A fermented traditional Chinese medicine preparation for treating porcine epidemic diarrhea virus infection and its application

Abstract

This invention discloses a fermented traditional Chinese medicine preparation for treating porcine epidemic diarrhea virus infection and its application. This fermented traditional Chinese medicine preparation is obtained by fermenting dandelion, isatis root, alisma, kudzu root, scutellaria, licorice, and hawthorn using a probiotic mother liquor. The probiotic mother liquor contains *Lactobacillus rhamnoides*, *Bacillus subtilis*, *Lactobacillus plantarum*, and *Bacillus coagulans*. The preparation method of this fermented traditional Chinese medicine preparation includes the following steps: (1) mixing the herbs, decocting, filtering, and sterilizing to obtain a mixed herbal liquid; (2) adding the probiotic mother liquor to the mixed herbal liquid for fermentation to obtain the fermented traditional Chinese medicine liquid. This fermented traditional Chinese medicine preparation can exert its anti-porcine epidemic diarrhea virus effect in vitro by preventing the virus from entering cells, inhibiting viral replication, and directly killing the virus. It has a significant preventive and therapeutic effect on diseases caused by this virus, is easy to prepare, can be mass-produced, and is beneficial to improving the economic benefits of pig farms.

Description

Technical Field

[0001] This invention belongs to the field of animal medicine technology, specifically relating to a traditional Chinese medicine fermentation preparation for treating porcine epidemic diarrhea virus infection and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDV), characterized by rapid disease progression and high mortality, causing serious economic losses to the pig industry.

[0003] Currently, there is no specific drug for the treatment of PEDV; treatment mainly relies on vaccination and symptomatic relief. Many broad-spectrum antiviral drugs have been banned. The vaccines available for PEDV prevention are primarily inactivated and live attenuated vaccines. However, due to the high mutation rate of PEDV and the existence of numerous wild-type strains, vaccine protection is not ideal. Several new types of vaccines have been developed (e.g., subunit vaccines, viral vector vaccines, live bacterial vector vaccines, transgenic plant vaccines, and nucleic acid vaccines). However, these new vaccines have several drawbacks, including weak immunogenicity, potential for secondary infections, low expression levels, long development cycles, and the possibility of hypersensitivity reactions.

[0004] Probiotics are single or well-defined mixtures of microorganisms that have health benefits. They can promote the digestion and absorption of nutrients, enhance immunity, maintain the balance of gut microbiota, improve meat quality, increase the body's antioxidant levels, inhibit intestinal inflammation, and protect the intestinal mucosal barrier.

[0005] Traditional Chinese medicine (TCM) contains various active ingredients that promote livestock and poultry growth, improve immune function, have antibacterial and antiviral effects, and increase feed utilization. TCM has advantages such as being natural, having few toxic side effects, being unlikely to leave residues in livestock and poultry, and not easily developing drug resistance with long-term use. Probiotic fermented TCM preparations can promote growth, enhance immune function and disease resistance, treat diarrhea, protect liver function, and reduce stress responses. Therefore, using several probiotics in co-fermentation, where strains are interdependent and mutually reinforcing, and where the combination of probiotics and TCM has a synergistic effect, can effectively improve production efficiency and the content of active ingredients. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems and to provide a fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea and its application.

[0007] 1. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea, made by fermenting traditional Chinese herbs using probiotic mother liquor;

[0008] The Chinese herbal medicines mentioned herein are in the following proportions by weight: 2-4 parts dandelion, 2-4 parts isatis root, 1-2 parts alisma, 1-2 parts kudzu root, 2-3 parts scutellaria, 1-3 parts licorice, and 1-3 parts hawthorn.

[0009] The probiotic mother liquor comprises, by weight: 2-4 parts of Lactobacillus rhamnosus mother liquor, 2-3 parts of Bacillus subtilis mother liquor, 2-3 parts of Lactobacillus plantarum mother liquor, and 1-2 parts of Bacillus coagulans mother liquor.

[0010] The Lactobacillus rhamnosus stock solution mentioned above is made by mixing 1×10 8 Lactobacillus rhamnosus at a concentration of CFU / mL was inoculated into MRS liquid medium at a 5% inoculum and cultured at 37°C and 180 rpm for 2 h.

[0011] The Bacillus subtilis stock solution: 1×10 8 Bacillus subtilis at CFU / mL was inoculated into LB liquid medium at an inoculum of 4% and cultured at 37°C and 200 rpm for 2 h.

[0012] The aforementioned *Lactobacillus plantarum* stock solution is made by mixing 1×10... 8 The *Lactobacillus plantarum* CFU / mL was inoculated into MRS liquid medium at a 4% inoculation rate and cultured at 37°C and 180 rpm for 2 h.

[0013] The Bacillus coagulans mother liquor is 1×10 8 The CFU / mL Bacillus coagulans was inoculated into LB liquid medium at an inoculum of 2.5% and cultured at 40°C and 180 rpm for 2 h.

[0014] The Chinese herbal medicines mentioned are in the following proportions by weight: 2 parts dandelion, 2 parts isatis root, 1 part alisma, 1 part kudzu root, 2 parts scutellaria, 1 part licorice, and 1 part hawthorn.

[0015] Two parts of the mother liquor of Lactobacillus rhamnosus, two parts of the mother liquor of Bacillus subtilis, two parts of the mother liquor of Lactobacillus plantarum, and one part of the mother liquor of Bacillus coagulans.

[0016] The Chinese herbal medicines mentioned are extracts of Chinese herbal medicines;

[0017] The herbal extract is prepared by the following method: add 400-500 times the volume of distilled water to the herbal medicine and soak for 30-60 minutes, boil for 15 minutes, then decoct for 1 hour, filter, add 200-300 times the volume of distilled water to the residue, decoct and filter in the same way, combine the two decoction filtrates, concentrate to a paste, dissolve the paste with distilled water to 1g / mL, and filter to remove bacteria.

[0018] Sterilization is achieved by filtration using a 0.22μm filter.

[0019] The fermentation is carried out at 35-40℃; the weight ratio of the probiotic mother liquor to the herbal extract is 1-2:10.

[0020] The fermentation process was carried out at 37°C with shaking at 180 rpm for 40-45 hours.

[0021] The application of a fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea in the prevention and treatment of swine epidemic diarrhea virus infection.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The traditional Chinese medicine fermentation preparation provided by this invention can improve diarrhea and electrolyte imbalance caused by porcine epidemic diarrhea virus infection, and has a significant preventive and therapeutic effect on the disease. It is easy to produce, can be mass-produced, and is conducive to improving the economic benefits of pig farms. Attached Figure Description

[0024] Figure 1 Results of Vero cytotoxicity test on fermented traditional Chinese medicine preparations;

[0025] Figure 2 Results of experiments on the effect of fermented traditional Chinese medicine preparations on PEDV inhibition rate under blocking effect;

[0026] Figure 3 Results of experiments on the effect of fermented traditional Chinese medicine preparations on PEDV inhibition rate under inhibitory effects;

[0027] Figure 4 Results of experiments on the effect of fermented traditional Chinese medicine preparations on the inhibition rate of PEDV under the killing effect. Detailed Implementation

[0028] Example 1: Four Probiotic Mother Liquids

[0029] The four types of probiotic stock solutions were prepared as follows:

[0030] (1) 1×10 8 Lactobacillus rhamnosus at a concentration of CFU / mL was inoculated into MRS liquid medium at a 5% inoculum and cultured at 37°C and 180 r / min for 2 h to obtain the mother liquor of Lactobacillus rhamnosus.

[0031] (2) 1×10 8 Bacillus subtilis at CFU / mL was inoculated into LB liquid medium at an inoculum of 4% and cultured at 37°C and 200 r / min for 2 h to obtain the mother liquor of Bacillus subtilis.

[0032] (3) 1×10 8CFU / mL of *Lactobacillus plantarum* was inoculated into MRS liquid medium at an inoculum volume of 4% and cultured at 37°C and 180 r / min for 2 h to obtain the mother liquor of *Lactobacillus plantarum*.

[0033] (4) 1×10 8 CFU / mL of Bacillus coagulans was inoculated into LB liquid medium at an inoculation rate of 2.5% and cultured at 40°C and 180 r / min for 2 h to obtain the mother liquor of Bacillus coagulans.

[0034] Example 2: A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea.

[0035] A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea includes the following steps: (1) After mixing the traditional Chinese herbs, add 400 times the volume of distilled water and soak for 30 min, boil over high heat for 15 min, then simmer over low heat for 1 h, filter, add 200 times the volume of distilled water to the residue, simmer and filter in the same way, combine the two decoction filtrates, concentrate to a paste with an alcohol lamp, dissolve the paste with distilled water to 1 g / mL, and filter with a 0.22 μm filter to remove bacteria; (2) Add probiotic mother liquor to the traditional Chinese medicine mixture at 35-40℃. The weight ratio of the probiotic mother liquor to the traditional Chinese medicine mixture is 1:10, ferment at 37℃ and 180 r / min for 42 h.

[0036] The weight parts of each component in the Chinese herbal medicine are as follows: 2 parts dandelion, 2 parts isatis root, 1 part alisma, 1 part kudzu root, 2 parts scutellaria, 1 part licorice, and 1 part hawthorn.

[0037] By weight, 2 parts of the mother liquor of Lactobacillus rhamnosus, 2 parts of the mother liquor of Bacillus subtilis, 2 parts of the mother liquor of Lactobacillus plantarum, and 1 part of the mother liquor of Bacillus coagulans prepared in Example 1 were mixed to obtain a probiotic mother liquor.

[0038] Example 3: A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea.

[0039] A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea includes the following steps: (1) After mixing the traditional Chinese herbs, add 500 times the volume of distilled water and soak for 40 min, boil over high heat for 15 min, then simmer over low heat for 1 h, filter, add 300 times the volume of distilled water to the residue, simmer and filter in the same way, combine the two decoction filtrates, concentrate to a paste with an alcohol lamp, dissolve the paste with distilled water to 1 g / mL, and filter with a 0.22 μm filter to remove bacteria; (2) Add probiotic mother liquor to the traditional Chinese medicine mixture at 35-40℃. The weight ratio of the probiotic mother liquor to the traditional Chinese medicine mixture is 2:10, ferment at 37℃ and 180 r / min for 45 h.

[0040] The weight parts of each component in the Chinese herbal medicine are as follows: 4 parts dandelion, 4 parts isatis root, 2 parts alisma, 2 parts kudzu root, 3 parts scutellaria, 3 parts licorice, and 3 parts hawthorn.

[0041] By weight, 4 parts of the mother liquor of Lactobacillus rhamnosus, 3 parts of the mother liquor of Bacillus subtilis, 3 parts of the mother liquor of Lactobacillus plantarum, and 2 parts of the mother liquor of Bacillus coagulans prepared in Example 1 were mixed to obtain the probiotic mother liquor.

[0042] Example 4: Therapeutic effect of fermented traditional Chinese medicine preparations on swine epidemic diarrhea.

[0043] Severe diarrhea was observed in 7-day-old piglets at a pig farm. Based on the clinical symptoms, pathological changes, and laboratory diagnosis, the pathogen was confirmed to be porcine epidemic diarrhea virus (PEDV) infection. Sixty piglets diagnosed with the disease were randomly divided into three groups of 20 piglets each: a control group, experimental group 1 (Example 2), and experimental group 2 (Example 3). The experimental period was 14 days, and the treatment methods for each group were as follows:

[0044] Control group: 5 mL of normal saline was administered by gavage once a day for 7 consecutive days.

[0045] Experiment 1: 5 mL of the fermented traditional Chinese medicine preparation obtained in Example 1 was administered by gavage once a day for 7 consecutive days.

[0046] Experiment 2: 5 mL of the fermented traditional Chinese medicine preparation obtained in Example 2 was administered by gavage once a day for 7 consecutive days.

[0047] Table 1. Effects of fermented traditional Chinese medicine preparations on porcine epidemic diarrhea virus infection in piglets.

[0048]

[0049] Table 2. Effects of fermented traditional Chinese medicine preparations on the growth performance of piglets.

[0050]

[0051] The experimental results showed that, compared with the control group, the cure rate of experimental groups 1 and 2 was improved, and the mortality rate was reduced, with a cure rate between 60% and 70% and a mortality rate between 30% and 40%. The weight of piglets in the control group decreased by 12.99% before and after the experiment, while the weight of piglets in the experimental groups increased by 17.94% and 6.61%, respectively. This indicates that the fermented traditional Chinese medicine preparation has an anti-diarrheal effect and shortens the course of the disease.

[0052] Example 5: Efficacy evaluation experiment of fermented traditional Chinese medicine preparations in inhibiting PEDV at the cellular level.

[0053] 1. CCK8 assay for the toxicity of fermented traditional Chinese medicine preparations to Vero cells (African green monkey kidney cells).

[0054] Cytotoxicity assays were performed using the Cell Counting Kit-8 (Beijing Solarbio Science & Technology Co., Ltd.). The toxicity of fermented traditional Chinese medicine preparations to Vero cells was evaluated by concentration-based experiments. Different concentrations (0 mg / mL, 1.95 mg / mL, 3.91 mg / mL, 7.81 mg / mL, 15.63 mg / mL, 31.25 mg / mL, 62.5 mg / mL, 125 mg / mL, 250 mg / mL, and 500 mg / mL) of the fermented traditional Chinese medicine preparation were added to 96-well cell plates with a cell density of 80-90%. Each concentration was tested in triplicate. A blank control group was prepared using cell culture medium instead of the test solution. After incubation at 37℃ and 5% CO2 for 24 h, the culture supernatant was discarded, and 100 μL of cell culture medium and 10 μL of CCK-8 reagent were added. The cells were incubated at 37℃ and 5% CO2 for 2 h, and the OD was measured using a microplate reader. 450 The absorbance value at nm was used to process the data using Graphpad Prism 9.0, and the CC value of the fermented traditional Chinese medicine preparation was calculated. 50 (The half-maximal cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become diseased in a cytotoxicity assay). The cell viability formula is as follows:

[0055] Cell viability = drug group OD 450 nm value / blank group OD 450 nm value × 100

[0056] The results are as follows Figure 1 As shown, high concentrations of fermented traditional Chinese medicine preparations have certain toxicity to Vero cells. The half-maximal inhibitory concentration (CMC) of the fermented traditional Chinese medicine preparations on Vero cells is [not specified]. 50 The concentration was 15.63 mg / mL.

[0057] 2. Effect of fermented traditional Chinese medicine preparations on PEDV inhibition rate under blocking effect.

[0058] The inhibition rate of fermented traditional Chinese medicine preparations against PEDV was evaluated using a method of adding the drug first and then the virus, under the blocking effect. The CC (correlation coefficient) of the fermented traditional Chinese medicine preparations was used as the criterion. 50 To test the drug stock solution, it was serially diluted 2-fold with cell culture medium, with concentrations denoted as C1-C5, and the concentrations were 15.63 mg / mL, 7.81 mg / mL, 3.91 mg / mL, 1.95 mg / mL, and 0.98 mg / mL respectively (the same applies below). 100 μL of the fermented traditional Chinese medicine preparation and 100 μL of cell culture medium were added to each well of a 96-well cell culture plate containing a monolayer of Vero cells. Each concentration was tested in triplicate. After 12 hours, the fermented traditional Chinese medicine preparation was aspirated, and 100 μL of 100 TCID50 solution was added. 50Viral fluid (TCID50 is 10) -5.67 / 0.1mL), and 100μL of cell culture medium. Simultaneously, three wells were set up as blank control (NC group, no fermented herbal preparation, no virus inoculation), three wells as virus control (PC group, no fermented herbal preparation, virus inoculation), and three wells as cell-free control (only cell culture medium added). After 72 h, the liquid was aspirated, and 100μL of cell culture medium and 10μL of CCK-8 solution were added. The mixture was incubated at 37℃ and 5% CO2 for 1 h in the dark, and the OD of each well was measured. 450 The nm value was used to calculate the inhibition rate of fermented traditional Chinese medicine preparations against PEDV, using the following formula (the same applies below):

[0059] Inhibition rate of fermented traditional Chinese medicine preparations against PEDV = (OD of drug group) 450 nm value – Virus control group OD 450 (nm value) / (OD of cell control group) 450 nm value – Virus control group OD 450 (nm value) × 100.

[0060] The results are as follows Figure 2 The results showed that the fermented traditional Chinese medicine preparation under the blocking effect had a certain inhibitory effect on PEDV, and the inhibition rate of PEDV at a concentration of 7.81 mg / mL could reach up to 111.47%.

[0061] 3. Effect of fermented traditional Chinese medicine preparations on PEDV inhibition rate under inhibitory effects.

[0062] The inhibition rate of fermented traditional Chinese medicine preparations against PEDV was evaluated using a method of adding the virus first and then the drug, with the inhibitory effect measured by CC (coronavirus concentration) of the fermented traditional Chinese medicine preparations. 50 To test the drug stock solution, it was serially diluted 2-fold with cell culture medium, and the concentrations were expressed as C1-C5. 100 μL of 100 TCID45 was added to each well of a 96-well cell culture plate containing a monolayer of Vero cells. 50 TCID virus 50 10 -5.67 / 0.1mL), after adsorption at 37 ℃ and 5% CO2 for 4 h, the virus solution was discarded. 100 μL of fermented traditional Chinese medicine preparation and 100 μL of cell culture medium were added to each well, with three replicates for each concentration. Simultaneously, three wells were set up as blank control groups (no fermented traditional Chinese medicine preparation, no virus inoculation), three wells as virus control PC groups (no fermented traditional Chinese medicine preparation, virus inoculation), and three wells as cell-free control groups (only cell culture medium added) for zeroing. After 72 h, the liquid was aspirated, and 100 μL of cell culture medium and 10 μL of CCK-8 solution were added. The mixture was incubated at 37 ℃ and 5% CO2 for 1 h in the dark, and the OD of each well was measured. 450 The nm value was used to calculate the inhibition rate of the traditional Chinese medicine solution against PEDV.

[0063] The results are as follows Figure 3 The results showed that fermented traditional Chinese medicine preparations under the inhibitory effect had a certain inhibitory effect on PEDV, and the inhibition rate of PEDV could reach up to 81.95% when the concentration was 7.81 mg / mL.

[0064] 4. Effect of fermented traditional Chinese medicine preparations on PEDV inhibition rate under bactericidal effect.

[0065] The inhibition rate of fermented traditional Chinese medicine preparations against PEDV was evaluated by simultaneously adding drugs and viruses. The CC (correlation coefficient) of the fermented traditional Chinese medicine preparations was used as the criterion for its inhibitory effect. 50 To test the drug stock solution, it was serially diluted 2-fold with cell culture medium, and the concentrations were expressed as C1-C5. 100 μL of the fermented traditional Chinese medicine preparation and 100 μL of 100 TCID45 were simultaneously added to each well of a 96-well cell culture plate containing a monolayer of Vero cells. 50 TCID virus 50 10 -5.67 / 0.1mL), with 3 replicates for each concentration. Simultaneously, 3 wells were set up as blank control groups (no fermented herbal preparation, no virus inoculation), 3 wells as virus control PC groups (no fermented herbal preparation, virus inoculation), and 3 wells as cell-free control groups (only cell culture medium added) for zeroing purposes. After 72 h, the liquid was aspirated, and 100 μL of cell culture medium and 10 μL of CCK-8 solution were added. The mixture was incubated at 37℃ and 5% CO2 for 1 h in the dark, and the OD of each well was measured. 450 The nm value was used to calculate the inhibition rate of the traditional Chinese medicine solution against PEDV.

[0066] The results are as follows Figure 4 The results showed that fermented traditional Chinese medicine preparations under the bactericidal effect had a certain inhibitory effect on PEDV, and the inhibition rate of PEDV could reach up to 91.70% when the concentration was 7.81 mg / mL.

[0067] The above results confirm that this fermented traditional Chinese medicine preparation can exert antiviral effects in vitro by preventing viruses from entering cells, inhibiting viral replication, and directly killing viruses.

Claims

1. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea, characterized in that, It is prepared by fermenting Chinese herbal extracts using probiotic mother liquor; The herbal extract is prepared from the following herbs in parts by weight: 2-4 parts dandelion, 2-4 parts isatis root, 1-2 parts alisma, 1-2 parts kudzu root, 2-3 parts scutellaria, 1-3 parts licorice, and 1-3 parts hawthorn. The probiotic stock solution comprises, by weight: 2-4 parts of Lactobacillus rhamnosus stock solution, 2-3 parts of Bacillus subtilis stock solution, 2-3 parts of Lactobacillus plantarum stock solution, and 1-2 parts of Bacillus coagulans stock solution.

2. The fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 1, characterized in that: The Lactobacillus rhamnosus stock solution mentioned above is made by mixing 1×10 8 Lactobacillus rhamnosus at a concentration of CFU / mL was inoculated into MRS liquid medium at a 5% inoculum and cultured at 37°C and 180 rpm for 2 h. The aforementioned Bacillus subtilis stock solution is made by mixing 1×10⁻⁶ Bacillus subtilis stock solution with 1×10⁻⁶ Bacillus subtilis stock solution. 8 Bacillus subtilis at CFU / mL was inoculated into LB liquid medium at an inoculum of 4% and cultured at 37°C and 200 rpm for 2 h. The aforementioned *Lactobacillus plantarum* stock solution is made by mixing 1×10... 8 The *Lactobacillus plantarum* CFU / mL was inoculated into MRS liquid medium at a 4% inoculation rate and cultured at 37°C and 180 rpm for 2 h. The aforementioned Bacillus coagulans mother liquor is made by mixing 1×10 8 The resulting solution was prepared by inoculating 2.5% of CFU / mL Bacillus coagulans into LB liquid medium and culturing at 40°C and 180 rpm for 2 hours.

3. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 1 or 2, characterized in that: The Chinese herbal medicines mentioned are in the following proportions by weight: 2 parts dandelion, 2 parts isatis root, 1 part alisma, 1 part kudzu root, 2 parts scutellaria, 1 part licorice, and 1 part hawthorn.

4. The fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 3, characterized in that: The aforementioned Lactobacillus rhamnosus mother liquor consists of 2 parts, Bacillus subtilis mother liquor consists of 2 parts, Lactobacillus plantarum mother liquor consists of 2 parts, and Bacillus coagulans mother liquor consists of 1 part.

5. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 1, 2, or 4, characterized in that: The herbal extract is prepared by the following method: the herbal extract is soaked in 400-500 times its volume of distilled water for 30-60 minutes, boiled for 15 minutes, decocted for 1 hour, filtered, and then 200-300 times its volume of distilled water is added to the residue. The decoction and filtration are carried out in the same way. The two decoction filtrates are combined and concentrated to a paste. The paste is dissolved in distilled water to a concentration of 1 g / mL and then filtered to remove bacteria.

6. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 5, characterized in that: Sterilization is achieved by filtration using a 0.22μm filter.

7. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 1, characterized in that: The fermentation is carried out at 35-40℃; the weight ratio of the probiotic mother liquor to the herbal extract is 1-2:

10.

8. A fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea according to claim 7, characterized in that: The fermentation process was carried out at 37°C with shaking at 180 rpm for 40-45 hours.

9. The application of the fermented traditional Chinese medicine preparation for treating swine epidemic diarrhea as described in claim 1 in the preparation of drugs for preventing and treating swine epidemic diarrhea virus infection.

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