A Clostridium paraputrificum JN807 and application thereof in preparation of a medicine for preventing and / or treating diarrhea of piglets

Abstract

This invention relates to the field of microbial technology, specifically to a double-fermented *Clostridium parahaemolyticus* JN807 and its application in the preparation of drugs for the prevention and / or treatment of diarrhea in piglets. Double-fermented *Clostridium parahaemolyticus* (JN807) Paraclostridium bifermentans JN807 is acid- and bile-resistant, and tolerates artificial gastrointestinal fluids well, thus it can easily pass through the stomach and enter the small intestine to exert its effects. Furthermore, the double-fermented *Clostridium paragallinarum* (… Paraclostridium bifermentans JN807 can effectively inhibit enterotoxin-producing Escherichia coli both in vivo and in vitro; and it can synergistically enhance the effect of preparing products for the prevention and / or treatment of piglet diarrhea with Clostridium butyricum BNCC337239.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a double-fermented Clostridium parasiticum JN807 and its application in the preparation of drugs for the prevention and / or treatment of diarrhea in piglets. Background Technology

[0002] Piglets are the core of modern pig production, and their health has a crucial impact on their later growth and development. In practice, to maximize economic benefits, intensive pig farming commonly employs early weaning techniques for piglets. However, because piglets have not yet established a stable intestinal micro-ecosystem, their resistance is low, making them sensitive to external stimuli and susceptible to various pathogens and stressors, thus making them prone to diarrhea. Escherichia coli is one of the most significant pathogens causing diarrhea in newborn and weaned piglets.

[0003] Currently, internationally recognized pathogenic Escherichia coli are mainly classified into five types, including pathogenic Escherichia coli, enterotoxigenic Escherichia coli (ETEC), invasive Escherichia coli, enterohemorrhagic Escherichia coli, and enterocytic Escherichia coli. Enterotoxigenic Escherichia coli is the primary cause of diarrhea and death in newborn or weaned piglets infected with this bacteria.

[0004] Antibiotics, as growth promoters, are widely used in livestock and poultry farming, making a significant contribution to the industry. However, the long-term and excessive use of antibiotics disrupts the body's microecological balance, leading to the emergence of drug-resistant bacteria and bacterial mutations. In recent years, the incidence of diarrhea in piglets caused by multidrug-resistant Escherichia coli has been increasing, seriously affecting piglet survival rates and easily causing endogenous or secondary infections. Therefore, there is an urgent need to research and develop a new formulation to effectively control piglet diarrhea.

[0005] Currently, the use of vaccines and probiotic preparations for prevention and treatment is the most promising approach. However, due to the numerous serotypes, high variability, and complex antigens of enterotoxigenic Escherichia coli, the actual effectiveness of vaccines varies, and they are costly, requiring stringent transportation and storage conditions. Furthermore, the use of live attenuated vaccines carries the risk of in vivo reversion to the virus; therefore, large-scale vaccine rollout faces significant challenges. Probiotics, as live bacterial preparations, promote nutrient metabolism and absorption, stimulate the animal immune system, and enhance disease resistance. In particular, intestinal probiotics have attracted widespread attention due to their advantages such as promoting the development of the animal's intestinal immune system and being environmentally friendly and residue-free.

[0006] Currently, there are studies on using probiotics as feed additives to reduce the diarrhea rate in piglets. However, probiotics themselves have some disadvantages, such as inconsistent antibacterial effects, intolerance to gastrointestinal fluids, and intolerance to bile. These problems mean that probiotic preparations may have good effects under laboratory conditions, but often fail to achieve the expected results under production conditions. Summary of the Invention

[0007] To overcome the above problems, the present invention provides a double-fermented Clostridium parasiticum JN807 and its application in the preparation of drugs for the prevention and / or treatment of diarrhea in piglets.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides a dual-fermentation Clostridium para-fermentans ( Paraclostridium bifermentans JN807 was deposited on August 11, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251813.

[0010] A second aspect of the present invention provides a probiotic composition comprising the double-fermented Clostridium parahaemolyticus described in the first aspect (… Paraclostridium bifermentans JN807 and Clostridium butyricum.

[0011] In one or more embodiments, the Clostridium butyricum is Clostridium butyricum BNCC337239.

[0012] A third aspect of the invention provides the dual-fermentation Clostridium para-fermentans described in the first aspect ( Paraclostridium bifermentans The use of the probiotic composition described in JN807 or the second aspect in the preparation of a medicine for the prevention and / or treatment of diarrhea in piglets.

[0013] In one or more embodiments, the application includes:

[0014] (1) Increase the weight of piglets with diarrhea;

[0015] (2) The diarrhea in piglets stopped;

[0016] (3) Reduce the expression of inflammatory factors in the serum of piglets with diarrhea, including IL-1β, TNF-α and IL-6;

[0017] (4) Reduce the expression of pro-inflammatory factors in the ileum of diarrheal piglets, including IL-6 and IL-1β;

[0018] (5) Enhance the expression of anti-inflammatory factors in the ileum of diarrheal piglets, wherein the anti-inflammatory factors include IL-10 and IL-4;

[0019] (6) Increase the content of secretory immunoglobulin A (sIgA) in the ileum of diarrheal piglets;

[0020] (7) Promotes the differentiation of CD4+ and CD8+ cells in the peripheral blood of diarrheal piglets;

[0021] (8) Increase the expression of tight junction proteins in the intestinal mucosal barrier of diarrheal piglets, wherein the tight junction proteins in the intestinal mucosal barrier include ZO-1 (zonula occludens-1) and Occludin;

[0022] (9) Maintain the integrity of the intestinal morphology and structure of piglets with diarrhea;

[0023] (10) Promotes the growth and differentiation of intestinal villi in piglets with diarrhea;

[0024] (11) Improve the absorption of intestinal nutrients in piglets with diarrhea.

[0025] In one or more embodiments, the piglet diarrhea is caused by Escherichia coli infection;

[0026] Preferably, the Escherichia coli is an enterotoxin-producing Escherichia coli.

[0027] A fourth aspect of the present invention provides a drug for the prevention and / or treatment of diarrhea in piglets, comprising the double-fermented Clostridium paragallinarum described in the first aspect (… Paraclostridium bifermentans The probiotic composition described in JN807 or the second aspect.

[0028] In one or more embodiments, the piglet diarrhea is caused by Escherichia coli infection;

[0029] Preferably, the Escherichia coli is an enterotoxin-producing Escherichia coli.

[0030] A fifth aspect of the present invention provides an animal feed additive comprising the dual-fermentation Clostridium paragallinarum described in the first aspect (… Paraclostridium bifermentans The probiotic composition described in JN807 or the second aspect.

[0031] A sixth aspect of the present invention provides an animal feed comprising the animal feed additives described in the fifth aspect.

[0032] The beneficial effects of this invention are as follows:

[0033] The dual-fermentation Clostridium parahaemolyticus provided by this invention ( Paraclostridium bifermentans JN807 is acid- and bile-resistant, and tolerates artificial gastrointestinal fluids well, thus it can easily pass through the stomach and enter the small intestine to exert its effects. Furthermore, the double-fermented *Clostridium paragallinarum* (… Paraclostridium bifermentans JN807 effectively inhibits enterotoxin-producing Escherichia coli both in vitro and in vivo. Specifically, in in vitro plate inhibition experiments, it inhibited the growth of double-fermented Clostridium paragallinarum (…). Paraclostridium bifermentans JN807 effectively inhibited the growth of enterotoxigenic Escherichia coli, with an average inhibition zone diameter of 2.5 cm. In in vivo experiments, the double-fermenting Clostridium paragallinarum (… Paraclostridium bifermentansBoth JN807 and the probiotic combination can significantly alleviate diarrhea in piglets caused by enterotoxigenic Escherichia coli infection, with the following effects: (1) increasing the weight of diarrheal piglets; (2) stopping diarrhea in diarrheal piglets; (3) reducing the expression of inflammatory factors in the serum of diarrheal piglets, including IL-1β, TNF-α and IL-6; (4) reducing the expression of pro-inflammatory factors in the ileum of diarrheal piglets, including IL-6 and IL-1β; (5) increasing the expression of anti-inflammatory factors in the ileum of diarrheal piglets, including IL-10 and IL-4; (6) increasing the content of secretory immunoglobulin A (sIgA) in the ileum of diarrheal piglets; (7) promoting the differentiation of CD4+ cells and CD8+ cells in the peripheral blood of diarrheal piglets; (8) increasing the expression of tight junction proteins in the intestinal mucosal barrier of diarrheal piglets, including ZO-1 (zonula) (9) Maintain the integrity of the intestinal morphology of diarrheal piglets; (10) Promote the growth and differentiation of intestinal villi in diarrheal piglets; (11) Improve the absorption of nutrients in the intestines of diarrheal piglets. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 Image showing Gram staining results of the isolated strain;

[0036] Figure 2 For double-fermenting Clostridium paraferi ( Paraclostridium bifermentans Growth curve of JN807;

[0037] Figure 3 For double-fermenting Clostridium paraferi ( Paraclostridium bifermentans The results of JN807's tolerance to artificial intestinal fluid and artificial gastric fluid are shown in the figure. In the figure, A is the curve of tolerance to artificial intestinal fluid and B is the curve of tolerance to artificial gastric fluid.

[0038] Figure 4 For double-fermenting Clostridium paraferi ( Paraclostridium bifermentans Results of in vitro plate antibacterial test of JN807;

[0039] Figure 5 The groups included a healthy group, a control group, a *Clostridium butyricum* BNCC337239 group, and a double-fermentation *Clostridium parahaemolyticus* group. Paraclostridium bifermentans JN807 group and probiotic composition group (double fermentation Clostridium parahaemolyticus) Paraclostridium bifermentans(JN807 and Clostridium butyricum BNCC337239) Piglet weight changes and survival status; where A is the graph of piglet weight change over time in each group; B is the survival curve of piglets in each group;

[0040] Figure 6 Diarrhea scores for each group of piglets;

[0041] Figure 7 The values ​​represent the levels of inflammatory factors in the serum of piglets in each group; where A represents IL-1β, B represents TNF-α, and C represents IL-6.

[0042] Figure 8 The relative contents of inflammatory factors in the ileum of piglets in each group are: A = IL-10, B = IL-4, C = IL-6, and D = IL-1β.

[0043] Figure 9 The sIgA content in the ileum of piglets in each group;

[0044] Figure 10 The differentiation of lymphocytes in the peripheral blood of piglets in each group is shown; where A represents CD4+ cells and B represents CD8+ cells.

[0045] Figure 11 The relative expression levels of tight junction proteins related to intestinal permeability in the intestines of piglets are shown in Figure 1; where A represents ZO-1 and B represents Occludin.

[0046] Figure 12 The villus status and pathological damage of the ileum in piglets in each group are shown. A is the control group, B is the healthy group, C is the *Clostridium butyricum* BNCC337239 group, and D is the double-fermented *Clostridium paraflavum* group. Paraclostridium bifermentans JN807 group and E is the probiotic composition group (double fermentation Clostridium parahaemolyticus ( Paraclostridium bifermentans (JN807 and Clostridium butyricum BNCC337239). Detailed Implementation

[0047] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration 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.

[0048] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0049] In this invention, *Clostridium parafertigrinum* (a double-fermenting bacterium) was isolated from fresh feces of Northeast China's wild boars. Paraclostridium bifermentans JN807 was deposited on August 11, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251813.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Deman-Rogosa-Sharp (MRS) liquid medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, Tween-80 1 mL / L, pH 6.0 ± 0.05, sterilized at 121 ℃ for 20 min. Solid medium requires the addition of 5 g / L agar powder.

[0052] Enterotoxin-producing Escherichia coli strain ATCC 25922 is preserved at the Jilin Provincial Engineering Research Center for Animal Microecological Preparations.

[0053] Clostridium butyricum BNCC337239 was purchased from Beina Biotechnology, product number BNCC337239.

[0054] Example 1

[0055] Take 5 g of fresh feces from Northeast China's native pigs and use a gradient dilution method to dilute the feces to 1, 10, and other concentrations. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 and 10 -9 The colonies were spread onto MRS liquid medium and incubated in a shaker at 37 ℃ for 18 h. After a single colony grew on the medium, it was picked and placed into MRS liquid medium. After incubation, Gram staining was performed. The experiment was repeated three times.

[0056] Add a drop of physiological saline to a glass slide, then select a single colony from the plate, transfer it to the saline, spread it evenly, fix it with a flame, and stain it after the liquid on the slide has evaporated. Cover with ammonium oxalate crystal violet, rinse with running water after 3 min, cover with Luger's iodine solution, rinse with running water after 2.5 min, decolorize with 95% alcohol, rinse with water after 10 s, and finally cover with carbolic acid fuchsin. After 3 min, blot dry with absorbent paper and observe under a microscope.

[0057] like Figure 1 As shown, Gram staining of the isolated strain revealed that it appeared purple under a microscope, indicating it was a Gram-positive bacterium.

[0058] DNA was extracted from the isolated strain using a DNA extraction kit, and the 16S rRNA gene was amplified. The sample was then sent to a biotechnology company for Sanger sequencing. The sequencing results, after BLAST comparison with NCBI, confirmed the strain as *Clostridium paraferi* (a double-fermentation bacterium). Paraclostridium bifermentans ).

[0059] Double-fermenting Clostridium parafolium was isolated. Paraclostridium bifermentans JN807 was deposited on August 11, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20251813.

[0060] Double-fermented Clostridium parahaemolyticus ( Paraclostridium bifermentans JN807, according to 1×10 7 A number of bacteria were added to 50 mL of MRS liquid culture medium, and the optical density OD value was measured using a UV spectrophotometer and recorded as 0 h. Subsequently, the optical density OD value was measured at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h and 24 h, and the results were plotted as a growth curve.

[0061] The results are as follows Figure 2 As shown, double-fermented Clostridium paraflavum ( Paraclostridium bifermentans JN807 was in the logarithmic growth phase from 4 h to 10 h, and in the plateau phase from 10 h to 15 h.

[0062] Example 2

[0063] Double-fermented Clostridium paraflavum ( Paraclostridium bifermentans Tolerance of JN807 to artificial intestinal fluid and artificial gastric fluid:

[0064] Preparation method of artificial gastric juice: Add 800 mL of water and 10 g of pepsin to 16.4 mL of 1 mol / L dilute hydrochloric acid, stir well, and then add water to make up to 1000 mL.

[0065] Preparation method of artificial intestinal fluid: Add 500 mL of water to 6.8 g of potassium dihydrogen phosphate, adjust the pH to 6.8 with 0.4 wt% sodium hydroxide solution to obtain solution A; add 50 mL of water to 10 g of pancreatic enzyme to obtain solution B; mix solutions A and B, and add water to make up to 1000 mL.

[0066] Double-fermented Clostridium parahaemolyticus ( Paraclostridium bifermentans JN807 strain was administered at a rate of 1×10⁻⁶. 9 CFU / mL of bacteria were added to simulated intestinal and gastric fluids, respectively. After anaerobic culture for 12 h, the bacteria were serially diluted and counted on MRS plates to determine the number of colonies.

[0067] The results are as follows Figure 3 As shown, from Figure 3 As can be seen, *Clostridium paragallinarum* exhibits strong tolerance to artificial intestinal fluid, with a survival rate as high as 80%. It also shows tolerance to artificial gastric fluid, with a survival rate of 85%.

[0068] Example 3

[0069] In vitro plate antibacterial test:

[0070] After the enterotoxin-producing Escherichia coli strain ATCC 25922 was activated and restored to activity in LB liquid medium for three generations, it was reinoculated into LB liquid medium at a rate of 2% (w / v). After incubation in a shaker at 37 ℃ for 12 h, the bacterial suspension was evenly spread onto MRS plates using a sterile disposable spreader. The medium was then allowed to air dry in a laminar flow hood. Holes were punched in the MRS plates using a 9 mm diameter punch. The plates were then sealed by heating them over the outer flame of an alcohol lamp for 3-5 s.

[0071] Double-fermented Clostridium parahaemolyticus ( Paraclostridium bifermentans JN807 and Clostridium butyricum BNCC337239 (as positive controls) were inoculated into MRS liquid medium and cultured for 12 h. The bacterial culture was centrifuged, the supernatant was discarded, and the culture was resuspended in phosphate-buffered saline (PBS). After washing twice, the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 8 CFU / mL, 150 μL of bacterial culture was pipetted into each well, and sterile PBS was added to the other well as a blank control. After the bacterial culture was completely absorbed into the culture medium, the wells were incubated at 37 ℃ for 24 h. The diameter of the transparent inhibition zone in each well was measured; a larger diameter indicated stronger antibacterial ability. Results are shown below. Figure 4 As shown.

[0072] from Figure 4 It can be seen from the double fermentation of Clostridium paraferii ( Paraclostridium bifermentans JN807 can significantly inhibit Escherichia coli (E. coli) P The growth of *Clostridium butyricum* (<0.0001) was inhibited, with an average inhibition zone diameter of 2.5 cm. *Clostridium butyricum* BNCC337239 also significantly inhibited the growth of *Escherichia coli*. P <0.001), and the average diameter of its inhibition zone is 1.2 cm.

[0073] Example 4

[0074] Double-fermented Clostridium paraflavum ( Paraclostridium bifermentans Both JN807 and the probiotic combination can significantly alleviate diarrhea in piglets caused by enterotoxigenic Escherichia coli infection.

[0075] Fifteen 2-day-old piglets (whose mothers had not been vaccinated against E. coli diarrhea) were randomly divided into 5 groups: a healthy group, a control group, a Clostridium butyricum BNCC337239 group, and a double-fermented Clostridium paragallinarum group. Paraclostridium bifermentans JN807 group and probiotic composition group (double fermentation Clostridium parahaemolyticus) Paraclostridium bifermentans (JN807 and Clostridium butyricum BNCC337239), 3 animals in each group.

[0076] Oral immunization was administered on days 2, 4, 6, 8, and 10 after grouping, with the immunization dose being 1×10⁻⁶ for the Clostridium butyricum BNCC337239 group. 10 2 mL of double-fermented Clostridium parafol (cells / mL) Paraclostridium bifermentans JN807 group (1×10) 10 2 mL of probiotic composition (each strain contains 1×10⁻⁶ cells / mL), 2 mL of probiotic combination group (each strain contains 1×10⁻⁶ cells / mL). 10 Double-fermented Clostridium parahaemolyticus (CFU / mL) Paraclostridium bifermentans A mixture of 1 mL each of JN807 and Clostridium butyricum BNCC337239 was used. On day 10, the bacteria were infected with enterotoxigenic Escherichia coli at a bacterial count of 5 × 10⁻⁶. 9 CFU / mL, 10 mL per pig.

[0077] The changes in body weight in each group after infection with enterotoxigenic Escherichia coli were recorded. Results are as follows: Figure 5 As shown, from Figure 5 As can be seen from A, Clostridium butyricum group BNCC337239 and double-fermented Clostridium para-fermentation ( Paraclostridium bifermentans JN807 group and probiotic composition group (double fermentation Clostridium parahaemolyticus) Paraclostridium bifermentans Both JN807 and Clostridium butyricum BNCC337239 can increase the weight of piglets with diarrhea, and the probiotic combination group (double-fermented Clostridium parasiticum) can also increase the weight of piglets with diarrhea. Paraclostridium bifermentansThe weight gain of piglets with *Clostridium parahaemolyticus* (JN807 and BNCC337239) was significantly higher than that of other groups, indicating that the double-fermented *Clostridium parahaemolyticus* (JN807 and BNCC337239) was significantly higher than that of other groups. Paraclostridium bifermentans JN807 and Clostridium butyricum BNCC337239 synergistically enhance the effect against enterotoxigenic Escherichia coli.

[0078] Figure 5 Figure B shows the survival curve for piglets. The results showed that one piglet in the control group died on day 5 after infection with E. coli. No piglets in other groups died.

[0079] After piglets are infected with enterotoxigenic Escherichia coli, the diarrhea status of the piglets is recorded daily, and the characteristics of the piglets' feces are also recorded. A fecal scoring standard is used, with a score below 0 considered diarrhea. The fecal scoring standard is shown in Table 1.

[0080] Table 1. Stool Scoring Criteria

[0081]

[0082] The results are as follows Figure 6 As shown, from Figure 6 It can be seen that the control group piglets showed obvious diarrhea, while the butyric acid clostridium BNCC337239 group and the double-fermented Clostridium parahaemolyticus group (…) Paraclostridium bifermentans JN807 group and probiotic composition group (double fermentation Clostridium parahaemolyticus) Paraclostridium bifermentans The diarrhea scores of piglets with JN807 and Clostridium butyricum BNCC337239 were both greater than 1, indicating that the feces were soft, formed, and there was no obvious diarrhea.

[0083] Flow cytometry was performed on day 14 after infection with enterotoxigenic Escherichia coli. Blood and feces were collected from piglets in each group. Blood samples were incubated at 4 °C for 2 h, centrifuged at 4 °C and 4000 rpm / min for 20 min, and the serum was transferred to PCR tubes and stored at -80 °C. Piglet feces were weighed, and 3 mL of 1 mmol / L PMSF solution was added per g of feces. The mixture was incubated at 4 °C for 2 h, centrifuged at 4 °C and 4000 rpm / min for 20 min, and the supernatant was transferred to PCR tubes and stored at -80 °C.

[0084] The levels of inflammatory factors, including IL-1β, TNF-α, and IL-6, in the serum of piglets in each group were detected by enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 7 As shown, on day 14 post-infection with enterotoxigenic Escherichia coli, the probiotic combination group (double-fermented Clostridium paragallinarum (...) Paraclostridium bifermentansThe levels of IL-1β, TNF-α, and IL-6 in piglets of *Clostridium butyricum* (JN807) and *Clostridium butyricum* (BNCC337239) were significantly lower than those in the control group, and the levels of *Clostridium paraferii* (dual fermentation) were also significantly lower. Paraclostridium bifermentans JN807 and Clostridium butyricum BNCC337239 can synergistically reduce inflammatory factors in piglet serum.

[0085] Intestinal tissues from each group of piglets were flash-frozen using liquid nitrogen and then ground into a powder with no obvious particles using liquid nitrogen. The relative levels of inflammatory factors in the ileum of each group of piglets were detected using real-time quantitative PCR (Q-PCR), including pro-inflammatory factors IL-6 and IL-1β and anti-inflammatory factors IL-10 and IL-4. Results are shown below. Figure 8 Compared with the control group, the probiotic composition group (double-fermented Clostridium parahaemolyticus) Paraclostridium bifermentans The anti-inflammatory factors IL-10 and IL-4 in piglets (JN807 and Clostridium butyricum BNCC337239) were significantly higher than those in the control group and the single-strain group, while the probiotic combination group (double-fermented Clostridium parahaemolyticus) showed significantly higher levels. Paraclostridium bifermentans The pro-inflammatory factors IL-6 and IL-1β in piglets of the JN807 and Butyric acid Clostridium butyricum BNCC337239 groups were significantly lower than those in other groups, indicating that the double-fermented Clostridium parahaemolyticus (JN807 and JN807) groups had significantly lower levels than other groups. Paraclostridium bifermentans JN807 and Clostridium butyricum BNCC337239 can synergistically reduce anti-inflammatory factors IL-10 and IL-4, and synergistically increase pro-inflammatory factors IL-6 and IL-1β.

[0086] The sIgA content in the ileum of piglets in each group was detected by enzyme-linked immunosorbent assay (ELISA). sIgA is a class of proteins produced and secreted into the intestinal lumen by plasma cells of the lamina propria to exert immunomodulatory effects. It plays a role in regulating the intestinal microbiota and neutralizing toxins, and is an important effector molecule of the intestinal mucosa. Working in conjunction with other immune factors, it can also prevent pathogenic microorganisms from adhering to the intestinal mucosa and effectively inhibit the occurrence of mucosal-associated inflammatory responses, thus protecting the intestine from damage. Results are as follows: Figure 9 As shown, the results revealed that the control group of piglets had the lowest sIgA content, while the probiotic combination group (double-fermented Clostridium parahaemolyticus) had the highest sIgA content. Paraclostridium bifermentans The sIgA content of *Clostridium butyricum* (JN807) and *Clostridium butyricum* (BNCC337239) was the highest in piglets, significantly higher than in other groups. *Clostridium paraferii* (dual fermentation)... Paraclostridium bifermentans JN807 and Clostridium butyricum BNCC337239 can synergistically increase sIgA content.

[0087] The effects of probiotics on peripheral blood lymphocytes in piglets were compared among different groups using flow cytometry. Blood was collected from piglets in each group, and peripheral blood lymphocytes were isolated and analyzed by flow cytometry. Figure 10 It was found that double-fermented Clostridium paraferii ( Paraclostridium bifermentansThe levels of CD4+ and CD8+ cells in the blood of piglets in the JN807 group and the probiotic combination group were significantly higher than those in the control group. (Double-fermented Clostridium paragallinarum) Paraclostridium bifermentans JN807 immunization promoted the differentiation of CD4+ and CD8+ cells in piglet blood (0.01 < 0.01). P <0.05). Probiotic composition group (double-fermented Clostridium parahaemolyticus ( Paraclostridium bifermentans The number of CD4+ and CD8+ cells in piglets of the JN807 and Butyric acid Clostridium butyricum BNCC337239 groups was significantly higher than that in piglets of other groups, indicating that the double-fermented Clostridium paraferii (JN807 and JN807) groups were significantly higher than those in piglets of other groups. Paraclostridium bifermentans JN807 and Clostridium butyricum BNCC337239 can synergistically activate the immune system of piglets and promote the production and differentiation of lymphocytes.

[0088] The relative expression levels of tight junction proteins related to intestinal permeability in the intestines of piglets in each group were quantified using Q-PCR. The effects of probiotics on intestinal permeability and the intestinal mucosal barrier in piglets were compared at the mRNA level. The experimental results are as follows: Figure 11 ZO-1 and Occludin are markers of tight junction proteins in the intestinal mucosal barrier. *Clostridium paragallinarum* (double-fermented) Paraclostridium bifermentans JN807 group of piglets and probiotic combination group (double fermentation Clostridium paragenes) Paraclostridium bifermentans The relative expression levels of ZO-1 and Occludin in *Clostridium butyricum* (JN807) and *Clostridium butyricum* (BNCC337239) were higher than those in the control group. The probiotic combination group (double-fermented *Clostridium parahaemolyticus*) showed significantly higher levels of these two expression levels. Paraclostridium bifermentans Piglets infected with enterotoxigenic Escherichia coli (JN807 and Clostridium butyricum BNCC337239) showed a significant increase in tight junction proteins in their intestines, effectively blocking the infection of enterotoxigenic Escherichia coli.

[0089] By preparing pathological sections and performing HE staining, the villus condition and pathological damage of the ileum in piglets from different groups were compared. The results are shown in [Figure 1]. Figure 12 The results showed that the ileum villi of the control group piglets were lost, and a large number of inflammatory mediators were present at the base of the villi. A large number of blood cells were also present in the intestines, indicating that enterotoxins damaged the intestines of the control group piglets after infection with *E. coli*, leading to bleeding. Separation of the intestinal lamina propria from the intestinal muscular layer may also have contributed to the diarrhea in the piglets. *Clostridium parafertigationum* (also known as *Clostridium parafertigationum*)... Paraclostridium bifermentans JN807 group of piglets and probiotic combination group (double fermentation Clostridium paragenes) Paraclostridium bifermentans The intestinal villi of piglets (JN807 and Clostridium butyricum BNCC337239) were similar to those of the healthy group, and the double-fermented Clostridium paraferii (JN807 and Clostridium butyricum BNCC337239) were similar to those of the healthy group. Paraclostridium bifermentansJN807 and Clostridium butyricum BNCC337239 have positive effects on maintaining the integrity of the intestinal morphology and structure, promoting the growth and differentiation of intestinal villi, and improving the absorption function of intestinal nutrients. At the same time, they expand the absorption area of ​​intestinal villi, which can better absorb and transform nutrients.

[0090] 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 double-fermentation Clostridium parahaemolyticus ( Paraclostridium bifermentans JN807, characterized in that, It was deposited on August 11, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251813.

2. A probiotic composition, characterized in that, Including the dual-fermentation Clostridium parahaemolyticus as described in claim 1 ( Paraclostridium bifermentans JN807 and Clostridium butyricum, wherein the Clostridium butyricum is Clostridium butyricum BNCC337239.

3. The dual-fermentation Clostridium parahaemolyticus as described in claim 1 ( Paraclostridium bifermentans The use of the probiotic composition according to JN807 or claim 2 in the preparation of a drug for preventing diarrhea in piglets, wherein the diarrhea in piglets is caused by Escherichia coli infection; wherein the Escherichia coli is an enterotoxin-producing Escherichia coli.

4. A drug for preventing diarrhea in piglets, characterized in that, Including the dual-fermentation Clostridium parahaemolyticus as described in claim 1 ( Paraclostridium bifermentans The probiotic composition according to JN807 or claim 2 is characterized in that the piglet diarrhea is caused by Escherichia coli infection; the Escherichia coli is enterotoxin-producing Escherichia coli.

5. An animal feed additive, characterized in that, Including the dual-fermentation Clostridium parahaemolyticus as described in claim 1 ( Paraclostridium bifermentans The probiotic composition as described in JN807 or claim 2.

6. An animal feed, characterized in that, Includes the animal feed additive as described in claim 5.

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