Traditional Chinese medicine fermentation liquor for resisting porcine epidemic diarrhea and preparation method thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a traditional Chinese medicine fermentation liquor for resisting swine epidemic diarrhea and a preparation method thereof. The application determines the treatment effect and safety of Andrographis paniculata on swine epidemic diarrhea through screening of different drugs, and then carries out fermentation on the basis of the Andrographis paniculata liquor combined with recombinant Lactobacillus plantarum, so as to obtain the traditional Chinese medicine fermentation liquor with the effect of resisting swine epidemic diarrhea. Piglet experiments show that the traditional Chinese medicine fermentation liquor can promote the activation of immune cells, improve the antibody level, reduce the viral load, and reduce the intestinal injury, thereby providing a comprehensive and effective solution for prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a traditional Chinese medicine fermentation broth for treating swine epidemic diarrhea and its preparation method. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a major disease challenge facing the pig farming industry, severely impacting pig health and economic benefits. Its pathogen, PEDV, is widely transmitted, exhibits distinct seasonal patterns of outbreaks, and can cause severe intestinal damage, leading to dehydration and death, especially in newborn piglets. Effective control measures are urgently needed to reduce its impact on the pig farming industry. Existing PEDV vaccines have limitations: live vaccines have poor safety profiles, and inactivated vaccines have weak immunogenicity and cannot effectively induce mucosal immunity. Furthermore, in exploring new control pathways, the construction of lactic acid bacteria vectors is difficult, the application of traditional Chinese medicine faces challenges such as complex components and a lack of standardized synergistic applications, and the synergistic technology of probiotics and traditional Chinese medicine is immature. Innovative technologies are urgently needed to overcome these bottlenecks. Summary of the Invention

[0003] To address the above problems, this invention provides a method for preparing traditional Chinese medicine fermentation broth, comprising the following steps:

[0004] (1) Preparation of Chinese medicine liquid: Wash and grind Andrographis paniculata into powder, add 8 times the volume of distilled water to soak for 2 hours and then boil. Filter through gauze, centrifuge at 4000 rpm for 15 minutes to take the supernatant, concentrate the supernatant to 1 g / mL, and finally autoclave at 115℃ for 25 minutes. After that, dispense into 1.5 EP tubes in a clean bench and store in a -20℃ refrigerator.

[0005] (2) Inoculation and fermentation: The above-mentioned Chinese medicine liquid was prepared into a Chinese medicine fermentation MRS medium. After high pressure sterilization, recombinant Lactobacillus plantarum WCFS-PEDV-S1 was inoculated into the Chinese medicine fermentation medium and fermented to obtain Chinese medicine fermentation liquid.

[0006] Furthermore, the method for constructing the recombinant Lactobacillus plantarum WCFS-PEDV-S1 is as follows:

[0007] (1) Plasmid modification: An EGFP fragment and a laboratory-constructed S-layer anchoring expression system were inserted into the 0537 position of the PHSP02 standard plasmid to obtain the modified plasmid PHSP02-EGFP;

[0008] (2) Plasmid modification: The EGFP portion of the modified plasmid PHSP02-EGFP was replaced with the S1 protein of the PEDVljx strain to obtain the modified plasmid PHSP02-PEDV-S1.

[0009] (3) Construction of competent cells: The helper plasmid PLH01 was transformed into Lactobacillus plantarum WCFS1 competent cells to obtain positive strain PW01;

[0010] (4) Screening of positive strains: The modified plasmids PHSP02-EGFP and PHSP02-PEDV-S1 were transformed into PW01 competent cells, the genome was extracted, and positive strains were screened by PCR.

[0011] (5) Verification of positive strains: The obtained positive strains were passaged for 10, 15, and 20 generations, and genomic PCR was performed to prove that they could be stably inherited. Bacterial smears were prepared, and green fluorescence was observed under an electron microscope, which can be considered that the novel recombinant lactic acid can autonomously express the target protein.

[0012] (6) PEDV-S1 protein expression verification: PEDV-S1 protein expression was verified by Western blot. The primary antibody was PEDV-S1 protein extracted from prokaryotic expression, and the secondary antibody was goat anti-rabbit protein.

[0013] Furthermore, the concentration of Andrographis paniculata in the fermentation MRS medium for traditional Chinese medicine is 12.5 mg / mL.

[0014] Furthermore, the inoculation amount of the recombinant Lactobacillus plantarum WCFS-PEDV-S1 is 2%.

[0015] Furthermore, the fermentation time is 20 hours.

[0016] Furthermore, the fermentation temperature is 37°C.

[0017] Furthermore, the pH of the fermentation is 6.5.

[0018] The present invention also provides a traditional Chinese medicine fermentation liquid, which is prepared by any of the above preparation methods.

[0019] This invention also provides the application of the above-mentioned traditional Chinese medicine fermentation liquid in the preparation of drugs for the prevention and treatment of swine epidemic diarrhea.

[0020] The present invention has the following beneficial effects:

[0021] Given the limitations of existing technologies in the prevention and control of porcine epidemic diarrhea (PEDD), this study aims to construct a recombinant lactic acid bacteria strain with stable genetic expression, integrating key PEDV antigens to overcome the challenges of constructing lactic acid bacteria vectors; to screen and optimize the co-fermentation process of traditional Chinese medicine (TCM) and recombinant lactic acid bacteria to address the application issues of TCM; and finally, to establish a comprehensive prevention and control strategy that integrates the advantages of vaccines, probiotics, and TCM fermentation to effectively improve the immune protection level of pigs against PEDV, reduce morbidity and mortality, fill existing technological gaps, and provide the pig industry with a safer, more efficient, and more comprehensive PEDD control solution. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Analysis of NK cell flow cytometry results in piglet PBL;

[0024] Figure 2 Flow cytometry analysis of macrophages in piglet PBL;

[0025] Figure 3 Flow cytometry analysis of CD4+ cells in piglet SP cells;

[0026] Figure 4 Expression of specific antibody IgG in piglet serum;

[0027] Figure 5 Expression of the specific antibody SIgA in piglet serum;

[0028] Figure 6 Viral load in piglet intestinal epithelial cells;

[0029] Figure 7 Pathological tissue section of piglet ileum;

[0030] Figure 8 Fluorescence of PEDV virus in the intestine of piglets. Detailed Implementation

[0031] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] 1. Materials and Methods

[0037] The study used WCFS1-PEDV-S1 (constructed previously), the WCFS1 strain preserved in the laboratory, and the PEDV LJX01 strain kindly provided by Researcher Liu Guangliang of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Twelve healthy 3-day-old newborn piglets were randomly divided into four groups of three and housed in isolation at the Small Animal Center of Jilin Agricultural University, fed with piglet-specific milk powder and drinking water at regular intervals. Flow cytometry reagents, containing various antibodies, were purchased from BD Biosciences. Bovine serum albumin (BSA) was purchased from Gibco. Other self-prepared reagents included erythrocyte lysis buffer (containing NH4Cl, KHCO3, EDTA, etc., autoclaved after volume adjustment) and ELISA reagents (coating buffer, washing buffer, blocking buffer, and stop buffer, each with specific formulations and preparation requirements). Major instruments include: CO2 incubator (ThermoScientific), fluorescence inverted microscope (Leica, Germany), microplate spectrophotometer (BioTek, USA), flow cytometer (BD, USA), 4℃ refrigerated centrifuge (Eppendorf, Germany), 411BR4513 electroporator (BIO-RAD, USA), fully automated paraffin sectioner (Leica, Germany), and -80℃ freezer (ThermoScientific).

[0038] 2 methods

[0039] 2.1 Immunization and challenge protocols for laboratory animals

[0040] 2.1.1 Piglet Immunization Grouping

[0041] Twelve 3-day-old piglets were randomly divided into four groups: a PBS control group, a WCFS1-PEDV-S1 experimental group, a WCFS1 experimental group, and a co-fermentation broth of Andrographis paniculata and WCFS1-PEDV-S1 experimental group. The piglets were immunized by oral gavage. The corresponding drugs (1×10⁹ CFU / ml recombinant lactic acid bacteria or fermentation drug per piglet) were administered to the piglets on days 1, 3, 5, 7, 9, and 11. The specific grouping and drug dosage are shown in the table. Each group consisted of 3 piglets, and the piglets were immunized every other day.

[0042] 2.1.2 Immunization challenge procedure and experimental design

[0043] In the piglet immunization experiment, PEDV was administered at a dose of 10⁴ TCID₅₀ for seven consecutive days starting from day 12. After all control group animals developed diarrhea, the piglets were euthanized. Blood, spleen, and mesenteric lymph node samples were collected for flow cytometry analysis. Hematologic malignancies (HE) sections of the small intestine were prepared for pathological observation. Simultaneously, ELISA was used to detect SIgA and IgG levels to assess immune marker changes. 1.2.2 Flow Cytometry

[0044] 2.1.3 Flow cytometry was used to detect changes in the expression levels of T lymphocytes, B lymphocytes, and helper T cells.

[0045] After euthanizing piglets, spleens and mesenteric lymph nodes were harvested. PBS was added, and the spleen was ground and transferred to centrifuge tubes. The mesenteric lymph nodes were treated similarly, and the supernatant was discarded after centrifugation. Red blood cells in the spleen were lysed with erythrocyte lysis buffer, and the supernatant was discarded after a series of operations. Peripheral blood was anticoagulated and cell density was adjusted. Spleens and mesenteric lymph nodes were diluted and counted. Cells were diluted, plated, and specifically stimulated. Flow cytometry antibodies were serially diluted, surface antibodies were premixed, and relevant flow cytometry tubes were set up. Intracellular staining was performed after special treatment, followed by multiple washes after antibody staining. Finally, buffer was added, mixed, and flow cytometry was performed for detection.

[0046] 2.1.4 ELISA Detection Method for Immune Indicators SIgA and IgG

[0047] First, prepare the coating solution, washing solution, stop solution, and blocking solution. Then, dilute the antigen according to the specified ratio, coat the ELISA plate, and incubate at 4°C overnight. Next, perform multiple washes, block the sample wells, and incubate overnight. Then, dilute the serum to be tested, incubate with the primary antibody, and wash again. Next, incubate with the diluted secondary antibody and wash again. Finally, develop the color with TBM chromogenic solution, add concentrated sulfuric acid at the appropriate time to stop the reaction, and read the OD450nm absorbance using an ELISA reader for data analysis.

[0048] 2.1.5 Real-time quantitative PCR detection of PEDV virus infection status

[0049] To infect piglet small intestinal epithelial cells with RNA, a 0.5 cm segment of piglet small intestine was first processed and the epithelial cells were collected and stored at 4°C (operated on ice to prevent RNA degradation). RNA was extracted and reverse transcribed into cDNA for storage. Next, primers were designed and synthesized based on the conserved sequence of the PEDV M gene, and the PEDV standard plasmid was diluted. Then, an RT-qPCR reaction system was prepared and the reaction was carried out according to a specific procedure. Finally, the viral copy number was calculated based on the concentration of the standard plasmid, a standard curve was plotted, and the viral copy number in the sample was calculated accordingly.

[0050] 2.1.6 HE staining for intestinal pathological changes

[0051] Samples of the middle segment of the small intestine from each group of piglets were fixed in 4% paraformaldehyde for at least two days. After trimming the fixed tissue, it was rinsed overnight with running water and dehydrated according to a specific procedure. After clearing with xylene, it was embedded in paraffin and cut into 3μm thick sections using a microtome. After dewaxing and rehydration by baking at 80℃, the sections were first stained with hematoxylin (including staining, washing, differentiation, washing again, ammonia treatment, and washing again), then stained with eosin. After dehydration and clearing according to the procedure, the sections were finally mounted with resin and observed and photographed under a microscope to record the lesions.

[0052] 2.1.7 Immunofluorescence detection of PEDV virus in the intestines of piglets

[0053] First, dehydration, embedding, and sectioning were performed (same steps as in the relevant section). Then, after antigen retrieval, PBS washing, and tissue selection, the tissue was blocked with PBS diluted with 5% goat serum and 0.3% Triton-100. Then, the tissue was incubated overnight at 4°C in the dark with primary antibody (PEDV N diluted 200 times), washed, incubated at 4°C in the dark with secondary antibody (Alexa Fluor 555 donkey anti-rabbit IgG (H+L) diluted 400 times), washed, stained with DAPI nuclei (PBS diluted 1:5000), washed, and finally mounted with an anti-fluorescence attenuator. The fluorescence channel was then adjusted for observation and imaging.

[0054] 3 Results

[0055] 3.1 Detection of immune cell activation in piglets

[0056] Natural killer cells (NK cells) are part of the innate immune system. This study detected NK cell activation levels by measuring CD11b+ in peripheral blood. It was found that the CD3-CD11b+ cell count in the group fed with recombinant lactic acid bacteria WCFS1-PEDV-S1 co-fermented with Andrographis paniculata and the group fed with recombinant lactic acid bacteria WCFS1-PEDV-S1 was significantly increased compared with the PBS group and the wild-type Lactobacillus plantarum WCFS1 group (P < 0.001). This indicates that feeding with the co-fermentation product of recombinant lactic acid bacteria WCFS1-PEDV-S1 and Andrographis paniculata can increase the number of NK cells in the peripheral blood of piglets.

[0057] 3.2 Recombinant Lactobacillus plantarum and fermentation products of traditional Chinese medicine promote macrophage activation

[0058] Phagocytosis is one of the main mechanisms of innate immunity, and macrophages are an important part of the first line of defense of the immune system. CD163 is a macrophage marker. In this study, we detected the expression of CD163+CD172+ in peripheral blood and found that compared with the PBS group and the WCFS1 group, the number of CD163+CD172+ cells in the co-fermentation group of recombinant Lactobacillus plantarum WCFS1-PEDV-S1 and Andrographis paniculata and the recombinant Lactobacillus plantarum WCFS1-PEDV-S1 group was significantly increased (P<0.001), indicating that feeding the corresponding co-fermentation products can promote the increase of macrophages in peripheral blood.

[0059] 3.3 Recombinant Lactobacillus plantarum and fermentation products of traditional Chinese medicine promote CD4+ T cell production

[0060] When the body is invaded by a virus, CD4 cells are the hub of the immune response, and CD8+ cells receive signals from them to fight the virus. In this study, CD3+ T cells and CD8- T cells were used to detect the status of CD4+ T cells in the spleen. Compared with the PBS group and the WCFS1 group, the number of CD4+ T cells in the recombinant Lactobacillus plantarum WCFS1-PEDV-S1 co-fermentation group with Andrographis paniculata and the recombinant Lactobacillus plantarum WCFS1-PEDV-S1 group was significantly increased (P<0.01), indicating that these two groups can promote the production of CD4+ T cells and have better immune function.

[0061] 3.4 Recombinant Lactobacillus plantarum and fermentation products of traditional Chinese medicine promote the activation of B cells

[0062] B cells play a crucial role in innate and adaptive immunity. This study, using a specific gate analysis strategy, found that after immune challenge, the percentage of CD45+CD21+ B cells in the mesenteric lymph nodes of piglets was significantly higher in both the PBS group and the WCFS1 group compared to the co-fermentation group of recombinant Lactobacillus plantarum WCFS1-PEDV-S1 and the recombinant Lactobacillus plantarum WCFS1-PEDV-S1 group. Furthermore, the co-fermentation group showed a higher percentage of B cells than the recombinant Lactobacillus plantarum WCFS1-PEDV-S1 group (P < 0.001), indicating that both of these conditions can promote the intestinal health of piglets.

[0063] 3.5 Co-fermentation products of recombinant lactic acid bacteria and traditional Chinese medicine promote the secretion of IgG and SIgA.

[0064] 3.5.1 Elevated levels of specific IgG in serum

[0065] IgG is a key component of total immunoglobulins in the body. Secreted by plasma cells, it is abundant and long-lasting, playing a vital role in maintaining immune function. When the body is infected by a virus, IgG can initiate an immune response to neutralize the toxin. IgG is a reactant of humoral immunity; therefore, detecting IgG in studies can assess the function of humoral immunity. Figure 4 As shown, compared with the PBS group, the specific antibody IgG in the Lactobacillus plantarum WCFS1 group, the recombinant lactic acid bacteria WCFS1-S1 group, and the recombinant lactic acid bacteria and Andrographis paniculata co-fermentation group all showed varying degrees of increase (P<0.01).

[0066] 3.5.2 Elevated levels of specific SIgA in serum

[0067] SIgA is abundant on the mucosal surface of the digestive tract and is a major secretory antibody in mucosal immunity, capable of activating mucosal immunity. After oral vaccination, novel recombinant lactic acid bacteria can produce specific SIgA. Detecting this specific antibody marker can accurately assess the body's mucosal immune function in suppressing PEDV infection. Figure 5 As shown, compared with the PBS group and the Lactobacillus plantarum WCFS1 group, the recombinant Lactobacillus plantarum WCFS1-S1 group and the recombinant Lactobacillus plantarum WCFS-S1 co-fermentation group with Andrographis paniculata showed enhanced expression of specific antibody SIgA, indicating that feeding recombinant Lactobacillus plantarum WCFS1-S1 and Andrographis paniculata fermentation products before porcine epidemic diarrhea virus infection can enhance the body's immune function and improve its resistance to the virus.

[0068] 3.6 Feeding with recombinant lactic acid bacteria and fermented Chinese medicine products significantly reduced viral load.

[0069] RT-qPCR can be used to quantitatively analyze gene expression levels, providing a more intuitive understanding of gene regulation mechanisms in physiological and pathological processes. By comparing gene expression levels under different conditions, key factors in regulatory networks can be identified, revealing the molecular mechanisms of disease occurrence and development. RT-qPCR is a commonly used method for detecting viral load. In this experiment, RNA was extracted from the intestinal epithelium of piglets, and the cDNA generated by reverse transcription was amplified and quantified. DNA-binding dyes were used to monitor the formation of PCR products in real time. A standard curve was established by performing qPCR on standards of known concentrations. Then, by comparing the fluorescence signal in the sample with the corresponding fluorescence signal on the standard curve, the viral load in the sample can be calculated. Figure 6 As shown, compared with the PBS group, the viral load of the Lactobacillus plantarum WCFS1 group, the recombinant Lactobacillus plantarum WCFS1-S1 group, and the group co-fermented with the traditional Chinese medicine Andrographis paniculata all decreased to varying degrees (P<0.001). Among them, the group co-fermented with the traditional Chinese medicine Andrographis paniculata had the lowest viral load and the best protective effect.

[0070] 3.7 Feeding recombinant Lactobacillus plantarum and fermented products of traditional Chinese medicine has a protective effect on the intestines.

[0071] like Figure 7 As shown, the PBS-challenged group exhibited more severe intestinal pathological changes, with significant hemorrhage in the lamina propria and submucosa of the ileum, fragmented intestinal villi tissue with inflammatory infiltration, and shortened intestinal villi. The empty vector WCFS1 group showed disordered arrangement of mucosal epithelial cells, inflammatory cell infiltration in the lamina propria, shortened intestinal villi, and destroyed glandular structure. The modified strain WCFS1-PEDV-S1 group showed milder symptoms, with scattered inflammatory cell infiltration and a small number of vacuoles. The WCFS1-PEDV-S1 co-fermentation group with traditional Chinese medicine showed intact intestinal villi structure, orderly glandular arrangement, and no obvious histopathological changes. The experimental results indicate that the WCFS1-PEDV-S1 co-fermentation group with traditional Chinese medicine can alleviate the intestinal pathological changes caused by PEDV infection of piglets with porcine epidemic diarrhea virus.

[0072] 3.8 The viral load in the intestines of piglets fed with recombinant lactic acid bacteria and fermented traditional Chinese medicine products was reduced.

[0073] like Figure 8 As shown, the PBS group showed the most red fluorescence on the intestinal villi, followed by the Lactobacillus plantarum WCFS group and the recombinant Lactobacillus plantarum WCFS1-PEDV-S1 group. The recombinant Lactobacillus plantarum WCFS1-PEDV-S1 co-fermented with Andrographis paniculata showed the least red fluorescence, indicating that feeding recombinant Lactobacillus plantarum WCFS1-PEDV-S1 co-fermented with Andrographis paniculata can produce a certain immune protection effect on piglets.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine fermentation broth, characterized in that, Includes the following steps: (1) Preparation of Chinese medicine liquid: Wash and grind Andrographis paniculata into powder, add 8 times the volume of distilled water to soak for 2 hours and then boil. Filter through gauze, centrifuge at 4000 rpm for 15 minutes to take the supernatant, then concentrate the supernatant to 1 g / mL, and finally autoclave at 115℃ for 25 minutes. After that, dispense into 1.5 EP tubes in a clean bench and store in a -20℃ refrigerator. (2) Inoculation and fermentation: The above-mentioned Chinese medicine liquid was prepared into a Chinese medicine fermentation MRS medium. After high pressure sterilization, recombinant Lactobacillus plantarum WCFS-PEDV-S1 was inoculated into the Chinese medicine fermentation medium and fermented to obtain Chinese medicine fermentation liquid. The method for constructing the recombinant Lactobacillus plantarum WCFS-PEDV-S1 is as follows: (1) Plasmid modification: An EGFP fragment and a laboratory-constructed S-layer anchoring expression system were inserted into the 0537 position of the PHSP02 standard plasmid to obtain the modified plasmid PHSP02-EGFP; (2) Plasmid modification: The EGFP portion of the modified plasmid PHSP02-EGFP was replaced with the S1 protein of the PEDVljx strain to obtain the modified plasmid PHSP02-PEDV-S1. (3) Construction of competent cells: The helper plasmid PLH01 was transformed into Lactobacillus plantarum WCFS1 competent cells to obtain positive strain PW01; (4) Screening of positive strains: The modified plasmids PHSP02-EGFP and PHSP02-PEDV-S1 were transformed into PW01 competent cells, the genome was extracted, and positive strains were screened by PCR. (5) Verification of positive strains: The obtained positive strains were passaged for 10, 15, and 20 generations, and genomic PCR was performed to prove that they could be stably inherited. The bacterial smears were prepared and green fluorescence was observed under an electron microscope, which can be considered that the novel recombinant Lactobacillus plantarum can autonomously express the target protein. (6) PEDV-S1 protein expression verification: PEDV-S1 protein expression was verified by Western blot. The primary antibody was PEDV-S1 protein extracted from prokaryotic expression, and the secondary antibody was goat anti-rabbit protein.

2. The preparation method according to claim 1, characterized in that, The concentration of Andrographis paniculata in the fermentation MRS medium for traditional Chinese medicine was 12.5 mg / mL.

3. The preparation method according to claim 1, characterized in that, The inoculation amount of the recombinant Lactobacillus plantarum WCFS-PEDV-S1 was 2%.

4. The preparation method according to claim 1, characterized in that, The fermentation time is 20 hours.

5. The preparation method according to claim 1, characterized in that, The fermentation temperature is 37°C.

6. The preparation method according to claim 1, characterized in that, The pH of the fermentation was 6.

5.

7. A traditional Chinese medicine fermentation liquid, characterized in that, It is prepared by any one of the preparation methods of claims 1-6.

8. The application of the traditional Chinese medicine fermentation liquid as described in claim 7 in the preparation of drugs for the prevention and treatment of porcine epidemic diarrhea.

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