effect of the complete scheme of this invention.

[0079] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preventing and controlling African swine fever in livestock farming, characterized in that, Includes the following steps: (I) Feed formulation: (1) Enzymatic hydrolysis pretreatment: Yucca extract with a steroidal saponin weight percentage of ≥85% is mixed with water to prepare an extract, and β-glucosidase is added for enzymatic hydrolysis to obtain enzymatic hydrolyzed yucca product; in the enzymatic hydrolyzed yucca product, the weight percentage of secondary saponins and aglycones is ≥40%, and the weight percentage of intact steroidal saponins is ≤30%; (2) Modified montmorillonite microparticles and the enzymatic hydrolyzed yucca product are added to the basic diet and mixed to obtain the control feed; the particle size of the modified montmorillonite microparticles is 50-200μm, and the zeta potential after surface modification with a cationic modifier is +15mV to +35mV; (ii) Feeding management: The pigs shall be fed the aforementioned control feed.

2. The method according to claim 1, characterized in that, The enzymatic hydrolysis treatment is carried out at a temperature of 40-60℃, a pH of 4.0-6.0, and a time of 2-8 hours.

3. The method according to claim 1, characterized in that, The β-glucosidase has an enzyme activity of 10,000-50,000 U / g, and the amount added is 0.1%-1.0% of the dry weight of the yucca extract.

4. The method according to claim 1, characterized in that, The enzymatic pretreatment also includes an enzyme inactivation step: heating the enzymatic hydrolysis product to 80-100℃ and holding for 5-15 minutes, then drying to obtain enzymatic hydrolyzed yucca product powder.

5. The method according to claim 1, characterized in that, The cationic modifier is chitosan, which has a degree of deacetylation of 80%-95% and a weight-average molecular weight of 50kDa-200kDa.

6. The method according to claim 1, characterized in that, The modified montmorillonite microparticles are added to the feed at a rate of 0.5-2.0 parts by weight, and the enzymatically hydrolyzed yucca product is added to the feed at a rate of 0.05-0.5 parts by weight.

7. The method according to claim 1, characterized in that, Step (II) further includes: adjusting the addition ratio of modified montmorillonite microparticles and enzymatically hydrolyzed yucca products in the prevention and control feed based on the monitoring results of African swine fever virus in the pig farm environment.

8. The method according to claim 7, characterized in that, The environmental monitoring of African swine fever virus involves regularly collecting cotton swab samples from the pigsty environment, using quantitative real-time PCR to detect African swine fever virus nucleic acid, and determining the environmental risk level based on the Ct value obtained from the test. A Ct value ≥ 37 indicates a low-risk state, while a Ct value < 37 indicates a high-risk state. Under high-risk conditions, the amount of modified montmorillonite microparticles and enzymatically hydrolyzed yucca products added is increased to 1.5-2.0 times the basic addition amount.

9. The method according to claim 1, characterized in that, In step (ii), a three-fixed feeding system of fixed time, fixed quantity, and fixed quality is adopted, feeding 2-4 times a day, with an interval of 4-6 hours between two consecutive feedings; when changing feed, a transition period of 3-5 days is set, during which the old and new feeds are replaced in a gradient of 20%-30% per day.

10. The method according to claim 1, characterized in that, It also includes drinking water management steps: using purified water for pigs to drink, and adding organic acids to adjust the pH of the drinking water to 3.5-4.5.

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