An antiviral agent against porcine reproductive and respiratory syndrome virus and its preparation method

Abstract

This invention discloses an anti-porcine reproductive and respiratory syndrome virus (PRRSV) preparation and its preparation method. The anti-PRRSV preparation comprises traditional Chinese medicine, excipients, and probiotics. The traditional Chinese medicine includes: mustard greens, thistle, bitter melon, and barley sprouts. The excipients include: soybean meal, wheat bran, and molasses. The probiotics include: Bacillus subtilis, Enterococcus faecium, and Saccharomyces cerevisiae. By feeding pigs suffering from PRRSV with the anti-PRRSV preparation of this invention, PRRSV can be antagonized, improving the recovery rate of PRRSV-affected pigs and reducing the mortality rate. The anti-PRRSV preparation of this invention can be used as an antagonist of PRRSV. Furthermore, the anti-PRRSV preparation of this invention can also be used as a feed additive for the prevention or treatment of PRRSV in pigs.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry technology, and in particular to an antiviral agent for porcine reproductive and respiratory syndrome virus and its preparation method. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). Pigs of all ages, breeds, and sexes can be infected. The disease is characterized by abortion, stillbirth, weak piglets, mummified fetuses in sows, and respiratory distress, septicemia, and high mortality in piglets. It causes significant economic losses to the pig farming industry. For a long time, PRRS has seriously threatened and damaged the interests of the pig farming industry, while also restricting its high-quality and healthy development.

[0003] Therefore, if a formulation with anti-porcine reproductive and respiratory syndrome virus efficacy can be developed, it will have broad application prospects in the field of pig farming. Summary of the Invention

[0004] This invention provides an anti-porcine reproductive and respiratory syndrome virus (PRRSV) formulation and its preparation method. Feeding pigs suffering from PRRSV with this anti-PRRSV formulation antagonizes PRRSV, improves the recovery rate of PRRS-affected pigs, and reduces mortality. This anti-PRRSV formulation can be used as an antagonist of PRRSV. It can also be used as a feed additive for the prevention or treatment of PRRS in pigs. Feeding pigs suffering from PRRSV with this anti-PRRSV formulation increases feed intake, reduces feed conversion ratio, and improves the body condition and growth performance of PRRS-affected pigs. This anti-PRRSV formulation can also be used as a body condition recovery agent for PRRS-affected pigs. Furthermore, this anti-PRRSV formulation can be used as an inhibitor or killer of Mycoplasma hyopneumoniae. This anti-PRRSV formulation also has a good therapeutic effect on Mycoplasma hyopneumoniae pneumonia.

[0005] The technical solution of this invention is as follows:

[0006] An anti-swine reproductive and respiratory syndrome virus (PRRSV) preparation, wherein the anti-PRRSV preparation comprises traditional Chinese medicine, excipients, and probiotics;

[0007] The Chinese herbal medicines mentioned include: mustard, thistle, bitter melon, and barley;

[0008] The auxiliary materials include: soybean meal, wheat bran, and molasses;

[0009] The probiotics include: Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae.

[0010] Preferably, the weight parts of each component of the Chinese herbal medicine are: 20-25 parts of mustard greens, 10-15 parts of thistle, 5-10 parts of bitter melon, and 3-7 parts of barley.

[0011] The weight parts of each component of the auxiliary material are: 5-10 parts soybean meal, 3-10 parts wheat bran, and 3-5 parts molasses.

[0012] The preparation method of the above-mentioned anti-porcine reproductive and respiratory syndrome virus preparation includes the following steps:

[0013] (1) The soybean meal is crushed to obtain soybean meal powder, the wheat bran is crushed to obtain wheat bran powder, and the obtained soybean meal powder, wheat bran powder and molasses are mixed to obtain a first mixture;

[0014] Water was added to the first mixture and mixed well to obtain a microbial solid fermentation culture medium;

[0015] (2) Add the probiotics to the microbial solid fermentation culture medium and mix well. After fermentation, the first fermentation material is obtained. The first fermentation material contains live bacteria of the probiotics and their metabolites.

[0016] (3) Add the Chinese herbal medicine to the first fermented material and mix well. After fermentation, the second fermented material is obtained.

[0017] During the process of adding the Chinese herbal medicine to the first fermentation material and mixing it, the live bacteria and their metabolites contained in the first fermentation material are evenly dispersed into the Chinese herbal medicine. The live bacteria and their active metabolites can act on the Chinese herbal medicine, promote the rupture of the cell walls of the Chinese herbal medicine, promote the release of small molecules, reduce the bitterness of the Chinese herbal medicine, and improve its palatability. At the same time, the Chinese herbal medicine promotes the production of antimicrobial peptides by Bacillus subtilis.

[0018] (4) The second fermentation material is dried to obtain an anti-swine reproductive and respiratory syndrome virus preparation.

[0019] Preferably, in step (1), when water is added to the first mixture, the weight ratio of the first mixture to the amount of water added is 10:4;

[0020] In step (1), after obtaining the microbial solid fermentation culture medium, steam is introduced into the obtained microbial solid fermentation culture medium to heat the material temperature of the microbial solid fermentation culture medium to 90°C and maintain it for 20 minutes. Then, the material temperature of the microbial solid fermentation culture medium is cooled to 30-35°C.

[0021] Preferably, in step (2), the method for preparing the probiotics includes:

[0022] The Bacillus subtilis was inoculated into the Bacillus subtilis culture medium at an inoculation rate of 0.5%, and activated at 28-32℃ for 4 hours to obtain the activated solution of Bacillus subtilis.

[0023] The Enterococcus faecalis was inoculated into a lactic acid bacteria culture medium at an inoculation rate of 0.5%, and activated at 28-32℃ for 4 hours to obtain the activated solution of the Enterococcus faecalis.

[0024] The brewer's yeast was inoculated into a fungal culture medium at an inoculation rate of 0.5% and activated at 28-32℃ for 4 hours to obtain the activated brewer's yeast solution.

[0025] The activated solutions of Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae were mixed at a weight ratio of 10:1:1 to obtain the probiotics.

[0026] Preferably, in step (2), the viable count of Bacillus subtilis in the probiotics obtained is (1-9) × 10⁻⁶. 8 CFU / ml, the viable count of Enterococcus faecalis was (1-9) × 10⁻⁶. 7 CFU / ml, viable count of Saccharomyces cerevisiae is (1-9)×10 7 CFU / ml.

[0027] Preferably, in step (2), the probiotics and the microbial solid fermentation culture medium are mixed at a weight ratio of (0.02-0.05):14, and the first fermented material is obtained after fermentation.

[0028] During fermentation, the fermentation temperature is 35-38℃ and the fermentation time is 48 hours.

[0029] Preferably, in step (3), the Chinese herbal medicine and the first fermented material are mixed at a weight ratio of 2:1, and the second fermented material is obtained after fermentation.

[0030] Preferably, in step (3), each component of the Chinese herbal medicine is crushed and then mixed to obtain a second mixture. Water is added to the second mixture and mixed to obtain a third mixture. The weight ratio of the second mixture to the added water is 2:1. Steam is introduced into the third mixture to heat the material temperature of the third mixture to 90°C and maintain it for 20 minutes. Then the material temperature of the third mixture is cooled to 30-35°C. The third mixture is then added to the first fermentation material and mixed to obtain a second fermentation material after fermentation. During the fermentation process of the first mixture, some water is consumed. Before fermentation after adding the Chinese herbal medicine to the first fermentation material and mixing, water is added to the second mixture and mixed to obtain a third mixture. This can replenish the water in the material of the Chinese herbal medicine in a timely manner and promote the continuous deep fermentation of the material by microorganisms.

[0031] During fermentation, the fermentation temperature is 35-38℃ and the fermentation time is 72 hours.

[0032] Preferably, in step (4), the second fermented material is dried at a temperature of 250±10℃ for 10-15 seconds.

[0033] The drying method for the second fermentation material can be drum drying, tube bundle drying, airflow drying, or a combination of multiple drying methods.

[0034] The beneficial effects of this invention are:

[0035] In the preparation process of the anti-swine reproductive and respiratory syndrome virus formulation of this invention, probiotics are fermented in the early stage using soybean meal, wheat bran, and molasses as substrates. The probiotics grow and multiply rapidly, producing abundant enzymes such as cellulase, amylase, pectinase, protease, and ligninase. In the later stage, the probiotics are fermented using traditional Chinese medicine as substrates. These enzymes produced by metabolism can rupture the cell walls of the traditional Chinese medicine plants, promoting the release of effective components in the traditional Chinese medicine. The synergistic effect of bacteria and enzymes can enhance the degradation of macromolecules, promote intestinal digestion and absorption, thereby improving the efficacy of the traditional Chinese medicine and enhancing its antagonistic effect against porcine reproductive and respiratory syndrome virus.

[0036] In the preparation of the anti-swine reproductive and respiratory syndrome virus formulation of the present invention, the traditional Chinese medicine described in the present invention can significantly promote the metabolism of Bacillus subtilis to produce antimicrobial peptides, thereby enhancing the inhibitory and killing effect of the anti-swine reproductive and respiratory syndrome virus formulation of the present invention on Gram-positive pathogens.

[0037] The anti-swine reproductive and respiratory syndrome virus formulation of the present invention has a strong inhibitory effect on both Gram-positive and Gram-negative bacteria, and the inhibitory effect on Gram-positive bacteria is stronger.

[0038] Feeding pigs with porcine reproductive and respiratory syndrome virus (PRRSV) of this invention can antagonize PRRSV, improve the recovery rate of PRRS-affected pigs, achieve a cure rate of 66.7%-86.7%, and reduce mortality. This PRRSV preparation can be used as an antagonist of PRRSV and as a feed additive for the prevention or treatment of PRRS in pigs.

[0039] Feeding pigs with porcine reproductive and respiratory syndrome virus (PRRSV) of the present invention to them can increase their feed intake, reduce their feed conversion ratio, and improve their body condition and growth performance. The anti-PRRSV preparation of the present invention can be used as a treatment for PRRS and a body condition recovery agent for PRRS-affected pigs.

[0040] The anti-porcine reproductive and respiratory syndrome virus formulation of the present invention can also be used as an inhibitor or killer of porcine mycoplasma pneumoniae.

[0041] The anti-porcine reproductive and respiratory syndrome virus formulation of the present invention has a good therapeutic effect on porcine mycoplasma pneumonia. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below.

[0043] Example 1

[0044] An anti-swine reproductive and respiratory syndrome virus (PRRSV) preparation, wherein the anti-PRRSV preparation comprises traditional Chinese medicine, excipients, and probiotics;

[0045] The Chinese herbal medicines mentioned include: mustard, thistle, bitter melon, and barley;

[0046] The auxiliary materials include: soybean meal, wheat bran, and molasses;

[0047] The probiotics include: Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae.

[0048] The weight parts of each component of the Chinese herbal medicine are as follows: 20 parts of mustard greens, 13 parts of thistle, 8 parts of bitter melon, and 3 parts of barley.

[0049] The weight parts of each component of the auxiliary material are: 5 parts soybean meal, 8 parts wheat bran, and 5 parts molasses.

[0050] The preparation method of the above-mentioned anti-porcine reproductive and respiratory syndrome virus preparation includes the following steps:

[0051] (1) The soybean meal is crushed to obtain soybean meal powder, the wheat bran is crushed to obtain wheat bran powder, and the obtained soybean meal powder, wheat bran powder and molasses are mixed to obtain a first mixture;

[0052] Water was added to the first mixture and mixed well to obtain a microbial solid fermentation culture medium;

[0053] (2) Add the probiotics to the microbial solid fermentation culture medium and mix well. After fermentation, the first fermentation material is obtained. The first fermentation material contains live bacteria of the probiotics and their metabolites.

[0054] (3) Add the Chinese herbal medicine to the first fermented material and mix well. After fermentation, the second fermented material is obtained.

[0055] (4) The second fermentation material is dried to obtain an anti-swine reproductive and respiratory syndrome virus preparation.

[0056] In step (1), when water is added to the first mixture, the weight ratio of the first mixture to the amount of water added is 10:4;

[0057] In step (1), after obtaining the microbial solid fermentation culture medium, steam is introduced into the obtained microbial solid fermentation culture medium to heat the material temperature of the microbial solid fermentation culture medium to 90°C, maintain it for 20 minutes, and then cool the material temperature of the microbial solid fermentation culture medium to 30°C.

[0058] In step (2), the method for preparing the probiotics includes:

[0059] The Bacillus subtilis was inoculated into the Bacillus subtilis culture medium at an inoculation rate of 0.5%, and activated at 28°C for 4 hours to obtain the activated solution of Bacillus subtilis.

[0060] The Enterococcus faecalis was inoculated into a lactic acid bacteria culture medium at an inoculation rate of 0.5%, and activated at 32°C for 4 hours to obtain the activated solution of the Enterococcus faecalis.

[0061] The brewer's yeast was inoculated into a fungal culture medium at an inoculation rate of 0.5% and activated at 32°C for 4 hours to obtain the activated brewer's yeast solution.

[0062] The activated solutions of Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae were mixed at a weight ratio of 10:1:1 to obtain the probiotics.

[0063] In step (2), the viable count of Bacillus subtilis in the prepared probiotics is 1 × 10⁻⁶. 8 CFU / ml, viable count of Enterococcus faecalis was 9 × 10⁻⁶. 7CFU / ml, viable count of brewer's yeast is 1×10⁻⁶. 7 CFU / ml.

[0064] In step (2), the probiotics and the microbial solid fermentation culture medium are mixed at a weight ratio of 0.04:14, and the first fermented material is obtained after fermentation.

[0065] During the fermentation process, the fermentation temperature was 38℃ and the fermentation time was 48 hours.

[0066] In step (3), the Chinese herbal medicine and the first fermented material are mixed at a weight ratio of 2:1, and the second fermented material is obtained after fermentation.

[0067] In step (3), each component of the Chinese herbal medicine is crushed and then mixed to obtain a second mixture. Water is added to the second mixture and mixed to obtain a third mixture. The weight ratio of the second mixture to the added water is 2:1. Steam is introduced into the third mixture to heat the material temperature to 90°C and maintain it for 20 minutes. Then the material temperature of the third mixture is cooled to 32°C. The third mixture is then added to the first fermented material and mixed to obtain a second fermented material after fermentation. During the fermentation process, the fermentation temperature is 36°C and the fermentation time is 72 hours.

[0068] In step (4), the second fermented material is dried at a temperature of 250°C for 12 seconds.

[0069] Example 2

[0070] An anti-swine reproductive and respiratory syndrome virus (PRRSV) preparation, wherein the anti-PRRSV preparation comprises traditional Chinese medicine, excipients, and probiotics;

[0071] The Chinese herbal medicines mentioned include: mustard, thistle, bitter melon, and barley;

[0072] The auxiliary materials include: soybean meal, wheat bran, and molasses;

[0073] The probiotics include: Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae.

[0074] The weight parts of each component of the Chinese herbal medicine are as follows: 22 parts of mustard greens, 15 parts of thistle, 5 parts of bitter melon, and 4 parts of barley.

[0075] The weight parts of each component of the auxiliary material are: 10 parts soybean meal, 10 parts wheat bran, and 4 parts molasses.

[0076] The preparation method of the above-mentioned anti-porcine reproductive and respiratory syndrome virus preparation includes the following steps:

[0077] (1) The soybean meal is crushed to obtain soybean meal powder, the wheat bran is crushed to obtain wheat bran powder, and the obtained soybean meal powder, wheat bran powder and molasses are mixed to obtain a first mixture;

[0078] Water was added to the first mixture and mixed well to obtain a microbial solid fermentation culture medium;

[0079] (2) Add the probiotics to the microbial solid fermentation culture medium and mix well. After fermentation, the first fermentation material is obtained. The first fermentation material contains live bacteria of the probiotics and their metabolites.

[0080] (3) Add the Chinese herbal medicine to the first fermented material and mix well. After fermentation, the second fermented material is obtained.

[0081] (4) The second fermentation material is dried to obtain an anti-swine reproductive and respiratory syndrome virus preparation.

[0082] In step (1), when water is added to the first mixture, the weight ratio of the first mixture to the amount of water added is 10:4;

[0083] In step (1), after obtaining the microbial solid fermentation culture medium, steam is introduced into the obtained microbial solid fermentation culture medium to heat the material temperature of the microbial solid fermentation culture medium to 90°C, maintain it for 20 minutes, and then cool the material temperature of the microbial solid fermentation culture medium to 33°C.

[0084] In step (2), the method for preparing the probiotics includes:

[0085] The Bacillus subtilis was inoculated into the Bacillus subtilis culture medium at an inoculation rate of 0.5%, and activated at 32°C for 4 hours to obtain the activated solution of Bacillus subtilis.

[0086] The Enterococcus faecalis was inoculated into a lactic acid bacteria culture medium at an inoculation rate of 0.5%, and activated at 28°C for 4 hours to obtain the activated solution of the Enterococcus faecalis.

[0087] The brewer's yeast was inoculated into a fungal culture medium at an inoculation rate of 0.5% and activated at 28°C for 4 hours to obtain the activated brewer's yeast solution.

[0088] The activated solutions of Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae were mixed at a weight ratio of 10:1:1 to obtain the probiotics.

[0089] The viable count of Bacillus subtilis in the prepared probiotics was 9 × 10⁻⁶. 8 CFU / ml, viable count of Enterococcus faecalis was 5 × 10⁻⁶. 7CFU / ml, viable count of brewer's yeast is 7×10⁻⁶. 7 CFU / ml.

[0090] In step (2), the probiotics and the microbial solid fermentation culture medium are mixed at a weight ratio of 0.05:14, and the first fermented material is obtained after fermentation.

[0091] During the fermentation process, the fermentation temperature was 37℃ and the fermentation time was 48 hours.

[0092] In step (3), the Chinese herbal medicine and the first fermented material are mixed at a weight ratio of 2:1, and the second fermented material is obtained after fermentation.

[0093] In step (3), each component of the Chinese herbal medicine is crushed and then mixed to obtain a second mixture. Water is added to the second mixture and mixed to obtain a third mixture. The weight ratio of the second mixture to the added water is 2:1. Steam is introduced into the third mixture to heat the material temperature to 90°C and maintain it for 20 minutes. Then the material temperature of the third mixture is cooled to 35°C. The third mixture is then added to the first fermented material and mixed to obtain a second fermented material after fermentation. During the fermentation process, the fermentation temperature is 35°C and the fermentation time is 72 hours.

[0094] In step (4), the second fermented material is dried at a temperature of 260°C for 10 seconds.

[0095] Example 3

[0096] An anti-swine reproductive and respiratory syndrome virus (PRRSV) preparation, wherein the anti-PRRSV preparation comprises traditional Chinese medicine, excipients, and probiotics;

[0097] The Chinese herbal medicines mentioned include: mustard, thistle, bitter melon, and barley;

[0098] The auxiliary materials include: soybean meal, wheat bran, and molasses;

[0099] The probiotics include: Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae.

[0100] The weight parts of each component of the Chinese herbal medicine are as follows: 25 parts of mustard greens, 10 parts of thistle, 10 parts of bitter melon, and 7 parts of barley.

[0101] The weight parts of each component of the auxiliary material are: 8 parts soybean meal, 3 parts wheat bran, and 3 parts molasses.

[0102] The preparation method of the above-mentioned anti-porcine reproductive and respiratory syndrome virus preparation includes the following steps:

[0103] (1) The soybean meal is crushed to obtain soybean meal powder, the wheat bran is crushed to obtain wheat bran powder, and the obtained soybean meal powder, wheat bran powder and molasses are mixed to obtain a first mixture;

[0104] Water was added to the first mixture and mixed well to obtain a microbial solid fermentation culture medium;

[0105] (2) Add the probiotics to the microbial solid fermentation culture medium and mix well. After fermentation, the first fermentation material is obtained. The first fermentation material contains live bacteria of the probiotics and their metabolites.

[0106] (3) Add the Chinese herbal medicine to the first fermented material and mix well. After fermentation, the second fermented material is obtained.

[0107] (4) The second fermentation material is dried to obtain an anti-swine reproductive and respiratory syndrome virus preparation.

[0108] In step (1), when water is added to the first mixture, the weight ratio of the first mixture to the amount of water added is 10:4;

[0109] In step (1), after obtaining the microbial solid fermentation culture medium, steam is introduced into the obtained microbial solid fermentation culture medium to heat the material temperature of the microbial solid fermentation culture medium to 90°C, maintain it for 20 minutes, and then cool the material temperature of the microbial solid fermentation culture medium to 35°C.

[0110] In step (2), the method for preparing the probiotics includes:

[0111] The Bacillus subtilis was inoculated into the Bacillus subtilis culture medium at an inoculation rate of 0.5%, and activated at 31°C for 4 hours to obtain the activated solution of Bacillus subtilis.

[0112] The Enterococcus faecalis was inoculated into a lactic acid bacteria culture medium at an inoculation rate of 0.5%, and activated at 29°C for 4 hours to obtain the activated solution of the Enterococcus faecalis.

[0113] The brewer's yeast was inoculated into a fungal culture medium at an inoculation rate of 0.5% and activated at 30°C for 4 hours to obtain the activated brewer's yeast solution.

[0114] The activated solutions of Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae were mixed at a weight ratio of 10:1:1 to obtain the probiotics.

[0115] In step (2), the viable count of Bacillus subtilis in the prepared probiotics is 8 × 10⁻⁶. 8 CFU / ml, viable count of Enterococcus faecalis was 1×10⁻⁶. 7CFU / ml, viable count of brewer's yeast is 9×10⁻⁶. 7 CFU / ml.

[0116] In step (2), the probiotics and the microbial solid fermentation culture medium are mixed at a weight ratio of 0.02:14, and the first fermented material is obtained after fermentation.

[0117] During the fermentation process, the fermentation temperature was 35℃ and the fermentation time was 48 hours.

[0118] In step (3), the Chinese herbal medicine and the first fermented material are mixed at a weight ratio of 2:1, and the second fermented material is obtained after fermentation.

[0119] In step (3), each component of the Chinese herbal medicine is crushed and then mixed to obtain a second mixture. Water is added to the second mixture and mixed to obtain a third mixture. The weight ratio of the second mixture to the added water is 2:1. Steam is introduced into the third mixture to heat the material temperature to 90°C and maintain it for 20 minutes. Then the material temperature of the third mixture is cooled to 30°C. The third mixture is then added to the first fermented material and mixed to obtain a second fermented material after fermentation. During the fermentation process, the fermentation temperature is 38°C and the fermentation time is 72 hours.

[0120] In step (4), the second fermented material is dried at a temperature of 240°C for 15 seconds.

[0121] Experiment Example 1: Experiment to promote the production of antimicrobial peptides by Bacillus subtilis

[0122] In the process of preparing the anti-porcine reproductive and respiratory syndrome virus formulations in Examples 1-3 above, samples were taken from the first fermentation material obtained in step (2) of Examples 1-3 respectively. Two groups of samples were taken from each identical sample, namely: one group as the experimental group and the other group as the control group, for a total of six groups. The specific sample numbers are as follows:

[0123] Two groups of samples were taken from the first fermentation material prepared in Example 1 and numbered as: experimental group 1 and control group 1;

[0124] Two groups of samples were taken from the first fermentation material obtained in Example 2 and numbered as: experimental group 2 and control group 2;

[0125] Two groups of samples were taken from the first fermentation material prepared in Example 3 and numbered as: experimental group 3 and control group 3.

[0126] Among them, the above-mentioned test groups 1-3 are respectively processed according to the preparation methods of Examples 1-3, and the second fermentation materials of test groups 1-3 are respectively obtained in step (3), and the obtained second fermentation materials are respectively numbered as S1A1, S2A1, and S3A1;

[0127] Among them, the control groups 1-3 were respectively treated according to the preparation method of Examples 1-3. The difference between the treatment of control groups 1-3 and the treatment of experimental groups 1-3 is that the Chinese herbal medicines were not added in step (3) of control groups 1-3. After the treatment of control groups 1-3, the second fermentation materials of control groups 1-3 were respectively prepared, and the second fermentation materials were respectively numbered as S1A2, S2A2, and S3A2.

[0128] Weigh 5g of each of the above-mentioned samples numbered S1A1, S2A1, S3A1, S1A2, S2A2, and S3A2. Add 40mg / mL EDTA-Na2 solution to each sample at a weight ratio of 1:2. Extract for 2 hours. Filter through a 0.45 μm organic filter membrane to remove impurities before use. After powering on and baseline running, the high-performance liquid chromatograph (HPLC) sequentially injected standard and sample S1A1, S2A1, S3A1, S1A2, S2A2, and S3A2. The peak times and peak areas of the standard and sample S1A1, S2A1, S3A1, S1A2, S2A2, and S3A2 were recorded. The concentrations of the test samples S1A1, S2A1, S3A1, S1A2, S2A2, and S3A2 were calculated based on the peak areas and concentrations of the standard. Based on the concentration and peak area of ​​the antimicrobial peptide standard, the regression equation was calculated as y = 0.0012x - 1.1243, with a correlation coefficient of 0.9995, where y represents the antimicrobial peptide concentration and x represents the peak area. The antimicrobial peptide content of the sample was calculated based on the regression equation and the peak area of ​​the sample. The results are shown in Table 1.

[0129] Table 1. Detection of antimicrobial peptide content

[0130]

[0131] Antimicrobial peptides are secondary metabolites produced during the growth of Bacillus and have a strong inhibitory effect on Gram-positive bacteria.

[0132] In this invention, the traditional Chinese medicine is added to the first fermented material and mixed evenly. After fermentation, a second fermented material is obtained. Compared with the fermentation treatment of this invention without the addition of the traditional Chinese medicine, the antimicrobial peptide content in the second fermented materials obtained in Examples 1-3 of this invention is significantly increased by 51.70%, 49.23%, and 41.42%, respectively. The traditional Chinese medicine in this invention significantly promotes the metabolism of Bacillus subtilis to produce antimicrobial peptides, thereby enhancing the inhibitory and killing effect of the anti-swine reproductive and respiratory syndrome virus preparation of this invention on Gram-positive pathogens.

[0133] Test Example 2: Antibacterial Activity Test

[0134] Weigh 5g of the anti-porcine reproductive and respiratory syndrome virus preparations obtained in Examples 1-3 respectively, add sterile distilled water at a weight ratio of 1:4, extract in a boiling water bath for 1 hour, adjust the pH to neutral, filter through a 0.22μm microporous membrane, and obtain the supernatant of test groups 1-3 for later use. Streak Escherichia coli, Staphylococcus aureus, Salmonella, and Clostridium perfringens on agar plates and incubate at 36℃ for 20 hours; pick single colonies on LB broth and incubate at 37℃ with shaking for 16 hours. Adjust the bacterial count of the cultured solution to 1×10⁻⁶. 7 CFU / mL. Take 100 μL of indicator bacterial suspension and drop it onto an LB agar plate. Spread it evenly with a spreader. Use sterile forceps to pick up a sterile Oxford cup and gently place it on the surface of the LB solid medium. Use a micropipette to pipette 200 μL of the prepared supernatant from test groups 1-3 and inject it into the stable Oxford cup. Place the medium containing the supernatant from test groups 1-3 in a 4°C refrigerator for 8 hours. Then, place the plate after complete diffusion treatment in a 37°C incubator and incubate for 16-24 hours. Record the results. (Note:)

[0135] The supernatant of test group 1 is corresponding to the test group 1 supernatant.

[0136] The supernatant of test group 2 is corresponding to the test group 2 supernatant.

[0137] The supernatant of test group 3 is the same as that of test group 3.

[0138] Table 2 Inhibitory effect on pathogenic bacteria (inhibition zone diameter, unit: mm)

[0139]

[0140] Two Gram-positive bacteria (Staphylococcus aureus and Clostridium perfringens) and two Gram-negative bacteria (Escherichia coli and Salmonella) were selected as indicator bacteria to study the antibacterial effect of the preparation of the present invention against porcine reproductive and respiratory syndrome virus. The results showed that the preparation of the present invention against porcine reproductive and respiratory syndrome virus had a strong inhibitory effect on both Gram-positive bacteria (Staphylococcus aureus and Clostridium perfringens) and Gram-negative bacteria (Escherichia coli and Salmonella), and the inhibitory effect on Gram-positive bacteria was stronger.

[0141] Experiment Example 3: Effects on pigs infected with porcine reproductive and respiratory syndrome (PRRS)

[0142] Piglets at a large-scale pig farm developed severe symptoms such as high fever, difficulty breathing, and coughing. Based on the clinical symptoms, pathological changes, and laboratory diagnosis, the pathogen was identified as porcine reproductive and respiratory syndrome virus (PRRSV), and the pigs were diagnosed with porcine reproductive and respiratory syndrome (PRRS).

[0143] Sixty piglets diagnosed with porcine reproductive and respiratory syndrome (PRRS) were selected and randomly divided into four groups: a control group, experimental group 1, experimental group 2, and experimental group 3, with 15 piglets in each group. They were fed the control group for 21 consecutive days. The treatments for each group were as follows:

[0144] Control group: fed with basal diet;

[0145] Experimental group 1: fed with basal diet + 0.5% of the anti-porcine reproductive and respiratory syndrome virus preparation prepared in Example 1;

[0146] Experimental group 2: fed with basal diet + 0.5% of the anti-porcine reproductive and respiratory syndrome virus preparation prepared in Example 2;

[0147] Experimental group 3: Feeding basal diet + 0.5% of the anti-swine reproductive and respiratory syndrome virus preparation prepared in Example 3.

[0148] The experimental results showed that, compared with the control group, the cure rate of diseased pigs in experimental groups 1-3 was improved and the mortality rate was reduced. The cure rate was 66.7%-86.7% and the mortality rate was 13.3%-33.3%, a reduction of about 60%. This effectively reduced the mortality rate of piglets suffering from porcine reproductive and respiratory syndrome virus (PRRSV). This indicates that feeding pigs suffering from PRRSV with the preparation of this invention can antagonize PRRSV, improve the recovery rate of pigs suffering from PRRSV, and reduce the mortality rate.

[0149] The experimental results showed that, compared with the control group, the average daily feed intake of the diseased pigs in experimental groups 1-3 increased by 6.58%-10.29%, the average final weight increased by 9.71%-12.62%, and the feed conversion ratio decreased. This indicates that feeding pigs with porcine reproductive and respiratory syndrome virus (PRRSV) of this invention can improve the feed intake, body condition, and growth performance of the diseased pigs. The PRRSV preparation of this invention can be used as a body condition recovery agent for pigs with PRRS.

[0150] Table 3. Effects of the treatment on porcine reproductive and respiratory syndrome (PRRS) in piglets.

[0151]

[0152] Table 4. Changes in feed intake and growth performance of surviving piglets

[0153]

[0154] The anti-swine reproductive and respiratory syndrome virus preparation of the present invention can be added to feed at a dosage of 0.5% and mixed for use by pigs, or it can be added to drinking water and mixed for use by pigs. 5g of the anti-swine reproductive and respiratory syndrome virus preparation of the present invention is added to each kilogram of drinking water.

[0155] By applying the anti-porcine reproductive and respiratory syndrome virus preparation of the present invention to pigs, the cure rate of pigs with porcine reproductive and respiratory syndrome virus can reach 66.7%-86.7%. The anti-porcine reproductive and respiratory syndrome virus preparation of the present invention can be used as a feed additive for the prevention or treatment of porcine porcine reproductive and respiratory syndrome virus.

[0156] The anti-porcine reproductive and respiratory syndrome virus preparation of the present invention can be used as a treatment for porcine reproductive and respiratory syndrome (PRRS) and as a condition recovery agent for PRRS-affected pigs.

[0157] Experimental Example 4: Inhibition Test of Mycoplasma

[0158] Mycoplasma hyopneumoniae is one of the most common diseases in pig herds. After pigs are infected with Mycoplasma hyopneumoniae, the bronchial epithelial cells and goblet cells are damaged, which makes it easier for other bacteria, especially those that enter the body through the mucous membrane, to invade. Mycoplasma hyopneumoniae has a broad inhibitory effect on cellular immunity. The harm caused by mixed infection of Mycoplasma hyopneumoniae and Porcine reproductive and respiratory syndrome virus is more serious than that of infection alone.

[0159] The test method is as follows:

[0160] Weigh 5g of the anti-porcine reproductive and respiratory syndrome virus (PRRSV) preparation obtained in the embodiments of this invention, add sterile distilled water at a weight ratio of 1:4, extract in a boiling water bath for 1 hour, adjust the pH to neutral, filter through a 0.22μm microporous membrane, and set aside (the concentration of the anti-PRRSV preparation extract is 250mg / mL). Using an inoculation loop, add 5mL of lyophilized porcine mycoplasma CVCC360 (purchased from the China Institute of Veterinary Drug Control's Culture Collection Center) to a modified Frey's liquid medium, and incubate at 37℃ in a 5% CO2 incubator for approximately 7 days, observing the color change daily. When the culture medium turns yellow, the mycoplasma activation solution is obtained. The activation solution is then stored at 4℃. The mycoplasma color change units (CCU / mL) are determined in a 96-well plate. The mycoplasma activation solution is diluted 10-fold, with modified Frey's liquid medium as a blank control, to determine the highest dilution concentration of mycoplasma culture that causes the color change in the medium, which is 1 CCU / mL. Take a sterile 96-well plate. Add 100 μL of modified Frey's medium to each well in the first three rows and first ten columns. Add 100 μL of the extract of the anti-porcine reproductive and respiratory syndrome virus (PRRSV) preparation prepared in Examples 1-3 to the first well in each of the first three rows. Dilute the anti-PRRSV preparation using a two-fold dilution method until the 10th well in each column. After mixing thoroughly in the 10th well, aspirate 100 μL of the mixture and discard it. Add 10% of the solution to each well in the first ten columns. 4 100 μL of mycoplasma (CCU / mL) was added to each well in column 11 as a negative control (200 μL of modified Frey's medium), and 100 μL of modified Frey's medium and 100 μL of mycoplasma were added to each well in column 12 as a positive control. The extract concentrations of the anti-porcine reproductive and respiratory syndrome virus (PRRSV) preparation were 10 dilutions: 125, 62.5, 31.25, 15.625, 7.81, 3.91, 1.95, 0.98, 0.49, and 0.24 mg / mL. The mixture was covered and incubated in a CO2 incubator at 37°C for 7 days before the results were determined. The minimum drug concentration at which no color change occurred was the minimum inhibitory concentration (MIC). An MIC ≤ 7.81 mg / mL was considered sensitive, an MIC of 7.81-250 mg / mL was considered moderately sensitive, and an MIC ≥ 250 mg / mL was considered insensitive.

[0161] According to the above experimental method, the preparations against porcine reproductive and respiratory syndrome virus obtained in Examples 1-3 of this invention were weighed and subjected to the above experiments, wherein:

[0162] Experimental group 1 corresponds to the anti-porcine reproductive and respiratory syndrome virus preparation obtained in Example 1;

[0163] Experimental group 2 corresponds to the anti-porcine reproductive and respiratory syndrome virus preparation obtained in Example 2;

[0164] Experimental group 3 corresponds to the anti-porcine reproductive and respiratory syndrome virus preparation obtained in Example 3.

[0165] Table 5. MIC determination results for mycoplasma

[0166]

[0167] The experimental results showed that the extracts of the anti-porcine reproductive and respiratory syndrome virus preparations obtained in Examples 1-3 had different degrees of inhibitory and killing effects on Mycoplasma hyopneumoniae, which were in the moderately sensitive range. This indicates that the anti-porcine reproductive and respiratory syndrome virus preparations of the present invention can be used as inhibitors or killers of Mycoplasma hyopneumoniae.

[0168] Experimental Example 5: Effect on porcine mycoplasmal pneumonia

[0169] In a large-scale pig farm in Hubei Province, 60 cases were randomly selected from pigs diagnosed with mycoplasma pneumonia in the past two years and divided into 4 groups of 15 pigs each. The pigs were fed continuously for 7 days according to the following feeding method.

[0170] Control group: fed with basal diet;

[0171] Experimental group 1: fed with basal diet + 0.45% of the anti-porcine reproductive and respiratory syndrome virus preparation prepared in Example 1;

[0172] Experimental group 2: fed with basal diet + 0.45% of the anti-porcine reproductive and respiratory syndrome virus preparation prepared in Example 2;

[0173] Experimental group 3: Feeding basal diet + 0.45% of the anti-swine reproductive and respiratory syndrome virus preparation prepared in Example 3.

[0174] Criteria for evaluating therapeutic efficacy:

[0175] (1) Cured: After administration, the clinical symptoms of wheezing, coughing and abdominal breathing in the pigs completely disappeared, and their spirit and appetite returned to normal. No recurrence was found within 7 days after stopping the medication.

[0176] (2) Effective: After administration, the clinical symptoms of wheezing, coughing and abdominal breathing in the pigs were reduced or temporarily disappeared, but the disease recurred within 7 days after the drug was stopped.

[0177] (3) Ineffective: After administration, the clinical symptoms of the pigs did not change significantly, or even worsened or resulted in death.

[0178] Table 6. Therapeutic efficacy against porcine mycoplasma pneumonia

[0179]

[0180] Overall effective rate (%) = (Number of cured patients + Number of effective patients) / Total number of patients treated × 100

[0181] The results showed that the total effective rate of treatment in the control group was 6.7%, while the total effective rate of treatment in experimental groups 1-3 increased to 66.7%-80.0%, and the total effective rate of treatment in experimental groups 1-3 was significantly higher than that in the control group. This indicates that the anti-porcine reproductive and respiratory syndrome virus preparation of the present invention has a good therapeutic effect on porcine mycoplasma pneumonia.

[0182] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An agent against porcine reproductive and respiratory syndrome virus, characterized by, The preparation against the porcine reproductive and respiratory syndrome virus is made of Chinese herbal medicine, auxiliary material and probiotic bacteria; The Chinese herbal medicine is: Borage, Cirsium japonicum DC, Momordica charantia L, Amsonia; The auxiliary material is: soybean meal, bran, molasses; The probiotic bacteria is: Bacillus subtilis, Enterococcus faecium, Saccharomyces cerevisiae; The method for preparing the preparation against the porcine reproductive and respiratory syndrome virus comprises the following steps: (1) crushing the soybean meal to prepare soybean meal powder, crushing the bran to prepare bran powder, mixing the prepared soybean meal powder, bran powder and molasses to prepare a first mixture; adding water to the first mixture and mixing uniformly to prepare a microbial solid fermentation medium; (2) adding the probiotic bacteria to the microbial solid fermentation medium and mixing uniformly, and preparing a first fermentation material after fermentation; (3) adding the Chinese herbal medicine to the first fermentation material and mixing uniformly, and preparing a second fermentation material after fermentation; (4) drying the second fermentation material to prepare the preparation against the porcine reproductive and respiratory syndrome virus; The weight parts of each component of the Chinese herbal medicine are: 20-25 parts of Borage, 10-15 parts of Cirsium japonicum DC, 5-10 parts of Momordica charantia L and 3-7 parts of Amsonia.

2. The preparation of claim 1, wherein the preparation is an inactivated vaccine. The weight parts of each component of the auxiliary material are: 5-10 parts of soybean meal, 3-10 parts of bran and 3-5 parts of molasses.

3. The preparation of claim 1, wherein the preparation is an inactivated vaccine. In step (1), when water is added to the first mixture, the weight ratio of the first mixture to the amount of water added is 10:4; In step (1), after the microbial solid fermentation medium is prepared, steam is introduced into the prepared microbial solid fermentation medium, the material temperature of the microbial solid fermentation medium is heated to 90℃, maintained for 20 min, and then the material temperature of the microbial solid fermentation medium is cooled to 30-35℃.

4. The preparation of claim 1, wherein the preparation is an inactivated vaccine.

5. The preparation of claim 1, wherein the preparation is a live vaccine. In step (2), the preparation method of the probiotic bacteria comprises: The Bacillus subtilis is inoculated into a Bacillus subtilis culture medium at an inoculation amount of 0.5%, incubated and activated at 28-32℃ for 4 h to obtain an activation liquid of the Bacillus subtilis; The Enterococcus faecium is inoculated into a lactic acid bacteria culture medium at an inoculation amount of 0.5%, incubated and activated at 28-32℃ for 4 h to obtain an activation liquid of the Enterococcus faecium; The Saccharomyces cerevisiae is inoculated into a fungus culture medium at an inoculation amount of 0.5%, incubated and activated at 28-32℃ for 4 h to obtain an activation liquid of the Saccharomyces cerevisiae; The prepared activation liquid of the Bacillus subtilis, activation liquid of the Enterococcus faecium and activation liquid of the Saccharomyces cerevisiae are mixed uniformly at a weight ratio of 10:1:1 to prepare the probiotic bacteria.

5. The preparation of claim 4, wherein the preparation is an inactivated vaccine. In step (2), the viable cell count of Bacillus subtilis in the prepared probiotic is (1-9) x 10 8 CFU / ml, the viable cell count of Enterococcus faecium is (1-9) x 10 7 CFU / ml, and the viable cell count of Saccharomyces cerevisiae is (1-9) x 10 7 CFU / ml.

6. The preparation of claim 1, wherein the preparation is an anti-PRRSV preparation. In step (2), the probiotic bacteria and the microbial solid fermentation medium are mixed uniformly at a weight ratio of (0.02-0.05):14 to prepare the first fermentation material after fermentation; During the fermentation process, the fermentation temperature is 35-38℃ and the fermentation time is 48 hours.

7. The preparation of claim 1, wherein the preparation is an anti-PRRSV preparation. In step (3), the Chinese herbal medicine and the first fermentation material are mixed uniformly at a weight ratio of 2:1 to prepare the second fermentation material after fermentation.

8. The preparation of claim 1, wherein the preparation is an anti-PRRSV preparation. In step (3), each component of the Chinese herbal medicine is ground, and then the components are mixed to obtain a second mixture. Water is added to the second mixture, which is mixed to obtain a third mixture. The weight ratio of the second mixture to the amount of water is 2:

1. Steam is introduced into the third mixture, and the material temperature of the third mixture is heated to 90°C for 20 min, and then cooled to 30-35°C. The third mixture is added to the first fermentation material and mixed, and then fermented to obtain a second fermentation material. During the fermentation, the fermentation temperature is 35-38°C, and the fermentation time is 72 h.

9. The preparation of claim 1, wherein the preparation is an anti-PRRSV preparation. In step (4), the second fermentation material is dried at a drying temperature of 250±10°C for 10-15 s.

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