Enterococcus durans C24, probiotic agent and application thereof

Abstract

The present application relates to the technical field of microorganism, and especially relates to a kind of Enterococcus durans C24, probiotic agent and application thereof.The present application provides Enterococcus durans C24, and its preservation number is CGMCC No.33995.The strain has broad-spectrum antibacterial activity, can inhibit Salmonella enteritidis, Salmonella pullorum and Salmonella typhimurium, has no hemolytic activity, and has good stress resistance, can survive in gastrointestinal environment, and also has high biological safety, and is expected to be used for clinical prevention and control of salmonellosis.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a robust Enterococcus C24, a probiotic agent, and their applications. Background Technology

[0002] The poultry industry plays a vital role in the food supply. Salmonella infection severely impacts the health and productivity of poultry, causing significant economic losses to the chicken farming industry. Currently, the main prevalent serotypes are *Salmonella enteritidis*, *Salmonella pullorum*, and *Salmonella typhimurium*. Salmonella can enter poultry bodies through contaminated feed or water. It can survive in the acidic environment of the stomach and trigger an acid tolerance response, subsequently attaching to and invading intestinal epithelial cells, causing inflammation.

[0003] The overuse of antibiotics has led to the emergence of numerous drug-resistant strains, making clinical prevention and control more challenging. Reducing antibiotic use and adopting alternative measures, such as optimizing nutrition and using probiotics, have become key strategies for controlling Salmonella infection. There are many types of probiotics, mainly including *Lactobacillus*, *Bifidobacterium*, *Enterococcus*, *Bacillus*, and *Saccharomyces*. However, research on probiotics mainly focuses on regulating the host's intestinal immune response, barrier function, and microbiota structure to promote intestinal development and health. For example, *Lactobacillus rhamnosus* can effectively reduce intestinal damage caused by heat stress and increase serum immune factor levels by regulating the intestinal microbiota of broilers, thus enhancing immune function. Furthermore, *Lactobacillus curvatureii* 2029 can effectively inhibit the activation of Caspase 9 and Caspase 3 and the occurrence of apoptosis in human intestinal epithelial cells induced by foodborne pathogens. Currently, there are few reports on the antibacterial and antibacterial effects of probiotics on bacterial diarrhea. There is an urgent need to develop a probiotic with broad-spectrum antibacterial activity against the main prevalent Salmonella serotypes (Salmonella enteritidis, Salmonella pullorum, and Salmonella typhimurium) for clinical prevention and control of salmonellosis. Summary of the Invention

[0004] The purpose of this invention is to provide a robust Enterococcus C24, a probiotic agent, and its application. The robust Enterococcus C24 provided by this invention has a good antibacterial effect against the three major circulating Salmonella serotypes and is expected to be used in clinical prevention and control of Salmonella infection.

[0005] This invention provides a robust Enterococcus durans C24 (also known as "durable enterococcus C24"), with accession number CGMCC No. 33995. It was deposited on March 27, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] In this invention, the strain was isolated from the feces of Dawu Jinfeng chicks in good condition at about 30 days old. The strain has inhibitory effects on Salmonella enteritidis, Salmonella pullorum, and Salmonella typhimurium, and has good stress resistance, can survive in the gastrointestinal environment, and has high biosafety, and is expected to be used for clinical prevention and control of salmonellosis.

[0007] In this invention, the microbiological characteristics of the robust enterococcus C24 are as follows: it is a round, raised white single colony; it is Gram-positive, and under microscopic examination, it can be seen as a non-spore-forming, non-flagellated coccus; under a scanning electron microscope, it appears as round or oval bacterial cells, with a cell size between 0.5 and 1.5 μm, existing as single cells, arranged in pairs, chains, or clusters.

[0008] Another object of the present invention is to provide a probiotic preparation comprising the *Enterococcus ferruginosa* C24 described in the above technical solution.

[0009] Optionally, the probiotic preparation includes pharmaceuticals or animal feed additives.

[0010] A third objective of this invention is to provide the use of the *Enterococcus ferruginosa* C24 or the probiotic preparation described in the above-described technical solutions in the preparation of drugs or feeds that inhibit pathogenic bacteria, wherein the pathogenic bacteria include at least one of *Salmonella enteritidis*, *Salmonella pullorum*, and *Salmonella typhimurium*. In this invention, the inhibition includes prevention or treatment.

[0011] Compared with existing technologies, the present invention has the following beneficial effects: The robust Enterococcus C24 provided by the present invention exhibits good inhibitory effects against various Salmonella species, including Salmonella enteritidis, Salmonella pullorum, and Salmonella typhimurium. It is non-hemolytic, possesses good resistance, can survive in the gastrointestinal environment, and also has high biosafety, making it promising for clinical control of salmonellosis. Experimental results show that the diameter of the inhibition zone of the robust Enterococcus C24 provided by the present invention against Salmonella enteritidis, Salmonella pullorum, and Salmonella typhimurium is greater than 17.5 mm; hemolytic test results show no hemolytic activity, indicating clinical safety; the strain can still survive to a certain extent under conditions of pH 2, bile salt concentration of 0.5%, or NaCl concentration of 0.5%; the strain can also reduce the cell adhesion of Salmonella enteritidis, Salmonella pullorum, and Salmonella typhimurium to a certain extent; in vivo experiments in chickens also show that the strain provided in this application can reduce the diarrhea rate. Attached Figure Description

[0012] The above and other objects, features, and advantages of the invention will be apparent from the following description of preferred embodiments illustrating the gist of the invention and its use, and the accompanying drawings, in which:

[0013] Figure 1 A circled map of the genome of Enterococcus fragilis C24.

[0014] Figure 2 A phylogenetic tree based on 16S rRNA is shown.

[0015] Figure 3 An optical microscope image of Enterococcus ferruginosa C24.

[0016] Figure 4 Gram-stained microscopic image of Enterococcus ferruginosa C24.

[0017] Figure 5 SEM image of Enterococcus ferruginosa C24.

[0018] Figure 6 The growth curve of Enterococcus ferruginosa C24 is shown.

[0019] Figure 7 The results of the hemolytic test for Enterococcus buergerianus C24 are shown, where A represents Enterococcus buergerianus C24 and B represents the positive control.

[0020] Figure 8 The results of antioxidant capacity tests in chicks under different treatment groups are shown, where A represents GSH-Px activity, B represents MDA content, and C represents SOD activity.

[0021] Figure 9 The results of intestinal permeability tests in chicks under different treatment groups are shown, where A represents DAO activity and B represents D-lactate content.

[0022] Figure 10 HE staining results of duodenal sections of chicks in different treatment groups are shown. A is the blank control group, B is the model group, C is the low-dose C24 group, D is the medium-dose C24 group, and E is the high-dose C24 group.

[0023] Figure 11 The results of the test on the relative expression levels of tight junction protein genes in the duodenum of chicks in different treatment groups are shown. A is the test result on the relative expression level of ZO-1 mRNA, B is the test result on the relative expression level of Claudin-1 mRNA, and C is the test result on the relative expression level of Occludin mRNA.

[0024] Figure 12The results of testing the expression levels of pro-inflammatory and anti-inflammatory factors in the duodenum of chicks in different treatment groups are shown. A represents the mRNA expression level of TNF-α, B represents the mRNA expression level of IL-1β, C represents the mRNA expression level of IL-10, and D represents the mRNA expression level of IL-18. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The embodiments of this application are only examples, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Strain Collection, Isolation and Identification

[0027] Fecal samples (1 g / chick) were aseptically collected from 40 healthy Dawu Jinfeng chicks (approximately 30 days old). The samples were placed in 10 mL centrifuge tubes containing 5 mL of sterile phosphate-buffered saline (PBS), vortexed, filtered through gauze, and thoroughly mixed. The samples were then serially diluted with sterile physiological saline (1% concentration). 10 samples were selected from each tube. -2 10 -3 10 -4 For the gradient solutions, take 100 μL of the diluted solution and spread it on LPS agar, then incubate at 37°C for 24 h. Pick a single colony and streak it on MRS solid medium at 37°C for 5 generations to obtain the purified C24 strain.

[0028] The purified C24 strain was cultured in MRS broth at 37°C for 18 h, and the bacterial cells were collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for whole-genome sequencing. Genomic DNA was extracted using the MagPure bacterial DNA extraction kit, and libraries were constructed using the NEXTFLEX Rapid DNA-Seq Kit. Paired-end sequencing was performed using the Illumina NovaSeq 6000 platform. The resulting genome map is shown below. Figure 1 As shown, whole-genome sequencing revealed that the C24 genome is 3,028,563 bp in size, with a GC content of 37.63%. A total of 2,858 protein-coding genes, 18 rRNAs, and 71 tRNAs were predicted, and it carries 7 plasmids.

[0029] 16S rRNA gene sequences were extracted and homology comparisons were performed in the NCBI database using the BLAST tool. Reference sequences with a similarity >95% were selected. A phylogenetic tree was constructed using Mega 6.0 software based on the neighbor-joining method (results are shown in Figure 1). Figure 2(As shown). Phylogenetic analysis showed that strain C24 was most closely related to the standard strain of Enterococcus durans, GCF_001544215.1, and based on morphological and molecular characteristics, it was identified as Enterococcus durans (E. durans).

[0030] Morphological characterization:

[0031] The purified C24 strain was inoculated onto MRS agar medium, and photographs of the purified C24 strain were taken, such as... Figure 3 As shown. By Figure 3 The strain can be seen as a round, raised, white single colony.

[0032] The purified C24 strain was Gram-stained on MRS agar medium and then its cell morphology was observed under an optical microscope (1000×). The results are as follows: Figure 4 As shown, the Gram staining is positive, and microscopic examination reveals cocci without spores or flagella.

[0033] The purified C24 strain was fixed in electron microscopy fixative for 2 h, then critically dried, sputter-coated with gold, and observed using scanning electron microscopy (SEM). The results are as follows: Figure 5 As shown, the right image is a magnified view of a portion of the left image. The bacteria are round or oval, with a diameter of 1.2–1.5 μm, and have a smooth surface. They are arranged singly, in pairs, in chains, or in clusters.

[0034] Growth characteristic test:

[0035] Purified C24 colonies were inoculated into MRS broth and incubated at 37°C for 24 h. Then, 1% of the colonies were inoculated into fresh MRS broth and incubated at 37°C. Absorbance was measured at 600 nm every 2 h using a spectrophotometer. The medium was vortexed before each measurement. OD values ​​were measured over a period of 0–24 h. 600 nm Values, recording data, with time as the horizontal axis, OD 600 nm The values ​​are plotted on the ordinate to create a growth curve for the strain. The results are as follows: Figure 6 As shown.

[0036] Depend on Figure 6 As can be seen, the purified C24 strain exhibits a lag phase during the first 0-2 hours of culture, a logarithmic growth phase during the first 2-8 hours, rapid cell proliferation, and a stationary phase after 8 hours of culture. The isolated strain maintains its peak growth from 8 to 24 hours, demonstrating good growth performance.

[0037] Example 2: In vitro antibacterial ability test

[0038] The inhibitory activity of the purified C24 strain (i.e., Enterococcus ferruginosa C24) in Example 1 against three serotypes of Salmonella (Salmonella enteritidis, Salmonella pullorum, and Salmonella typhimurium) was tested.

[0039] Pick a single colony and incubate it overnight at 37°C with shaking at 220 rpm in 10 mL of MRS liquid medium. Continue to expand the bacterial culture, transfer it to an autoclaved Erlenmeyer flask, add 200 mL of MRS liquid medium, and incubate with shaking until OD reaches [value missing]. 600 nm The absorbance value at 600 nm was 0.8, resulting in a bacterial suspension. The bacterial suspension was centrifuged at 4℃ and 4500 r / min for 10 min to obtain cell-free supernatant (abbreviated as CFS), which is Enterococcus tumefaciens C24 bacterial suspension.

[0040] Using Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis as indicator bacteria, 1×10⁻⁶ samples were prepared respectively. 8 CFU / mL bacterial suspension. The antibacterial activity against each indicator bacterium was determined using the Oxford cup method. The specific test method was as follows: 100 μL of indicator bacterial suspension was spread onto LB agar medium and evenly distributed into four sterile Oxford cups. 200 μL of CFS was added to three of the Oxford cups, and 200 μL of MRS liquid medium was added to one Oxford cup as a control. After overnight incubation at 37°C, the diameter of the inhibition zone was measured. The criteria for determining the antibacterial effect are shown in Table 1.

[0041] Table 1 Criteria for Judging Antibacterial Effect

[0042]

[0043] The test results are shown in Table 2. The robust Enterococcus C24 produced inhibition zones of approximately 18.12 mm, 17.54 mm, and 20.10 mm in diameter against Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis, respectively, all exhibiting high levels of inhibition. This demonstrates that the robust Enterococcus C24 has a high inhibitory effect on all prevalent Salmonella serotypes and possesses potential for clinical control of salmonellosis.

[0044] Table 2. Diameter of the inhibition zone of *Enterococcus ferruginosa* C24 against prevalent Salmonella serotypes.

[0045]

[0046] Example 3 Hemolytic Test

[0047] The blood agar plates used were prepared using Columbia blood agar base, defibrinated sheep blood, and vitamin K, model HBPM0124-15. The purified C24 strain obtained in Example 1 was streaked onto the blood agar plates and incubated at 37°C for 24 h. Enterotoxigenic Escherichia coli ETEC K88ac was used as a positive control to observe hemolysis. The appearance of a clear hemolysis ring indicated a positive hemolysis phenotype. The results are as follows: Figure 7 As shown.

[0048] Depend on Figure 7 As can be seen, the strain C24 isolated and purified in Example 1 grows into round, gray colonies on blood agar plates with a white center and a smooth, convex surface (see...). Figure 7 (A in the middle). Figure 7 B in the diagram is the positive control, and a distinct β-hemolytic ring is visible. *Enterococcus ferruginosa* C24 shows no hemolytic activity, and no erythrocyte lysis is observed around the colony, indicating that this strain is safe for use in animals and has clinical safety.

[0049] Example 4: Stress Resistance Test

[0050] (1) Acid resistance test: The C24 strain isolated and purified in Example 1 was inoculated into MRS broth medium with pH 2.0, 3.0, 4.0 and 5.0 respectively (control pH about 6.0).

[0051] (2) Bile salt tolerance test: The C24 strain isolated and purified in Example 1 was inoculated into MRS broth medium containing 0% (control group), 0.1%, 0.2%, 0.3%, 0.4% and 0.5% (w / v) bile salts, respectively;

[0052] (3) NaCl tolerance test: The C24 strain isolated and purified in Example 1 was inoculated into MRS broth medium containing 0% (control group), 0.1%, 0.2%, 0.3%, 0.4% and 0.5% (w / v) NaCl, respectively.

[0053] All the above experiments were conducted at 37 ℃ and 180 r / min for 8 h, and the absorbance (OD) was measured at 600 nm. 600 nm ), calculate the relative survival rate of the strains.

[0054] Survival rate (%) = (average OD of experimental groups) 600 nm Value / control group mean OD 600 nm Value) × 100%

[0055] The test results are shown in Table 3-5. Table 3-5 shows that the *Enterococcus resistant* C24 strain provided by this invention can survive in the pH range of 2.0-5.0, with a survival rate of 5%-60%; it can grow in bile salt concentrations of 0.1%-0.5%, with a survival rate of over 20% at 0.5% bile salt concentration; and it can grow in MRS medium with NaCl concentrations of 1.0%-5.0%, with a survival rate of 61.59% under 5.0% high-salt conditions. These results demonstrate that the *Enterococcus resistant* C24 strain exhibits good tolerance to the simulated gastrointestinal environment in vitro.

[0056] Table 3 Results of acid resistance test

[0057]

[0058] Table 4 Results of bile salt tolerance test

[0059]

[0060] Table 5. Results of NaCl tolerance test

[0061]

[0062] Example 5: Adhesion properties of *Enterococcus ferruginosa* C24 to Caco-2 cells

[0063] Caco-2 cells were seeded in MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrose antibody at a concentration of 1×10⁶ cells / mL. 5 The culture was inoculated into Transwell plates at a density of 90% per well and cultured at 37°C with 5% CO2 until confluence reached 90%. The *Enterococcus ferruginosa* C24 culture prepared according to the method in Example 2 was resuspended in MEM medium to a concentration of 1×10⁻⁶. 8 CFU / mL (labeled as *Enterococcus ferruginosus* C24 resuspension) was added to each well at a ratio of 100:1 to Caco-2 cells for co-incubation. After 2 h of co-incubation with cells, the cells were washed three times with PBS, digested with trypsin, and then plated on MRS plates for counting. Each group was divided into three replicates (n=3), and the average value was taken. The adhesion rate of the isolated strain to Caco-2 cells was calculated using the following formula.

[0064] Adhesion rate (%) = (V1 / V0) × 100%

[0065] In the formula, V0 is the initial number of viable bacteria inoculated; V1 is the number of viable bacteria adhering to the inoculum.

[0066] The culture medium for the isolated strain of Enterococcus ferruginosa C24 was prepared according to the method in Example 2, and exclusion, competition, and displacement adhesion experiments were performed. Details are as follows:

[0067] Exclusion test: 500 μL of resuspended Enterococcus faecalis C24 (1×10⁻⁶) was used as the culture medium. 8 (CFU / mL) was added to Caco-2 cells to achieve a bacterial-to-cell ratio of approximately 100:1, and incubated at 37°C and 5% CO2 for 60 min; subsequently, a Salmonella suspension (Salmonella typhimurium, Salmonella enteritidis, or Salmonella pullorum) (500 μL, 1×10⁻⁶ CFU / mL) was added to the cells. 8 (CFU / mL), and continue incubation for 60 min under the same conditions.

[0068] Competitive experiment: Equal volumes and concentrations of *Enterococcus ferruginosa* C24 resuspension and *Salmonella* suspension (500 μL each, 1×10⁻⁶) were prepared. 8 (CFU / mL) was added to Caco-2 monolayer cells to make the ratio of each bacterium to the cells approximately 100:1, and incubated at 37°C and 5% CO2 for 120 min.

[0069] Displacement test: Salmonella suspension (500 μL, 1×10⁻⁶) was added to the culture medium. 8 (CFU / mL) was added to Caco-2 cells to make the bacteria-to-cell ratio approximately 100:1, and incubated at 37°C and 5% CO2 for 60 min; then, 500 μL of Enterococcus ferrugin C24 resuspension (1×10⁻⁶ CFU / mL) was added. 8 (CFU / mL), continue incubation for 60 min.

[0070] The control group was set up as follows: The control group consisted of 500 μL of Salmonella suspension (1×10⁻⁶ cells / mL). 8 CFU / mL was added to Caco-2 monolayer cells to make the ratio of bacteria to cells approximately 100:1, and the cells were incubated at 37°C and 5% CO2 for 120 min.

[0071] After incubation, the samples were washed three times with PBS, digested with trypsin, serially diluted, and spread onto LB agar for Salmonella counting. Each experiment was performed in triplicate and independently three times (n=3), and the average value was taken. The adhesion inhibition rate of the isolated strain against Salmonella was calculated using the following formula.

[0072] Adhesion inhibition rate (%) = (B0 - B1) / B2 × 100%

[0073] In the formula, B0 is the number of Salmonella adhering to the control group; B1 is the number of Salmonella adhering after co-incubation; and B2 is the total number of live Salmonella bacteria initially inoculated.

[0074] The test results are shown in Table 6. As can be seen from Table 6, in different adhesion models, *Enterococcus steadfast* strain C24 exhibited adhesion inhibition against all three serotypes of Salmonella. The adhesion rate of *Enterococcus steadfast* strain C24 alone to Caco-2 cells was approximately 21.11%. Under different experimental conditions, *Enterococcus steadfast* strain C24 showed significant competitive, exclusionary, and displacement inhibition against *Salmonella pullorum* (P < 0.05); significant competitive, exclusionary, and displacement inhibition against *Salmonella typhimurium* stem cells (P < 0.05), with the strongest exclusionary inhibition; and significant competitive and displacement inhibition against *Salmonella enteritidis* (P < 0.05).

[0075] Table 6 Results of cell adhesion performance test

[0076]

[0077] "-" indicates that exclusive, competitive, and displacement adhesion assays were not performed on durable enterococci alone; different letters under the same column indicate significant differences (P < 0.05); the same letter under the same column indicates no significant differences (P > 0.05).

[0078] Example 6: Probiotic Effects of Enterococcus faecalis C24 on Chicks

[0079] (1) Prepare the bacterial suspension according to the method in Example 2, and dilute it with MRS liquid medium to a concentration of 2×10⁻⁶. 4 CFU / mL, 2×10 5 CFU / mL, 2×10 6 CFU / mL suspension of Enterococcus fulva C24.

[0080] Forty one-day-old SPF Golden Phoenix chicks were randomly divided into 5 groups (n=8). The blank control group and the model group were administered PBS by gavage. The low, medium, and high doses of Enterococcus faecalis C24 treatment groups (denoted as low-dose C24 group, medium-dose C24 group, and high-dose C24 group, respectively) were administered PBS by gavage at a concentration of 2×10⁻⁶. 4 CFU / mL, 2×10 5 CFU / mL, 2×10 6 CFU / mL of *Enterococcus fragilis* C24 suspension was administered to each group by gavage at a rate of 1 mL daily for 25 consecutive days. From day 26 onwards, the model group and each dose group of *Enterococcus fragilis* C24 were administered 500 μL of a mixed Salmonella bacterial suspension by gavage daily. (This mixed bacterial suspension was prepared by separately culturing and adjusting *Salmonella pullorum*, *Salmonella typhimurium*, and *Salmonella enteritidis* to the same concentration, then mixing them in equal volumes (1:1:1) to obtain a total bacterial concentration of 5 × 10⁻⁶.) 9A mixture of three Salmonella strains (CFU / mL) was cultured to the above concentration and then challenged. A blank control group was administered an equal volume of PBS by gavage for 5 consecutive days. Diarrhea was recorded daily. The results are shown in Table 7.

[0081] Table 7. Diarrhea rate in chicks after Salmonella challenge.

[0082]

[0083] Different letters in the same column of data indicate significant differences (P < 0.05); the same letter in the same column of data indicates no significant differences (P > 0.05).

[0084] Table 7 shows that the diarrhea rate in the model group after Salmonella challenge reached 75.0%. Compared with the model group, the diarrhea rate in all C24 dosage groups was significantly reduced (P < 0.05), with the medium-dose group decreasing to 25.0%, and the low and high-dose groups decreasing to 37.5%. The low and high-dose groups decreased to 12.5%, and the medium-dose group decreased to 25.0%. The results indicate that C24 can effectively alleviate diarrhea in chicks caused by Salmonella infection and has a good in vivo protective effect.

[0085] (2) After the challenge, chicks in each group were euthanized according to animal ethics guidelines, and blood samples were immediately collected from the carotid artery. Whole blood samples were collected from the carotid artery, placed in sterile centrifuge tubes, and centrifuged at 3000 r / min for 10 min at room temperature to separate serum. The serum was separated, aliquoted, and stored at -80℃. The duodenum and cecum were dissected. The duodenum was washed with PBS at 4℃, and a portion was fixed in 4% paraformaldehyde for subsequent hematoxylin-eosin (HE) staining. The other portion was stored at -80℃ for subsequent detection of antioxidant expression levels. The cecal tissue was immediately flash-frozen in liquid nitrogen and transferred to a -80℃ freezer for later use. The cecal contents were collected, flash-frozen with dry ice, and stored at -80℃ for intestinal microbial detection. The duodenum was collected, washed with PBS at 4℃, and a portion was fixed in 4% paraformaldehyde for histological observation. The other portion was flash-frozen in liquid nitrogen and stored at -80℃ for gene expression analysis.

[0086] ① Determination of the effect of bacterial strains on the antioxidant capacity of chicks

[0087] The serum samples isolated and preserved above were used to determine serum antioxidant indicators, including glutathione peroxidase (GSH-Px) activity, total superoxide dismutase (T-SOD) activity, and malondialdehyde (MDA) content, using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 8As shown, "Control" represents the control group, "Model" represents the model group, "C24-L" represents the low-dose C24 group, "C24-M" represents the medium-dose C24 group, and "C24-H" represents the high-dose C24 group (the control group, model group, and each dose group in the figures of this application all use this abbreviation). The symbols in the figures are as follows: Compared with the blank group, This indicates that P < 0.05. This indicates that P < 0.01, meaning no The symbol indicates P > 0.05; for model group comparison, # indicates P < 0.05, ## indicates P < 0.01, and no # indicates P > 0.05.

[0088] Depend on Figure 8 Compared with the control group, the model group showed a highly significant decrease in GSH-Px and SOD activities and a highly significant increase in MDA content (P < 0.001). Compared with the model group, the medium- and high-dose Enterococcus survivingans C24 treatment groups significantly increased GSH-Px (P < 0.01), highly significantly increased SOD (P < 0.001), and significantly decreased MDA content (P < 0.01). This indicates that Enterococcus survivingans effectively improved the antioxidant capacity of chicks and mitigated oxidative stress damage caused by Salmonella challenge by restoring the activity of antioxidant enzymes such as GSH-Px and SOD and reducing the accumulation of lipid peroxidation products such as MDA.

[0089] ② Determination of the effect of the strain on intestinal permeability in chicks:

[0090] The serum samples isolated and preserved above were used to determine serum intestinal barrier function indicators, including diamine oxidase (DAO) activity and D-lactate content, using ELISA. The results are as follows: Figure 9 As shown in the figure, the symbols in the figure represent the following: compared with the blank group, This indicates that P < 0.05. This indicates that P < 0.01, meaning there is no... The symbol indicates P > 0.05; for model group comparison, # indicates P < 0.05, ## indicates P < 0.01, and no # indicates P > 0.05.

[0091] like Figure 9 As shown, compared with the blank group, DAO and D-lac were significantly upregulated in the model group (P < 0.001), indicating that Salmonella infection leads to impaired intestinal barrier function and increased permeability. Compared with the model group, DAO and D-lac were significantly downregulated in the high-dose Enterococcus ferruginosa C24 treatment group (P < 0.001), indicating that Enterococcus ferruginosa C24 exerts its intestinal protective effect by downregulating serum DAO and D-lac levels and improving intestinal permeability.

[0092] ③ Hematoxylin-eosin staining (i.e., HE staining) and quantitative analysis of chick duodenum

[0093] Duodenal tissue was fixed with 4% paraformaldehyde, dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Cross-sectional sections were prepared and stained with hematoxylin and eosin (HE). The stained sections were then observed under an optical microscope to examine the tissue morphology (e.g., ...). Figure 10 As shown in Table 8), the villi height and crypt depth of 5 complete villi were randomly measured in each slice, and the villi height / crypt depth ratio (villi-crypt ratio) was calculated (the results are shown in Table 8).

[0094] Depend on Figure 10 It can be seen that in the blank control group, the duodenal villi of chicks showed finger-like projections and clear crypts; in the model group, the villi were disordered and the tips were atrophied and detached; after intervention with each dose of C24, the morphology of both villi and crypts improved. According to the quantitative analysis results in Table 8, the villus height / crypt depth ratio (V / C) in the model group was significantly lower than that in the blank group (P < 0.05), indicating damage to intestinal morphology. Compared with the model group, the villus height and V / C ratio in each dose of C24 treatment group were significantly increased (P < 0.05), while there was no significant difference in crypt depth (P > 0.05), indicating that the damage to intestinal villi in chicks treated with C24 was reduced, suggesting that it maintains barrier function by protecting the intestinal structure.

[0095] Table 8. Duodenal villus height, crypt depth, and villus-crypt ratio in chicks of different treatment groups

[0096]

[0097] Different letters in the same column of data indicate significant differences (P < 0.05); the same letter in the same column of data indicates no significant differences (P > 0.05).

[0098] ④ Duodenal RNA extraction and real-time quantitative PCR

[0099] The relative expression levels of tight junction protein genes and the mRNA expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-18) and anti-inflammatory factors (IL-10) in the duodenum of chicks in each treatment group were tested using the following methods:

[0100] Total RNA was extracted from the duodenum using the TRIzol method and stored at -80℃. 1 μg of total RNA was reverse transcribed into cDNA. Using the cDNA as a template, the relative expression levels of tight junction protein genes (ZO-1, Claudin-1, Occludin) were detected by qRT-PCR using the SYBR Green fluorescence method. β-actin was used as an internal control gene. Primers were designed using NCBI Primer-BLAST (as shown in Table 9) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reaction mixture (20 μL) consisted of: 2 μL cDNA, 0.4 μL each of forward and reverse primers, 10 μL 2×Taq Pro Universal SYBR Qpcr Master Mix, and 7.2 μL ddH2O. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, annealing and extension at 60℃ for 30 s, for a total of 40 cycles. 2 -△△Ct Legal analysis, results as follows Figure 11 and Figure 12 As shown; symbols in the figure: comparison between the model group and the blank group. The difference is significant (P < 0.05). This indicates a highly significant difference (P < 0.01), with no... The difference is not significant (P>0.05); compared with the model group, the difference is significant (P<0.05) for the durable enterococcus C24 treatment group, the difference is extremely significant (P<0.01) for the # group, and the difference is not significant (P>0.05) for the # group.

[0101] Table 9 Primer Information

[0102]

[0103] Depend on Figure 11 As can be seen, compared with the control group, the mRNA expression levels of ZO-1, Claudin-1, and Occludin in the model group were significantly downregulated (P < 0.01). Compared with the model group, the mRNA expression levels of ZO-1, Occludin, and Claudin-1 in each C24 dose group were significantly increased, with the high-dose C24 group showing even higher mRNA expression levels of ZO-1, Occludin, and Claudin-1 than the control group (P < 0.01 or P < 0.05). This result indicates that Enterococcus steadfast C24 can protect intestinal barrier function by regulating tight junction protein expression.

[0104] Depend on Figure 12As can be seen, compared with the control group, the pro-inflammatory factors (TNF-α, IL-1β, IL-18) were significantly upregulated in the model group (P < 0.01), and IL-10 was significantly downregulated (P < 0.05). Compared with the model group, low, medium, and high doses of *Enterococcus steadfast* C24 significantly downregulated TNF-α (P < 0.01), medium and high doses of *Enterococcus steadfast* C24 significantly downregulated IL-1β (P < 0.01) and significantly upregulated IL-10 (P < 0.01), medium and high doses of *Enterococcus steadfast* C24 highly significantly upregulated IL-18 (P < 0.001), and low doses of *Enterococcus steadfast* C24 significantly upregulated IL-18 (P < 0.01). Therefore, *Enterococcus steadfast* C24 can improve intestinal inflammatory responses by regulating the expression of pro-inflammatory and anti-inflammatory factors.

[0105] Although preferred embodiments of the invention have been shown and described, it is conceivable that those skilled in the art can devise various modifications to the invention within the spirit and scope of the appended claims.

Claims

1. A robust Enterococcus durans C24, with accession number CGMCC No. 33995.

2. A probiotic preparation containing the Enterococcus ferruginosa C24 as described in claim 1.

3. The probiotic preparation according to claim 2, characterized in that, The probiotic preparation includes pharmaceuticals or animal feed additives.

4. The use of the *Enterococcus ferruginosa* C24 of claim 1 or the probiotic preparation of claim 2 or 3 in the preparation of drugs or feeds that inhibit pathogenic bacteria, wherein the pathogenic bacteria include at least one of *Salmonella enteritidis*, *Salmonella pullorum*, and *Salmonella typhimurium*.

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