Lactobacillus reuteri and application thereof in enhancing intestinal barrier

Abstract

The invention relates to the technical field of probiotics, in particular to lactobacillus reuteri and application thereof in enhancing intestinal barriers. The lactobacillus reuteri BCre4 provided by the invention is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.34891. The lactobacillus reuteri BCre4 provided by the invention has the advantages that the lactobacillus reuteri BCre4 is preserved in the China General Microbiological Culture Collection Center; the bacterial strain has high safety, good gastrointestinal fluid tolerance and other probiotic properties, can improve the intestinal barrier, enhance the activity of damaged intestinal cells and promote intestinal injury repair, can be used for preventing, relieving or treating intestinal barrier injury, has excellent performance in the aspects of improving the integrity of the intestinal barrier and improving intestinal health, and can be used for preventing, relieving or treating the intestinal barrier injury. The method has a good application prospect in the development of probiotic preparations related to intestinal health.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and more particularly to a strain of *Lactobacillus reuteri* and its application in enhancing the intestinal barrier. Background Technology

[0002] In recent years, with changes in people's dietary habits and lifestyles, the incidence of a series of intestinal diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and colorectal cancer, has shown a year-on-year upward trend. The intestine is one of the largest and most important barriers in the human body, protecting the host from harmful substances and pathogenic microorganisms present in the intestinal lumen. In the development of intestinal-related diseases, the intestinal barrier plays a crucial "gatekeeper" role. Studies have found that disruption of the intestinal barrier integrity leads to increased susceptibility to colitis in the host, and in severe cases, can further develop into colorectal cancer.

[0003] The human gut is home to a large number of microorganisms (~10). 12 -10 13 These abundant microorganisms constitute the gut's first biological defense against external aggressors, playing a crucial role in maintaining gut homeostasis and health. Existing research has found that antibiotic use depletes the gut microbiota, leading to increased intestinal barrier permeability, and gut microbiota dysbiosis can also cause intestinal barrier dysfunction. This indicates that maintaining a healthy gut microbiota ecosystem is essential for maintaining stable intestinal barrier function.

[0004] The World Health Organization (WHO) defines probiotics as live microorganisms with positive health benefits. Currently, probiotics are mainly used in agriculture, food, health products, and the medical field. Studies have found that *Lactobacillus johnsonii* can promote the polarization of M2 macrophages by secreting extracellular vesicles, alleviating intestinal inflammatory damage and repairing intestinal barrier function. *Bacillus subtilis* can enhance intestinal barrier integrity by promoting the expression of tight junction proteins between epithelial cells. Studies by Wang et al. have shown that *Bifidobacterium lactis* (…) Bifidobacteria lactisIt can restore symptoms of ulcerative colitis (UC), such as colonic shortening. *Lactobacillus rhamnosus* GG (LGG) is a probiotic strain isolated from the intestines of healthy individuals in the early 1980s. It has been granted "safety certification" by the European Food Safety Authority (EFSA) and was included in China's "List of Strains that Can Be Used in Infant Formula" in 2011. It possesses various probiotic properties, and can alleviate inflammatory bowel disease by maintaining intestinal barrier function, promoting tissue repair, regulating gut microbiota, and exerting immunomodulatory effects. These research results indicate that probiotics play an important role in improving intestinal barrier function and maintaining intestinal health.

[0005] Currently, while the academic community has extensively and deeply studied the positive effects of probiotics in repairing damaged intestinal barriers and alleviating related intestinal diseases, the mechanisms by which different probiotics exert their effects vary considerably. Given the complexity of living environments and the diversification of dietary structures, the stresses and challenges faced by the intestinal system differ among individuals, and the specific mechanisms of intestinal barrier damage vary widely. This undoubtedly poses an obstacle to the widespread application of probiotic therapy. Therefore, developing new probiotics is of great significance for preventing and treating intestinal barrier damage, protecting intestinal health, and reducing disease risk. Summary of the Invention

[0006] This invention provides a strain of *Lactobacillus reuteri* and its application in enhancing the intestinal barrier.

[0007] This invention isolates and screens a probiotic strain of *Lactobacillus reuteri* that improves the intestinal barrier. Through comprehensive strain safety evaluation and probiotic characteristic testing, combined with in vitro cell experiments and animal experiments, the potential role of this strain in maintaining intestinal barrier health is systematically verified, providing a foundation for the development and application of related probiotic products.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides *Lactobacillus reuteri* (… Limosilactobacillus reuteri The strain BCare4 was deposited on June 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) and classified as *Lactobacillus reuteri*. Limosilactobacillus reuteri The accession number is CGMCCNo.34891.

[0010] The present invention contains *Lactobacillus reuteri* ( Limosilactobacillus reuteriBCare4 is isolated and purified from the intestines of healthy infants and young children. It has high safety and good gastrointestinal fluid tolerance, and can improve the intestinal barrier, enhance the vitality of damaged intestinal cells, and promote the repair of intestinal damage. It can be used to prevent, alleviate or treat intestinal barrier damage.

[0011] Secondly, the present invention provides a microbial preparation comprising the aforementioned *Lactobacillus reuteri* (…). Limosilactobacillus reuteri )BCare4 or by the aforementioned *Lactobacillus reuteri* ( Limosilactobacillus reuteri BCare4 was prepared.

[0012] The above-mentioned microbial preparations may be liquid or solid preparations, and their active ingredients include Lactobacillus reuteri (…). Limosilactobacillus reuteri In addition to BCare4, it may also contain *Lactobacillus reuteri* (…). Limosilactobacillus reuteri Metabolites of BCare4 (e.g., cell-free supernatant) may also contain other probiotics (e.g., probiotics that improve intestinal barrier function); in addition to the active ingredient, excipients permitted in the field of microbial preparations may also be included.

[0013] Thirdly, the present invention provides a probiotic preparation, wherein the probiotic preparation comprises the aforementioned *Lactobacillus reuteri* (… Limosilactobacillus reuteri )BCare4.

[0014] The above-mentioned probiotic preparations can be in liquid or solid form, and their active ingredients include Lactobacillus reuteri (…). Limosilactobacillus reuteri In addition to BCare4, it may also contain other probiotics (such as probiotics that improve intestinal barrier function), and in addition to the active ingredient, it may also contain excipients permitted in the field of probiotic preparations.

[0015] Preferably, in the probiotic preparation, the *Lactobacillus reuteri* (…) Limosilactobacillus reuteri BCare4 exists in the form of live bacteria.

[0016] Fourthly, the present invention provides a method for preparing the above-described microbial preparation or the probiotic preparation, the method comprising: processing the *Lactobacillus reuteri* (… Limosilactobacillus reuteri )BCare4 was cultured to obtain a culture.

[0017] The above culture can be performed using the commonly used culture media and conditions for *Lactobacillus reuteri*.

[0018] In some embodiments of the present invention, MRS medium is used, and the culture is carried out at 35-37°C under anaerobic conditions.

[0019] For the microbial preparation, the culture may include Lactobacillus reuteri (… Limosilactobacillus reuteri One or more of the following: BCare4 cells, its metabolites, its cell-free supernatant, and directly collected cultures containing cells and supernatant.

[0020] For the probiotic preparation, the culture is preferably *Lactobacillus reuteri* (…). Limosilactobacillus reuteri )BCare4 cells.

[0021] Fifthly, the present invention provides the above-described *Lactobacillus reuteri* (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation or the probiotic preparation in enhancing the intestinal barrier or in the preparation of products for enhancing the intestinal barrier.

[0022] The aforementioned enhancement of the intestinal barrier includes improving and strengthening the intestinal barrier in a non-disease state.

[0023] Sixthly, the present invention provides the above-described *Lactobacillus reuteri* (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation or the probiotic preparation in the preparation of products for the prevention, relief or treatment of intestinal barrier damage.

[0024] In the above applications, the intestinal barrier damage includes structural or functional impairment of the intestinal mucosal barrier (including intestinal epithelial cells, mucus layer, tight junction proteins, immune cells, etc.), leading to increased intestinal permeability and subsequently causing inflammation or disease.

[0025] This invention demonstrates through experiments that *Lactobacillus reuteri* (… Limosilactobacillus reuteri BCare4 can effectively alleviate intestinal barrier damage, specifically by alleviating weight loss, inflammation, and changes in intestinal structure or function caused by intestinal barrier damage.

[0026] In a seventh aspect, the present invention provides the above-described *Lactobacillus reuteri* (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation or the probiotic preparation in the preparation of products for improving the vitality of damaged intestinal cells.

[0027] Eighthly, the present invention provides the above-described *Lactobacillus reuteri* (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation or the probiotic preparation in the preparation of products for promoting the repair of intestinal damage.

[0028] This invention discovers that *Lactobacillus reuteri* (… Limosilactobacillus reuteriBCare4 can promote intestinal cell migration, thereby promoting the repair of intestinal damage.

[0029] Ninthly, the present invention provides the above-described *Lactobacillus reuteri* (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation or the probiotic preparation in the preparation of a product for the prevention or relief of weight loss, intestinal congestion, colonic shortening and / or intestinal inflammation caused by intestinal barrier damage.

[0030] In this invention, the product is preferably a drug.

[0031] The active ingredient of the product includes *Lactobacillus reuteri* (…). Limosilactobacillus reuteri BCare4 may also contain other probiotics that enhance the intestinal barrier or have other functional probiotics. In addition to the active ingredient, the product may also contain pharmaceutically permitted excipients.

[0032] In this invention, the product is applied to mammals that require strengthening the intestinal barrier or preventing, alleviating or treating intestinal barrier damage, preferably humans.

[0033] In a tenth aspect, the present invention provides a medicament comprising the above-described *Lactobacillus reuteri* (…). Limosilactobacillus reuteri )BCare4 or the microbial preparation or the probiotic preparation.

[0034] Eleventhly, the present invention provides a method for enhancing the intestinal barrier, the method comprising: administering the *Lactobacillus reuteri* (… Limosilactobacillus reuteri )BCare4, the microbial preparation, the probiotic preparation, or the drug was administered to the test subject.

[0035] In a twelfth aspect, the present invention provides a method for preventing, alleviating, or treating intestinal barrier damage, the method comprising: administering the *Lactobacillus reuteri* (… Limosilactobacillus reuteri )BCare4, the microbial preparation, the probiotic preparation, or the drug was administered to the test subject.

[0036] Preferably, the test subject is a mammal, more preferably a human.

[0037] The beneficial effects of the present invention include at least the following: the *Lactobacillus reuteri* provided by the present invention (… Limosilactobacillus reuteriBCare4 has high safety and good gastrointestinal fluid tolerance, among other probiotic properties. It can improve the intestinal barrier, enhance the vitality of damaged intestinal cells, and promote the repair of intestinal damage. It can be used to prevent, alleviate, or treat intestinal barrier damage. It performs excellently in improving intestinal barrier integrity and intestinal health, and has good application prospects in the development of probiotic preparations related to intestinal health. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 This is a technical roadmap for the experimental content involved in this invention.

[0040] Figure 2 This is the phylogenetic tree of *Lactobacillus reuteri* BCare4 in Example 1 of the present invention.

[0041] Figure 3 The images show the colony morphology of *Lactobacillus reuteri* BCare4 in Example 1 of this invention. a is a complete image of the plate, and b is a partial image of the plate.

[0042] Figure 4 This is the growth curve of *Lactobacillus reuteri* BCare4 in Example 1 of the present invention.

[0043] Figure 5 This refers to the tolerance of *Lactobacillus reuteri* Bcare4 to artificial gastrointestinal fluid in Example 1 of this invention.

[0044] Figure 6 This is the hemolytic analysis result of *Lactobacillus reuteri* Bcare4 in Example 1 of this invention. a is the positive control bacteria: *Staphylococcus aureus* subsp. *goldenii* (… Staphylococcusaureus subsp. aureus Rosenbach ATCC43300); b is the strain to be tested: Lactobacillus reuteri BCare4.

[0045] Figure 7 This is a schematic diagram of the HT-29 cell scratch experiment in Example 2 of the present invention.

[0046] Figure 8 This invention relates to the effect of *Lactobacillus reuteri* BCare4 on the activity of DSS-treated Caco-2 cells in Example 2 of this invention.

[0047] Figure 9The effect of *Lactobacillus reuteri* BCare4 on scratch healing of HT-29 cells in Example 2 of this invention (48h): a) Comparison of scratch damage under a microscope at 0h and 48h; b) Cell migration rate after 48h compared to 0h.

[0048] Figure 10 This is a schematic diagram of the animal experiment design in Embodiment 3 of the present invention.

[0049] Figure 11 For the mouse experiment of *Lactobacillus reuteri* BCare4 in Example 3 of the present invention: a) is the percentage of mouse body weight during DSS modeling to the starting day 0 of modeling; b) is the disease activity index of mice during DSS modeling; c) is a representative photograph of the colon of each group of mice at the end of the experiment.

[0050] Figure 12 The following are the histopathological results of mouse colon tissue in Example 3 of the present invention: a) HE-stained section of colon tissue; b) Histological damage score of colon tissue. Detailed Implementation

[0051] In a specific embodiment of the present invention, the research on *Lactobacillus reuteri* BCare4 mainly includes the following aspects: 1. Isolation, purification, and identification of bacterial strains A bacterial strain was isolated and purified from the intestines of healthy infants and identified as *Lactobacillus reuteri* by 16S rDNA sequence analysis.

[0052] 2. *Lactobacillus reuteri* BCare4 exhibits high safety and gastrointestinal fluid tolerance. The safety of the strain was assessed using hemolytic agar plates and drug susceptibility tests. The results showed that *Lactobacillus reuteri* BCare4 had good safety.

[0053] The gastrointestinal tolerance of the strain was assessed using artificial gastrointestinal fluid. The results showed that Lactobacillus reuteri BCare4 had good tolerance to artificial gastric acid and artificial bile salts, and could pass through the gastrointestinal tract in a live state, safely reaching the colon to exert its probiotic effects.

[0054] 3. Lactobacillus reuteri BCare4 can significantly promote intestinal cell vitality and intestinal injury healing. The effect of the strain on cell viability was detected using the Caco-2 cell model. The results showed that the cell-free supernatant of the BCare4 strain could significantly improve the cell viability of Caco-2 cells damaged by dextran sulfate sodium salt (DSS).

[0055] In addition, a cell scratch model was constructed using the HT-29 cell model, and it was found that cell-free supernatant of BCare4 strain could significantly promote the scratch healing rate of HT-29 cell monolayer in the context of DSS injury.

[0056] 4. Lactobacillus reuteri BCare4 can significantly alleviate intestinal barrier damage. This invention combines two models: cells and mice. First, intestinal epithelial cell lines are used to screen for strains that can potentially alleviate intestinal damage, and then mice are used to verify their function.

[0057] Using a mouse model of intestinal injury induced by gavage followed by DSS induction, and with LGG as a positive control strain, the efficacy of the BCare4 strain in preventing DSS-induced intestinal injury was evaluated. The main parameters measured were changes in mouse body weight, disease activity index, colon length, and colonic pathology. Results showed that the BCare4 strain effectively alleviated intestinal barrier damage.

[0058] This invention demonstrates through experiments that Lactobacillus reuteri BCare4 has the function of preventing and alleviating intestinal barrier damage, and its effect is superior to LGG.

[0059] The technical route diagram of this invention is shown below. Figure 1 As shown.

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0061] Example 1: Isolation and identification of probiotics, and assessment of their tolerance and safety in gastrointestinal fluids. This invention isolates and purifies multiple strains of bacteria from the intestines of infants and young children, and then screens one of the probiotic strains to evaluate its tolerance to gastrointestinal fluids and its safety.

[0062] 1.1 Strain Isolation *Lactobacillus reuteri* BCare4 was isolated from the feces of healthy infants in Chengdu, China in 2024. Fresh fecal samples were immediately placed in sterile containers and then in anaerobic bags at 4°C for storage, ensuring delivery to the laboratory within 12 hours. 2.0 g of fecal sample was added to 20 mL of sterile PBS buffer, thoroughly vortexed, and 0.5 mL was added to 5 mL of MRS liquid culture medium (Hangzhou Best Biotechnology Co., Ltd., catalog number: BS1137). Enrichment was performed by anaerobic incubation at 37°C for 24 hours. Subsequently, 0.5 mL of the enriched sample was added to 4.5 mL of sterile physiological saline to prepare 10... -1 Diluent; then add 0.5 mL of the diluent to 4.5 mL of physiological saline, and prepare 10 [units] in sequence. -4 Up to 10 -6 Diluents. Take 100 μL of each serially diluted solution and spread it onto MRS solid medium (Hangzhou Best Biotechnology Co., Ltd., catalog number: BS1138) using the three-zone streak method. Incubate anaerobicly at 37℃ for 48 h to obtain single colonies. Select single colonies exhibiting typical characteristics of *Lactobacillus reuteri* and showing good growth (white or milky white, moist and smooth surface, with neat edges).

[0063] 1.2 Strain Identification 1.2.1 Strain purification Selected single colonies were picked from MRS solid medium, streaked with a sterile inoculating loop, and anaerobically cultured at 37°C for 48 h to obtain purified single colonies. These purified single colonies were then inoculated into 5 mL of MRS liquid medium and anaerobically cultured at 37°C for 24 h to prepare a bacterial suspension. Gram staining was performed on the bacterial suspension, and rod-shaped, Gram-positive strains were selected.

[0064] 1.2.2 Strain Preservation The enriched bacterial suspension was centrifuged at 3000 rpm for 15 minutes at 4°C. After collecting the cell pellet, the cells were washed twice with PBS buffer and then resuspended in fresh MRS medium. The cell suspension was mixed with sterile 30% glycerol at a 1:1 volume ratio and stored at -80°C.

[0065] 1.2.3 Preparation of bacterial suspension after secondary activation of the strain Take the BCare4 strain frozen at -80℃, quickly thaw it in a 37℃ water bath, and then use a sterile inoculation loop to take a small amount of bacterial suspension. Spread the suspension onto ordinary MRS solid medium using the three-zone streak method, and incubate anaerobically at 37℃ for 48 hours. Pick a single colony and inoculate it onto ordinary MRS liquid medium, incubating anaerobically at 37℃ for 18-24 hours. Then, inoculate 1% of the culture onto fresh MRS liquid medium, subculture twice overnight to obtain an activated bacterial suspension. Use this activated suspension for subsequent experiments.

[0066] Five mL of fresh BCare4 activated bacterial culture was taken, and total DNA was extracted from the strain. The DNA was then amplified using the universal 16S rRNA gene primers 27F / 1492R to obtain the PCR product of the 16S rRNA gene. Sequencing revealed the 16S rDNA sequence of the strain (SEQ ID NO.1), as shown below: SEQ ID NO.1:

[0067] The 16S rDNA sequence of the strain was aligned to the NCBI database using BLAST software. The alignment results were then used to construct a phylogenetic tree using MEGA software. Figure 2 After comparison, BCare4 was identified as *Lactobacillus reuteri*. Limosilactobacillus reuteri ), which is a strain listed in the "List of Microbial Strains that Can Be Used in Food".

[0068] 1.3 Preservation of bacterial strains Lactobacillus reuteri BCare4 was deposited on June 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) and classified as Lactobacillus reuteri. Limosilactobacillus reuteri The accession number is CGMCC No. 34891.

[0069] 1.4 Colony morphology observation Dip an inoculation loop into a small amount of bacterial suspension and streak it onto an MRS solid medium plate using the three-zone streak method. After anaerobic incubation at 37°C for 48 hours, remove the plate and observe the colony morphology.

[0070] On MRS solid medium, colonies of *Lactobacillus reuteri* BCare4 appear milky white, round, with a raised center, a moist and smooth surface, and neat edges. Figure 3 ).

[0071] 1.5 Determination of growth curve Bacterial growth typically involves four phases: lag phase, logarithmic phase, stationary phase, and decline phase. Understanding the growth characteristics of bacterial strains helps in culturing them at the appropriate phase. To observe the growth characteristics of *Lactobacillus reuteri* BCare4, its growth curve was determined using the following method: Fresh, twice-activated BCare4 bacterial suspension was prepared and inoculated into 15 mL of MRS liquid medium at a 1% inoculum. The suspension was incubated at 37°C for 32 h. Every 2 h, 200 μL of the mixed bacterial suspension was collected and used as a control. The absorbance (OD) at 600 nm was measured. 600nm The data were measured in triplicate, with three replicates to ensure accuracy. A growth curve was then plotted based on the results.

[0072] Growth curve results showed that *Lactobacillus reuteri* BCare4 entered the logarithmic growth phase after 8 hours of culture and entered the stationary phase after about 16 hours. Figure 4 ).

[0073] 1.6 Tolerance to artificial gastrointestinal fluids Probiotics need to colonize the gastrointestinal tract before they can exert their beneficial effects. Therefore, the tolerance of *Lactobacillus reuteri* BCare4 in simulated gastric fluid (pH approximately 3.0) (Beijing Regen Biotechnology Co., Ltd., catalog number CZ0211) and simulated small intestinal fluid (pH approximately 6.8) (Beijing Regen Biotechnology Co., Ltd., catalog number CZ0201) was tested. The BCare4 bacterial culture, activated twice, was centrifuged at 3000 rpm for 15 minutes, washed three times with sterile PBS buffer, and the bacterial concentration was adjusted to 2 × 10⁻⁶. 8 CFU / mL, 1 mL of bacterial culture was inoculated into 9 mL of simulated gastric / intestinal fluid. A blank culture medium was used as a negative control, and each treatment was performed in triplicate. The culture was incubated anaerobically at 37°C for 3 h. Viable bacteria were counted at 0 h, 2 h, and 3 h, and the survival rate was calculated based on the viable bacterial count. The formula for calculating bacterial survival rate is: .

[0074] The results showed that *Lactobacillus reuteri* BCare4 exhibited good tolerance to artificial gastrointestinal fluids. Figure 5 This indicates that it can safely pass through the human digestive tract and reach the intestines in the form of live bacteria.

[0075] 1.7 Security Assessment 1.7.1 Hemolytic Analysis The safety of the tested bacterial strains was assessed using a microbial hemolysis detection method. The BCare4 strain, activated twice, was diluted and inoculated onto Columbia blood agar plates. After anaerobic incubation at 37°C for 36 hours, the presence or absence of hemolysis was observed. Staphylococcus aureus was selected as a positive control. Figure 6 As shown, *Lactobacillus reuteri* BCare4 forms white colonies and does not exhibit hemolysis on blood agar plates, while *Staphylococcus aureus* colonies show a clear transparent hemolytic zone. This indicates that the target strain is not significantly pathogenic and is a safe lactic acid bacterium.

[0076] 1.7.2 Drug sensitivity assessment The drug susceptibility of the strains was assessed using antimicrobial susceptibility testing. The tests were conducted according to the standard methods in ISO 10932 / IDF223. The resistance of the strains was expressed as the minimum inhibitory concentration (MIC).

[0077] (1) Preparation of the bacterial suspension to be tested Take fresh, twice-activated BCare4 bacterial culture and centrifuge at 3000 rpm for 15 min at 4°C, discarding the supernatant. Wash the bacteria three times with PBS buffer to remove residual culture medium. Finally, adjust the bacterial concentration to 6 × 10⁻⁶ using LSM liquid medium. 5CFU / mL, for later use.

[0078] Preparation of LSM medium: LSM medium consists of 90% IST liquid medium (ISO-SENSITEST Broth) (Shandong Top Biotechnology Co., Ltd., catalog number: M8120C) and 10% MRS liquid medium.

[0079] (2) Preparation and gradient dilution of antibiotic working solutions The antibiotic working solution was prepared with an initial concentration of 512 μg / mL using LSM liquid medium. A two-fold serial dilution method was used: 100 μL of the working solution was mixed with 100 μL of LSM medium, and the solution was diluted sequentially until the final concentration was 1 μg / mL.

[0080] (3) 96-well plate loading and bacterial suspension inoculation Add 100 μL of bacterial culture to each well of a 96-well microplate. Then add 100 μL of different concentrations of antibiotic working solution to each well (to dilute both the antibiotic and bacterial culture by 2-fold in each well, resulting in an antibiotic concentration gradient of 256–0.5 μg / mL and a final bacterial concentration of 3 × 10⁻⁶). 5 (CFU / mL). Three replicates were set for each antibiotic concentration to ensure experimental reliability.

[0081] (4) Control group setup Positive control (3 replicates): 100 μL LSM medium + 100 μL bacterial suspension (antibiotic-free).

[0082] Negative control (3 replicates): 200 μL LSM medium (without antibiotics and bacterial culture).

[0083] (5) Cultivation conditions The microplate reader reads the OD values ​​of each well at 0 h. 600 nm After the value was determined, the 96-well plate was placed in an anaerobic incubator at 37°C and incubated statically for 24 h.

[0084] (6) Result reading After 24 hours of incubation, the growth of the positive and negative control wells was examined, and the minimum inhibitory concentration (MIC) at which each antimicrobial agent completely inhibited the growth of the test strain was recorded.

[0085] The seven classes of antibiotics selected covered various resistance mechanisms. MIC test results (Table 1) showed that, except for ampicillin, *Lactobacillus reuteri* BCare4 was sensitive to all other antibiotics. Since the vast majority of lactic acid bacteria are resistant to ampicillin, this indicates that *Lactobacillus reuteri* BCare4 is a safe lactic acid bacterium.

[0086] Table 1. Antimicrobial susceptibility test results of *Lactobacillus reuteri* BCare4

[0087] Note: The cutoff value is based on the EFSA standard (Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance). A MIC value > the cutoff value indicates a resistant (R) type, and a MIC value ≤ the cutoff value indicates a sensitive (S) type.

[0088] Based on the combined hemolytic activity assessment and drug susceptibility results, *Lactobacillus reuteri* BCare4 is a safe lactic acid bacterium.

[0089] Example 2: Protective effect of cell-free supernatant from BCare4 strain on intestinal cell model. To verify the protective effect of Lactobacillus reuteri BCare4 on intestinal cells, Caco-2 cell models with structures and functions similar to the small intestine and HT-29 cell models with intestinal epithelial morphology and function were selected as in vitro test models.

[0090] 2.1 Experimental Methods for Damage Protection Based on Caco-2 Cell Model The Caco-2 cell viability assay consisted of six groups: a normal control group (Control), a DSS model group (DSS), and five intervention groups (LGG, MN15925, MN12934, MN02044, and BCare4). Except for the Control group, all other groups received 3% DSS treatment. The Control and DSS groups received additional MRS treatment solution, while the five intervention groups received their respective supernatants.

[0091] The specific method is as follows: 2.1.1 Preparation of cell-free supernatant from the strain (1) Prepare fresh bacterial suspensions of BCare4, LGG, MN15925, MN12934 and MN02044 strains that have been activated twice, and inoculate them into 15 mL of MRS liquid medium at an inoculation rate of 1% and culture them anaerobically at 37℃ until the plateau phase.

[0092] (2) Determine the OD of the bacterial culture during the plateau phase. 600nm Value, via OD 600nm - The CFU standard curve was used to dilute the bacterial culture to 1×10⁻⁶. 9 CFU / mL, take 10 mL of bacterial culture, centrifuge at 4℃ and 3000 rpm for 15 minutes, and collect the supernatant.

[0093] (3) The collected supernatant was placed in a vacuum freeze dryer (Ningbo Xinzhi Freeze Drying Equipment Co., Ltd., model: SCIENTZ-30ND) and freeze-dried according to the standard procedure to prepare cell-free supernatant freeze-dried powder of the strain.

[0094] (4) Prepare an equal amount of MRS liquid culture medium lyophilized powder under the same conditions. It was added as a control group in subsequent experiments to avoid potential interference from residual MRS culture medium components in the supernatant on the experimental results.

[0095] (5) Preparation of supernatant / MRS treatment solution: When using, the lyophilized supernatant powder of the strain and the lyophilized MRS liquid culture medium were dissolved in 10 mL of Caco-2 cell-specific culture medium (Suzhou Haixing Biotechnology Co., Ltd., product number: TCH-G146), filtered through a 0.22 μm filter membrane for sterilization, and then diluted 10 times with Caco-2 cell-specific culture medium (approximately 1×10⁻⁶). 8 (CFU / mL bacterial metabolites), after adjusting the pH, supernatant and MRS treatment solution were obtained for subsequent cell experiments.

[0096] 2.1.2 Caco-2 cell culture and relative survival rate test (1) Take Caco-2 cells in the logarithmic growth phase and adjust the cell density to 2×10⁻⁶ using Caco-2 cell-specific culture medium. 5 Cells were seeded at a rate of 100 μL per well in 96-well microplates and incubated at 37°C in a 5% CO2 incubator for 24 h until the cells were fully adhered.

[0097] (2) After the cells adhered to the wall, the original culture medium was removed. 100 μL of 3% DSS solution prepared with Caco-2 cell culture medium containing 10% fetal bovine serum was added to each experimental group, and the cells were cultured for another 24 h to establish a cell damage model (the Control group used Caco-2 cell culture medium with 10% fetal bovine serum without DSS).

[0098] (3) After the induced injury is completed, the original DSS-containing medium is discarded. The medium is replaced with Caco-2 cell-specific medium (supernatant or MRS). Each group is divided into 6 replicates and cultured for 24 h.

[0099] (4) After 24 h of intervention, the original culture medium was discarded, and 110 μL of DMEM basal medium (Gibco, USA, catalog number: C11995500BT) containing 10 μL of CCK-8 reagent (GlpBio, USA, catalog number: GK10001) was added to each well. The medium was then incubated at 37°C in the dark for 2 h.

[0100] (5) The absorbance (OD) of each well at 450 nm was measured using an ELISA reader. 450 nm The cell viability of Caco-2 cells is calculated using the following formula: .

[0101] (6) The effect of the strain on the repair of intestinal epithelial cell injury was evaluated by the statistical difference in cell viability among different treatment groups.

[0102] 2.2 Cell Scratch Assay Method Based on HT-29 Cell Model The experiment was divided into four groups: a normal control group (Control), a DSS model group (DSS), and two intervention groups (LGG and BCare4). Except for the Control group, all other groups received 3% DSS treatment. The Control and DSS groups were supplemented with MRS treatment solution, while the two intervention groups were supplemented with their respective supernatants.

[0103] The specific method is as follows: 2.2.1 Preparation of cell-free supernatant for the strain: (1) Prepare fresh BCare4 and LGG strain suspensions that have been activated twice, and inoculate them into 15 mL of MRS liquid medium at an inoculation rate of 1%, and culture them anaerobically at 37°C until the plateau phase.

[0104] (2) Determination of OD in bacterial culture during the plateau phase 600nm Value, via OD 600nm - The CFU standard curve was used to dilute the bacterial culture to 1×10⁻⁶. 9 CFU / mL, take 10 mL of bacterial culture, centrifuge at 4℃ and 3000 rpm for 15 minutes, and collect the supernatant.

[0105] (3) Place the collected supernatant in a vacuum freeze dryer and freeze dry it according to the standard procedure to prepare cell-free supernatant freeze-dried powder of the strain.

[0106] (4) Prepare an equal amount of MRS liquid culture medium lyophilized powder under the same conditions. It was added as a control group in subsequent experiments to avoid potential interference from residual MRS culture medium components in the supernatant on the experimental results.

[0107] (5) Preparation of supernatant / MRS treatment solution: When using, the lyophilized supernatant powder of BCare4 / LGG strain and the lyophilized MRS liquid culture medium were dissolved in 10 mL of RPMI 1640 medium (Gibco, USA, catalog number: 11875-093) with 2% FBS, filtered through a 0.22 μm filter membrane for sterilization, and then diluted 10 times with RPMI 1640 medium containing 2% FBS (the concentration of metabolites of BCare4 / LGG strain is approximately 1×10). 8 (CFU / mL), and after adjusting the pH, supernatant and MRS treatment solutions were obtained for subsequent cell experiments.

[0108] 2.2.2 HT-29 cell scratch test (1) Draw grid lines on the back of the 12-well cell culture plate beforehand with a marker to accurately locate the same field of view during subsequent microscopic observation. For example Figure 7 As shown, the gray area on the 12-well cell culture plate represents the cell monolayer, the black horizontal dashed line is the marker line on the back of the well plate, and the white vertical line is the scratch wound on the cell monolayer. The intersection of the scratch wound and the horizontal marker line on the back of the well plate is taken as the observation and photography point. Two observation points are selected for each well, and pictures are taken at 0 and 48 hours to statistically analyze the changes in the scratch area.

[0109] (2) Take HT-29 cells in the logarithmic growth phase and use 2×10 5 The cells were seeded at a density of 1 cell / mL in 12-well plates, and 1 mL of RPMI 1640 medium containing 10% fetal bovine serum was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator until the cells reached approximately 90% confluence.

[0110] (3) Discard the culture medium, gently wash the cells three times with PBS buffer pre-warmed at 37°C, 1 mL each time, discard the PBS, and then replace it with serum-free RPMI 1640 medium for serum starvation treatment for 12 h.

[0111] (4) After serum starvation treatment, the culture medium was discarded, and 1 mL of serum-free RPMI 1640 medium containing 3% DSS was added. The culture was continued for 6 h to establish an intestinal epithelial barrier injury model.

[0112] (5) Damage treatment: Using a 10 μL sterile pipette tip perpendicular to the bottom of the well plate, apply constant pressure to draw two parallel straight lines. Immediately wash three times with PBS buffer to remove exfoliated cells.

[0113] (6) Replace each treatment solution (supernatant treatment solution or MRS treatment solution) with RPMI 1640 medium containing 2% FBS, and add 1 mL to each well.

[0114] (7) Images of the scratched area were taken at pre-marked fixed positions using an inverted phase contrast microscope (10× objective lens) at 0 h and 48 h after scratching. Two fields of view were selected per well at each time point for image acquisition, and a total of 6 fields of view were obtained from three parallel wells in each treatment group.

[0115] (8) The images were analyzed using ImageJ software (Version 1.53), and the scratch area (μm) at each time point was calculated using threshold setting and region measurement functions. 2 The formula for calculating cell scratch healing migration rate is: .

[0116] (9) The effect of the strain on the repair of intestinal epithelial cell wounds was evaluated by the statistical difference in the cell scratch healing rate among different treatment groups.

[0117] 2.3 Statistical Analysis Methods Cellular experimental data were statistically analyzed and plotted using GraphPad Prism (Version=10.4.0) software. Ordinary one-way ANOVA was performed on the experimental data to determine if there were significant differences between the experimental groups. After confirming significant differences, Tukey's multiple comparison test was further employed, using single pooled variance to perform pairwise comparisons to determine if the differences between specific pairs of groups were statistically significant. The significance level was set at p<0.05, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0118] 2.4 Experimental Results 2.4.1 Lactobacillus reuteri BCare4 can enhance the viability of DSS-treated Caco-2 cells. The results of the Caco-2 cell viability assay showed that, compared with the Control group, the viability of Caco-2 cells in the DSS group was significantly reduced after 24 hours of DSS treatment (p<0.0001). However, in the BCare4 group, which also received DSS treatment, the viability of Caco-2 cells was significantly higher than that in the DSS group (p<0.05), and there was no significant difference between the BCare4 group and the LGG group (p>0.05). This indicates that *Lactobacillus reuteri* BCare4 can significantly improve the cell viability of DSS-treated Caco-2 cells, suggesting its potential to enhance the viability of damaged intestinal cells. Figure 8 Other bacterium such as Bifidobacterium bifidum MN15925, Bifidobacterium bifidum MN12934, and Lactobacillus reuteri MN02044 did not significantly improve Caco-2 cell activity (p>0.05). 2.4.2 *Lactobacillus reuteri* BCare4 significantly promoted DSS-induced scratch healing in HT-29 cells. Cell scratch assay results are as follows Figure 9 As shown, DSS treatment can lead to inhibited migration of HT-29 cells and poor cell condition. The BCare4 group can significantly repair the damage caused by DSS (p<0.01), with no significant difference compared with the normal control group and the LGG group (p>0.05). That is, Lactobacillus reuteri BCare4 can significantly alleviate the inhibition of HT-29 cell migration by DSS and accelerate scratch healing, indicating that it can repair the damage caused by DSS to intestinal cells and has the potential to promote intestinal damage repair.

[0119] Example 3: The alleviating effect of strain BCare4 on DSS-induced intestinal barrier damage 3.1 Preparation method of BCare4 / LGG bacterial suspension (1) Prepare fresh suspensions of BCare4 / LGG strains that have been activated twice, and inoculate them into 15 mL of MRS liquid medium at an inoculation rate of 1%, and culture them anaerobically at 37°C until the logarithmic growth phase.

[0120] (2) OD was measured using a spectrophotometer 600nm Value, based on pre-established OD 600nm - The concentration of bacterial solution is calculated using the CFU standard curve.

[0121] (3) Based on the required viable bacterial count for the experiment, transfer the appropriate volume of bacterial suspension to a sterile centrifuge tube and centrifuge at 3000 rpm for 15 minutes at 4°C to collect the bacterial cells. Discard the supernatant, gently resuspend the suspension in sterile PBS buffer, and wash three times. Finally, dissolve the bacterial cells in 200 μL of PBS buffer (containing 2 × 10⁻⁶ viable cells). 8 CFU was then administered using a 1 mL sterile syringe and a gavage needle.

[0122] 3.2 Experimental Methods for DSS-Induced Intestinal Barrier Injury in Mice (1) Preparation of experimental animals ( Figure 10 ): Six-week-old SPF-grade male C57BL / 6 mice were selected and acclimatized in a standard environment for one week.

[0123] (2) Grouping and intervention regimen: Mice were randomly divided into 4 groups (n=8): control group, DSS model group, BCare4 intervention group, and LGG intervention group. The treatment lasted for 21 days. Control group: Free access to sterile water throughout the treatment, and 200 μL of sterile PBS buffer was administered by gavage daily. Model group: Free access to sterile water for the first 14 days, and sterile water containing 2.5% DSS for the last 7 days, and 200 μL of sterile PBS buffer was administered by gavage daily. Intervention group (BCare4 / LGG): Sterile water for the first 14 days, and sterile water containing 2.5% DSS for the last 7 days, and 200 μL of sterile PBS buffer containing 2×10⁻⁶ DSS was administered by gavage daily. 8 CFU in PBS bacterial suspension.

[0124] (3) Observation indicators: Daily assessment of the disease activity index (DAI): including changes in weight, stool characteristics, and rectal bleeding. Record changes in food intake and water intake.

[0125] (4) Sample collection: On day 21 of the experiment, mice were euthanized by isoflurane inhalation anesthesia, and the colon tissue (from the anus to the cecum) was completely separated and its length was measured. The tissue samples were fixed with 4% paraformaldehyde and then embedded in paraffin. Subsequently, 5-micrometer-thick paraffin-embedded colon sections were prepared and stained with hematoxylin and eosin (H&E) for tissue staining.

[0126] 3.3 Statistical Analysis Methods Mouse experimental data were statistically analyzed and plotted using GraphPad Prism (Version=10.4.0) software. Ordinary one-way ANOVA was performed on the experimental data to determine whether there were significant differences between the experimental groups. After showing significant differences, Tukey's multiple comparison test was further used, employing single pooled variance to perform pairwise comparisons to determine whether the differences between specific pairs of groups were statistically significant. The significance level was set at p<0.05 (* indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001). Experimental values ​​are expressed as mean ± standard error (SEM).

[0127] 3.4 Experimental Results Mouse experiments showed that after administration of 2.5% DSS, the body weight of mice in the DSS model group began to decrease rapidly from day 4, reaching its lowest value on day 7, and was significantly lower than that of the control group (p<0.0001). Figure 11(a). The BCare4 intervention group significantly alleviated the weight loss in mice caused by DSS (p<0.05), and the allergic effect was significantly better than that of the LGG intervention group (p<0.05).

[0128] The Disease Activity Index (DAI) was calculated based on weight loss, stool characteristics, and fecal blood in mice. Starting from day 5 of DSS treatment, the DAI of the DSS model group mice significantly increased, reaching its peak on day 7. Figure 11 (b). The DAI score of mice in the BCare4 intervention group increased from day 6, but was significantly lower than that in the DSS model group and the LGG intervention group (p<0.0001). After the experiment, the colonic tissue and intestinal lumen of mice in the DSS model group showed severe congestion, and the average colon length was 3.6±0.13 cm. In contrast, the congestion of the colonic tissue and intestinal lumen of mice in the BCare4 intervention group was significantly improved, and the average colon length was 4.0±0.13 cm, significantly longer than that in the DSS model group (p<0.05). Figure 11 (c).

[0129] In summary, strain BCare4 can significantly alleviate DSS-induced intestinal damage and related symptoms in mice, and alleviate DSS-induced intestinal barrier injury.

[0130] Histological staining results showed that after DSS treatment, the colonic mucosa of mice in the DSS model group (DSS group) was severely damaged, with extensive ulceration, extensive neutrophil infiltration in the lamina propria, severe villus necrosis, and loss of epithelial structure. Figure 12 (a) Compared with the DSS model group, the colonic tissue damage in the BCare4 intervention group mice was significantly restored, the inflammation was reduced, the crypt structure was clearer, and its histopathological score was significantly lower than that of the DSS model group (p<0.001), and also significantly lower than that of the LGG intervention group (p<0.001). Figure 12 (b).

[0131] In summary, the BCare4 strain can effectively alleviate intestinal barrier damage caused by DSS, and its effect is significantly better than that of the LGG strain.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. *Lactobacillus reuteri* ( Limosilactobacillus reuteri BCare4, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34891.

2. A microbial preparation, characterized in that, The microbial preparation comprises *Lactobacillus reuteri* as described in claim 1. Limosilactobacillus reuteri )BCare4 or Lactobacillus reuteri as described in claim 1 ( Limosilactobacillus reuteri BCare4 was prepared.

3. A probiotic preparation, characterized in that, The probiotic preparation comprises *Lactobacillus reuteri* as described in claim 1. Limosilactobacillus reuteri )BCare4.

4. The method for preparing the microbial preparation according to claim 2 or the probiotic preparation according to claim 3, characterized in that, The method includes: processing the *Lactobacillus reuteri* (as described in claim 1) Limosilactobacillus reuteri )BCare4 was cultured to obtain a culture.

5. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation of claim 2 or the probiotic preparation of claim 3 in enhancing the intestinal barrier or in the preparation of products for enhancing the intestinal barrier.

6. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation of claim 2 or the probiotic preparation of claim 3 in the preparation of products for the prevention, relief or treatment of intestinal barrier damage.

7. The *Lactobacillus reuteri* as described in claim 1 ( Limosilactobacillus reuteri The use of BCare4 or the microbial preparation of claim 2 or the probiotic preparation of claim 3 in the preparation of products for improving the vitality of damaged intestinal cells.

8. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation of claim 2 or the probiotic preparation of claim 3 in the preparation of products for promoting the repair of intestinal damage.

9. The *Lactobacillus reuteri* as described in claim 1 (… Limosilactobacillus reuteri The use of BCare4 or the microbial preparation of claim 2 or the probiotic preparation of claim 3 in the preparation of a preparation for the prevention or relief of weight loss, intestinal congestion, colonic shortening and / or intestinal inflammation caused by intestinal barrier damage.

10. A drug, characterized in that, The drug contains *Lactobacillus reuteri* as described in claim 1. Limosilactobacillus reuteri )BCare4 or the microbial preparation of claim 2 or the probiotic preparation of claim 3.

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