A fermentation liquor for inhibiting bovine viral diarrhea virus and a preparation method and application thereof

Abstract

The application provides a fermentation liquor for inhibiting bovine viral diarrhea virus and a preparation method and application thereof, and belongs to the field of virus inhibiting drugs. The fermentation liquor is a co-fermentation product of patrinia and saccharomyces cerevisiae. The co-fermentation product of saccharomyces cerevisiae and patrinia can inhibit the infection of bovine viral diarrhea virus and has a good prevention and treatment effect on intestinal damage caused by bovine viral diarrhea virus infection. Compared with patrinia or saccharomyces cerevisiae alone, the co-fermentation product shows a stronger synergistic effect, can effectively prevent and inhibit bovine viral diarrhea virus infection, and is not prone to drug resistance. The preparation method is simple, raw materials are easy to obtain, and is suitable for developing antibacterial drugs and feed additives.

Description

Technical Field

[0001] This invention belongs to the field of virus inhibitory drug technology, specifically relating to a fermentation broth that inhibits bovine viral diarrhea virus, its preparation method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Bovine viral diarrhea virus (BVDC) Bovine Viral Diarrhea Virus Bovine viral diarrhea virus (BVDV) is a serious pathogen that severely damages the cattle industry, causing various clinical symptoms in cattle and resulting in enormous economic losses to the global livestock sector. Cattle are the primary susceptible animals, and cattle of all ages and breeds can be infected, with young cattle being more susceptible. After invading the body, BVDV replicates first in the nasal and oral mucosa, then spreads throughout the body via the blood and lymphatic system to all organs and tissues. The virus can damage the body's immune system, leading to immunosuppression and reducing the body's resistance to other pathogens, thus triggering secondary infections. Acutely infected cattle typically exhibit fever (temperature can rise to 40-42°C), lethargy, loss of appetite or anorexia, rapid breathing, and respiratory symptoms such as coughing. Diarrhea is one of the typical symptoms of BVDV infection; feces are watery or pasty, sometimes containing blood and mucus. Severe diarrhea can lead to dehydration, electrolyte imbalance, and acid-base imbalance in affected cattle. Some sick cattle may also develop symptoms such as oral mucosal ulcers and laminitis.

[0004] Currently, there are no specific antiviral drugs for bovine viral diarrhea virus infection. Clinical treatment mainly focuses on symptomatic and supportive care, controlling secondary infections, and strengthening nursing care, aiming to alleviate the clinical symptoms of sick cattle, improve their resistance, and reduce mortality. Intravenous injections of compound sodium chloride solution and glucose saline can be administered, while electrolyte supplementation is adjusted according to blood biochemical indicators to correct acid-base imbalance. If necessary, nutritional solutions can be administered orally or intravenously to supplement the sick cattle with energy and nutrients and enhance their resistance.

[0005] In her article "Clinical Symptom Prevention and Treatment of Bovine Viral Diarrhea," Zhu Hui mentions that a suitable traditional Chinese medicine formula can be used, consisting of 200g of rhubarb leaves, 200g of isatis root, 300g of *Senecio scandens*, 300g of *Patrinia scabiosaefolia*, 300g of *Lysimachia christinae*, 150g of *Citrus aurantium*, 130g of *Carthamus tinctorius*, 150g of *Scutellaria baicalensis*, 160g of rhubarb, 130g of *Coptis chinensis*, 130g of *Phellodendron chinense*, 130g of *Gardenia jasminoides*, and 120g of white peony root. This formula should be decocted in water and used once daily. The remaining dregs should be added to the feed, allowing the cattle to graze freely. This is beneficial for enhancing the cattle's antiviral capabilities and preventing the outbreak of viral diarrhea. However, as traditional Chinese medicine, the bitter taste of this formula may affect the cattle's willingness to eat.

[0006] Therefore, in order to prevent and treat a large number of calf infections of bovine viral diarrhea virus, it is imminent to find a safe and effective new type of anti-calf viral diarrhea drug or treatment method. SUMMARY

[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a fermentation broth for inhibiting bovine viral diarrhea virus and a preparation method and application thereof. The fermentation broth is a co-fermentation broth of Saccharomyces cerevisiae and Patrinia, which significantly improves the prevention and inhibition effect on bovine viral diarrhea virus infection, enhances the immune function of the body, and protects the integrity of the intestinal barrier, thereby providing a new technical solution to solve the problems in the prior art.

[0008] As a first aspect of the present application, a fermentation broth for inhibiting bovine viral diarrhea virus is provided, and the fermentation broth is a co-fermentation product of Patrinia and Saccharomyces cerevisiae. The Saccharomyces cerevisiae uses a Saccharomyces cerevisiae strain Saccharomyces cerevisiae Hansen In an embodiment, the Saccharomyces cerevisiae is a strain purchased from North China Biological Engineering Center of Industrial Microorganisms, Henan Province (BNCC), with the number BNCC336054; the number of viable bacteria is 10 8 CFU / mL~10 9 CFU / mL.

[0009] The present application experiment found that, compared with other types of traditional Chinese medicine, the inhibition effect of Patrinia on bovine viral diarrhea virus after fermentation is much higher than that of other types of traditional Chinese medicine.

[0010] The Patrinia is a Patrinia decoction.

[0011] As a second aspect of the present application, a preparation method of the fermentation broth for inhibiting bovine viral diarrhea virus is provided, and Patrinia and Saccharomyces cerevisiae are co-fermented to obtain a fermentation product.

[0012] Further, the preparation method is as follows: preparing a Patrinia decoction, mixing the Patrinia decoction with a liquid medium, inoculating Saccharomyces cerevisiae, and co-fermenting.

[0013] In some embodiments of the present application, the mixing ratio of the Patrinia decoction and the liquid medium is 1:4-20 by volume, and preferably 6 vol% of the Patrinia decoction is added to the medium.

[0014] In some embodiments of the present application, the inoculation conditions of Saccharomyces cerevisiae are as follows: the inoculation amount is 5%-10%, and preferably the inoculation amount is 5%. Preferably, the inoculated bacterial liquid is a logarithmic growth phase bacterial liquid activated for 24-36 hours.

[0015] In some embodiments of the present invention, the co-fermentation process parameters include: a pH range of 6.5-7.2, preferably 6.8-7.0; a fermentation temperature range of 30℃-40℃, preferably 37℃; a fermentation time range of 24h-96h, preferably 72h; and an anaerobic static fermentation method. Experimental comparisons of the present invention have shown that the virus inhibition rate under these conditions is significantly higher than under other conditions.

[0016] In some embodiments of the present invention, the preparation method of the Patrinia scabiosifolia decoction includes: crushing Patrinia scabiosifolia into coarse powder, sieving it, adding distilled water, soaking it, placing it in an extraction tank, heating it to boiling, maintaining a gentle boil while decocting, filtering it after decoction, and collecting the filtrate; repeating the decoction of the residue according to the above method, and combining the filtrates; concentrating the combined filtrate under reduced pressure to obtain the concentrated Chinese medicine liquid, which is the Patrinia scabiosifolia decoction.

[0017] In some embodiments of the present invention, the preparation method of Patrinia scabiosifolia decoction includes the following steps:

[0018] S1. Raw material pretreatment:

[0019] Crush Patrinia scabiosifolia into coarse powder and pass it through a 40-mesh sieve; add distilled water at a material-to-liquid ratio of 1:10 (g / mL) and soak for at least 12 hours;

[0020] S2. Extraction process: Place the soaking liquid in an extraction tank; heat to boiling and then maintain a gentle boil; decoct for 1.5-2 hours for the first time; filter with gauze and collect the filtrate; add water to the residue and decoct again 1-2 times, then combine the filtrates;

[0021] S3. Concentration treatment: The combined filtrate prepared in step S2 is concentrated under reduced pressure, with the temperature controlled at 60℃±5℃, until the relative density is 1.2±0.1;

[0022] Finally, sterilize the concentrated Chinese medicine solution at 121℃ for 20-25 minutes and cool it to room temperature for later use.

[0023] As a third aspect of the invention, the use of the fermentation broth that inhibits bovine viral diarrhea virus in the preparation of a product for inhibiting bovine viral diarrhea virus is provided, the product being selected from pharmaceuticals or feed additives.

[0024] As a fourth aspect of the invention, the use of the fermentation broth that inhibits bovine viral diarrhea virus in the preparation of products for the prevention, relief and / or treatment of diseases caused by bovine viral diarrhea virus is provided, said products being selected from pharmaceuticals or feed additives.

[0025] In some embodiments of the present invention, the fermentation broth has a preventive effect, which is manifested in preventing bovine viral diarrhea virus infection, enhancing animal immunity, improving intestinal health, and improving production performance.

[0026] In some embodiments of the present application, the fermentation broth has a therapeutic effect, which is manifested in alleviating symptoms of infection, reducing inflammatory response, repairing intestinal damage, improving clinical prognosis, etc.

[0027] In some embodiments of the present application, the product is in the form of a liquid preparation, such as an injection, an oral solution, etc., or is in the form of a solid preparation, such as a lyophilized powder, a granule, etc.

[0028] In some embodiments of the present application, the composition of the product can be added with suitable excipients according to the type of preparation. For example, a protective agent can be added as needed, an excipient and a stabilizer can be added as needed, and the content of the active ingredient can be adjusted as needed when using excipients.

[0029] In some embodiments of the present application, the drug can be a veterinary drug, an anti-infective drug, an intestinal drug, an immunomodulator, etc.

[0030] In some embodiments of the present application, the feed additive can be a microecological preparation, a prophylactic additive, a nutrition-promoting agent, an immune-enhancing agent, etc.

[0031] The beneficial effects achieved by one or more embodiments of the present application are as follows:

[0032] 1. The present application provides a fermentation broth for inhibiting bovine viral diarrhea virus. In the treatment of bovine viral diarrhea virus, the fermentation broth prepared by co-fermentation of traditional Chinese medicine and probiotics achieves a synergistic effect, overcoming the problem of limited effect when used alone. Meanwhile, the fermentation broth can be prepared into various dosage forms, facilitating administration to livestock.

[0033] 2. The fermentation broth prepared by co-fermentation of Saccharomyces cerevisiae and Patrinia scabiosae Fisch. provided by the present application exhibits significant technical effects and excellent immunomodulatory effects. In terms of enhancing immune function of the body, the fermentation broth can significantly increase secretion of antiviral factor IFN-β and reduce production of pro-inflammatory factor TNF-α. In terms of immune cell regulation, the present application can enhance CD4 + IL-4 + T cells to enhance humoral immunity and maintain CD8 + IFN-γ T cells to enhance cellular immunity, exhibiting stronger immunomodulatory capacity than using either component alone. In terms of protecting the intestinal barrier, the present application can significantly increase expression of tight junction proteins ZO-1 and Occludin, reduce viral load in the intestinal tract of mice, maintain the integrity of the intestinal tissue morphology, and reduce congestion, edema, and mucosal damage. The above effects are superior to using either component alone.

[0034] 3, In addition, the application has the characteristics of simple and feasible preparation method and wide raw material sources, and can be used for drug development and suitable for the development of feed additives. The above technical effects and excellent effects have been fully verified by weight change, survival rate, immune index, histopathology and other experimental indexes, indicating that the application has significant practical value. BRIEF DESCRIPTION OF DRAWINGS

[0035] The drawings constituting a part of the specification illustrate the present application and, together with the description, serve to explain the principles of the application. They are intended solely for purposes of illustration and should not be construed as a limitation of the present application.

[0036] Figure 1 The figure is a weight change curve of mice.

[0037] Figure 2 The figure is a fecal diarrhea score chart of mice.

[0038] Figure 3 The figure is a survival curve of mice.

[0039] Figure 4 The figure is a comparison chart of the damage of the spleen of mice in each group.

[0040] Figure 5 The figure is a chart of the change of cytokines in the serum of mice after infection with bovine viral diarrhea virus, A: comparison chart of the content of IFN-β in the serum of mice; B: comparison chart of the content of IL-1β in the serum of mice; wherein, P <0.0001; *** P <0.001; ns (no significance) P >0.05.

[0041] Figure 6 The figure is a comparison chart of the number of CD3 + CD4 + IL-4 T cells in the spleen of mice in each group; wherein, P <0.0001; ** P <0.01.

[0042] Figure 7 The figure is a comparison chart of the number of CD3 + CD8 + IFN-γ T cells in the spleen of mice in each group; wherein, P <0.01; ns (no significance) P >0.05.

[0043] Figure 8The following are comparisons of the relative expression levels of tight junction proteins in the intestines of mice in different experimental groups: A: Comparison of the relative expression levels of ZO-1 in the small intestine; B: Comparison of the relative expression levels of Occludin in the small intestine; among them, **** P <0.0001; *** P <0.001;** P <0.01; ns (no significance) P >0.05.

[0044] Figure 9 This image shows a comparison of viral load in the small intestine and colon of mice in each group. **** P <0.0001; *** P <0.001;* P <0.05.

[0045] Figure 10 The images show pathological tissue sections of the small intestine of mice in each group. A: Pathological tissue section of the small intestine of mice in the PBS group; B: Pathological tissue section of the small intestine of mice in the Patrinia scabiosifolia group; C: Pathological tissue section of the small intestine of mice in the Saccharomyces cerevisiae group; D: Pathological tissue section of the small intestine of mice in the co-fermentation group; E: Pathological tissue section of the small intestine of mice in the control group. Detailed Implementation

[0046] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] Example 1

[0048] Screening of fermentation broths that inhibit the growth of bovine viral diarrhea virus

[0049] 1. Experimental Materials: Five Chinese medicinal herbs were selected as research subjects: Patrinia scabiosifolia, Chrysanthemum indicum, Sophora flavescens, Indigo naturalis, and Euphorbia humifusa. These herbs were all purchased from local, reputable Chinese medicine pharmacies and were identified as meeting the relevant standards of the Chinese Pharmacopoeia.

[0050] Strains: Bovine viral diarrhea virus (BVDV) Bovine Viral Diarrhea Virus The virus strain used was isolated and identified by our team. This strain was stored in liquid nitrogen and used to infect MDCK cells every 1-2 weeks to maintain its activity. Before the experiment, bovine viral diarrhea virus (BVDV) was inoculated into 6-well MDCK cells and cultured in a carbon dioxide anaerobic incubator for 24-48 hours to observe cytopathic effects. In subsequent experiments, it will be referred to as BVDV.

[0051] The brewing yeast used in this invention is a brewing yeast strain ( Saccharomyces cerevisiae Hansen) was purchased from North China Industrial Microbial Strain Engineering Technology Research Center (BNCC), Henan Province, with the number BNCC336054. The strain was preserved on a yeast culture medium slope and stored in a refrigerator at 4°C, with monthly subculturing. When used, the S. cerevisiae was inoculated in a yeast liquid medium and incubated at 30°C for 24-36 h, then transferred to fresh yeast liquid medium at an inoculation amount of 2%-3% and further incubated to the logarithmic growth phase for fermentation experiments.

[0052] 2. Main reagents: yeast culture medium (containing malt extract, glucose, yeast extract powder, peptone, sodium chloride, sodium hydroxide, etc.), all of which were analytical pure reagents purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., and were prepared into yeast liquid medium by adding water.

[0053] 3. Main instruments: HZQ-F160 full-temperature shaking incubator (Harbin Donglian Electronic Technology Development Co., Ltd.), SPX-25B biochemical incubator (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), SW-CJ-2FD double single-side clean bench (Suzhou Purification Equipment Co., Ltd.), LDZX-50KBS vertical pressure steam sterilizer (Shanghai Shen'an Medical Instrument Factory), TDL-5-A centrifuge (Shanghai Anting Scientific Instrument Factory), RE-52AA rotary evaporator (Shanghai Yalong Biochemical Instrument Factory), and Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, USA).

[0054] 4. Experimental method

[0055] 1) Preparation of traditional Chinese medicine liquid

[0056] Five kinds of traditional Chinese medicines, including Herba Patriniae, Flos Chrysanthemi Indici, Radix Sophorae, Indigo Naturalis, and Herba Euphorbiae Humifusae, were ground into coarse powder by a grinder and passed through a 40-mesh sieve. A certain amount of traditional Chinese medicine coarse powder was weighed and added with distilled water according to a solid-liquid ratio of 1:10 (g / mL) for 24 h of soaking. Then, the soaking liquid was placed in a multifunctional extraction tank and heated to boiling, and kept at a state of slight boiling for 2 h of decoction. After the decoction was completed, the filtrate was collected by filtering through 4 layers of gauze. The filter residue was repeatedly decocted once according to the above method, and the filtrates were combined. The combined filtrate was concentrated under reduced pressure to a relative density of 1.1 (60°C), about 1 / 5 of the original volume, to obtain a traditional Chinese medicine concentrate. Finally, the traditional Chinese medicine concentrate was sterilized at 121°C for 20 min, cooled to room temperature, and used as a reserve for co-fermentation with S. cerevisiae and in vitro antiviral experiments in the examples.

[0057] 2) Cultivation and fermentation of S. cerevisiae with traditional Chinese medicine

[0058] S. cerevisiae preserved on the slant of yeast culture medium was inoculated into the liquid medium of yeast, and incubated at 30°C for 24 h for activation. The activated S. cerevisiae was inoculated into fresh liquid medium of yeast at a volume ratio of 2%, and incubated until the logarithmic growth phase to obtain the seed liquid of S. cerevisiae. The prepared concentrated liquid of traditional Chinese medicine was mixed with the liquid medium of yeast at a volume ratio of 10%, and the pH value was adjusted to 6.5. Then, the S. cerevisiae seed liquid was inoculated at a volume ratio of 10%, and anaerobic fermentation was carried out at 37°C for 72 h. During the fermentation process, samples were taken every 24 h to determine the number of S. cerevisiae bacteria and the pH value of the fermentation liquid to monitor the fermentation process.

[0059] 3) In vitro antiviral experiment

[0060] The virus load of the isolated bovine viral diarrhea virus was determined by real-time fluorescent quantitative RT-PCR method. First, a bottle of well-grown MDCK cells was taken. The cell culture solution was first poured out, washed once with PBS, and then dispersed by trypsin to prepare a cell suspension. The cells were inoculated into a 96-well culture plate, 100 µL of cell suspension was added to each well, and then placed in a 37°C, 5% CO2 cell incubator for culture. When the MDCK cells were cultured to 70% of the cell monolayer, they could be used for the experiment. The virus was diluted 10 times by cell maintenance solution, and added to the MDCK cell monolayer of the 96-well plate, with 8 repeated holes for each dilution. Eight control holes were set and cell maintenance solution was added. Incubate in a 37°C, 5% CO2 cell incubator. Observe the cytopathic effect daily and record the results until no cytopathic effect occurs. The Reed-Muench formula was used to calculate the tissue culture infectious dose 50% (TCID 50 .

[0061] Then, well-grown MDCK cells were prepared in a 96-well plate, and when the MDCK cells were cultured to 70% of the cell monolayer, they could be used for the experiment. The prepared traditional Chinese medicine liquid was added to the 96-well plate, with 6 repeated holes for each traditional Chinese medicine liquid, 100 µL of traditional Chinese medicine liquid was added to each well, and virus control group and normal MDCK cell control were set. 100 TCID 50 of virus liquid was added to the virus control holes; cell maintenance solution was added to the control holes containing normal MDCK cells. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 12 h. After taking it out, the virus was added to the cells and adsorbed at 37°C for 2 h. Then the virus liquid was discarded, and the drug-containing maintenance solution was added to the cell holes. Put it into a 37°C, 5% CO2 incubator for continuous culture, observe and record the cytopathic effect daily, and do MTT test after the cells do not have cytopathic effect. According to the resistance of traditional Chinese medicine liquid to virus infection, the traditional Chinese medicine with the strongest antiviral effect was screened.

[0062] The results of the pre-fermentation of Saccharomyces cerevisiae and different Chinese medicines are shown in Table 1, and the pH value changes are shown in Table 2:

[0063] Table 1 Monitoring of the change in the number of bacteria in the fermentation of Saccharomyces cerevisiae and Chinese medicines (10 9 CFU / mL)

[0064]

[0065] Table 2 Monitoring of the change in the pH value in the fermentation of Saccharomyces cerevisiae and Chinese medicines

[0066]

[0067] As shown in Tables 1 and 2, under the co-fermentation of S. cerevisiae and patrinia, S. cerevisiae can grow and reproduce in patrinia, and the growth and reproduction are far superior to those of other Chinese medicine species; at the same time, after the co-fermentation of patrinia and S. cerevisiae, the pH value is significantly reduced.

[0068] 4) In vitro inhibition effect of Chinese medicine on bovine viral diarrhea virus

[0069] The MTT method uses a microplate reader to detect the absorbance at OD560, so as to determine the virus inhibition rate. The virus inhibition rate calculation formula is: virus inhibition rate (%) = (drug treatment value OD value - virus control group OD value) / (normal cell group OD value - virus control group OD value) x 100%. The results are shown in Table 3.

[0070] Table 3 Inhibition of bovine viral diarrhea virus by each fermented Chinese medicine liquid

[0071]

[0072] As shown in Table 3, under the same fermentation conditions, the inhibition of bovine viral diarrhea virus by patrinia after fermentation is much higher than that of other Chinese medicine species, and the virus inhibition rate is 92.5%. Therefore, in the subsequent fermentation of Chinese medicine by S. cerevisiae, patrinia is used for fermentation.

[0073] Example 2

[0074] Comparison of different fermentation conditions

[0075] 1. Fermenting Chinese medicinal materials with S. cerevisiae, and comparing fermentation time, fermentation temperature, and strain inoculation amount to obtain the best fermentation method, and performing bovine viral diarrhea virus inhibition experiments on the fermentation liquid of the strain obtained by fermentation.

[0076] 1) Comparison of fermentation time:

[0077] In 50 mL of yeast liquid medium, 6% by volume of patrinia Chinese medicine liquid was added, the pH value was adjusted to 6.8, 121℃ high pressure sterilization for 25 min, and 5% of the bacterial liquid (5 x 109 CFU). Incubate at 37℃ for 24h, 48h, 72h, 96h. Take 100μL of the fermented Chinese medicine liquid and add to MDCK cells, and infect 100 TCID50 of BVDV at the same time. Determine the virus inhibition rate by MTT method using an enzyme marker to detect the absorbance at OD560. 50 9 CFU). Incubate at 37℃ for 24h, 48h, 72h, 96h. Take 100μL of the fermented Chinese medicine liquid and add to MDCK cells, and infect 100 TCID50 of BVDV at the same time. Determine the virus inhibition rate by MTT method using an enzyme marker to detect the absorbance at OD560.

[0078] 2) Comparison of fermentation temperature:

[0079] Add 6% volume of the Chinese medicine liquid of Herba Patriniae to 50mL of yeast liquid medium, adjust the pH value to 6.8, sterilize at 121℃ for 25min, inoculate 5% of the bacterial liquid (5x10 9 CFU), adjust the pH value to 6.8. Incubate at 30℃, 35℃, 37℃, 40℃, 42℃ according to the optimal fermentation time selected. Take 100μL of the fermented Chinese medicine liquid and add to MDCK cells, and infect 100 TCID50 of BVDV at the same time. Determine the virus inhibition rate by MTT method using an enzyme marker to detect the absorbance at OD560.

[0080] 3) Comparison of inoculum size:

[0081] Add 10% volume of the Chinese medicine liquid of Herba Patriniae to 50mL of yeast liquid medium, adjust the pH value to 6.8, sterilize at 121℃ for 25min, inoculate 0.01%, 0.1%, 1%, 10% of the bacterial liquid. Take 100μL of the fermented Chinese medicine liquid and add to MDCK cells, and infect 100 TCID50 of BVDV at the same time. Determine the virus inhibition rate by MTT method using an enzyme marker to detect the absorbance at OD560. The results are shown in Tables 4-6.

[0082] Table 4 Inhibition rate of Chinese medicine fermented liquid on bovine viral diarrhea virus at different fermentation times

[0083]

[0084] As shown in Table 4, the determination results of the inhibition rate of Chinese medicine fermented liquid on bovine viral diarrhea virus at different fermentation times showed that the inhibition rate increased first and then decreased with the extension of fermentation time. The inhibition rate of the fermentation group was 84.1% at 48h of fermentation; the inhibition rate was the largest at 72h of fermentation, which was 92.5%; the inhibition rate was 89.9% at 96h of continuous fermentation. This shows that the fermentation time has a significant effect on the inhibition effect of the Chinese medicine fermented liquid, and the inhibition effect of the Chinese medicine fermented liquid on bovine viral diarrhea virus is the strongest at about 72h of fermentation.

[0085] Table 5 Inhibition rate of traditional Chinese medicine fermentation broth on bovine viral diarrhea virus at different fermentation temperatures

[0086]

[0087] As shown in Table 5, the determination results of the inhibition rate of traditional Chinese medicine fermentation broth on bovine viral diarrhea virus at different fermentation temperatures show that, with the increase of fermentation temperature, the inhibition rate presents a trend of first increasing and then decreasing. When the fermentation temperature is 37℃, the inhibition rate of the co-fermentation group is 92.3%, and the inhibition rate reaches the maximum. When the fermentation temperature is increased to 42℃, the inhibition rate is reduced to 89.3%. This shows that the fermentation temperature has a significant influence on the inhibition effect of traditional Chinese medicine fermentation broth, and the inhibition effect of traditional Chinese medicine on bovine viral diarrhea virus is the strongest at about 37℃.

[0088] Table 6 Inhibition rate of traditional Chinese medicine fermentation broth on bovine viral diarrhea virus at different inoculation amounts of bacteria liquid

[0089]

[0090] As shown in Table 6, the determination results of the inhibition rate of traditional Chinese medicine fermentation broth on bovine viral diarrhea virus at different inoculation amounts of Saccharomyces cerevisiae bacteria liquid show that, with the increase of the inoculation amount of bacteria liquid, the inhibition rate presents a trend of first increasing and then decreasing. When the inoculation amount of bacteria liquid is 1%, the inhibition rate is 85.4%. When the inoculation amount of bacteria liquid is 10%, the inhibition rate is the maximum, which is 92.1%. This shows that the inoculation amount of bacteria liquid has a significant influence on the inhibition effect of traditional Chinese medicine fermentation broth, and the inhibition effect of traditional Chinese medicine on bovine viral diarrhea virus is the strongest at about 10%.

[0091] Example 3

[0092] Inhibition of Saccharomyces cerevisiae fermented traditional Chinese medicine preparation on bovine viral diarrhea virus infection in mice

[0093] In this example, Saccharomyces cerevisiae and Herba Patriniae were co-fermented (co-fermentation group), and the mice were fed with PBS group, Saccharomyces cerevisiae group, and Herba Patriniae group, respectively, and the protection test was carried out. Through ELISA, Flow Cytometry and H&E staining detection methods, the changes of mouse body weight, the contents of IFN-β, TNF-α, IL-1β and IL-10 in serum, the activation of immune organs and immune cells in the body, and the pathological changes of mouse small intestine were detected and analyzed, and the protection effect of co-fermentation product of Herba Patriniae and Saccharomyces cerevisiae on bovine viral diarrhea virus infection in mice was studied.

[0094] 1. Experimental animals

[0095] Fifty healthy 30-day-old mice were selected and randomly divided into 5 groups, 10 mice in each group, and were isolated and fed. The mice were fed with water at 8:00, 14:00 and 20:00 every day.

[0096] 2. Reagents

[0097] Mouse interleukin 1 beta (IL-1β), mouse interferon beta (IFN-β), mouse tumor necrosis factor alpha (TNF-α), mouse interleukin 10 (IL-10) ELISA kits were purchased from Enzyme Free (Jiangsu) Industrial Co., Ltd. Mouse CD3-PE-Cy7 antibody, CD4-PE antibody, CD8-FITC antibody (BD company); 1640 cell culture medium (Hyclone); blocking mouse serum was stored in the laboratory; antibody diluent: 1% BSA; red blood cell lysis solution, Fluor 488 labeled goat anti-rabbit IgG (H + L) (Biyun Tian Biotechnology Co., Ltd.); FACS solution (1000 mL of PBS for cell culture, 10 mL of FBS, 0.9 g of sodium azide); AXYGEN PCRSTRIP TUBES (American Corning Company); Mini BEST Viral RNA / DNA Extraction Kit Ver5.0 (Code No. 9766 Takara); blocking goat serum; DAPI staining solution; anti-quenching mounting agent; 4% paraformaldehyde solution; sodium citrate antigen retrieval solution.

[0098] 3. Main experimental equipment:

[0099] ABI Prism 7500 QT-qPCR instrument (USA) ABI Company; Heparin sodium anticoagulation tube, coagulation tube purchased from (Jiangsu) Kangjian Medical Supplies Co., Ltd.; Tissue embedding machine, paraffin section machine, DMi8 fluorescence inverted microscope purchased from (Germany) Leica Instrument Co., Ltd.; Tissue homogenizer is domestic.

[0100] 4. Experimental method:

[0101] 1) Experimental scheme

[0102] Experimental animals were divided into groups: PBS group (n=10), Saccharomyces cerevisiae group (n=10), Herba Patriniae group (n=10), co-fermentation group (n=10), control group (n=10); male and female were randomly allocated, and the grouping is shown in Table 7 below.

[0103] Table 7 Animal experiment grouping

[0104]

[0105] Immunization and challenge procedure:

[0106] Each group of mice was immunized at 1d, 3d, 5d, 7d, 9d, 11d, and 13d. Except that the PBS group and the control group were given 5mL PBS by gavage, the other groups were given the corresponding drugs by gavage. The Saccharomyces cerevisiae group was given 1×10 9CFU / mL, 0.5 mL of Baijiaocao per mouse, 0.5 mL of Baijiaocao fermented liquid per mouse.

[0107] The preparation method of the Saccharomyces cerevisiae fermentation liquid is as follows: 5% of the Saccharomyces cerevisiae (2.5 mL) is inoculated in 50 mL of yeast liquid medium, and fermentation is carried out at 37°C for 72 h. After the fermentation is completed, the whole fermentation liquid is taken after being fully shaken, and the viable cell count is ensured to be 1×10 9 CFU / mL.

[0108] The preparation method of the Baijiaocao medicinal liquid is as follows: Baijiaocao is crushed into coarse powder with a pulverizer, and the coarse powder is sieved through a 40-mesh sieve. 25 g of the coarse powder is added to 250 mL of distilled water according to the solid-liquid ratio of 1:10 (g / mL), and the mixture is soaked for 12 h. Then, the soaking liquid is placed in a multifunctional extraction tank, and heated to boiling. The mixture is kept at a state of gentle boiling for 1.5 h. After the boiling is completed, the mixture is filtered through 4 layers of gauze, and the filtrate is collected. The residue is boiled again according to the above method, and the filtrate is collected. The combined filtrate is concentrated under reduced pressure to a relative density of 1.1 (60°C), and the volume is reduced to about 1 / 5 of the original volume, to obtain 50 mL of a medicinal concentrated liquid. Finally, the medicinal concentrated liquid is sterilized at 121°C for 20 min, and cooled to room temperature. The Baijiaocao medicinal liquid is obtained, and is fully shaken before use.

[0109] The preparation method of the Baijiaocao medicinal liquid is as follows: Baijiaocao is crushed into coarse powder with a pulverizer, and the coarse powder is sieved through a 40-mesh sieve. 25 g of the coarse powder is added to 250 mL of distilled water according to the solid-liquid ratio of 1:10 (g / mL), and the mixture is soaked for 12 h. Then, the soaking liquid is placed in a multifunctional extraction tank, and heated to boiling. The mixture is kept at a state of gentle boiling for 1.5 h. After the boiling is completed, the mixture is filtered through 4 layers of gauze, and the filtrate is collected. The residue is boiled again according to the above method, and the filtrate is collected. The combined filtrate is concentrated under reduced pressure to a relative density of 1.1 (60°C), and the volume is reduced to about 1 / 5 of the original volume, to obtain 50 mL of a medicinal concentrated liquid. Finally, the medicinal concentrated liquid is sterilized at 121°C for 20 min, and cooled to room temperature. The Baijiaocao medicinal liquid is obtained, and is fully shaken before use. 9 CFU / mL.

[0110] After immunization, the body weight of the mice is measured once a day, and the average daily weight gain is calculated. On day 14, the mice in the PBS group are given 0.5 mL of PBS by gavage, and the mice in the other groups are given 0.5 mL of bovine viral diarrhea virus by gavage (the virus titer is 100 TCID 50 ).

[0111] 2) Growth performance index detection

[0112] Body weight: The mice are weighed once a day, and the body weight change data is recorded and the body weight change curve is drawn. The feces of the mice are recorded every day, and the feces are scored. The feces scoring method is as follows: the rectal feces of each mouse are collected and evaluated, and the photos are used by professional veterinarians to score the feces consistently. The feces consistency score ranges from 1 to 4, wherein 1 = normal; 2 = mild diarrhea; 3 = moderate diarrhea; and 4 = severe watery diarrhea.

[0113] 3) The detection method of immune indicators IFN-β, TNF-α, IL-1β, IL-10

[0114] After infection with bovine viral diarrhea virus, blood was taken from the eyeball of each mouse. 1 mL of blood was collected and centrifuged in a centrifuge tube to separate the serum and store it at -80. The content of IFN-β, TNF-α, IL-1β, IL-10 in the serum was detected by ELISA method.

[0115] 4) Preparation of single cell suspension

[0116] Preparation of spleen single cell suspension: part of the spleen was gently ground to obtain a single cell suspension, which was passed through a 200 mesh filter into a corresponding 15 mL centrifuge tube, balanced, centrifuged at 1650 rpm, 4°C for 5 min. Discard the supernatant, resuspend the cells with 1 mL of red blood cell lysis solution, lyse for 10 min on ice, take out after 5 min, shake for 30 s. Add 10 mL of PBS buffer to terminate, balance, centrifuge at 1650 rpm, 4°C for 5 min. Discard the supernatant, resuspend the cells with 1 mL of FACS buffer, and count.

[0117] Preparation of mesenteric lymph node single cell suspension: the lymph nodes were gently ground into a single cell suspension, which was passed through a 200 mesh filter into a corresponding 15 mL centrifuge tube, balanced, centrifuged at 1650 rpm, 4°C for 5 min. Discard the supernatant, resuspend the cells with 0.2 mL of FACS buffer, and count.

[0118] 5) Flow cytometry detection

[0119] Flow antibody staining

[0120] (1) Divide the above single cell suspensions into tubes, ensuring 1×10 6 cells per tube (5×10 5 for peripheral blood), with a total volume of 100 μL.

[0121] (2) According to the antibody titer, add the corresponding volume of flow cytometry antibody of cell surface marker.

[0122] (3) After shaking and mixing, avoid light, place at 4°C for 30 min.

[0123] (4) After labeling, add 3.5 mL of ice-cold FACS buffer in the tube, centrifuge at 1650 rpm, 4°C for 5 min, discard the supernatant, resuspend the cells with a small amount of FACS buffer.

[0124] (5) Repeat step (4) once to completely remove unbound antibodies and reduce non-specific staining on the cell surface. The sample is detected using a BD flow cytometer, and the data analysis and graphics processing are performed using FlowJo_v10.6.2 software.

[0125] 6) Fluorescent quantitative PCR to detect intestinal tight junction proteins

[0126] The extraction of RNA from sample tissues or cells usually includes the following steps: first, add the sample into the lysis buffer and crush it to release the RNA. Then, separate the RNA from the supernatant by phenol / chloroform mixture and precipitate the RNA with isopropanol or ethanol. Subsequently, wash the RNA precipitate to remove impurities and finally dissolve the RNA with RNase-free water or buffer. The extracted RNA can be detected for its concentration and purity by colorimetric method or fluorescence analyzer, and stored in a refrigerator at -80°C to prevent degradation. These steps can be adjusted according to the specific experimental purpose and sample type. The relative quantitative q-PCR method is used to detect the transcription level of two barrier proteins ZO-1 and Occludin in mouse small intestine.

[0127] 7) H&E staining to detect intestinal pathological changes

[0128] In order to further observe the pathological changes of the mouse small intestine, the middle part of the mouse small intestine is taken, and the same part of the intestine is taken from each group. The sample is placed in 4% paraformaldehyde solution for fixation, and the fixation time is more than two days. Then the fixed tissue is cut into a neat shape, and the steps of embedding, sectioning and H&E staining are as follows:

[0129] (1) Dehydration: put the sample into 70% alcohol, 80% alcohol, 85% alcohol, 90% alcohol, 95% I alcohol and 95% II alcohol for 2h, 90% alcohol overnight, 95% I alcohol and 95% II alcohol for 1h, and 100% I alcohol and 100% II alcohol for 1h.

[0130] (2) Transparency: put the sample into xylene I and xylene II in order, and the transparency time is 5min each time, until the red skin sample can be seen with the naked eye, which means the transparency is finished.

[0131] (3) Wax immersion: put the tissue block into wax I, wax II and wax III in order, and immerse in wax for 1h at 56°C, then put the tissue block into the embedding box for embedding.

[0132] (4) Sectioning: cut out a tissue slice with a thickness of 3.5pm from each embedding block, and spread the slice on a clean glass slide in a water bath at 41°C. Put the glass slide in a dry box at 80°C to bake the glass slide for 1h, and then perform H&E staining.

[0133] (5) H&E staining procedure as follows: the sample was placed in xylene I and xylene II for 8 min, then in 100% alcohol I, 100% alcohol II, 95% alcohol, 80% alcohol and 70% alcohol for 1 min, and in ultrapure water to wash off the excess alcohol. Then stained with hematoxylin, washed with ultrapure water, differentiated in 0.5% hydrochloric acid in alcohol bath for 5 s, then washed, then in light ammonia water for 2 min, then washed with water, placed in 0.5% aqueous solution of eosin for 5 min, then washed with water, washed in 80% alcohol bath, observed the eosin staining, and then the sample was sequentially placed in 95%, 100% I, 100% II alcohol bath for 5 times, then sequentially placed in xylene I and xylene II for 1-2 min, and mounted with neutral balsam.

[0134] 5. Experimental results:

[0135] 1) Growth performance index test results

[0136] Body weight changes were recorded during the experiment, and the results are shown in Figure 1 , it was found that the co-fermentation liquid of Saccharomyces cerevisiae and Patrinia scabiosaefolia could protect mice from infection of bovine viral diarrhea virus and reduce the decrease in body weight.

[0137] Specifically, during the immunization period of 0-14 days, the body weight of mice in all groups showed a stable growth trend, and the growth conditions of each group were similar. After the challenge treatment on the 14th day, the growth of the PBS group was basically not affected, and the body weight of the control group decreased greatly, reflecting the negative impact of bovine viral diarrhea virus infection. The Patrinia scabiosaefolia group and the Saccharomyces cerevisiae group could alleviate the sudden decrease in body weight to a certain extent, and the body weight change of the mice in the co-fermentation group was significantly different. The above changes in body weight indicated that the mice fed with the co-fermentation liquid of Saccharomyces cerevisiae and Patrinia scabiosaefolia could resist the infection of bovine viral diarrhea virus to a certain extent, and played a preventive protection role.

[0138] When scoring the diarrhea of mice in each group, the results are shown in Figure 2 , it can be seen that the PBS group occasionally increased, but basically maintained at about 1 point, indicating normal fecal state; the score of the control group gradually increased to 2-3 points, indicating obvious diarrhea symptoms, reaching a relatively severe degree on the 6th-7th day; the score of the Patrinia scabiosaefolia group basically maintained at about 2, indicating mild diarrhea; the score of the Saccharomyces cerevisiae group basically maintained at about 1-2 points, occasionally with mild diarrhea; this indicated that the Patrinia scabiosaefolia group and the Saccharomyces cerevisiae group could alleviate the degree of diarrhea to a certain extent compared with the control group; while the score of the co-fermentation group maintained at 1 point, indicating normal fecal state, which indicated that the co-fermentation liquid of Saccharomyces cerevisiae and Patrinia scabiosaefolia could alleviate the diarrhea of mice caused by bovine viral diarrhea virus infection.

[0139] Figure 3The survival of each group is shown, and the survival rate of each group is 100% during the 0-14 day immunization period, indicating that there is no death phenomenon during the immunization period, and the drug safety is good. After the 14th day of challenge, the PBS group and the co-fermentation group maintained a 100% survival rate, indicating that the mice fed with the co-fermentation liquid of Saccharomyces cerevisiae and Herba Patriniae showed excellent preventive protection against bovine viral diarrhea virus infection; the survival rate of the Herba Patriniae group and the Saccharomyces cerevisiae group decreased from the 15th day, and decreased to 40% on the 16th day, but was still better than that of the control group, and the survival rate of the control group decreased significantly after challenge and decreased to 0 on the 16th day. The results are shown in Figure 3 .

[0140] Figure 4 The damage to the spleen of each group of mice is shown, and it can be seen from the figure that the spleen of the control group of mice has a deep purple necrosis at the edge, which is the damage caused by BVDV virus infection of the spleen, and the spleen of the Herba Patriniae group and the Saccharomyces cerevisiae group is significantly enlarged, which may be related to viral infection; and the PBS group and the co-fermentation group do not show obvious viral damage changes.

[0141] 2) Detection results of immune indicators IFN-β and TNF-α

[0142] The collected serum was used to detect the secretion of cytokines IFN-β and TNF-α by EILSA, and the results are shown in Figure 5 .

[0143] As shown in Figure 5 A, after immunization, each group of mice was infected with bovine viral diarrhea virus, and the serum IFN-β content of the control group did not change significantly (ns) compared with the PBS group; the Herba Patriniae group and the Saccharomyces cerevisiae group did not differ significantly (ns) from the control group, indicating that the Herba Patriniae group and the Saccharomyces cerevisiae group did not significantly improve the decrease of IFN-β caused by bovine viral diarrhea virus infection; the IFN-β of the co-fermentation group differed extremely significantly (**** P <0.0001) from that of the control group, indicating that the co-fermentation liquid can significantly increase the secretion of IFN-β, and the secretion amount is also increased compared with the PBS group, which indicates that the co-fermentation liquid can significantly improve the immunity of mice and resist bovine viral diarrhea virus infection.

[0144] As shown in Figure 5 B, after immunization, each group of mice was infected with bovine viral diarrhea virus, and the serum TNF-α content of the control group increased extremely significantly (**** P <0.0001) compared with the PBS group, which indicates that after infection with bovine viral diarrhea virus, a large amount of pro-inflammatory factor TNF-α is induced in the body of mice; the TNF-α level of the Herba Patriniae group decreased (*** P <0.001) compared with that of the control group, and the Saccharomyces cerevisiae group showed a more significant decrease (*** P<0.001), indicating that both treatments had anti-inflammatory effects, and the effect of Saccharomyces cerevisiae was better than that of Patrinia scabiosifolia, but both were still significantly different from the PBS group; the difference in TNF-α between the co-fermentation group and the control group was extremely significant (****). P The co-fermentation broth showed the lowest TNF-α level (<0.0001), which was close to and slightly lower than the PBS level, indicating that the co-fermentation broth had the strongest anti-inflammatory effect and could significantly inhibit the production of TNF-α induced by bovine viral diarrhea virus infection, further confirming its excellent anti-inflammatory effect.

[0145] 3) Effects of co-fermentation products of Saccharomyces cerevisiae and Patrinia scabiosifolia on TH2 response

[0146] TH2-type cellular immunity participates in humoral immunity through the secretion of IL-4. Therefore, the activation level of TH2 cells in the spleen was examined. The results showed that infection with bovine viral diarrhea virus (BVPD) could activate CD4 cells in *Saccharomyces cerevisiae*, *Patrinia scabiosifolia* herbal extract, and co-fermentation broth of *Saccharomyces cerevisiae* and *Patrinia scabiosifolia*. + T cells secrete IL-4, and importantly, the co-fermentation broth of *Saccharomyces cerevisiae* and *Patrinia scabiosifolia* stimulates higher levels of IL-4, with the synergistic fermentation showing the best immune protective effect. The results are as follows: Figure 6 As shown.

[0147] according to Figure 6 It can be seen that, compared with the PBS group, the spleen of the control group had CD4+. + The proportion of IL-4 T cells decreased extremely significantly (****) P <0.0001), indicating that infection with bovine viral diarrhea virus significantly inhibits the Th2 immune response in mice; compared with the control group, the *Patrinia scabiosifolia* group and the *Saccharomyces cerevisiae* group showed significantly lower CD4 counts. + The proportion of IL-4 T cells was significantly increased (**) P <0.01), indicating that both treatments can maintain the Th2 immune response to some extent; the difference between the co-fermentation group and the control group was extremely significant (****). P <0.0001), its CD4 + The proportion of IL-4 T cells was close to that of the PBS group, indicating that the co-fermentation broth could significantly improve the Th2 immune response. This suggests that the co-fermentation broth can effectively maintain the body's immune balance and enhance its defense against bovine viral diarrhea virus infection.

[0148] 4) Effects of co-fermentation products of Saccharomyces cerevisiae and Patrinia scabiosifolia on TH1 response

[0149] Killer T cells generate a TH1 immune response by secreting IFN-γ, therefore the secretion level of IFN-γ in the spleen was measured. The results showed that after infection with bovine viral diarrhea virus, the fermentation broth of *Saccharomyces cerevisiae*, *Patrinia scabiosifolia*, and co-fermentation broth of *Saccharomyces cerevisiae* and *Patrinia scabiosifolia* could activate CD3.+ CD8 + T cells secreted higher levels of IFN-γ, enhancing the cellular immune response, and the Saccharomyces cerevisiae and Patrinia scabiosaefolia liquid co-fermentation effect was more significant, as shown in Figure 7

[0150] As shown in Figure 7 , compared with the PBS group, the CD8 + IFN-γ T cell ratio was very significantly reduced (** P <0.01), indicating that bovine viral diarrhea virus infection significantly inhibited the cellular immune response; the CD8 + IFN-γ T cell ratio of the Patrinia scabiosaefolia group and the Saccharomyces cerevisiae group was not significantly different (ns) compared with the PBS group, indicating that the use of these two treatments alone had limited effect on maintaining cellular immunity; the CD8 + IFN-γ T cell level of the co-fermentation group was not significantly different (ns) from the PBS group, but was very significantly higher than the control group (** P <0.01), indicating that the co-fermentation liquid could effectively maintain the level of CD8 + IFN-γ T cells, which showed that the co-fermentation liquid had the strongest immune protection effect and could effectively prevent the decline in cellular immune function caused by bovine viral diarrhea virus infection.

[0151] 5) Quantitative analysis results of mouse small intestinal tight junction proteins

[0152] The tight junction proteins ZO-1 (Zonula occludens-1) and Occludin of the small intestine of mice in each group were quantified. ZO-1 is a scaffolding protein located on the inside of the cell and is primarily responsible for connecting other proteins to the cytoskeleton. Occludin is a transmembrane protein that spans the cell membrane and interacts with Occludin of adjacent cells. These two proteins together form the intestinal barrier, which works in concert to maintain tight junctions between cells and control the selective passage of substances through the intercellular space, thereby protecting the intestine from harmful substances. When the expression of these proteins decreases, it usually means that the intestinal barrier function is impaired. Therefore, by detecting the expression levels of ZO-1 and Occludin, the integrity and functional status of the intestinal barrier can be evaluated.

[0153] After immunization of mice with Saccharomyces cerevisiae, Patrinia scabiosaefolia liquid, and Saccharomyces cerevisiae and Patrinia scabiosaefolia co-fermentation liquid, compared with the control group, the relative expression of ZO-1 and Occludin was significantly increased, indicating that bovine viral diarrhea virus can damage the small intestine and reduce the expression of small intestinal tight junction proteins, as shown in Figure 8 .

[0154] Specifically, as shown in Figure 8 ​As shown in Figure A, the relative expression level of ZO-1 mRNA in the small intestine was significantly lower in the control group compared to the PBS group (***). P <0.0001), indicating that infection with bovine viral diarrhea virus significantly disrupts the expression of the small intestinal tight junction protein ZO-1; the expression levels of ZO-1 in the *Patrinia scabiosifolia* and *Saccharomyces cerevisiae* groups were not significantly different from the control group (ns); the co-fermentation group showed a highly significant difference compared to the control group (**). P The co-fermentation broth showed a value <0.01), and its ZO-1 expression level was closest to that of the PBS group, indicating that the co-fermentation broth could effectively maintain the expression of the small intestinal tight junction protein ZO-1, which suggests that the co-fermentation broth has the strongest intestinal barrier protection effect.

[0155] like Figure 8 As shown in Figure B, compared with the PBS group, the relative expression level of Occludin mRNA in the small intestine of the control group was significantly decreased (****). P <0.0001), indicating that infection with bovine viral diarrhea virus significantly disrupts the expression of the small intestinal tight junction protein Occludin; there were no significant differences (ns) between the *Patrinia scabiosifolia* group and the *Saccharomyces cerevisiae* group and the control group, indicating that the effects of these two treatments alone on maintaining intestinal barrier integrity are limited; the co-fermentation group showed a highly significant difference (***) compared with the control group. P The co-fermentation broth showed a value <0.001), and its Occludin expression level was closest to that of the PBS group, indicating that the co-fermentation broth could effectively maintain the expression of the small intestinal tight junction protein Occludin, which suggests that the co-fermentation broth has the strongest intestinal barrier protection effect.

[0156] Viral load can visually indicate the amount of virus in tissues. This was achieved by statistically analyzing the BVDV viral load in mouse small and colon tissues. The results are as follows: Figure 9 As shown in Figure A, the co-fermentation group had the lowest viral load in the mouse small intestine (****). P <0.0001), and the *Patrinia scabiosifolia* group and the *Saccharomyces cerevisiae* group showed significant antiviral effects. The viral load in the *Patrinia scabiosifolia* group was significantly different from the control group (****). P <0.0001), the difference between the brewer's yeast group and the control group was extremely significant (***). P The value <0.001 indicates that *Patrinia scabiosifolia* and *Saccharomyces cerevisiae* have a certain inhibitory effect on viral infection in the small intestine. For example... Figure 9 As shown in Figure B, the viral load of BVDV in the mouse colon was statistically analyzed. The results showed that the viral load in the co-fermentation group was significantly different from that in the control group (****). P <0.0001), while the difference between the Patrinia scabiosifolia group and the control group was extremely significant (***). P The value <0.001 indicates that Patrinia scabiosifolia and Saccharomyces cerevisiae have a certain inhibitory effect on viral infection in the colon.

[0157] 6) Pathological section results of small intestine tissue

[0158] like Figure 10 As shown, observations of intestinal lesions revealed that the PBS group ( Figure 10 (A) shows normal intestinal tissue structure, with neatly arranged villi, intact mucosa, and no obvious pathological changes. (Control group) Figure 10 In mice (group B), the intestines showed obvious lesions such as congestion, edema, and mucosal necrosis; the intestinal wall was thinned, and the intestines were filled with gas and bloody contents. The Patrinia scabiosifolia group ( Figure 10 (C) and brewer's yeast group ( Figure 10 In the D group, the intestinal tissues of both groups showed some degree of lesions, but the severity was milder than that of the control group, with mild tissue damage and structural changes observed. In contrast, the co-fermentation group (… Figure 10 The intestinal lesions in the E mice were relatively mild, with relatively intact intestinal mucosa, minimal congestion and edema, and less gas and bloody contents in the intestines. These pathological results indicate that bovine viral diarrhea virus infection can cause severe damage to the intestinal tissue of mice, and the co-fermentation broth of Saccharomyces cerevisiae and Patrinia scabiosifolia can significantly alleviate this damage and has a significant protective effect on intestinal tissue, which is better than using Patrinia scabiosifolia or Saccharomyces cerevisiae alone.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation broth for inhibiting bovine viral diarrhea virus, characterized in that, The fermentation broth is a co-fermentation product of a mixture of Patrinia scabiosaefolia decoction and liquid culture medium, inoculated with Saccharomyces cerevisiae; the Saccharomyces cerevisiae strain used is ( ). Saccharomyces cerevisiae Hansen (Number of samples: BNCC336054; viable count: 10) 8 CFU / mL ~10 9 CFU / mL.

2. The method for preparing fermentation broth that inhibits bovine viral diarrhea virus according to claim 1, characterized in that, A decoction of Patrinia scabiosifolia was prepared by mixing the decoction with a liquid culture medium and then co-fermenting it with Saccharomyces cerevisiae. The co-fermentation conditions were: pH 6.5-7.2, fermentation temperature 30℃-40℃ for 24-96 hours.

3. The method for preparing fermentation broth that inhibits bovine viral diarrhea virus according to claim 2, characterized in that... The inoculation amount of brewer's yeast is 5%-10%.

4. The method for preparing fermentation broth that inhibits bovine viral diarrhea virus according to claim 3, characterized in that, The volume ratio of the aqueous decoction of Patrinia scabiosifolia to the liquid culture medium is 1:4-20.

5. The method for preparing fermentation broth that inhibits bovine viral diarrhea virus according to claim 4, characterized in that, The preparation method of the Patrinia scabiosifolia decoction includes: crushing Patrinia scabiosifolia into coarse powder, sieving it, adding distilled water, soaking it, placing it in an extraction tank, heating it to boiling, maintaining a gentle boil while decocting, filtering it after decoction, and collecting the filtrate; repeating the decoction of the residue according to the above method, and combining the filtrates; concentrating the combined filtrate under reduced pressure to obtain the concentrated Chinese medicine liquid, which is the Patrinia scabiosifolia decoction.

6. The use of the fermentation broth for inhibiting bovine viral diarrhea virus as described in claim 1 in the preparation of a product for inhibiting bovine viral diarrhea virus, characterized in that, The product is selected from pharmaceuticals or feed additives.

7. The use of the fermentation broth for inhibiting bovine viral diarrhea virus as described in claim 1 in the preparation of products for the prevention, relief, and / or treatment of diseases caused by bovine viral diarrhea virus, characterized in that, The product is selected from pharmaceuticals or feed additives.

8. The use of the fermentation broth for inhibiting bovine viral diarrhea virus according to claim 7 in the preparation of products for the prevention, relief, and / or treatment of diseases caused by bovine viral diarrhea virus, characterized in that, The product also includes auxiliary materials.

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