Calf-derived lactobacillus reuteri and application thereof in preparation of product for preventing and treating colitis injury

Abstract

The invention discloses calf-derived lactobacillus reuteri and application thereof in preparation of products for preventing and treating colitis injury, the calf-derived lactobacillus reuteri has been preserved in China General Microbiological Culture Collection Center on October 9, 2025, the preservation address is No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. The preservation number of the lactobacillus reuteri is CGMCC No.36229, the lactobacillus reuteri is classified and named as lactobacillus reuteri KY-1, and the lactobacillus reuteri can directly inhibit growth of pathogenic escherichia coli, relieve colitis, remarkably relieve inflammation caused by the pathogenic escherichia coli and relieve deep mucosa muscular layer ulcer, gland structure damage and inflammatory cell infiltration caused by the pathogenic escherichia coli. Therefore, the lactobacillus reuteri has a good treatment effect in treating colitis caused by pathogenic escherichia coli, and a theoretical support is provided for medicine development of calf-derived lactobacillus reuteri in the aspect of treating intestinal inflammation.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a calf-derived Lactobacillus reuteri and its application in the preparation of products for the prevention and treatment of colitis-related injuries. Background Technology

[0002] Lactobacillus reuteri ( Lactobacillus reuteri , L.reuteri Lactobacillus is an important component of the normal gut flora and one of the most extensively studied strains in the field of probiotics. As a representative strain of the genus Lactobacillus, it plays an important role in gut colonization and metabolic activity.

[0003] L.reuteri It can bind to adhesion receptors on intestinal epithelial cells through adhesion proteins (such as adhesin A) on its surface, competitively inhibiting the colonization of pathogenic bacteria (such as pathogenic Escherichia coli and Salmonella), while promoting the expression of tight junction proteins (occludin, ZO-1) in intestinal epithelial cells and enhancing the physical barrier function of the intestine. L.reuteri It can ferment carbohydrates to produce short-chain fatty acids (SCFAs) such as lactic acid and acetic acid, which lower the pH value of the intestine and inhibit the growth of pathogenic bacteria. Its unique metabolite, reuterin (a broad-spectrum antibacterial substance with the chemical nature of 3-hydroxypropionaldehyde), has an inhibitory effect on Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0004] Currently, in existing research, L.reuteri Most sources of *Lactobacillus reuteri* are human, swine, or rodent, and its applications are mainly focused on improving human intestinal dysfunction (such as diarrhea and irritable bowel syndrome) and intestinal diseases in pigs and poultry. However, there are few reports on *Lactobacillus reuteri* from calves. Because *Lactobacillus reuteri* from calves is isolated from the calf intestine, it may have a stronger adhesion and adaptation to calf intestinal epithelial cells, and its metabolites (such as reuterin and short-chain fatty acids) have more targeted inhibitory activity against common pathogenic bacteria in calves (such as pathogenic *Escherichia coli*). However, at present, *Lactobacillus reuteri* from calves has limited applications in the treatment of colitis. Summary of the Invention

[0005] The purpose of this application is to provide a calf-derived Lactobacillus reuteri and its application in the preparation of products for the prevention and treatment of colitis. Through in vitro and in vivo dual verification, the therapeutic effect of calf-derived Lactobacillus reuteri in treating colitis caused by pathogenic Escherichia coli and its effect in preventing colitis in calves are determined.

[0006] To address the aforementioned technical problems, this application provides a *Lactobacillus reuteri* strain derived from calves. This *Lactobacillus reuteri* strain was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36229, and is classified as *Lactobacillus reuteri*. Lactobacillus reuteri .

[0007] To address the aforementioned technical problems, this application also provides the application of the above-mentioned calf-derived Lactobacillus reuteri in the preparation of products for the prevention and treatment of colitis-related injuries.

[0008] As a preferred embodiment, the application of *Lactobacillus reuteri* derived from calves in the preparation of products for the prevention and treatment of colitis-related injuries is described, wherein the intervention dose of *Lactobacillus reuteri* derived from calves is 1.0 × 10⁻⁶. 8 The intervention period was 1 week for mice and 28 days for calves, with CFU / day.

[0009] The plan requires detailed explanation of the application of *Lactobacillus reuteri* derived from calves in the preparation of products for the prevention and treatment of colitis-related injuries. Products containing *Lactobacillus reuteri* from calves at least include the following effects: It alleviated E. coli-induced weight loss; It inhibits the growth of pathogenic Escherichia coli and alleviates colitis caused by pathogenic Escherichia coli; It alleviated colonic developmental delay caused by pathogenic Escherichia coli; It can alleviate deep mucosal muscular layer ulcers, glandular structure destruction, and inflammatory cell infiltration caused by pathogenic Escherichia coli.

[0010] The plan requires further detailed explanation of the application of a calf-derived Lactobacillus reuteri in the preparation of products for the prevention and treatment of colitis-related injuries, wherein the product is a live bacterium.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects: Through dual in vitro and in vivo validation, it was determined that *Lactobacillus reuteri* isolated from the intestines of calves failed to downregulate the mRNA expression levels of pro-inflammatory cytokines IL-8 and IL-1β after LPS treatment, and failed to significantly upregulate the mRNA expression levels of anti-inflammatory cytokines IL-10 and TGF-β. It can directly inhibit the growth of pathogenic *Escherichia coli*, alleviate colitis, significantly relieve inflammation caused by pathogenic *E. coli*, reduce deep mucosal muscularis ulcers, glandular structure destruction, and inflammatory cell infiltration caused by pathogenic *E. coli*, and significantly increase crypt depth. Therefore, it has good therapeutic effects in treating colitis caused by pathogenic *E. coli*, providing theoretical support for the future development of drugs using *Lactobacillus reuteri* from calves for the treatment of intestinal inflammation. Attached Figure Description

[0012] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0013] Figure 1 This is a schematic diagram illustrating the effect of *Lactobacillus reuteri* from calves on the transcriptional levels of inflammatory factors, provided in an embodiment of this application. Figure 2 This is a schematic diagram of a mouse experiment provided in an embodiment of this application; Figure 3 This is a schematic diagram illustrating the weight change of a mouse, provided as an embodiment of this application. Figure 4 This is a schematic diagram of a DAI scoring method provided in an embodiment of this application; Figure 5 This is a schematic diagram of a mouse spleen index provided in an embodiment of this application; Figure 6 A comparative schematic diagram of mouse colon length provided in an embodiment of this application; Figure 7 A schematic diagram illustrating the effect of Lactobacillus reuteri from calves on the morphology of colon tissue in mice with colitis, provided in an embodiment of this application. Figure 8 This is a schematic diagram illustrating the effect of Lactobacillus reuteri on the morphology of calf colon tissue, provided in an embodiment of this application. Figure 9 This is a schematic diagram illustrating the effect of Lactobacillus reuteri on the colonic development of calves, as provided in an embodiment of this application. Detailed Implementation

[0014] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0015] The core of this application is to provide a calf-derived Lactobacillus reuteri and its application in the preparation of products for the prevention and treatment of colitis. Through in vitro and in vivo dual verification, the therapeutic effect of calf-derived Lactobacillus reuteri in treating colitis caused by pathogenic Escherichia coli is determined.

[0016] Figure 1 This is a schematic diagram illustrating the effect of *Lactobacillus reuteri* of calf origin on the transcriptional levels of inflammatory factors, provided in an embodiment of this application. Figure 2 This is a schematic diagram of a mouse experiment provided in an embodiment of this application. Figure 3This is a schematic diagram illustrating the weight change of a mouse, provided in an embodiment of this application. Figure 4 This is a schematic diagram of a DAI scoring method provided in an embodiment of this application. Figure 5 This is a schematic diagram of a mouse spleen index provided in an embodiment of this application. Figure 6 This is a comparative schematic diagram of mouse colon length provided in an embodiment of this application. Figure 7 This is a schematic diagram illustrating the effect of *Lactobacillus reuteri* derived from calves on the morphology of colon tissue in mice with colitis, provided in an embodiment of this application. Figure 8 This is a schematic diagram illustrating the effect of Lactobacillus reuteri on the morphology of calf colon tissue, provided in an embodiment of this application. Figure 9 A schematic diagram illustrating the effect of *Lactobacillus reuteri* on calf colon development, provided in an embodiment of this application, is shown below. Figures 1 to 9 As shown.

[0017] A *Lactobacillus reuteri* strain derived from calves, deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36229, and classified as *Lactobacillus reuteri*. Lactobacillus reuteri .

[0018] The application of *Lactobacillus reuteri* of calf origin as described in the above embodiments in the preparation of products for the prevention and treatment of colitis-related injuries.

[0019] Based on the above embodiments, the application of *Lactobacillus reuteri* derived from calves in the preparation of products for the prevention and treatment of colitis-related injuries is described, wherein the intervention dose of *Lactobacillus reuteri* derived from calves is 1.0 × 10⁻⁶. 8 The intervention period was 1 week for mice and 28 days for calves, with CFU / day.

[0020] Based on the above embodiments, the application of *Lactobacillus reuteri* of calf origin in the preparation of products for the prevention and treatment of colitis-related injuries, wherein the products containing *Lactobacillus reuteri* of calf origin have at least the following effects: It alleviated E. coli-induced weight loss; It inhibits the growth of pathogenic Escherichia coli and alleviates colitis caused by pathogenic Escherichia coli; It alleviated colonic developmental delay caused by pathogenic Escherichia coli; It can alleviate deep mucosal muscular layer ulcers, glandular structure destruction, and inflammatory cell infiltration caused by pathogenic Escherichia coli.

[0021] Based on the above embodiments, the application of a calf-derived Lactobacillus reuteri in the preparation of a product for the prevention and treatment of colitis-related injuries, wherein the product is a live bacterium.

[0022] To enable those skilled in the art to better understand this solution, the following provides a detailed description of this application from two aspects: in vitro experiments and in vivo experiments. In vitro assay: Caco-2 cells: a commonly used cell line for studying colon inflammation.

[0023] (1) A cell inflammation model was established using lipopolysaccharide (LPS). The mRNA expression levels of inflammatory factors (IL-8, IL-1β, TNF-α, IL-10, TGF-β) and cell morphology were detected by real-time quantitative PCR (RT-qPCR) to determine whether the modeling was successful. (2) The established cell inflammation model was treated with supernatant of Lactobacillus reuteri from calves and Lactobacillus reuteri from broken calves, respectively. Then, the mRNA expression levels of inflammatory factors (IL-8, IL-1β, TNF-α, IL-10, TGF-β) were detected by real-time fluorescence quantitative PCR to determine the therapeutic effect of Lactobacillus reuteri from calves on inflammation. Culture of Caco-2 cells: (1) Cell resuscitation: 1. Preheat the culture medium before removing the cells from liquid nitrogen: Preheat the culture medium and culture flasks in a 37°C water bath for 30 minutes, and then remove the frozen cells from the liquid nitrogen.

[0024] 2. Wearing gloves and taking necessary protective measures, use long forceps to remove the cell cryovial from the liquid nitrogen container and transfer it to a -80°C freezer. Let it sit for several minutes to allow the residual liquid nitrogen to evaporate. Place it in a 37°C water bath to thaw rapidly, keeping the cap above the liquid level while gently shaking the cryovial to accelerate thawing. (When there is still a small piece of ice in the cryovial, remove it from the water bath).

[0025] 3. After wiping with alcohol, place in the cabinet. Take a 15 ml centrifuge tube, add 9 ml of incomplete culture medium, quickly transfer the cell cryopreservation suspension to the centrifuge tube, gently pipette the suspension, centrifuge at 1000 rpm for 5 min at room temperature, and discard the supernatant.

[0026] 4. Add 5 ml of complete culture medium containing 10% fetal bovine serum (FBS) to the centrifuge tube. Gently pipette to thoroughly mix the complete culture medium with the cell pellet at the bottom of the centrifuge tube. Resuspend the cells until they are evenly distributed and single cells. Seed the cell suspension into a cell culture flask and incubate in a constant temperature cell culture incubator (37℃, 5% CO2). After 24 hours, observe the cells under a microscope for contamination and adherence. Replace the culture medium if necessary. Passage the cells after 2-3 days.

[0027] (2) Cell passage: 1. Before removing cells from the incubator, sterilize the biosafety cabinet and preheat the culture medium.

[0028] 2. Caco-2 cells can be passaged when their density reaches 70%-90%. In a biosafety cabinet, pour the culture medium from the culture flask into the waste container, wash the cells 1-2 times with PBS, then aspirate the PBS and add trypsin solution.

[0029] 3. Place the culture flask in an incubator and incubate for 3-5 minutes. When you observe under a microscope that most of the cells have become round and no longer adhere to the wall, and a large number of cells detach when you gently shake and tap the sides of the culture flask, stop the digestion immediately.

[0030] 4. Add 2 times the volume of complete DMEM medium to stop digestion, and gently pipette the cells several times to ensure that all cells are completely detached from the cell wall. Then, transfer the cells to a centrifuge tube and centrifuge (1000 rpm, 5 min).

[0031] 5. Discard the supernatant and resuspend the cells in 1-3 mL of complete culture medium.

[0032] 6. After resuspending the cells, passage them at a ratio of 1:2 to 1:4, and add fresh complete culture medium to 8-10 mL / bottle.

[0033] (3) Cell cryopreservation: Before cryopreserving cells, prepare cryopreservation solution pre-cooled to 4°C, and label cryopreservation tubes with cell name, cell passage number, quantity, and cryopreservation date.

[0034] Following the cell passage method, the cells in the culture flasks were digested into a single-cell suspension in a biosafety cabinet, and culture medium was added to terminate the reaction. All liquid was transferred to 15 mL centrifuge tubes, mixed thoroughly by pipetting, and the cell morphology and number were observed under a microscope.

[0035] Centrifuge at 1000 rpm for 4 minutes at room temperature. After centrifugation, open the cap and discard the supernatant. Resuspend the cells in 1-2 mL of pre-cooled cryopreservation solution at 4°C.

[0036] Dispense the cell suspension into cryovials at a rate of 1 mL per tube and tighten the caps.

[0037] Place the cryovials in a pre-cooled programmed cooling box at 4°C, and place the programmed cooling box in an ultra-low temperature freezer within 15 minutes of the end of the cryopreservation process.

[0038] After overnight storage, the frozen cells were transferred to a liquid nitrogen tank for preservation.

[0039] ②Establishment of inflammation model: Human colon adenocarcinoma cell line Caco-2 was cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics, and incubated at 37°C in a 5% CO2 incubator. The medium was changed regularly, and cell growth was observed to ensure stable experimental conditions. When cells adhered well, exhibited regular morphology, and reached 80% confluence, they were passaged and seeded into 6-well cell culture plates.

[0040] Culture for 18 h to allow cells to fully adhere. After cell adhesion, discard the original culture medium and add DMEM medium containing 1 mg / mL LPS to achieve a LPS concentration of 10 μg / mL. Continue culturing for 6 h, then collect the cells, wash three times with PBS, centrifuge to collect the cell pellet for subsequent experiments.

[0041] ③ Co-culture of Lactobacillus reuteri from calves The study was divided into four groups: control group (CON group), LPS group (LPS group), Lactobacillus reuteri supernatant group from calves (LRS group), and Lactobacillus reuteri group from broken calves (BLR group).

[0042] Preparation of supernatant from *Lactobacillus reuteri* strain in calves: Single colonies of *Lactobacillus reuteri* strain were picked and inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24 h to obtain seed culture. The seed culture was then inoculated into fresh MRS medium at a 3% inoculum and anaerobically cultured at 37°C until the concentration of *Lactobacillus reuteri* from calves reached 10⁻⁶. 9 After centrifuging at 8000 g / min for 15 min, the supernatant was filtered through a 0.22 μm microporous membrane. 1 mL of the supernatant was then mixed with 9 mL of LDM MEMS complete culture medium to obtain 10 mL of the supernatant. 8 cfu / mL Lactobacillus reuteri supernatant.

[0043] Preparation of Lactobacillus reuteri suspension from calf: Take 2 mL of bacterial suspension with a concentration of 10... 8 A suspension of *Lactobacillus reuteri* cfu / mL was centrifuged at 8000 g / min for 15 min, then 2 mL of DMEM complete culture medium was added. After ultrasonic disruption, the suspension was filtered through a 0.22 μm microporous membrane to obtain the disrupted *Lactobacillus reuteri* suspension from calves.

[0044] CON group: Normal culture until cell density reaches 80% (10% DMEM medium takes about 24 h).

[0045] LPS group: 6 h before the cell density reached 80%, the culture medium was changed and a complete culture medium containing LPS was added to make the LPS concentration in the culture medium 10 μg / mL.

[0046] Supernatant of Lactobacillus reuteri from calves: Caco-2 cells were mixed with 10 8 The supernatant of cfu / mL Lactobacillus reuteri was pre-incubated at 37°C for 4 hours, then the bacterial culture was aspirated, and complete medium containing LPS was added to make the LPS concentration in the medium 10 μg / mL.

[0047] Broken calf-derived Lactobacillus reuteri group: Caco-2 cells were suspended with Lactobacillus reuteri. Pre-incubate at 37°C for 4 hours, then remove the bacterial culture and add complete culture medium containing LPS to make the LPS concentration in the culture medium 10 μg / mL.

[0048] ④ RNA extraction and quantitative reverse transcription PCR analysis RNA was extracted from collected Caco-2 cells using the EASYspin Plus kit (Aidlab, Beijing, China). Total RNA was treated with DNase I, and 500 ng of total RNA was used for cDNA synthesis via reverse transcription using the PrimeScript RT kit with gDNA eraser (Takala, Beijing, China). Real-time PCR was performed using IL-8, IL-1β, IL-10, and TGF-β (primers are shown in Table 1), and the products were detected using a QuantStudio 6 Flex real-time PCR instrument (Applied Biosystems, Foster City, California, USA). PCR was performed in a 20 μL system containing 2 μL cDNA, 10 μL 2×SYBR green Premix ExTaq (Takala Biosciences, Japan), and 0.5 μM of gene-specific primers. The thermal cycling parameters were 95 °C for 2 minutes, followed by 42 cycles of 95 °C for 15 seconds, 60 °C for 15 seconds, and 72 °C for 15 seconds, and one cycle of 95 °C for 30 seconds, 60 °C for 30 seconds, and 95 °C for 30 seconds. Finally, PCR product melting curves were obtained to determine amplification specificity. Each sample was run in triplicate on a 96-well plate, and the expression level of each gene was calculated based on GAPDH gene expression.

[0049] Table 1 Primer Table

[0050] according to Figure 1As shown, real-time PCR analysis of the collected cells revealed that both the supernatant of *Lactobacillus reuteri* from calves and the fragmented *Lactobacillus reuteri* from calves downregulated the mRNA expression levels of pro-inflammatory cytokines IL-8 and IL-1β after LPS treatment, but failed to upregulate the mRNA expression levels of anti-inflammatory cytokines IL-10 and TGF-β. This indicates that neither the supernatant of *Lactobacillus reuteri* from calves nor the fragmented *Lactobacillus reuteri* from calves significantly alleviated LPS-induced inflammation. Therefore, it is concluded that *Lactobacillus reuteri* from calves can alleviate colitis by directly inhibiting the growth of pathogenic *Escherichia coli*.

[0051] In vivo experiments: Mouse experiments Grouping Group 1 was the control group (CON group), with each mouse fed distilled water; Group 2 was the infection group (E. coli group), with each mouse fed a solution containing 10... 10 CFU Escherichia coli suspension, Group 3 calf-derived Lactobacillus reuteri group (LR group), each mouse was fed with 10 10 CFU E. coli suspension and 10 8 CFU suspension of Lactobacillus reuteri from calves.

[0052] The specific steps are as follows: BALB / c mice aged 6-8 weeks were acclimatized for one week, receiving 200 μL of distilled water daily via gavage. From day 3 to 6, mice in each group had free access to drinking water containing 5 g / L streptomycin. Twenty-four hours before the formal experiment, drinking water was used instead of streptomycin. Mice were grouped according to body weight, with mice of similar weight grouped together. The control group received distilled water via gavage from day 8 to day 14, while the infection group received an Escherichia coli suspension via gavage from day 8 to day 14. The calf-derived Lactobacillus reuteri group received a suspension of both Escherichia coli and calf-derived Lactobacillus reuteri via gavage from day 8 to day 14. Daily changes in mouse body weight and DAI index were observed and recorded. On day 15, all mice were dissected, colon tissue samples were collected, and colon length and spleen index were measured for each group.

[0053] DAI Index: The degree of weight loss, fecal viscosity and blood in mice were observed daily. Scores were assigned according to Table 1. The sum of the scores was the Disease Activity Index (DAI). The higher the score, the higher the degree of intestinal inflammation in mice.

[0054] Spleen index: Spleen wet weight (mg) / mouse body weight (g).

[0055] Table 2 DAI Scoring Criteria

[0056] according to Figure 3It was found that the initial body weight (100%) was the body weight on the day before the start of the formal trial (day 7), and the daily body weight thereafter (days 8-15) was compared with the initial body weight. Compared with the CON group, the E. coli group showed a decrease in body weight, and the LR group significantly alleviated the E. coli-induced weight loss.

[0057] according to Figure 4 It can be seen that, compared with the CON group, the DAI score of the E. coil group was significantly higher, while the DAI score of the LR group was not significantly different.

[0058] In inflammatory states, spleen indices will increase. According to... Figure 5 It can be seen that, compared with the CON group, the spleen index of the E. coli group was significantly increased, while the spleen index of the LR group showed no significant change, indicating that gavage administration of Lactobacillus reuteri from calves can significantly alleviate inflammation caused by pathogenic Escherichia coli.

[0059] according to Figure 6 It was found that, compared with the CON group, the colon length of mice in the E. coli group and the LR group was significantly shortened, which is a manifestation of the body's inflammatory response. After gavage administration of Lactobacillus reuteri from calves, colonic developmental delay caused by pathogenic Escherichia coli was alleviated, but not significantly.

[0060] according to Figure 7 Histological analysis showed that, 7 days after infection, gavage administration of Lactobacillus reuteri from calves could alleviate deep mucosal muscularis ulcers, glandular structural damage, and inflammatory cell infiltration caused by pathogenic Escherichia coli.

[0061] The mucosa of the intestinal tissue in group CON protrudes into the intestinal lumen to form folds, which are abundant. The mucosal epithelium consists of a single layer of columnar epithelium and goblet cells. The lamina propria contains a large number of intestinal glands, which are straight tubes, densely arranged, and contain a large number of goblet cells and a small number of columnar cells. The muscularis mucosae separates the lamina propria from the submucosa. The submucosa is loose connective tissue, and the muscularis mucosae structure is clear, with no obvious erosion or ulceration.

[0062] In the E. coil group, small focal erosions were observed in the intestinal tissue, with damage extending to the mucosal layer. Intestinal glands disappeared and were replaced by proliferating fibrous connective tissue, with occasional lymphocyte infiltration. The muscular layer structure was clear.

[0063] In the LR group, the mucosal layer of the intestinal tissue protrudes into the intestinal lumen to form folds, which are abundant. The mucosal epithelium consists of a single layer of columnar epithelium and goblet cells. The lamina propria contains a large number of intestinal glands, which are straight tubes, densely arranged, and contain a large number of goblet cells and a small number of columnar cells. The muscularis mucosae separates the lamina propria from the submucosa. The submucosa is loose connective tissue, and the muscular layer structure is clear, with no obvious erosion or ulceration.

[0064] Therefore, it can be seen that the calf-derived Lactobacillus reuteri provided in this application can directly inhibit pathogenic Escherichia coli and alleviate Escherichia coli-induced colitis in mice.

[0065] To further verify that the calf-derived Lactobacillus reuteri provided in this application can prevent calf colitis, a detailed description is provided below using calf experiments.

[0066] In vivo experiments: Calf experiments: The Holstein calves used in this experiment came from a ranch in Ningxia Hui Autonomous Region, China. All calves were treated without antibiotics and were individually examined to ensure they were free of injury, disease, and dehydration. A total of 60 newborn calves were included in the experiment: 40 female calves and 20 male calves. They were randomly divided into two groups, with 20 females and 10 males in each group. The control group (CON) was fed normal feed, while the experimental group (LR) was fed a supplemental 10... 8 Milk containing CFU of Lactobacillus reuteri. The feeding trial lasted 21 days, with weighing every week. At the end of the trial, three bulls from each group were randomly selected for dissection, and intestinal tissue samples were collected. All experimental calves were given colostrum via gavage according to the "1-2-4" principle after birth: 4 liters of colostrum were administered within one hour of birth, and the colostrum from the calving cow was emptied within two hours. Calves were housed individually in calf islands to avoid direct contact. Feeding amounts: 2-6 days old, 6 L / calves; 7-12 days old, 7 L / calves; 13-19 days old, 8 L / calves; 20-21 days old, 9 L / calves; all calves had free access to water. Each calf was individually housed in a naturally ventilated calf island with a water bucket and free access to water.

[0067] CON: The intestinal mucosa shows folds, and the mucosal epithelium is mainly composed of a single layer of columnar epithelial cells and goblet cells; the intestinal glands in the lamina propria are loosely arranged, appearing as long tubules, and there are abundant goblet cells; the submucosa is composed of loose connective tissue; the muscular layer has a clear structure and the muscle cells are arranged regularly; no obvious inflammatory cell infiltration was observed.

[0068] LR: Folds are visible in the intestinal mucosa. The mucosal epithelium is mainly composed of a single layer of columnar epithelial cells and goblet cells. The intestinal glands in the lamina propria are loosely arranged and appear as long tubules, with an abundance of goblet cells. The submucosa is composed of loose connective tissue. The muscular layer has a clear structure and the muscle cells are arranged regularly. No obvious inflammatory cell infiltration is observed.

[0069] Depend on Figure 9 It is known that feeding Lactobacillus reuteri can significantly increase the crypt depth and the number of crypts per unit area in the calf colon.

[0070] Conclusion: Lactobacillus reuteri may alleviate colitis and promote colonic development in calves by directly inhibiting pathogenic Escherichia coli.

[0071] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and incorporate common knowledge or customary techniques in the art disclosed herein. The specification and examples are to be considered exemplary only, and the true scope of this application is indicated by the claims.

[0072] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The embodiments of this application described above do not constitute a limitation on the scope of protection of this application.

Claims

1. A type of Lactobacillus reuteri derived from calves, characterized in that, The *Lactobacillus reuteri* derived from calves was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36229, and classified as *Lactobacillus reuteri*. Lactobacillus reuteri.KY-1 .

2. The use of the *Lactobacillus reuteri* of calf origin as described in claim 1 in the preparation of products for the prevention and treatment of inflammatory damage to the colon.

3. The application according to claim 2, characterized in that, The intervention dose of *Lactobacillus reuteri* from calves was 1.0 × 10⁻⁶. 8 The intervention period was 1 week for mice and 28 days for calves, with CFU / day.

4. The application according to claim 2, characterized in that, Products containing the aforementioned calf-derived Lactobacillus reuteri have at least the following effects: It alleviated E. coli-induced weight loss; It inhibits the growth of pathogenic Escherichia coli and alleviates colitis caused by pathogenic Escherichia coli; It alleviated colonic developmental delay caused by pathogenic Escherichia coli; It can alleviate deep mucosal muscular layer ulcers, glandular structure destruction, and inflammatory cell infiltration caused by pathogenic Escherichia coli.

5. The application according to claim 2, characterized in that, The product contains live bacteria.

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