Use of areca products as the sole active ingredient in the preparation of biological feed and drinking water agents for preventing and controlling African swine fever

Abstract

The application belongs to the technical field of biological feed and specifically relates to application of betel nut product as a sole active ingredient in preparation of biological feed and drinking water agent for prevention and control of African swine fever. The application takes the betel nut product as the sole active ingredient, mixes the betel nut product with an African swine fever virus suspension, inoculates cells, and finds, through in-vitro cell experiment evaluation, that the betel nut product as the sole active ingredient has a good killing effect on the African swine fever virus, can kill 100% of the African swine fever virus with a virus titer of 5x10 3 HAD 50 / mL in 30 minutes, and can kill 100% of the African swine fever virus with a virus titer of 5x10 4 HAD 50 / mL within 2 hours. Adding the betel nut product as an additive to feed or water can block the transmission of the African swine fever virus through feed and drinking water, the method is simple, the cost is controllable, and the method provides an effective measure for biological safety of African swine fever prevention and control.

Description

[0001] This application claims priority to Chinese Patent Application No. CN202310231797.5, filed on March 13, 2023, entitled "Application of Areca Product as Sole Active Ingredient in the Preparation of Biological Feed and / or Drinking Water Agent for the Prevention and Control of African Swine Fever", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biological feed technology, specifically relating to the application of areca nut products as the sole active ingredient in the preparation of biological feed and / or drinking water for the prevention and control of African swine fever. Background Technology

[0003] African swine fever (ASF) is an acute, febrile, and highly contagious zoonotic disease of pigs caused by the African swine fever virus (ASFV). The morbidity and mortality rates in infected pigs can reach 100%. Currently, several low-fatality variants of the ASFV have emerged, exhibiting insidious clinical manifestations, but still posing a risk of horizontal transmission, leading to persistent infection, chronic disease progression, and even death, further increasing the complexity and difficulty of its control. For viral diseases, vaccination has always been the preferred control strategy; however, to date, there is no commercially available vaccine to prevent African swine fever.

[0004] The primary mode of transmission for African swine fever virus (ASFV) is oral transmission, with contaminated pig feed and water sources being the main transmission vectors. Currently, the most effective biosafety method to block ASFV transmission via feed is high-temperature pelleting at 85°C for 3 minutes to kill the virus in the feed. However, this method requires significant investment, significantly reduces production efficiency, increases costs by approximately 80 yuan per ton of feed, and also destroys heat-sensitive nutrients in the feed, such as various vitamins. Compared to feed-borne transmission, ASFV transmission via drinking water has a lower infection threshold and carries a higher risk. Therefore, contaminated water sources in pig farms are more likely to cause infection and disease in pig herds. The common method to prevent the spread of African swine fever virus through drinking water is to add disinfectants (such as potassium persulfate or sodium hypochlorite) or acidifiers to the drinking water. However, no research has yet confirmed that acidifiers are effective in completely killing the African swine fever virus. Disinfectants need to reach a certain concentration to kill the virus. Long-term addition of high concentrations of chemical disinfectants to drinking water will inevitably damage the oral and intestinal mucosa of pigs and disrupt the intestinal flora, making pigs more susceptible to infection and disease.

[0005] Areca nut is an evergreen tree belonging to the Areca genus of the palm family. It mainly grows in the tropical and subtropical regions of southern my country, as well as other South and Southeast Asian countries. It is one of my country's traditional medicinal materials and important medicinal plants, with its seeds, pericarp, and flowers all used medicinally. The raw areca nut contains various physiologically active components, mainly including alkaloids, polyphenols, polysaccharides, and fatty acids. Modern research shows that areca nut has a wide range of pharmacological effects, possessing various biological activities such as promoting digestion, antioxidation, anti-inflammation, antiparasitic activity, and antibacterial activity, and has certain effects on the human digestive, nervous, cardiovascular, and endocrine systems. However, research on the effectiveness of areca nut and its extracts in killing African swine fever virus has not yet been found. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of areca nut products as the sole active ingredient in the preparation of biological feed and / or drinking water agents for the prevention and control of African swine fever. By adding areca nut products to feed or water sources, African swine fever virus in feed and drinking water can be killed, thus cutting off the transmission of African swine fever through feed and drinking water.

[0007] This invention provides the application of areca nut products as the sole active ingredient in the preparation of biological feed and / or drinking water agents for the prevention and control of African swine fever.

[0008] Preferably, the areca product includes areca extract and / or areca ultrafine powder.

[0009] Preferably, the polyphenol content in the areca nut extract is 2% to 30%;

[0010] The areca nut ultrafine powder is the material that passes through a sieve with a mesh size of ≥80 mesh.

[0011] Preferably, the preparation method of the areca nut extract includes the following steps:

[0012] Mix the areca nut raw material with the extraction solvent, extract, and collect the areca nut extract;

[0013] The areca nut extract is dried to obtain the areca nut extract.

[0014] Preferably, the mass-to-volume ratio of the areca nut raw material to the extraction solvent is 1g:(8-35)mL;

[0015] The drying process is carried out at a temperature of 65–75°C for 72–96 hours.

[0016] Preferably, the extraction solvent is any one of water, methanol, and hydrochloric acid aqueous solution with a mass concentration of 0.2-0.5%, more preferably water or hydrochloric acid aqueous solution with a mass concentration of 0.2-0.5%, and even more preferably water.

[0017] When the extraction solvent is water, the extraction method includes water bath extraction. The water bath extraction temperature of the present invention is preferably 60–90°C, more preferably 90°C; the water bath extraction time is preferably 1–3 hours, more preferably 2 hours.

[0018] When the extraction solvent is methanol, the extraction method includes ultrasonic extraction. The preferred frequency of the ultrasonic extraction in this invention is 60–100 Hz, and the preferred time is 15–30 min.

[0019] When the extraction solvent is an aqueous solution of hydrochloric acid with a mass concentration of 0.2-0.5%, the extraction method includes: mixing the areca nut raw material with an aqueous solution of hydrochloric acid with a mass concentration of 0.2-0.5%, soaking for 1 hour, loading it into a percolation column, and extracting it by percolation with an aqueous solution of hydrochloric acid with a mass concentration of 0.2-0.5%.

[0020] This invention also provides a biological feed for the prevention and control of African swine fever, which consists of areca nut products and basic feed;

[0021] The basic feed includes any one or more of complete feed, concentrated feed, and premixed feed.

[0022] Preferably, the areca product includes areca extract and / or areca ultrafine powder;

[0023] The polyphenol content in the areca nut extract is 2% to 30%;

[0024] When the areca product is areca extract, 25g to 400g of the areca extract shall be added per ton of complete feed;

[0025] When the areca nut product is areca nut ultrafine powder, 125g to 2000g of the areca nut ultrafine powder shall be added per ton of complete feed;

[0026] When the areca nut product is areca nut ultrafine powder and areca nut extract, 60-1000g of the areca nut ultrafine powder and 10-200g of the areca nut extract shall be added per ton of complete feed.

[0027] The present invention also provides a drinking water agent for preventing and controlling African swine fever, which is composed of areca products and water.

[0028] Preferably, the areca product includes areca extract and / or areca ultrafine powder;

[0029] The polyphenol content in the areca nut extract is 2% to 30%;

[0030] When the areca product is areca extract, 25g to 400g of the areca extract shall be added per ton of water;

[0031] When the areca product is areca ultrafine powder, 125g to 2000g of the areca ultrafine powder is added per ton of water;

[0032] When the areca product is areca ultrafine powder and areca extract, 60-1000g of the areca ultrafine powder and 10-200g of the areca extract are added per ton of water.

[0033] Beneficial effects:

[0034] This invention simulates the feed and water conditions of a pig farm. After mixing areca nut products with an African swine fever virus suspension and inoculating cells, in vitro cell experiments showed that areca nut products, as the sole active ingredient, have a good killing effect on African swine fever virus, achieving 100% virus eradication at a titer of 5 × 10⁻⁶ within 30 minutes. 3 HAD 50 / mL African swine fever virus, can kill 100% of the virus titer of 5×10⁹ / mL within 2 hours. 4 HAD 50 / mL African swine fever virus. Using areca products as an additive in feed industrial enterprises or farms for self-prepared feed, drinking water, and environmental disinfection does not require the addition of expensive high-temperature pelleting equipment or modification of water supply facilities. It is convenient to use and can effectively cut off the transmission of African swine fever virus through feed and drinking water, providing an important biosecurity measure for the prevention and control of African swine fever. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0036] Figure 1 The positive control for HAD detection is specifically the HAD detection result of the fifth group in Example 7 after directly incubating cells with the viral stock solution;

[0037] Figure 2 The negative control for HAD detection is specifically the HAD detection result of the seventh group of cells incubated with PBS solution for 2 hours in Example 7;

[0038] Figure 3 The HAD detection results of the third group in Example 7 were obtained by inactivating the virus with BL-5, a areca nut extract at a concentration of 50 ppm (actual working concentration of 25 ppm after mixing), at 4°C for 2 hours and then incubating the cells for 2 hours.

[0039] Figure 4 The results of HAD detection in the fourth group of Example 7 are as follows: the virus was inactivated by BL-5, a 50 ppm areca nut extract (actual working concentration of 25 ppm after mixing), at 25°C for 2 hours, followed by cell incubation for 2 hours.

[0040] Figure 5 This serves as a positive control for IFA detection, specifically the IFA detection results of the fifth group in Example 7 after directly incubating cells with the viral stock solution;

[0041] Figure 6 This serves as a negative control for IFA detection, specifically the IFA detection results of the seventh group of cells incubated with PBS solution for 2 hours in Example 7;

[0042] Figure 7 The IFA test results of the third group in Example 7 are obtained by inactivating the virus with BL-5, a areca nut extract at a concentration of 50 ppm (actual working concentration of 25 ppm after mixing), at 4°C for 2 hours and then incubating the cells for 2 hours.

[0043] Figure 8 This refers to the IFA detection results of the fourth group in Example 7, which used 50 ppm areca extract BL-5 (actual working concentration after mixing 25 ppm) to inactivate the virus at 25°C for 2 hours and then incubated the cells for 2 hours.

[0044] in, Figures 5 to 8 From left to right: DAPI staining results, African swine fever virus P30 protein fluorescence staining results, and a fusion image of DAPI staining and fluorescence staining, scale bar 100um. Detailed Implementation

[0045] This invention provides the application of areca nut products as the sole active ingredient in the preparation of biological feed and / or drinking water agents for the prevention and control of African swine fever.

[0046] In this invention, the areca nut product preferably comprises areca nut extract and / or areca nut ultrafine powder, and more preferably areca nut extract. The areca nut extract of this invention is preferably an areca nut aqueous extract; the polyphenol content in the areca nut extract is preferably 2%–30%, more preferably 8.8%–30%, even more preferably 10.72%–24.01%, more preferably 13.67%–19.56%, and most preferably 18.80%; the areca nut ultrafine powder is preferably the undersize material from a sieve with a mesh size ≥ 80 mesh.

[0047] The present invention does not have strict requirements on the method of adding the areca nut product to feed or water. It can be added to feed or daily drinking water in the form of premix or powder.

[0048] This invention does not have strict requirements regarding the source of the areca nut extract or areca nut ultrafine powder; it can be purchased directly or prepared in-house. Preferably, this invention uses a self-preparation method to obtain the areca nut extract. The preparation method of the areca nut extract of this invention preferably includes the following steps:

[0049] Mix the areca nut raw material with the extraction solvent, extract, and collect the areca nut extract;

[0050] The areca nut extract is dried to obtain the areca nut extract.

[0051] This invention involves mixing areca nut raw material with an extraction solvent, extracting, and collecting the areca nut extract. In this invention, the areca nut raw material preferably includes fresh areca nuts and / or dried areca nuts; the invention does not have strict requirements on the maturity of the fresh or dried areca nuts, and any maturity level is acceptable; the preferred mass-to-volume ratio of the areca nut raw material to the extraction solvent is 1g:(8-35)mL, more preferably 1g:(10-20)mL, more preferably 1g:(15-20)mL, and most preferably 1g:20mL. This invention preferably uses a pulverizer to pulverize the areca nut raw material, and then mixes the pulverized areca nut with water for extraction. The preferred method for collecting the areca nut extract is filtration.

[0052] In this invention, the extraction solvent is preferably any one of water, methanol, and an aqueous solution of hydrochloric acid with a mass concentration of 0.2-0.5%, more preferably water or an aqueous solution of hydrochloric acid with a mass concentration of 0.2-0.5%, and even more preferably water. The methanol used in this invention is preferably methanol with a volume concentration of 50%.

[0053] When the extraction solvent is water, the extraction method of the present invention preferably includes water bath extraction; the temperature of the water bath extraction is preferably 60-90°C, more preferably 90°C; the time of the water bath extraction is preferably 1-3 hours, more preferably 2 hours.

[0054] When the extraction solvent is methanol, the frequency of the ultrasonic extraction in this invention is preferably 60-100 Hz, more preferably 70 Hz, and the time is preferably 15-30 min, more preferably 20 min.

[0055] When the extraction solvent is an aqueous hydrochloric acid solution with a mass concentration of 0.2-0.5%, the extraction method of the present invention preferably includes: mixing the areca nut raw material with an aqueous hydrochloric acid solution with a mass concentration of 0.2-0.5%, soaking for 1 hour, loading it into a percolation column, and extracting it by percolation with an aqueous hydrochloric acid solution with a mass concentration of 0.2-0.5%.

[0056] After obtaining the areca nut extract, the present invention preferably performs a drying treatment on the areca nut concentrate. In the present invention, the drying temperature is preferably 65-75°C, more preferably 70°C; the drying time is preferably 72-96 hours, more preferably 75-90 hours, and more preferably 80-85 hours.

[0057] Before drying the areca nut extract, the present invention preferably concentrates the areca nut extract. The concentration method is preferably vacuum concentration. The concentration factor is preferably 30-35 times, more preferably 32 times. The present invention does not have strict requirements on the specific parameters of the concentration, as long as it can be concentrated to 1 / 30 to 1 / 35 of the filtrate volume. When the extraction solvent is an aqueous solution of hydrochloric acid with a mass concentration of 0.2-0.5%, the present invention preferably passes the areca nut extract through a 732 type cation adsorption column before concentration to obtain the cation adsorption column effluent.

[0058] This invention simulates the situation where feed and drinking water in the pig farming industry are contaminated with high viral loads. Using areca nut products as the sole active ingredient, the product was subjected to an African swine fever virus inactivation experiment, and it was found that areca nut products can have a good killing effect on African swine fever virus.

[0059] The present invention also provides a biological feed for the prevention and control of African swine fever, which consists of areca nut products and a basic feed; the basic feed includes any one or more of complete feed, concentrated feed and premixed feed.

[0060] In this invention, the basic feed includes any one or more of complete feed, concentrated feed and premixed feed, and is more preferably any one of complete feed, concentrated feed and premixed feed.

[0061] When the base feed is a complete feed, the present invention preferably adds the areca nut product directly to the complete feed to obtain the biological feed. The areca nut product of the present invention preferably includes areca nut extract and / or areca nut ultrafine powder. When the areca nut product is areca nut extract, the present invention preferably adds 25g to 400g of the areca nut extract per ton of complete feed, more preferably 125g to 300g, and even more preferably 150g to 200g. When adding areca nut extract, the preferred dosage is any value within the range of 25g to 400g, for example, 25g, 28g, 30g, 40g, 50g, 55g, 60g, 80g, 100g, 125g, 130g, 145g, 150g, 180g, 195g, 200g, 230g, 250g, 260g, 280g, 300g, 350g, 360g, 380g, or 400g. When the areca nut product is areca nut ultrafine powder, the preferred dosage is 125g to 2000g of the areca nut ultrafine powder per ton of complete feed, more preferably 250g to 1500g, more preferably 400g to 1200g, and most preferably 800g to 1200g. When adding areca nut ultrafine powder, the preferred amount is any value within the range of 125g to 2000g. When the areca nut product is areca nut extract and areca nut ultrafine powder, the preferred amount is 60-1000g of areca nut ultrafine powder and 10-200g of areca nut extract per ton of complete feed. The preferred amount of areca nut ultrafine powder per ton of complete feed is 100-800g, more preferably 200-600g, and even more preferably 400-600g; the preferred amount of areca nut extract per ton of complete feed is 40-180g, more preferably 60-140g, and even more preferably 80-140g or 120g.

[0062] When the base feed is a concentrated feed, the present invention preferably adds areca nut product to the concentrated feed according to the concentration ratio of the concentrated feed and the mass of areca nut product to be added to the complete feed to obtain the biological feed. When the concentration ratio is N, and when Xg of areca nut product needs to be added to 1 ton of complete feed, NXg of areca nut product is added per ton of concentrated feed to obtain the biological feed. The concentration ratio of the present invention is preferably 3 to 5 times. The biological feed of the present invention can be sold alone or mixed with other ingredients, such as energy feed, to prepare complete feed for sale. When preparing complete feed according to the present invention, it is preferred to add Xg of areca nut product per ton of complete feed.

[0063] When the base feed is a premixed feed, the present invention preferably adds areca nut product to the premixed feed according to the concentration ratio of the premixed feed and the mass of areca nut product to be added to the complete feed to obtain the biological feed. When the concentration ratio of the premixed feed is N, and when Xg of areca nut product needs to be added to 1 ton of complete feed, NXg of areca nut product is added per ton of premixed feed to obtain the biological feed. The concentration ratio of the present invention is preferably 20 to 25 times. The biological feed of the present invention can be sold separately or mixed with other ingredients to prepare complete feed for sale. When preparing complete feed, the present invention preferably adds Xg of areca nut product per ton of complete feed.

[0064] The present invention does not have strict requirements on the source of the complete feed, concentrated feed and premixed feed, which can be purchased or prepared by oneself.

[0065] The present invention preferably provides a method for preparing the above-mentioned biological feed, which preferably includes the following steps: mixing the areca nut product with a basic feed and letting it stand for 0.5h to 3h to obtain the feed, and more preferably letting it stand for 1h to 2h.

[0066] This invention adds areca nut products to basic feed, which can kill African swine fever virus that may be contaminating the feed, block the spread of African swine fever virus through feed, and prevent and control African swine fever.

[0067] The present invention also provides a drinking water agent for preventing and controlling African swine fever, which is composed of areca products and water.

[0068] In this invention, the areca nut product preferably includes areca nut extract and / or areca nut ultrafine powder. When the areca nut product is areca nut extract, this invention preferably adds 25g to 400g of the areca nut extract per ton of water, more preferably 125g to 200g, and even more preferably 150g. When adding areca nut extract, this invention preferably selects any value within the range of 25g to 400g, for example, 25g, 28g, 30g, 40g, 50g, 55g, 60g, 80g, 100g, 125g, 130g, 145g, 150g, 180g, 195g, 200g, 230g, 250g, 260g, 280g, 300g, 350g, 360g, 380g, or 400g.

[0069] When the areca nut product is areca nut ultrafine powder, the present invention preferably adds 125g to 2000g of the areca nut ultrafine powder per ton of water, more preferably 250g to 1500g, more preferably 400g to 1200g, and most preferably 800g to 1200g. When adding areca nut ultrafine powder, the present invention preferably selects any value within the range of 125g to 2000g.

[0070] When the areca product is areca extract and areca ultrafine powder, the present invention preferably adds 60-1000g of areca ultrafine powder and 10-200g of areca extract per ton of water. The amount of areca ultrafine powder added per ton of drinking water is preferably 100-800g, more preferably 200-600g, and even more preferably 400-600g; the amount of areca extract added per ton of drinking water is preferably 40-180g, more preferably 60-140g, and even more preferably 80-140g or 120g.

[0071] The present invention preferably provides a method for preparing the above-mentioned drinking water agent, which preferably includes the following steps: mixing areca nut extract with water and letting it stand for 0.5 to 3 hours to obtain the drinking water agent, more preferably letting it stand for 1 to 2 hours; or preferably including the following steps: putting areca nut ultrafine powder into a filter bag and soaking it in water for 12 hours or more to obtain the drinking water agent.

[0072] This invention adds areca nut products to water, and the resulting aqueous solution can be used directly as drinking water for animals, killing any African swine fever virus that may be contaminated in the drinking water, blocking the spread of African swine fever virus through water, and preventing and controlling African swine fever; the aqueous solution can also be sprayed on the pigsty environment to block the spread of African swine fever virus through environmental media and prevent and control African swine fever.

[0073] To further illustrate the present invention, the application of the areca nut product provided by the present invention as the sole active ingredient in the preparation of biological feed and / or drinking water agents for the prevention and control of African swine fever is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] The preparation of areca nut extract consists of the following steps:

[0076] (1) After crushing the areca slices with a pulverizer, the areca powder is obtained;

[0077] (2) Mix the areca nut powder and 50% methanol at a mass-volume ratio of 1g:20mL, and sonicate at 70Hz for 20min to obtain ultrasonic extract. Filter the ultrasonic extract to obtain areca nut extract.

[0078] (3) The areca nut extract was concentrated to 1 / 30 of the filtrate volume by vacuum concentration and dried at 70°C for 72-96 hours to obtain the areca nut extract.

[0079] Example 2

[0080] The preparation of areca nut extract consists of the following steps:

[0081] (1) Crush the dried areca nuts with a grinder to obtain areca nut powder;

[0082] (2) Mix the areca nut powder and water at a mass-volume ratio of 1g:25mL, extract in a water bath at 80℃ for 1h, filter, and obtain areca nut extract.

[0083] (3) The areca nut extract was concentrated to 1 / 35 of the filtrate volume by vacuum concentration and dried at 70°C for 72-96 hours to obtain the areca nut extract.

[0084] Example 3

[0085] The preparation of areca nut extract consists of the following steps:

[0086] (1) Crush the dried areca nuts with a grinder to obtain areca nut powder;

[0087] (2) Mix the areca nut powder and 0.5% hydrochloric acid water at a mass-volume ratio of 1g:10mL, soak for 1h, then pack into a percolation column and extract with 3000mL of 0.5% hydrochloric acid water to obtain areca nut hydrochloric acid filtrate.

[0088] (3) Pass the areca hydrochloric acid filtrate through a 400mL 732 type cation adsorption column to obtain the cation adsorption column effluent;

[0089] (4) Adjust the pH of the effluent to 7 with 1% sodium hydroxide solution, concentrate the effluent to 1 / 40 of the total volume by vacuum concentration, and dry at 70℃ for 72-96 hours to obtain areca nut extract.

[0090] Example 4

[0091] Same as Example 2, except that in step (2), the areca nut powder and water are mixed at a mass-volume ratio of 1g:15mL.

[0092] Example 5

[0093] Same as Example 3, except that in step (2), the areca nut powder and hydrochloric acid water with a mass concentration of 0.2% are mixed and soaked for 1 hour at a mass-volume ratio of 1g:10mL, and then loaded into a percolation column and extracted by percolation with 3000mL of hydrochloric acid water with a mass concentration of 0.2% to obtain areca nut hydrochloric acid filtrate.

[0094] Test Example 1

[0095] The contents of the areca nut extracts obtained in Examples 1-5 were analyzed by liquid chromatography and the Folin-Ciocalteu method. The results are shown in Table 1.

[0096] Table 1. Results of the detection of components and content of areca nut extracts obtained in Examples 1-5

[0097]

[0098] As can be seen from Table 1, the total phenol content of the areca nut extract obtained by this invention is 10.72% to 24.01%, and the total alkali content is 0.044% to 1.57%.

[0099] Example 6

[0100] To simulate the situation where feed and drinking water in the pig farming industry are contaminated with high viral loads, the areca nut extract prepared in Example 1 was prepared into three concentrations of 400ppm, 800ppm, and 1600ppm using water as a solvent. Here, 1ppm means that 1mg of areca nut extract is added to 1L of water.

[0101] In collaboration with the National African Swine Fever Regional Laboratory (Guangzhou), we conducted an African swine fever virus inactivation experiment using areca nut extract. The concentration of African swine fever virus in this experiment was 10... 4 HAD 50 / mL.

[0102] The specific experimental steps are as follows:

[0103] (1) Experimental grouping: Areca nut extract was reacted with virus suspension, with 4 replicate experiments in each group.

[0104] Group 1: At 4℃, areca nut extract and African swine fever virus suspension were mixed for 30 min, 1 h, 2 h and 3 h to obtain samples with different virus inactivation times. The samples with different virus inactivation times were diluted 100 times and inoculated into cells.

[0105] Group 2: At 25℃, areca nut extract and African swine fever virus suspension were mixed for 30 min, 1 h, 2 h and 3 h to obtain samples with different virus inactivation times. The samples with different virus inactivation times were diluted 100 times and inoculated into cells.

[0106] Group 3: African swine fever virus suspension stock solution was diluted 10 times, 100 times, 1000 times and 10000 times at 4℃ to obtain virus solutions of different concentrations, which were then inoculated into cells;

[0107] Group 4: At 25℃, African swine fever virus suspension stock solution was diluted 10 times, 100 times, 1000 times and 10000 times to obtain virus solutions of different concentrations, which were then inoculated into cells.

[0108] Group 5: Cells were inoculated with PBS solution at 4°C;

[0109] Group 6: Cells were inoculated with PBS solution at 25°C.

[0110] (2) PAMs cells were seeded into 96-well plates. For the first group, 0.5 mL of areca nut extract at concentrations of 400 ppm, 800 ppm, and 1600 ppm were reacted with 0.5 mL of virus suspension at 4°C. After the reaction was carried out for the predetermined virus inactivation time (i.e., 30 min, 1 h, 2 h, and 3 h), the reaction product was diluted 100 times. The product was seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well. After incubation for 2 h, the reaction product was removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0111] In the second group, 0.5 mL of areca nut extract at concentrations of 400 ppm, 800 ppm, and 1600 ppm were reacted with 0.5 mL of virus suspension at 25°C. After the reaction was carried out for the predetermined virus inactivation time (i.e., 30 min, 1 h, 2 h, and 3 h), the reaction product was diluted 100 times. The product was then seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well and incubated for 2 h. The reaction product was then removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0112] The third group was inoculated with virus solutions of different concentrations into 96-well plates containing PAMs cells at an inoculation rate of 200 μL / well at 4°C. After incubation for 2 hours, the reaction products were removed, cell maintenance culture medium was added, and the plates were incubated in a 37°C incubator with 5% CO2.

[0113] In the fourth group, at an inoculation rate of 200 μL / well, different concentrations of virus solution were inoculated into 96-well plates containing PAMs cells at 25°C. After incubation for 2 hours, the reaction products were removed, cell maintenance culture medium was added, and the plates were incubated in a 37°C incubator with 5% CO2.

[0114] In the fifth group, PBS solution was seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well and incubated at 4°C for 2 h. The reaction products were removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0115] In the sixth group, PBS solution was seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well and incubated at 25°C for 2 hours. The reaction products were then removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0116] The above cell maintenance culture medium consists of 10% fetal bovine serum and 100 U / mL. -1 Penicillin, 100 U / mL -1The medium consisted of streptomycin and the remainder of RPMI-1640 medium, in which fetal bovine serum (FBS), RPMI-1640 medium, penicillin (cell culture grade), and streptomycin (cell culture grade) were all purchased from Gibco.

[0117] (3) After culturing for 48 hours in step (2), the concentration of PAMs cells was approximately 70%–80% in the 96-well plate. The red blood cell adsorption assay (HAD) was performed by the African Swine Fever Regional Laboratory (Guangzhou), and the results are shown in Table 2.

[0118] Table 2 Results of Hemocyte Adsorption (HAD)

[0119]

[0120]

[0121] Note: "-" and "+" indicate HAD negative and positive, respectively.

[0122] As shown in Table 2, the lowest actual working concentration of areca nut extract, at 200 ppm, can effectively kill viruses with a titer of 5 × 10⁻⁶ within 30 minutes. 3 HAD 50 / mL African swine fever virus, the hemocytosis adsorption (HAD) test result showed that the virus was negative.

[0123] Example 7

[0124] To simulate a scenario where feed and drinking water in the pig farming industry are contaminated with high viral loads, the areca nut extract prepared in Example 2 (denoted as BL-1) was prepared into three concentrations of 400 ppm, 200 ppm, and 100 ppm using water as a solvent; the areca nut extract prepared in Example 3 (denoted as BL-5) was prepared into three concentrations of 200 ppm, 100 ppm, and 50 ppm. In collaboration with the National African Swine Fever Regional Laboratory (Guangzhou), the areca nut extracts were subjected to African swine fever virus inactivation experiments. The concentration of African swine fever virus in this experiment was 10... 5 HAD 50 / mL.

[0125] The specific experimental steps are as follows:

[0126] (1) Experimental grouping: Areca nut extract was reacted with virus suspension, with 4 replicate experiments in each group.

[0127] Group 1: Areca nut extract BL-1 and African swine fever virus suspension were mixed for 2 hours at 4℃, diluted 100 times, and inoculated into cells;

[0128] Group 2: At 25°C, areca nut extract BL-1 and African swine fever virus suspension were mixed for 2 hours, diluted 100 times, and inoculated into cells;

[0129] Group 3: Areca nut extract BL-5 and African swine fever virus suspension were mixed for 2 hours at 4℃, diluted 100 times, and inoculated into cells;

[0130] Group 4: At 25℃, areca nut extract BL-5 and African swine fever virus suspension were mixed for 2 hours, diluted 100 times, and inoculated into cells;

[0131] Group 5: African swine fever virus suspension stock solution, diluted 10 times, 100 times, 1000 times, and 10000 times at 4℃, was inoculated into cells;

[0132] Group 6: African swine fever virus suspension stock solution, diluted 10 times, 100 times, 1000 times, and 10000 times, was inoculated into cells at 25℃.

[0133] Group 7: Cells were inoculated with PBS solution at 4°C;

[0134] Group 8: Cells were inoculated with PBS solution at 25°C.

[0135] (2) PAMs cells were seeded into 96-well plates. For the first group, 0.5 mL of different concentrations of areca nut extract were reacted with 0.5 mL of virus suspension at 4°C. After the reaction was carried out for the predetermined virus inactivation time (i.e., 2 h) for each group, the reaction product was diluted 100 times. The product was seeded into 96-well plates containing PAMs cells at an inoculation rate of 200 μL / well. After incubation for 2 h, the reaction product was removed, and cell maintenance culture medium was added. The plates were then placed in a 37°C incubator with 5% CO2 for incubation.

[0136] In the second group, 0.5 mL of different concentrations of areca nut extract were reacted with 0.5 mL of virus suspension at 25°C. After the reaction was carried out for the predetermined virus inactivation time (i.e., 2 h), the reaction product was diluted 100 times. The product was then seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well. After incubation for 2 h, the reaction product was removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0137] The third group took 0.5 mL of areca nut extract at different concentrations and reacted it with 0.5 mL of virus suspension at 4℃. After the reaction was carried out for the predetermined virus inactivation time (i.e., 2 h) of the above groups, the reaction product was diluted 100 times. It was then seeded into a 96-well plate containing PAMs cells at an inoculation rate of 200 μL / well, incubated for 2 h, the reaction product was removed, cell maintenance culture medium was added, and the plate was placed in a 37℃ incubator with 5% CO2.

[0138] In the fourth group, 0.5 mL of different concentrations of areca nut extract were reacted with 0.5 mL of virus suspension at 25°C. After the reaction was carried out for the predetermined virus inactivation time (i.e., 2 h), the reaction product was diluted 100 times. The product was then seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well. After incubation for 2 h, the reaction product was removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0139] In the fifth group, at an inoculation rate of 200 μL / well, different concentrations of virus solution were inoculated into 96-well plates containing PAMs cells at 4°C. After incubation for 2 hours, the reaction products were removed, cell maintenance culture medium was added, and the plates were incubated in a 37°C incubator with 5% CO2.

[0140] In the sixth group, at an inoculation rate of 200 μL / well, different concentrations of virus solution were inoculated into 96-well plates containing PAMs cells at 25°C. After incubation for 2 hours, the reaction products were removed, cell maintenance culture medium was added, and the plates were incubated in a 37°C incubator with 5% CO2.

[0141] In the seventh group, PBS solution was seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well and incubated at 4°C for 2 h. The reaction products were removed, cell maintenance culture medium was added, and the plates were incubated in a 37°C incubator with 5% CO2.

[0142] In the eighth group, PBS solution was seeded into 96-well plates containing PAMs cells at a seeding rate of 200 μL / well and incubated at 25°C for 2 hours. The reaction products were removed, and cell maintenance culture medium was added. The plates were then incubated in a 37°C incubator with 5% CO2.

[0143] Cell maintenance culture medium consists of 10% fetal bovine serum and 100 U / mL -1 Penicillin, 100 U / mL -1 The medium consisted of streptomycin and the remainder of RPMI-1640 medium, which included fetal bovine serum (FBS), RPMI-1640 medium, penicillin (cell culture grade), and streptomycin (cell culture grade), all of which were purchased from Gibco.

[0144] (3) After culturing for 48 hours in step (2), the concentration of PAMs cells was approximately 70%-80% in the 96-well plate. The erythrocyte adsorption assay (HAD) and indirect immunofluorescence assay (IFA) were performed by the African Swine Fever Regional Laboratory (Guangzhou). The results are shown in Table 3.

[0145] Because the results of BL-1 treatment and BL-5 treatment at different temperatures and three concentration groups were similar, and due to the large number of groups, for space limitations, only partial HAD detection results are provided (e.g. Figures 1 to 4(as shown) and some IFA test results (such as) Figures 5 to 8 ),in Figure 1 and Figure 5 The positive control is the result of the fifth group after cells were incubated directly with the virus stock solution (4℃ - without areca nut extract); Figure 2 and Figure 6 This serves as a negative control, specifically the result of the seventh group of cells after incubation in PBS solution for 2 hours (4℃ - virus-free); Figure 3 and Figure 7 The results for the third group are as follows: the virus was inactivated for 2 hours using areca nut extract BL-5 at a concentration of 50 ppm (actual working concentration after mixing was 25 ppm), followed by cell incubation for 2 hours (inactivation at 4℃ for 2 hours). Figure 4 and Figure 8 Specifically, the fourth group used the results of the detection of areca nut extract BL-5 at a concentration of 50 ppm (actual working concentration of 25 ppm after mixing) and the virus inactivated for 2 hours and then incubated the cells for 2 hours (25℃-2h inactivation). Figures 5 to 8 From left to right: DAPI staining results, African swine fever virus P30 protein fluorescence staining results, and a fusion image of DAPI staining and fluorescence staining.

[0146] Table 3. Results of Hemocyte Adsorption (HAD)

[0147]

[0148]

[0149] Note: "-" indicates that both HAD and IFA are negative, and "+" indicates that both HAD and IFA are positive.

[0150] according to Figures 5 to 8 It can be seen that green fluorescence was present in the nuclei of PAMs cells under the fifth treatment, indicating the presence of African swine fever virus. No fluorescence signal was generated under other treatments. Combined with Table 3, it can be seen that under conditions of 4℃ and 25℃, the lowest actual working concentration of areca nut extract BL-1 (50 ppm) or the lowest actual working concentration of areca nut extract BL-5 (25 ppm) could 100% kill the virus titer of 5 × 10⁻⁶ within 2 hours. 4 HAD 50 / mL African swine fever virus.

[0151] Example 8

[0152] Adding 25g of any one of the areca nut extracts from Examples 1-5 to each ton of complete feed yields five types of biological feeds, as detailed below:

[0153] Type 1 biological feed: Add 25g of areca nut extract from Example 1 to each ton of complete feed;

[0154] The second type of biological feed: Add 25g of areca nut extract from Example 2 to each ton of complete feed;

[0155] The third type of biological feed: Add 25g of areca nut extract from Example 3 to each ton of complete feed;

[0156] The fourth type of biological feed: Add 25g of areca nut extract from Example 4 to each ton of complete feed;

[0157] Fifth type of biological feed: Add 25g of areca nut extract from Example 5 to each ton of complete feed;

[0158] The same description is used in all the following examples.

[0159] Example 9

[0160] Adding 200g of any one of the areca nut extracts from Examples 1 to 5 to each ton of complete feed yields 5 kinds of biological feeds.

[0161] Example 10

[0162] Adding 400g of any one of the areca nut extracts from Examples 1 to 5 to each ton of complete feed yields five kinds of biological feeds.

[0163] Example 11

[0164] Adding 125g of areca nut ultrafine powder (80 mesh and above) to each ton of complete feed yields 5 kinds of biological feed.

[0165] Example 12

[0166] Adding 1000g of areca nut ultrafine powder (80 mesh and above) to each ton of complete feed yields 5 kinds of biological feed.

[0167] Example 13

[0168] Adding 2000g of areca nut ultrafine powder (80 mesh and above) to each ton of complete feed yields 5 kinds of biological feed.

[0169] Example 14

[0170] Add 125g of any areca nut extract from Examples 1 to 5 to each ton of concentrated feed with a concentration ratio of 5 to obtain 5 kinds of biological feed. When the biological feed and energy feed are mixed to form a complete feed, the proportion of biological feed in the complete feed is 1 / 5, that is, 25g of areca nut extract is added to each ton of complete feed.

[0171] Example 15

[0172] Add 625g of any areca nut extract from Examples 1 to 5 to each ton of concentrated feed with a concentration ratio of 5 to obtain 5 kinds of biological feed. When the biological feed and energy feed are mixed to form a complete feed, the proportion of biological feed in the complete feed is 1 / 5, that is, 125g of areca nut extract is added to each ton of complete feed.

[0173] Example 16

[0174] Add 2000g of any areca nut extract from Examples 1 to 5 to every ton of concentrated feed with a concentration ratio of 5 to obtain 5 kinds of biological feed. When the biological feed and energy feed are mixed to form a complete feed, the proportion of biological feed in the complete feed is 1 / 5, that is, 400g of areca nut extract is added to every ton of complete feed.

[0175] Example 17

[0176] Add 625g of areca nut ultrafine powder (80 mesh or higher) to each ton of concentrated feed with a concentration ratio of 5 to obtain biological feed. When biological feed and energy feed are mixed to form complete feed, the proportion of biological feed in complete feed is 1 / 5, that is, 125g of areca nut ultrafine powder (80 mesh or higher) is added to each ton of complete feed.

[0177] Example 18

[0178] Add 3125g of areca nut ultrafine powder (80 mesh or above) to each ton of concentrated feed with a concentration ratio of 5 to obtain biological feed. When biological feed and energy feed are mixed to form complete feed, the proportion of biological feed in complete feed is 1 / 5, that is, 625g of areca nut ultrafine powder (80 mesh or above) is added to each ton of complete feed.

[0179] Example 19

[0180] Add 10,000g of areca nut ultrafine powder (80 mesh or above) to each ton of concentrated feed with a concentration ratio of 5 to obtain biological feed. When the biological feed and energy feed are mixed to form a complete feed, the proportion of biological feed in the complete feed is 1 / 5, that is, 2,000g of areca nut ultrafine powder (80 mesh or above) is added to each ton of complete feed.

[0181] Example 20

[0182] Add 625g of any areca nut extract from Examples 1 to 5 to each ton of premixed feed with a concentration ratio of 25 times to obtain 5 kinds of biological feeds. When the biological feeds are mixed with energy feeds and protein feeds to form a complete feed, the proportion of biological feeds in the complete feed is 1 / 25, that is, 25g of areca nut extract is added per ton of complete feed.

[0183] Example 21

[0184] Add 10,000g of any of the areca nut extracts from Examples 1 to 5 to each ton of premixed feed with a concentration ratio of 25 times to obtain 5 kinds of biological feeds. When the biological feeds are mixed with energy feeds and protein feeds to form a complete feed, the proportion of biological feeds in the complete feed is 1 / 25, that is, 400g of areca nut extracts are added per ton of complete feed.

[0185] Example 22

[0186] Add 3125g of any areca nut extract from Examples 1 to 5 to each ton of premixed feed with a concentration ratio of 25 times to obtain 5 kinds of biological feeds. When the biological feeds are mixed with energy feeds and protein feeds to form a complete feed, the proportion of biological feeds in the complete feed is 1 / 25, that is, 125g of areca nut extract is added per ton of complete feed.

[0187] Example 23

[0188] Add 10,000g of areca nut ultrafine powder (80 mesh or higher) to each ton of premixed feed with a concentration ratio of 25 times to obtain biological feed. When the biological feed is mixed with energy feed and protein feed to form a complete feed, the proportion of biological feed in the complete feed is 1 / 25, that is, 400g of areca nut ultrafine powder (80 mesh or higher) is added to each ton of complete feed.

[0189] Example 24

[0190] Add 3125g of areca nut ultrafine powder (80 mesh or above) to each ton of premixed feed with a concentration ratio of 25 times to obtain biological feed. When the biological feed is mixed with energy feed and protein feed to form complete feed, the proportion of biological feed in the complete feed is 1 / 25, that is, 125g of areca nut ultrafine powder (80 mesh or above) is added to each ton of complete feed.

[0191] Example 25

[0192] Add 50,000g of areca nut ultrafine powder (80 mesh or higher) to each ton of premixed feed with a concentration ratio of 25 to obtain biological feed. When the biological feed is mixed with energy feed and protein feed to form a complete feed, the proportion of biological feed in the complete feed is 1 / 25, that is, 2,000g of areca nut ultrafine powder (80 mesh or higher) is added to each ton of complete feed.

[0193] Example 26

[0194] Add 25g of any areca nut extract from Examples 1-5 to each ton of water, let stand for 2 hours, and then drink freely.

[0195] Example 27

[0196] Add 400g of any areca nut extract from Examples 1-5 to each ton of water, let stand for 2 hours, and then drink freely.

[0197] Example 28

[0198] Mix 125g of areca nut ultrafine powder (80 mesh or higher) with each ton of water. Place the areca nut ultrafine powder in a filter bag and soak it in the water for 12 hours or more. It is then ready to drink.

[0199] Example 29

[0200] Mix 2000g of areca nut ultrafine powder (80 mesh and above) with each ton of water. Put the areca nut ultrafine powder into a filter bag and soak it in the water for 12 hours or more. It is then ready to drink.

[0201] Example 30

[0202] Add 25g of any areca nut extract from Examples 1-5 to each ton of water and spray it into the pigsty environment.

[0203] Example 31

[0204] Add 400g of any areca nut extract from Examples 1-5 to each ton of water and spray it into the pigsty environment.

[0205] As can be seen from the above, using areca nut products as the sole active ingredient can block the spread of African swine fever virus through feed and drinking water. The method is simple and cost-effective, providing an effective measure for the prevention and control of African swine fever.

[0206] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of areca nut products as the sole active ingredient in the preparation of biological feed and drinking water agents for the prevention and control of African swine fever, characterized in that, The areca product is areca nut extract; The areca nut extract kills the African swine fever virus in biological feed and drinking water, thus cutting off the transmission of African swine fever through feed and drinking water and preventing African swine fever. The preparation method of the areca nut extract consists of the following steps: mixing areca nut raw material with an extraction solvent, extracting, and collecting the areca nut extract; drying the areca nut extract to obtain the areca nut extract. The extraction solvent is any one of water, methanol, and hydrochloric acid aqueous solution with a mass concentration of 0.2-0.5%; When the extraction solvent is water, the extraction method is water bath extraction; the temperature of the water bath extraction is 60~90℃, and the time is 1~3h. When the extraction solvent is methanol, the extraction method is ultrasonic extraction; the frequency of ultrasonic extraction is 60~100Hz, and the time is 15~30min. When the extraction solvent is a hydrochloric acid aqueous solution with a mass concentration of 0.2-0.5%, the extraction method is as follows: the areca nut raw material is mixed with a hydrochloric acid aqueous solution with a mass concentration of 0.2-0.5%, soaked for 1 hour, then packed into a percolation column, and extracted by percolation with a hydrochloric acid aqueous solution with a mass concentration of 0.2-0.5%. The mass-to-volume ratio of the areca nut raw material to the extraction solvent is 1g:(8~35)mL; Before drying the areca nut extract, the areca nut extract is concentrated. When the extraction solvent is a hydrochloric acid aqueous solution with a mass concentration of 0.2~0.5%, before the concentration treatment, the areca nut extract is passed through a 732 type cation adsorption column to obtain the effluent from the cation adsorption column. The drying process is carried out at a temperature of 65-75°C for 72-96 hours. The areca nut extract contains 10.72% to 19.56% polyphenols and 0.044% to 1.57% total alkalis.

2. The application according to claim 1, characterized in that, The biological feed consists of areca nut products and basic feed; the areca nut products are areca nut extract. The basic feed includes any one or more of complete feed, concentrated feed, and premixed feed; Add 25g to 400g of the areca nut extract per ton of complete feed.

3. The application according to claim 1, characterized in that, The drinking solution is composed of areca nut product and water; the areca nut product is areca nut extract. Add 25g to 400g of the areca nut extract per ton of water.

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