An oral preparation and its use in the prevention of porcine epidemic diarrhea

Abstract

The application relates to the technical field of veterinary biological products, in particular to an oral preparation and application thereof in prevention of porcine epidemic diarrhea, which comprises a freeze-dried powder of Lactobacillus plantarum LP-1 metabolites and / or medicinal grade ursodeoxycholic acid, supplemented with an oral excipient carrier. The preparation is suitable for prevention of piglets of different ages, has broad-spectrum antiviral activity, no antibiotic residue and convenient administration, and cooperates with double-cavity packaging to ensure stability, thereby providing a new strategy for green breeding.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, and specifically relates to an oral preparation and its application in the prevention of porcine epidemic diarrhea. Background Technology

[0002] In swine herds, porcine epidemic diarrhea virus (PEDV) infection is a highly contagious disease characterized by acute watery diarrhea, vomiting, and dehydration. The pathogen belongs to the Coronaviridae family and primarily attacks the epithelial cells of the small intestine, causing villus atrophy and malabsorption.

[0003] PEDV, as a single-stranded positive-sense RNA virus, has a high mutation frequency, approximately 10 times that of DNA viruses. 3 -10 4 The genomic differences between different circulating strains can reach 5%–8%, leading to frequent antigenic drift and antigenic switching. Furthermore, the specific cellular receptor for PEDV is currently unclear, resulting in a lack of target basis for receptor-blocking intervention strategies and limiting the development of subunit vaccines and targeted drugs. Currently widely used vaccines offer limited cross-protection against emerging variant strains, and maternal antibodies have short half-lives, insufficient retention time in piglets to cover the high-risk infection window, potentially interfering with active immunization. Antibiotics have no direct inhibitory effect on the virus, and long-term use may actually promote the spread of drug-resistant bacteria; while interferon and other biological agents possess some antiviral potential, their stability and bioavailability under oral administration conditions make them ineffective in the critical 24 hours of early infection.

[0004] In recent years, *Lactobacillus plantarum*, as a Generally Recognized As Safe (GRAS) probiotic, has attracted attention in the field of animal virus control. Previous studies have shown that it has inhibitory effects on HIV, rotavirus, influenza virus, and transmissible gastroenteritis virus (TGEV), and can enhance host defense capabilities by regulating the expression of local antiviral and inflammatory factors in the intestine. Our team previously isolated a strain of *Lactobacillus plantarum* LP-1 (GenBank accession number: MH727586.1) from the cecum of piglets in a pig farm in Chongqing, and its metabolites showed significant inhibitory activity against TGEV. Meanwhile, ursodeoxycholic acid (UDCA), as a secondary bile acid, in addition to its traditional choleretic and hepatoprotective uses, has been found in recent studies to interfere with SARS-CoV-2 invasion by regulating FXR receptors and affecting ACE2 expression. It also possesses multiple functions, including regulating immune responses, inhibiting excessive inflammatory responses, and reducing intestinal epithelial cell damage.

[0005] Given that PEDV, TGEV, and SARS-CoV-2 belong to the Coronaviridae family and share certain commonalities in their pathogenic mechanisms and pathological manifestations, this study systematically evaluated the inhibitory effects of *Lactobacillus plantarum* metabolites (LPM) and UDCA on PEDV and analyzed their mechanisms of action. The results showed that LPM mainly blocks the adsorption of viral particles to host cells by physically adhering to them, while downregulating the expression of pro-inflammatory factors such as IL-1β and IL-6 and inducing early apoptosis in damaged intestinal epithelial cells. UDCA, on the other hand, inhibits viral replication by activating the IRF3-IFN-α / β-JAK-STAT1 signaling axis and upregulating the expression of interferon-stimulated genes (ISGs) such as MX1, ISG15, OASL, and ZAP. The combined use of these two agents can achieve a synergistic effect at different stages of viral infection, simultaneously addressing viral blockade, immune activation, and inflammatory regulation.

[0006] Currently, most PEDV control products focus on a single mechanism, and there is a lack of compound oral formulations that can adapt to the physiological characteristics and clinical needs of piglets of different ages. Therefore, developing a novel oral formulation based on the synergistic effect of multiple targets of "virus blocking-immune regulation-inflammation control" and matched to different application scenarios such as newborn, weak piglet emergency care, and weaning groups is of great significance for improving the efficiency of PEDV control, reducing breeding losses, and promoting the green and high-quality development of the pig industry. Summary of the Invention

[0007] The present invention aims to provide an oral formulation for the prevention of porcine epidemic diarrhea (PED) and its application. This oral formulation comprises *Lactobacillus plantarum* metabolites and / or ursodeoxycholic acid. The *Lactobacillus plantarum* metabolites are derived from a strain of *Lactobacillus plantarum* with anti-coronavirus activity, and the ursodeoxycholic acid is a secondary bile acid with immunomodulatory and intestinal epithelial protective functions. By combining the two in a specific formulation, a compound oral formulation is constructed that can synergistically exert its effects at different stages of viral infection. This formulation, without relying on traditional vaccines or antibiotics, achieves effective control of PED virus through a triple mechanism of physically blocking viral adsorption, activating the host's innate immune response, and regulating intestinal inflammatory response. It also opens up a new technical path for the combined application of probiotic metabolites and bile acid derivatives in the prevention and control of animal viral diseases.

[0008] Technical solution: An oral preparation for the prevention of porcine epidemic diarrhea, composed of Lactobacillus plantarum metabolites and / or ursodeoxycholic acid, wherein the Lactobacillus plantarum metabolites are freeze-dried powder of Lactobacillus plantarum culture supernatant after sterile filtration, and the Lactobacillus plantarum is Lactobacillus plantarum LP-1 strain with the accession number corresponding to GenBank accession number MH727586.1; the ursodeoxycholic acid is pharmaceutical grade pure product with a purity not lower than a preset purity threshold.

[0009] The above-mentioned Lactobacillus plantarum metabolites were prepared by the following method: Lactobacillus plantarum LP-1 strain was inoculated into MRS liquid medium, cultured under anaerobic conditions within a predetermined temperature range to the late logarithmic growth stage, centrifuged to remove bacterial cells, and the supernatant was collected, filtered through a filter membrane with a pore size not greater than a preset pore size threshold for sterilization, and then freeze-dried to prepare lyophilized powder, which was then sealed and stored in a light-proof and dry environment.

[0010] The above-mentioned oral preparations are used in the preparation of oral liquids or powders for the prevention of PEDV in piglets of different ages.

[0011] The above-mentioned oral formulation for the prevention of porcine epidemic diarrhea includes the following components: 1) Lactobacillus plantarum metabolites; 2) ursodeoxycholic acid; 3) oral excipients; wherein the mass ratio of Lactobacillus plantarum metabolites to ursodeoxycholic acid is within a preset ratio range, and the oral excipients are selected from one or more combinations of microcrystalline cellulose, lactose, mannitol, trehalose, and sodium carboxymethyl cellulose, and the amount added is adjusted according to the physical stability requirements of the final dosage form of the formulation.

[0012] The dosage form of the above-mentioned oral preparation is an oral solution, an oral suspension, an oral lyophilized powder, or an oral granule; when the dosage form is an oral lyophilized powder, the preparation also includes a lyophilization protectant, which is a mixture of trehalose and glycine, and its total mass accounts for a predetermined mass percentage of the total mass of the solution before lyophilization.

[0013] The reagent composition of the above oral formulation is as follows: Base carrier solution: prepared by dissolving an oral excipient carrier in sterile purified water; Active component I: lyophilized powder of Lactobacillus plantarum metabolite, pre-packaged in individual aluminum foil bags according to the amount of active ingredient required per unit dose; Active component II: ursodeoxycholic acid powder, pre-packaged in individual aluminum foil bags according to the amount of active ingredient required per unit dose; When using, add active component I and / or active component II to the base carrier solution, mix thoroughly to form a ready-to-use oral solution.

[0014] The method of administration for oral preparations used for the prevention of porcine epidemic diarrhea (PED) for non-therapeutic purposes is as follows: The dosage for a single administration is determined based on the age and weight of the target piglets. The age is divided into three categories: newborn piglets, high-risk weak piglets intervention group, and weaning transition group. Each category corresponds to a different dosage specification. Take the base carrier solution, add the corresponding doses of active component I and / or active component II, and stir at a predetermined speed for no less than a predetermined time at room temperature until a uniform dispersion system is formed; The prepared oral formulation is administered to the target piglets via gastric tube, bottle, or drinking system. The administration frequency is once daily or every other day, and the continuous administration period covers the peak season of PEDV or the period of risk of disease exposure. During the administration period, the fecal characteristics, feeding behavior, and survival status of piglets were monitored simultaneously as auxiliary evaluation indicators of the formulation's effectiveness.

[0015] In the above application methods, the administration of the newborn piglet group started within twelve hours after birth and continued for no less than the predetermined number of days; the administration of the high-risk weak piglet intervention group started within six hours after the appearance of suspected clinical symptoms and continued until the symptoms were relieved for no less than the predetermined number of days; the administration of the weaning transition group started one day before weaning and continued until no less than the predetermined number of days after weaning.

[0016] In the above-mentioned oral formulations, the mechanism of action of Lactobacillus plantarum metabolites includes: specific physical adhesion of porcine epidemic diarrhea virus particles to their surface polysaccharide and protein components, blocking the binding of the virus to potential receptors on the surface of small intestinal epithelial cells; at the same time, downregulating the expression levels of local intestinal pro-inflammatory factors IL-1β and IL-6, and promoting infected or damaged intestinal epithelial cells to enter the programmed apoptosis pathway, thereby limiting the spread and replication of the virus in the intestine.

[0017] In the aforementioned oral formulations, the mechanism of action of ursodeoxycholic acid includes: activating the IRF3 signaling molecule in intestinal epithelial cells, thereby inducing the expression of type I interferon IFN-α / β, initiating the JAK-STAT1 signaling pathway, and ultimately upregulating the transcriptional levels of interferon-stimulated genes MX1, ISG15, OASL, and ZAP, forming an antiviral state to inhibit the RNA replication process of porcine epidemic diarrhea virus; in addition, ursodeoxycholic acid also alleviates virus-induced intestinal villus atrophy and increased epithelial permeability by regulating the expression of intestinal barrier-related proteins.

[0018] In the preparation of the above oral formulation, the mixing order of Lactobacillus plantarum metabolites and ursodeoxycholic acid is as follows: first, ursodeoxycholic acid is dissolved in a base carrier solution containing a small amount of solubilizer. After it is completely dissolved, the lyophilized powder of Lactobacillus plantarum metabolites is added to avoid the inactivation of active proteins in the metabolites due to sudden changes in pH or ionic strength.

[0019] The above-mentioned oral formulation is packaged in a dual-chamber, independently sealed structure. The first chamber contains the base carrier solution, and the second chamber contains the mixed lyophilized powder of active component I and active component II. Before use, the two chambers are connected by pressing or rotating to achieve instant mixing and ensure that the active ingredients maintain maximum stability before administration.

[0020] Compared with existing technologies, this invention has the following advantages: For the first time, this invention scientifically combines the metabolites of *Lactobacillus plantarum* LP-1 with ursodeoxycholic acid to construct a compound oral formulation based on a three-pronged mechanism of action: virus adsorption blocking, innate immune activation, and inflammatory response regulation. This formulation does not rely on the definitive identification of virus-specific receptors, nor is it affected by viral antigenic drift, and maintains broad-spectrum inhibitory activity against currently prevalent highly variable PEDV strains. *Lactobacillus plantarum* metabolites directly neutralize virus particles through physical adhesion, acting rapidly and exerting a barrier effect in the early stages of viral infection; ursodeoxycholic acid activates endogenous antiviral pathways in host cells, establishing a durable immune defense state. The two complement each other synergistically in terms of time and target. The formulation is administered orally, conforming to the physiological characteristics of piglets and avoiding injection stress, making it particularly suitable for newborn piglets under 7 days old who cannot effectively develop active immunity. By designing differentiated dosing regimens for different age groups, it achieves full-cycle coverage from immediate protection at birth, emergency intervention for weak piglets, to the transition to weaning stress. All ingredients used are recognized as safe substances with no risk of antibiotic residues, aligning with green farming and antibiotic reduction policies. The formulation employs a dual-chamber packaging design separating the lyophilized powder and liquid, significantly improving the stability of the active ingredients during storage and transportation, and overcoming the technical bottleneck of low oral bioavailability of bioactive substances. The overall solution is simple to operate, cost-effective, and highly safe, providing a new, scalable strategy for the precise prevention and control of porcine epidemic diarrhea (PED). Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the early apoptosis of damaged cells induced by LPM as described in this invention. Among them: A. caspase-3 mRNA and protein levels at different time points under physiological conditions; B. caspase-3 mRNA and protein levels at different time points after LPM and PEDV treatment; C. immunofluorescence results of caspase-3 protein after LPM and PEDV treatment; D. STAT1 protein level after LPM and PEDV treatment; E. antiviral factor transcription levels after LPM and PEDV treatment; Figure 2 This is a schematic diagram illustrating the changes in IRF3 transcription levels under PEDV infection conditions after UDCA treatment as described in this invention. Figure 3 This is a schematic diagram of the Western blot detection results of p-IRF3 protein expression as described in this invention; Figure 4 This is a schematic diagram of the grayscale analysis results of p-IRF3 quantified protein after UDCA pretreatment according to the present invention; Figure 5This is a schematic diagram showing the changes in IRF3 nuclear shift in IPEC-J2 cells under PEDV infection conditions after UDCA treatment, as described in this invention. Figure 6 This is a schematic diagram illustrating the detection of IFN-a transcription levels at different time points using RT-qPCR as described in this invention. Figure 7 This is a schematic diagram illustrating the IFN-β transcription level detection at different time points using RT-qPCR as described in this invention. Figure 8 This is a schematic diagram illustrating the ELISA detection of IFN-α secretion in IPEC-J2 cells according to the present invention; Figure 9 This is a schematic diagram illustrating the ELISA detection of IFN-β secretion in IPEC-J2 cells according to the present invention; Figure 10 This is a schematic diagram illustrating the changes in STAT1 transcription levels at different time points detected by RT-qPCR as described in this invention. Figure 11 This is a schematic diagram of the Western blot detection of p-STAT1 protein expression results described in this invention; Figure 12 This is a schematic diagram of the protein grayscale analysis results described in this invention; Figure 13 This is a schematic diagram showing the changes in STAT1 nuclear shift in IPEC-J2 cells under PEDV infection conditions after UDCA treatment, as described in this invention. Figure 14 This diagram illustrates the transcriptional levels of MX1, ISG15, OASL, and ZAP in IPEC-J2 cells under PEDV infection conditions following UDCA treatment, as described in this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments.

[0023] Example 1 An oral preparation for the prevention of porcine epidemic diarrhea, comprising the following components: The preparation method of the basic carrier solution is as follows: Oral excipient: prepared by dissolving 5 wt.% trehalose and 0.5 wt.% sodium carboxymethyl cellulose in sterile purified water, with a total volume of 10 mL; The preparation method of active component I is as follows: The freeze-dried powder of *Lactobacillus plantarum* metabolites is derived from *Lactobacillus plantarum* LP-1 (GenBank accession number: MH727586.1). It was obtained by anaerobic culture in MRS liquid medium to the late logarithmic growth stage, centrifugation to remove bacterial cells, filtration through a 0.22 μm filter membrane for sterilization, and then freeze-drying. Each sachet contains the equivalent of 1 × 10⁻⁶ effective metabolites. 9 The active ingredient of the original culture medium at CFU / mL is pre-packaged into individual aluminum foil bags according to a single-dose dosage. The above-mentioned active component I and the base carrier solution are placed in different chambers of the dual-chamber packaging structure. When in use, the two chambers are connected by rotating the opening device, and after mixing, a ready-to-use oral liquid is formed.

[0024] The method of use for oral preparations is as follows: Healthy newborn piglets weighing 1.2–1.8 kg within 6 hours of birth were selected as the target group; Take 10 mL of the basic carrier solution, add one packet of active component I, and stir at 300 rpm for 5 minutes at room temperature to form a uniform dispersion system; The prepared oral solution is administered via bottle, with a single dose of 2 mL per head, once daily for 5 consecutive days; Record the piglets' daily fecal score (0=normal formed, 1=soft stool, 2=watery diarrhea), feed intake and survival status during the administration period; On day 5 of drug administration, ileal tissue samples were collected, and the relative expression levels of IL-1β, IL-6 and MX1 genes were detected by qRT-PCR. Meanwhile, an untreated control group and a commercially available probiotic control group were set up to compare the morbidity and mortality rates of each group after PEDV challenge. On day 7 after viral challenge, the survival rate of each group was calculated, and the intestinal tissue was pathologically scored to assess the villus height / crypt depth ratio (V / C ratio).

[0025] The experimental results are shown in Table 1. Table 1 compares the clinical symptoms, expression of inflammatory factors and histopathological indicators of different treatment groups after PEDV challenge.

[0026] Table 1

[0027] Example 2 An oral preparation for the prevention of porcine epidemic diarrhea, comprising the following components: The preparation method of the basic carrier solution is as follows: Oral excipient: prepared by dissolving 4 wt.% mannitol and 1 wt.% microcrystalline cellulose in sterile purified water, with a total volume of 10 mL; The preparation method of active component II is as follows: Ursodeoxycholic acid powder, pharmaceutical grade, purity ≥99%, each bag contains 50mg, pre-packaged in individual aluminum foil bags according to single-dose dosage; The active component II and the base carrier solution were placed in different chambers of the dual-chamber packaging structure and mixed before use.

[0028] The oral formulation described above was tested using the method described in Example 1. The target group was piglets weighing 6–8 kg on the first day after weaning. The medication was administered continuously for 7 days, followed by a 10-day observation period after viral challenge. The experimental results are shown in Table 2.

[0029] Table 2

[0030] Example 3 An oral preparation for the prevention of porcine epidemic diarrhea, comprising the following components: The preparation method of the basic carrier solution is as follows: Oral excipient: prepared by dissolving 3 wt.% lactose, 2 wt.% trehalose and 0.3 wt.% sodium carboxymethyl cellulose in sterile purified water, with a total volume of 10 mL; The preparation method of active component I is as follows: Lyophilized powder of Lactobacillus plantarum metabolites, each sachet contains the equivalent of 5 × 10⁻⁶ effective metabolites. 8 CFU / mL of the active ingredient in the original culture medium; The preparation method of active component II is as follows: Ursodeoxycholic acid powder, each sachet contains 30mg; Active component I and active component II are encapsulated together in the same aluminum foil bag, and placed separately from the base carrier solution in two independent chambers of the dual-chamber packaging structure.

[0031] The oral formulation described above was tested using the method described in Example 1. The target population was 7-day-old weak pups exhibiting mild diarrhea symptoms (fecal score of 1). Administration began within 4 hours of symptom onset and continued for 5 consecutive days. The experimental results are shown in Table 3.

[0032] Table 3

[0033] As shown in Tables 1, 2, and 3, the oral formulation described in this invention significantly reduced the mortality rate and clinical symptom severity caused by PEDV infection in different application scenarios. The treatment group using the combined Lactobacillus plantarum metabolite and ursodeoxycholic acid showed significantly higher levels of induction of interferon-stimulated genes (such as MX1, ISG15, OASL, and ZAP) than the single-component treatment group. Simultaneously, the expression of pro-inflammatory factors IL-1β and IL-6 was effectively inhibited, and the intestinal epithelial cell apoptosis index was moderately increased, indicating that damaged cells were promptly cleared. Furthermore, the maintenance of the V / C ratio indicates that the integrity of the intestinal villus structure was protected, which is consistent with the regulatory effect of ursodeoxycholic acid on intestinal barrier function.

[0034] The above description is merely an analysis of the action path of this invention in a typical application scenario. In actual implementation, the timing, dosage, and ratio of administration can be adaptively adjusted according to the breeding environment, viral load, and health status of piglets. All equivalent implementation schemes based on the core mechanism of this invention should be included in the scope of patent protection.

Claims

1. An oral preparation for the prevention of porcine epidemic diarrhea, characterized in that: The product includes *Lactobacillus plantarum* metabolites and / or ursodeoxycholic acid, wherein the mass ratio of *Lactobacillus plantarum* metabolites to ursodeoxycholic acid is 1:0.1 to 1:10, wherein the *Lactobacillus plantarum* metabolites are lyophilized powder of *Lactobacillus plantarum* LP-1 strain culture supernatant after aseptic filtration, wherein the *Lactobacillus plantarum* is *Lactobacillus plantarum* LP-1 strain with the accession number corresponding to GenBank accession number MH727586.1, and wherein the ursodeoxycholic acid is pharmaceutical grade pure product.

2. The oral preparation for preventing porcine epidemic diarrhea as described in claim 1, characterized in that... The Lactobacillus plantarum metabolites were prepared by inoculating Lactobacillus plantarum LP-1 strain into MRS liquid medium, culturing it under anaerobic conditions at 37°C to the late logarithmic growth stage, centrifuging to remove the bacterial cells, collecting the supernatant, filtering it through a 0.22 μm pore size filter membrane for sterilization, and then freeze-drying it to prepare lyophilized powder.

3. The oral preparation for preventing porcine epidemic diarrhea as described in claim 1 or 2, characterized in that... It also includes oral excipients selected from one or more combinations of microcrystalline cellulose, lactose, mannitol, trehalose, and sodium carboxymethyl cellulose.

4. The oral preparation for preventing porcine epidemic diarrhea as described in claim 3, characterized in that... Dosage forms include oral solutions, oral suspensions, oral lyophilized powders, or oral granules; When the dosage form is an oral lyophilized powder, it also includes a lyophilization protectant, which is a mixture of trehalose and glycine, and its total mass accounts for 5-10 wt.% of the total mass of the solution before lyophilization.

5. The oral preparation for preventing porcine epidemic diarrhea as described in claim 3, characterized in that... The reagent composition is as follows: Basic carrier solution: prepared by dissolving an oral excipient in sterile purified water; Active ingredient I: Lyophilized powder of Lactobacillus plantarum metabolites, pre-packaged in individual aluminum foil bags according to the amount of active ingredient required per unit dose; Active ingredient II: Ursodeoxycholic acid powder, pre-packaged in individual aluminum foil bags according to the amount of active ingredient required per unit dose; When using, add active component I and / or active component II to the base carrier solution, mix well, and then form a ready-to-use oral solution.

6. The oral preparation for preventing porcine epidemic diarrhea as described in claim 5, characterized in that... The concentration of the oral excipient in the base carrier solution is: trehalose 3 wt.% to 5 wt.%, sodium carboxymethyl cellulose 0.3 wt.% to 0.5 wt.%, or mannitol 4 wt.%, microcrystalline cellulose 1 wt.%, or a combination of lactose 3 wt.%, trehalose 2 wt.%, and sodium carboxymethyl cellulose 0.3 wt.%.

7. The oral preparation for preventing porcine epidemic diarrhea as described in claim 5, characterized in that... During the preparation process, ursodeoxycholic acid powder is first dissolved in a base carrier solution containing a small amount of solubilizer. After it is completely dissolved, lyophilized Lactobacillus plantarum metabolite powder is added.

8. The oral preparation for preventing porcine epidemic diarrhea as described in claim 5, characterized in that... It adopts a dual-chamber independent sealed packaging structure. The first chamber contains the base carrier solution, and the second chamber contains the mixed lyophilized powder of active component I and active component II or the separately packaged lyophilized powder. Before use, the two chambers are connected by pressing or rotating.

9. The use of the oral formulation according to any one of claims 1 to 8 in the preparation of an oral liquid or oral powder for the prevention of porcine epidemic diarrhea virus in piglets of different ages.

10. The method of application of an oral preparation for the prevention of porcine epidemic diarrhea (PED) for non-therapeutic purposes, characterized in that... Includes the following steps: The dosage for a single administration is determined based on the age and weight of the target piglets, with the age group divided into newborn piglet group, high-risk weak piglet intervention group, and weaning transition group. Take the base carrier solution, add the corresponding doses of active component I and / or active component II, and stir at 300 rpm for no less than 5 minutes at room temperature to form a uniform dispersion system; The prepared oral formulation is administered to the target piglets via a gastric tube, feeding bottle, or drinking system. The administration frequency is once daily or once every other day, and the continuous administration period covers the peak season of porcine epidemic diarrhea virus or the period of risk of disease exposure. Monitor the fecal characteristics, feeding behavior, and survival status of piglets during the drug administration period.

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