A strain of *Lactobacillus reuteri* PFL-2501, its bacterial agent, and its application in alleviating ulcerative colitis.

Abstract

This invention belongs to the field of bacterial agent preparation and biomedical technology, specifically relating to a strain of *Lactobacillus reuteri*, a bacterial agent, and its application in treating ulcerative colitis. This invention isolated a strain of *Lactobacillus reuteri*, named PFL-2501, and preserved it biologically. The *Lactobacillus reuteri* PFL-2501 exhibits strong gastrointestinal tolerance and high safety, with no hemolytic properties. It can inhibit harmful pathogenic bacteria in the intestine, regulate immune balance, reduce the content of pro-inflammatory factors in colonic tissue, decrease peroxidase activity, increase the level of anti-inflammatory factors, and improve the expression of intestinal tight junction proteins. The results of the examples show that *Lactobacillus reuteri* PFL-2501 and its bacterial agent can reduce weight loss in ulcerative colitis model mice, reduce the disease activity index, prolong colon length, protect the integrity of the colonic mucosa, and reduce inflammatory damage, thereby achieving a significant effect in alleviating ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of bacterial agent preparation and biomedical technology, specifically involving a strain of *Lactobacillus reuteri*, a bacterial agent, and its application in the treatment of ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a type of inflammatory bowel disease characterized by continuous inflammation of the colonic mucosa and submucosa, typically starting in the rectum and spreading throughout the colon. Clinical manifestations include chronic or subacute diarrhea, bloody and purulent stools, and abdominal pain. UC has a long course and is prone to relapse, impacting patients' quality of life and increasing their risk of colorectal cancer. Currently, the pathogenesis of UC is unclear, but research suggests it is primarily related to immune, genetic, and environmental factors.

[0003] Current treatment for ulcerative colitis (UC) primarily focuses on inducing and maintaining clinical remission and mucosal healing, preventing complications, improving patients' quality of life, and strengthening long-term management. However, most patients experience recurrent episodes, making a complete cure difficult. Existing drug therapies, such as aminosalicylic acids, can reduce the expression of inflammatory signaling factors, but are only effective in mild to moderate cases. Corticosteroids act on immune cells and upper-layer cells, proving effective in severe cases, but these drugs have side effects and can easily lead to drug dependence. Biologics, such as infliximab, can inhibit the expression of inflammatory signaling factors, but their efficacy diminishes over time, and they are ineffective in one-third of patients. Immunosuppressants, such as azathioprine delivery systems, alleviate inflammation by limiting T-cell numbers, but simultaneously reduce the patient's resistance. Therefore, developing novel alternative therapies is a crucial need in UC treatment.

[0004] Lactobacillus reuteri ( Lactobacillus reuteri Lactobacillus reuteri is a probiotic widely found in the intestines of humans and animals. It has good gastrointestinal tolerance and adhesion ability and can produce a variety of antibacterial substances, such as reuterin, which has a broad inhibitory effect on harmful microorganisms such as Gram-positive bacteria, Gram-negative bacteria, yeast, fungi, and pathogens. However, Lactobacillus reuteri, which has anti-ulcerative colitis effects, still needs further development. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Lactobacillus reuteri* PFL-2501, its bacterial agent, and its application in relieving ulcerative colitis. The *Lactobacillus reuteri* PFL-2501 and its bacterial agent can effectively relieve ulcerative colitis.

[0006] This invention provides a strain of *Lactobacillus reuteri* PFL-2501, with accession number CGMCC No. 35503.

[0007] The present invention also provides a microbial agent, comprising *Lactobacillus reuteri* PFL-2501 as described in the above technical solution.

[0008] Preferably, the bacterial agent comprises a bacterial suspension of *Lactobacillus reuteri* PFL-2501.

[0009] Preferably, the formulation of the microbial agent includes liquid or powder.

[0010] Preferably, when the bacterial agent includes *Lactobacillus reuteri* PFL-2501 cells, the effective viable concentration of *Lactobacillus reuteri* PFL-2501 is ≥1×10⁻⁶. 8 CFU / mL.

[0011] The present invention also provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating Lactobacillus reuteri PFL-2501 into a culture medium for fermentation culture or scale-up culture to obtain the bacterial agent.

[0012] Preferably, the fermentation or scale-up culture conditions include: a temperature of 35-37°C, a pH of 6.6-7.0, and a time of 24-48 hours.

[0013] This invention also provides the application of *Lactobacillus reuteri* PFL-2501 described in the above-described technical solutions, or the bacterial agent described in the above-described technical solutions, or the bacterial agent prepared by the preparation method described in the above-described technical solutions, in the preparation of products for relieving ulcerative colitis.

[0014] Preferably, the relief of ulcerative colitis includes at least one of reducing inflammatory damage, protecting the integrity of the colonic mucosa, lengthening the colon, reducing the disease activity index, and increasing body weight.

[0015] Preferably, the product includes pharmaceuticals.

[0016] Beneficial effects: This invention provides a strain of *Lactobacillus reuteri* PFL-2501, its bacterial agent, and their application in alleviating ulcerative colitis. The present invention isolates and names *Lactobacillus reuteri* PFL-2501, which has been biopreserved. *Lactobacillus reuteri* PFL-2501 exhibits strong gastrointestinal tolerance and high safety, lacks hemolytic properties, is sensitive to multiple antibiotics without antibiotic resistance, and does not produce toxins. It can inhibit harmful pathogenic bacteria in the intestine, regulate immune balance, reduce the content of pro-inflammatory factors (IL-6, IL-1β) in colonic tissue, reduce the activity of peroxidase (MPO), increase the level of anti-inflammatory factor (IL-10), and improve the expression of intestinal tight junction proteins. The results of the examples show that *Lactobacillus reuteri* PFL-2501 and its bacterial agent can reduce weight loss in ulcerative colitis model mice, reduce the disease activity index (DAI), prolong colon length, protect the integrity of the colonic mucosa, and reduce inflammatory damage, thereby achieving a significant effect in alleviating ulcerative colitis.

[0017] Biological Preservation Information: Lactobacillus reuteri PFL-2501, biologically classified as Limosilactobacillus reuteri It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 4, 2025, with accession number CGMCC No. 35503, and the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a colony characteristic diagram of *Lactobacillus reuteri* PFL-2501 on MRS solid medium in Example 1; Figure 2 This is a Gram-stained electron micrograph of *Lactobacillus reuteri* PFL-2501 from Example 1. Figure 3 This is the phylogenetic tree of *Lactobacillus reuteri* PFL-2501 in Example 1; Figure 4 This is a growth curve of *Lactobacillus reuteri* PFL-2501 in Example 2; Figure 5 This is a blood agar colony diagram of *Lactobacillus reuteri* PFL-2501 from Example 2; Figure 6 The graph shows the body weight and DAI score results of each group of mice in Example 3; Figure 7 The image shows the colon length results for each group of mice in Example 3; Figure 8 The results of ELISA assays for cytokine levels in the colon of mice in each group, as shown in Example 3; exist Figures 6-8 middle" "", "and" "" indicates no significant difference, significant difference, and extremely significant difference, respectively. Detailed Implementation

[0020] This invention provides a strain of *Lactobacillus reuteri* PFL-2501, with accession number CGMCC No. 35503.

[0021] The *Lactobacillus reuteri* PFL-2501 described in this invention was isolated from the feces of a healthy woman aged 20 years and identified as *Lactobacillus reuteri* through colony characteristics, physicochemical properties, and 16S rDNA sequence. The morphological characteristics of *Lactobacillus reuteri* PFL-2501 described in this invention are: Gram-positive bacillus, growing under anaerobic conditions, non-spore-forming, and non-hemolytic; short rod-shaped with blunt ends, mostly arranged in pairs or short chains; forming opaque white circular colonies on MRS solid medium, with a raised surface and neat edges. The 16S rDNA sequence of *Lactobacillus reuteri* PFL-2501 described in this invention is shown in SEQ ID NO:1. As one embodiment, *Lactobacillus reuteri* PFL-2501 can be stored for a long time in a 50% anaerobic glycerol system at -80°C.

[0022] The *Lactobacillus reuteri* PFL-2501 described in this invention exhibits strong gastrointestinal tolerance, specifically its ability to withstand acidic and bile salt environments, as well as artificial gastric and intestinal fluids, facilitating colonization and functional exertion in the intestines. Furthermore, *Lactobacillus reuteri* PFL-2501 demonstrates high safety, exhibiting no hemolytic properties, sensitivity to multiple antibiotics, no antibiotic resistance, and no toxin production, meeting probiotic safety standards. Additionally, *Lactobacillus reuteri* PFL-2501 rapidly produces lactic acid in MRS liquid culture medium without producing acetic acid, propionic acid, or butyric acid, and can rapidly utilize propionic acid in the culture medium.

[0023] This invention also provides a microbial agent comprising *Lactobacillus reuteri* PFL-2501 as described in the above-mentioned technical solution. As one embodiment, the microbial agent comprises a bacterial suspension of *Lactobacillus reuteri* PFL-2501. As one embodiment, the dosage form of the microbial agent includes a liquid or a powder; further, the liquid can be a bacterial suspension, and the powder can be a lyophilized powder. As one embodiment, when the microbial agent comprises *Lactobacillus reuteri* PFL-2501 cells, the effective viable concentration of *Lactobacillus reuteri* PFL-2501 is ≥1×10⁻⁶. 8 Furthermore, the CFU / mL can be 1×10 8 ~1×10 10 CFU / mL, or 1×10 9 CFU / mL.

[0024] This invention also provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating *Lactobacillus reuteri* PFL-2501 into a culture medium for fermentation or scale-up culture to obtain the bacterial agent. As one embodiment, the conditions for the fermentation or scale-up culture include: a temperature of 35-37°C, or 36°C; a pH of 6.6-7.0, or 6.7-6.9; and a time of 24-48 h, or 30-36 h. As one embodiment, the culture medium is MRS liquid medium.

[0025] This invention also provides the application of *Lactobacillus reuteri* PFL-2501 or the bacterial agent prepared by the method described above in the preparation of a product for relieving ulcerative colitis. As one embodiment, the relief of ulcerative colitis includes at least one of reducing inflammatory damage, protecting the integrity of the colonic mucosa, prolonging colon length, reducing the disease activity index, and increasing body weight. As one embodiment, the product can be a pharmaceutical product.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] The biomaterials and their preparation methods used in the following examples: MRS solid medium: 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 5.0g sodium acetate, 2.0g diammonium hydrogen citrate, 1.0g Tween 80, 2.0g K2HPO4, 0.2g MgSO4, 0.05g MnSO4, 0.5g cysteine, 0.25g L-cysteine ​​hydrochloride, 15g agar, and 1.0L distilled water.

[0028] MRS solid culture medium preparation steps: After mixing the above components, autoclave at 121℃ for 15 minutes. After sterilization, remove and cool to about 50℃ (not hot to the touch). Take a sterile culture dish and pour 20~25mL of culture medium into each dish, and wait for it to solidify.

[0029] Simply remove the agar when preparing MRS liquid culture medium.

[0030] Preparation of 50% anaerobic glycerol: Add 50mL of glycerol to 50mL of ultrapure water, add 0.05g of L-cysteine ​​hydrochloride and 0.025g of sodium sulfide, mix well, and autoclave at 121℃ for 15min before use.

[0031] Example 1 Screening and identification of Lactobacillus reuteri PFL-2501 1. Screening of Lactobacillus reuteri PFL-2501 Take approximately 0.1g of fresh feces from a healthy donor, perform serial dilution, and take 10... -5 10 -6 10 -7 Three gradient dilutions (100 μL each) were spread onto MRS solid medium and anaerobically cultured at 37°C for 48 h until single colonies formed. Single colonies were then picked and inoculated onto MRS liquid medium and cultured anaerobically at 37°C for 24–48 h.

[0032] The information of the above-mentioned healthy donor is as follows: age 20, female, BMI 20.3, healthy after physical examination and psychological testing, and has not taken antibiotics or probiotic products within the past month.

[0033] 2. Identification of *Lactobacillus reuteri* PFL-2501 2.1 Colony characteristics The strain obtained in step 1 was inoculated onto MRS solid medium and cultured for 48 hours. The colonies were round, smooth, with regular edges, and milky white in color. Figure 1 .

[0034] 2.2 Microscopic morphology The colonies obtained in step 1 were smeared and their morphology was observed under a microscope. The results showed that they were Gram-positive, did not produce spores, were short rod-shaped with blunt ends, and were mostly arranged in pairs or short chains. Figure 2 .

[0035] 2.3 16S rDNA Identification Genomic DNA of the target strain was extracted using MP's fecal DNA extraction kit. The extracted genomic DNA was used as a template for PCR amplification. The PCR experiment of 16S rDNA was carried out using the universal bacterial primers 27F and 1492R according to the PCR amplification system in Table 1 and the PCR amplification reaction procedure in Table 2. After the PCR reaction amplification was completed, the PCR product was taken for agarose gel detection and photography. The amplified fragment length was about 1500bp.

[0036] Table 1 PCR amplification system

[0037] Table 2 PCR amplification program

[0038] The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained 16S rDNA sequence is shown in SEQ ID NO:1, as follows:

[0039] The 16S rDNA shown in SEQ ID NO:1 was subjected to BLAST sequence alignment on the NCBI website, and the results showed that the sequence was similar to that of *Lactobacillus reuteri* (…). Limosilactobacillus reuteri The 16S rDNA sequence homology of the sample exceeds 95%, such as... Figure 3 As shown, in Figure 3 LR-1 is Lactobacillus reuteri PFL-2501.

[0040] 3. Gastrointestinal tolerance test: 3.1 Artificial gastric fluid: 0.2% NaCl + 0.32% pepsin, adjusted to pH 2.0 with hydrochloric acid, filtered through a 0.22μm filter membrane for sterilization before use.

[0041] Artificial intestinal fluid: 0.68% KH2PO4 + 0.1% trypsin + 0.3% bovine bile salt, adjusted to pH 8.0 with NaOH, filtered through a 0.22μm filter membrane for sterilization before use.

[0042] 3.2 Inoculate Lactobacillus reuteri PFL-2501 into MRS liquid medium and anaerobic culture at 37℃ for 24-36 h to bring the bacterial culture into the logarithmic growth phase.

[0043] 3.3 The bacterial culture was mixed with artificial gastric fluid and artificial intestinal fluid at a volume ratio of 1:9, and cultured at 37°C with shaking (150 rpm). After 3 hours, samples were taken to detect the viable bacterial count.

[0044] 3.4 After being exposed to artificial gastric fluid, the viable bacterial survival rate of *Lactobacillus reuteri* PFL-2501 was 82%, and after being exposed to artificial intestinal fluid, the viable bacterial survival rate of *Lactobacillus reuteri* PFL-2501 was 65%.

[0045] 4. Antibiotic susceptibility testing 4.1 Take cryopreserved tubes of *Lactobacillus reuteri* PFL-2501 and inoculate them into MRS broth at a 10% inoculum. Incubate anaerobically at 37°C for 18–24 h. Perform plate counting on the activated bacterial culture and dilute the culture to approximately 1 × 10⁻⁶ based on the counting results. 8 CFU / mL.

[0046] 4.3 Referring to the CLSI standard, the concentration gradient ranges of seven antibiotics—penicillin, ampicillin, meropenem, vancomycin, daptomycin, erythromycin, and clindamycin—were determined. The antibiotic powders were accurately weighed, dissolved in sterile water, and prepared as stock solutions. The stock solutions were then serially diluted with liquid culture medium to prepare 10 different concentrations of antibiotic working solutions.

[0047] 4.4 Take a sterile 96-well plate and add 100 μL of antibiotic working solution of different concentrations to each well. Add 100 μL of culture medium (negative control) to well 11 and 100 μL of bacterial solution (positive control) to well 12.

[0048] 4.5 Add 100 μL of the prepared bacterial suspension to each well, gently shake to mix, and place in a 96-well plate for anaerobic incubation at 37°C for 18–24 h.

[0049] 4.7 Observe the turbidity of each well to determine the minimum inhibitory concentration (MIC) of each antibiotic. Negative control wells should show no sterile growth (clear and transparent liquid), while positive control wells should be significantly turbid. The MIC is defined as the lowest antibiotic concentration that completely inhibits the growth of the bacterial strain, with clear liquid in the well and no visible sediment or turbidity. The results are shown in Table 3.

[0050] Table 3. Results of drug resistance testing for *Lactobacillus reuteri* PFL-2501

[0051] Example 2 Characterization of *Lactobacillus reuteri* PFL-2501 obtained in Example 1 1. Growth curve determination Activated Lactobacillus reuteri PFL-2501 bacterial culture was inoculated into MRS liquid medium at a 2% (v / v) inoculum and anaerobically cultured at 37℃ for 0h, 2h, 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h, and 72h. OD was measured. 600 Values, plot growth curves, see Figure 4 .

[0052] 2. Safety testing: Hemolytic activity test: *Lactobacillus reuteri* PFL-2501 was streaked onto Columbia blood agar medium containing 5% defibrinated sheep blood and anaerobically cultured at 37°C for 48 h. All samples showed γ-hemolysis, i.e., no hemolysis was observed. (See attached image) Figure 5 .

[0053] Based on the results of growth curve determination and safety testing, it can be concluded that Lactobacillus reuteri PFL-2501 has good growth performance, does not exhibit hemolysis, and has strong safety.

[0054] Example 3 Evaluation of the in vivo anti-inflammatory activity of the *Lactobacillus reuteri* PFL-2501 strain obtained in Example 1 1. Experimental Design Experimental grouping: After 7 days of adaptive feeding, 30 C57BL / 6J mice were randomly divided into 3 groups (n=10 per group): blank control group (NC group), DSS model group (DSS group), and Lactobacillus reuteri intervention group (LR group).

[0055] Model establishment: Mice in the blank control group had free access to sterile distilled water every day, while mice in the DSS model group and the Lactobacillus reuteri PFL-2501 intervention group had free access to 2% DSS solution every day. During the modeling period, fresh sterile water and 2% dextran sulfate sodium salt (DSS) solution were used every 2 days to establish a mouse model of ulcerative colitis.

[0056] Strains intervention: Starting on day 8, LR group mice were administered Lactobacillus reuteri PFL-2501 bacterial agent (containing 1×10⁻⁶ live bacteria) once daily. 9 Mice in the DSS and NC groups were given 200 μL of PBS solution per mouse (CFU / mL), while mice in the NC group were given the same volume of PBS solution by gavage daily. All other feeding conditions, such as temperature, humidity, light, and feed, were kept the same. After 7 days of intervention, mice were euthanized by cervical dislocation on day 8, and the colon was dissected and its length was measured.

[0057] The preparation steps for the *Lactobacillus reuteri* PFL-2501 bacterial agent in the intervention group were as follows: cryopreserved glycerol tubes were inoculated into MRS liquid medium at a 10% inoculum size, anaerobically cultured at 37°C for 1–2 days, and diluted to 1 × 10⁻⁶ after plate counting. 9 Take 1 mL of the CFU / mL solution, centrifuge at 12000 rpm for 5 min, discard the supernatant, resuspend the precipitate in 200 μL of PBS solution, and then administer it to mice by gavage.

[0058] 2. Detection indicators and results 2.1 Weight and DAI score During the modeling process, mice were weighed and recorded daily at regular intervals. Clinical symptoms such as changes in body weight, activity level, fecal condition, and bloody stool were observed and recorded. Scoring was performed using the criteria in Table 4, and the three factors were then summed to calculate the Disease Activity Index (DAI).

[0059] Table 4 Assessment criteria for disease activity index

[0060] Table 5 Disease activity index scores during mouse intervention

[0061] According to Table 5 and Figure 6 According to the records, during the modeling period, the DSS model group showed obvious bloody stools, loose stools, and weight loss. The mice's weight decreased significantly. In addition, the disease activity index of the DSS model group mice increased significantly from the first day of modeling. The intake of *Lactobacillus reuteri* PFL-2501 significantly reduced the weight loss of colitis mice, improved stool characteristics and bloody stools, and reduced DAI values. This indicates that *Lactobacillus reuteri* PFL-2501 of the present invention has the function of alleviating the disease symptoms of IBD mice.

[0062] 2.2 Colon length Mice were sacrificed after the experiment, and colonic tissue was collected to measure the length of the colon between the terminal cecum and the proximal rectum. DSS-induced ulcerative colitis in mice leads to colonic shortening; therefore, colonic length is an important indicator for evaluating the severity of inflammation in colitis-affected mice. The results are as follows: Figure 7 As shown.

[0063] in accordance with Figure 7 The results showed that the colon length of mice in the NC group was 8 cm, while that in the DSS group was shortened to 4.2 cm, significantly shorter than that in the normal group, indicating successful establishment of the colitis model. Compared with the DSS group, the colon length of mice in the LP group was significantly longer, at 6.8 cm. These experimental results indicate that *Lactobacillus reuteri* PFL-2501 can effectively reduce colon shortening.

[0064] 2.3 ELISA measurement of cytokine levels (1) Weigh about 0.1g of colon tissue, homogenize it with sterile physiological saline, centrifuge at 14000g for 15min and collect the supernatant.

[0065] (2) Add the standard and sample to the 96-well plate respectively, then add BCA working solution to each well and incubate at 37°C for 25 min. Use an enzyme-linked immunosorbent assay reader to measure the absorbance at a wavelength of 562 nm.

[0066] (3) Elisa operation steps: a) Soaking the microplate: Prepare the necessary reagents and standards. Add 300 μL of washing buffer to both the standard wells and the sample wells of the microplate and let it stand for 30 seconds. Discard the washing buffer and pat dry.

[0067] b) Add standards and samples: Add the corresponding volume of standards to the corresponding wells of the ELISA plate, add an appropriate volume of sample to the sample wells, and bring the volume of each well to 100 μL.

[0068] c) Antibody incubation: Add 50 μL of the corresponding diluted antibody to the standard and sample wells, shake at 100-300 rpm, and incubate at room temperature. The incubation time for IL-6 and MPO is 1.5 h, and the incubation time for IL-10 and IL-1β is 2 h.

[0069] d) Washing: After incubation, add 300 μL of washing buffer to each well to wash the microplate, for a total of 6 washes. After each wash, discard the washing buffer and pat dry on absorbent paper.

[0070] e) Enzyme incubation: After washing, add 100 μL of diluted horseradish peroxidase-labeled streptavidin to the standard and sample wells, shake at 100-300 r / min and incubate at room temperature.

[0071] f) Washing: Wash the microplate 6 times in the same manner as d).

[0072] g) Color development: After washing, add 100 μL of the color development substrate to the standard wells and the sample wells, and incubate at room temperature for 5-30 min in the dark to develop the color. After incubation, add 100 μL of stop solution to each well and mix thoroughly to stop the color development.

[0073] h) Reading: The reading time should be controlled within 30 minutes after adding the stop solution. Use an ELISA reader to perform dual-wavelength detection, measuring the absorbance (OD) at 450 nm and 630 nm.

[0074] The ELISA kits and antibodies used in the above experiments were all purchased from Thermo Fisher Scientific.

[0075] The measurement results are as follows Figure 8 As shown, the LR group significantly reduced the levels of IL-6 and IL-1β in colonic tissue and increased the level of IL-10. Peroxidase (MPO) activity: The LR group reduced MPO activity in colonic tissue. This indicates that LR intervention can significantly improve the inflammatory state of colonic tissue by regulating the balance of pro-inflammatory / anti-inflammatory factors and inhibiting neutrophil infiltration and activation, thereby alleviating colonic inflammation.

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri PFL-2501, accession number CGMCC No.35503.

2. A microbial agent, characterized in that, Includes *Lactobacillus reuteri* PFL-2501 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent includes a bacterial suspension of Lactobacillus reuteri PFL-2501.

4. The microbial agent according to claim 2 or 3, characterized in that, The formulation of the microbial agent includes liquid or powder.

5. The microbial agent according to claim 2, characterized in that, When the bacterial agent includes *Lactobacillus reuteri* PFL-2501 cells, the effective viable concentration of *Lactobacillus reuteri* PFL-2501 is ≥1×10⁻⁶. 8 CFU / mL.

6. The method for preparing the microbial agent according to any one of claims 2 to 5, characterized in that, The process includes the following steps: inoculating Lactobacillus reuteri PFL-2501 into a culture medium for fermentation or scale-up culture to obtain the bacterial agent.

7. The preparation method according to claim 6, characterized in that, The conditions for fermentation culture or scale-up culture include: temperature of 35~37℃, pH of 6.6~7.0, and time of 24~48h.

8. The use of the *Lactobacillus reuteri* PFL-2501 as described in claim 1, or the bacterial agent as described in any one of claims 2 to 5, or the bacterial agent prepared by the preparation method described in claim 6 or 7, in the preparation of products for relieving ulcerative colitis.

9. The application according to claim 8, characterized in that, The relief of ulcerative colitis includes at least one of reducing inflammatory damage, protecting the integrity of the colonic mucosa, lengthening the colon, reducing the disease activity index, and increasing body weight.

10. The application according to claim 8 or 9, characterized in that, The products include pharmaceuticals.

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