Application of Lactobacillus reuteri PFL-2501 and its inoculants in the treatment of Helicobacter pylori

Abstract

This invention belongs to the field of biological agents and biomedicine, specifically relating to the application of *Lactobacillus reuteri* PFL-2501 and its agents in the treatment of *Helicobacter pylori*. This invention discovers that *Lactobacillus reuteri* PFL-2501 and its agents can alleviate gastritis and systemic inflammatory responses caused by *Helicobacter pylori* infection, effectively improving gastritis symptoms and reducing the severity of infection. The results of the embodiments show that *Lactobacillus reuteri* PFL-2501 has comprehensive functions, can inhibit *Helicobacter pylori*, regulate immune balance, has a good inhibitory effect on urease activity, and can reduce the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in serum, as well as reduce the level of immunoglobulin G, laying a research foundation for the subsequent development of anti-*Helicobacter pylori* products.

Description

Technical Field

[0001] This invention belongs to the field of biological agents and biomedicine technology, specifically relating to the application of Lactobacillus reuteri PFL-2501 and its agents in the treatment of Helicobacter pylori. Background Technology

[0002] Helicobacter pylori (Hp) is a bacterium that resides in the stomach and is one of the most common human pathogens. Long-term Hp infection can lead to various gastrointestinal diseases, such as peptic ulcers and chronic gastritis, and in severe cases, even stomach cancer. In fact, Hp infection is the most common cause of stomach cancer, accounting for over 60% of all cases. Hp produces ammonia by breaking down urea, neutralizing stomach acid and creating a relatively neutral microenvironment around it. Simultaneously, ammonia damages the gastric mucosal barrier, inducing apoptosis of gastric mucosal cells, leading to tissue damage and ulcer formation. Reactive oxygen species (ROS) directly produced by Hp or induced by epithelial cells can damage DNA, suggesting a role in the development of stomach cancer. Furthermore, various virulence factors produced by Hp can directly damage the host's stomach tissue, thereby triggering a variety of gastric diseases.

[0003] Clarithromycin-based triple therapy is one of the earliest and most effective eradication therapies, with initial eradication rates exceeding 95%. However, the increasing drug resistance of Helicobacter pylori limits the use of antibiotics, and antibiotic overuse can cause numerous side effects, such as gut microbiota dysbiosis and gastrointestinal dysfunction.

[0004] *Lactobacillus reuteri* has long been used as a probiotic due to its various beneficial effects in the human body, including antibacterial activity, regulation of gut microbiota, immune system training, and prevention of colon cancer. However, the number of strains currently available for use against *Helicobacter pylori* in clinical practice remains insufficient, thus necessitating the exploration of more strains capable of effectively inhibiting *Helicobacter pylori* infection. Summary of the Invention

[0005] The purpose of this invention is to provide the application of *Lactobacillus reuteri* PFL-2501 and its bacterial agent in the treatment of *Helicobacter pylori*. The *Lactobacillus reuteri* PFL-2501 and its bacterial agent can effectively combat *Helicobacter pylori* infection, alleviate gastritis and systemic inflammatory response caused by *Helicobacter pylori* infection, and the effect is significant.

[0006] This invention provides the application of *Lactobacillus reuteri* PFL-2501 in the preparation of products for treating *Helicobacter pylori*, wherein the preservation number of *Lactobacillus reuteri* PFL-2501 is CGMCC No. 35503.

[0007] The present invention also provides the application in products containing *Lactobacillus reuteri* PFL-2501; the preservation number of *Lactobacillus reuteri* PFL-2501 is CGMCC No. 35503.

[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 solid formulations.

[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] Preferably, the preparation method of the bacterial agent includes the following steps: inoculating Lactobacillus reuteri PFL-2501 into a culture medium for fermentation or scale-up culture to obtain the bacterial agent.

[0012] Preferably, the product includes pharmaceuticals.

[0013] Preferably, the anti-Helicobacter pylori treatment includes alleviating gastritis and / or systemic inflammation caused by Helicobacter pylori infection.

[0014] Preferably, the anti-Helicobacter pylori method includes at least one of inhibiting urease activity, reducing the content of pro-inflammatory factors, and reducing the content of immunoglobulin G.

[0015] The present invention also provides an anti-Helicobacter pylori product, the product comprising a bacterial agent and excipients containing *Lactobacillus reuteri* PFL-2501 or a derivative of *Lactobacillus reuteri* PFL-2501; the preservation number of *Lactobacillus reuteri* PFL-2501 is CGMCC No. 35503.

[0016] Beneficial effects: This invention provides the application of *Lactobacillus reuteri* PFL-2501 in the preparation of anti-*Helicobacter pylori* products. The *Lactobacillus reuteri* PFL-2501 has the CGMCC No. 35503. This invention discovers that *Lactobacillus reuteri* PFL-2501 and its bacterial agents can alleviate gastritis and systemic inflammatory responses caused by *Helicobacter pylori* infection, effectively improving gastritis symptoms and reducing the severity of infection. The results of the examples show that *Lactobacillus reuteri* PFL-2501 has comprehensive functions, can inhibit *Helicobacter pylori*, regulate immune balance, has a good inhibitory effect on urease activity, and can reduce the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in serum, as well as reduce the level of immunoglobulin G. This lays a research foundation for the subsequent development of anti-*Helicobacter pylori* products.

[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 This is a graph showing the statistical results of weight changes in each mouse in Example 3; Figure 7 The results of the determination of urease, IgG, tumor necrosis factor-α, interleukin-1β and interleukin-6 levels in the serum of mice in each group in Example 3 are as follows: Figure 7 Different letters in the bar chart indicate significant differences. Detailed Implementation

[0020] This invention provides the application of *Lactobacillus reuteri* PFL-2501 in the preparation of products for treating *Helicobacter pylori*, wherein the preservation number of *Lactobacillus reuteri* PFL-2501 is 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 the application of a bacterial agent containing *Lactobacillus reuteri* PFL-2501 in the preparation of products for treating *Helicobacter pylori*; the *Lactobacillus reuteri* PFL-2501 has the accession number CGMCC No. 35503.

[0024] In one embodiment, the bacterial agent comprises a bacterial suspension of *Lactobacillus reuteri* PFL-2501. In another embodiment, the dosage form of the bacterial agent includes a liquid or a solid; further, the liquid can be a bacterial suspension; the solid can be bacterial sludge or lyophilized powder. In one embodiment, 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; more specifically, when the bacterial agent is a fermentation broth, the effective viable count of *Lactobacillus reuteri* PFL-2501 is ≥1×10⁻⁶. 8 CFU / mL; when the bacterial agent is fermented bacterial sludge, the effective viable count of *Lactobacillus reuteri* PFL-2501 is ≥1×10⁻⁶. 9 CFU / g; when the bacterial agent is a lyophilized powder, the effective viable count of *Lactobacillus reuteri* PFL-2501 is ≥1×10⁻⁶.10 CFU / g; the moisture content of the freeze-dried powder is ≤5%.

[0025] In one embodiment, the preparation method of the bacterial agent includes the following steps: inoculating *Lactobacillus reuteri* PFL-2501 into a culture medium for fermentation or scale-up culture to obtain the bacterial agent. In 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 hours, or 30-36 hours. In one embodiment, the culture medium is MRS liquid medium. This invention does not specifically limit the post-processing method; conventional post-processing methods in the art can be used.

[0026] In one embodiment, the product includes a pharmaceutical product. In one embodiment, the anti-Helicobacter pylori treatment includes alleviating gastritis and / or systemic inflammation caused by Helicobacter pylori infection; in another embodiment, the anti-Helicobacter pylori treatment can inhibit Helicobacter pylori and / or regulate immune balance; in yet another embodiment, the anti-Helicobacter pylori treatment can involve inhibiting urease activity, reducing the content of pro-inflammatory factors, and reducing the content of immunoglobulin G; in yet another embodiment, reducing the content of pro-inflammatory factors can involve reducing the content of at least one of the pro-inflammatory factors tumor necrosis factor-α, interleukin-1β, and interleukin-6 in serum.

[0027] This invention also provides an anti-Helicobacter pylori product, comprising an agent containing *Lactobacillus reuteri* PFL-2501 or a derivative of *Lactobacillus reuteri* PFL-2501 and excipients; the *Lactobacillus reuteri* PFL-2501 has the accession number CGMCC No. 35503. As one embodiment, this invention does not specifically limit the type and amount of the excipients; they can be conventionally selected according to need.

[0028] 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.

[0029] The biomaterials and their preparation methods used in the following examples: 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.

[0030] 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.

[0031] Simply remove the agar when preparing MRS liquid culture medium.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 .

[0036] 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 .

[0037] 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.

[0038] Table 1 PCR amplification system

[0039] Table 2 PCR amplification program

[0040] 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:

[0041] 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.

[0042] 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 .

[0043] 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 .

[0044] 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.

[0045] Example 3 The in vivo anti-Helicobacter pylori infection activity evaluation of *Lactobacillus reuteri* PFL-2501 obtained in Example 1 was conducted as follows: 1. Experimental Design Experimental grouping: Thirty SPF-grade male C57BL / 6J mice (6 weeks old, weighing 20-22g at purchase, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 7 days and then randomly divided into 3 groups (n=10 per group): blank control group (NC group), Helicobacter pylori infection control group (Ctrl group), and Lactobacillus reuteri intervention group (LR group).

[0046] Model establishment: Except for the NC group, mice in other groups were administered Helicobacter pylori bacterial suspension by gavage every other day. Each mouse was administered 100 μL of bacterial suspension (1×PBS) by gavage, and the inoculation amount was 5×10⁻⁶. 8 CFU was administered via gavage five times to establish an infection model. Patients fasted for 12 hours before infection and were fed 2 hours after infection.

[0047] 2. Strains intervention Starting from day 11, LR group mice were administered Lactobacillus reuteri PFL-2501 (containing 1×10⁶ live bacteria) once daily. 9 Mice in the NC and Ctrl groups were given 200 μL of PBS (CFU / mL) daily via gavage, while mice in the NC and Ctrl groups were given the same volume of PBS daily via gavage. All other feeding conditions, such as temperature, humidity, light, and feed, were kept the same. After 5 weeks of intervention, mice were euthanized by cervical dislocation, and blood was collected from the retroorbital venous plexus. After the blood samples were allowed to clot, they were centrifuged at 1000g for 10 min to separate the serum. The samples were stored at -20℃ for testing.

[0048] 3. Detection indicators and results 3.1 Changes in mouse body weight Mice were weighed and their weight recorded daily at regular intervals. Changes in body weight and activity levels were also observed and recorded. The weight measurement results are as follows: Figure 6 As shown.

[0049] according to Figure 6 The records show that there was no significant difference in the weight change of mice during the experiment.

[0050] 3.2 The levels of serum urease (ELISA kit and corresponding antibody purchased from Shanghai Keabob Biotechnology Co., Ltd.), IgG, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) (kit for detecting IgG, TNF-α, and IL-1β purchased from Jiangsu Enzyme Immunoassay Co., Ltd.) in mouse serum were determined using an ELISA kit. The steps are as follows: 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.

[0051] b) Add standards and serum 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.

[0052] c) Antibody incubation: Add 50 μL of the corresponding diluted antibody to the standard and sample wells, shake at 100~300 r / min, and incubate at room temperature.

[0053] 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.

[0054] 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.

[0055] f) Washing: Wash the microplate 6 times in the same manner as d).

[0056] 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.

[0057] h) Reading: The reading time should be controlled within 30 minutes after adding the stop solution. Use an ELISA reader for dual-wavelength detection and measure the absorbance (OD) at 450 nm.

[0058] Test results as follows Figure 7 As shown, by Figure 7 The results showed that Helicobacter pylori infection significantly increased serum urease and IgG levels. Serum urease and IgG levels in the intervention group were significantly lower than those in the control group. Helicobacter pylori infection also significantly increased serum TNF-α, IL-1β, and IL-6 levels, leading to a systemic inflammatory state. Intervention treatment significantly restored these cytokines to normal levels.

[0059] 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. *Lactobacillus reuteri* ( Limosilactobacillus reuteri The application of PFL-2501 in the preparation of products against Helicobacter pylori, wherein the preservation number of Lactobacillus reuteri PFL-2501 is CGMCC No. 35503.

2. Application of a bacterial agent containing *Lactobacillus reuteri* PFL-2501 in the preparation of products for treating *Helicobacter pylori*; the preservation number of *Lactobacillus reuteri* PFL-2501 is CGMCC No. 35503.

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

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

5. The application 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 application according to claim 2, characterized in that, The preparation method of the bacterial agent 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 application according to claim 1 or any one of claims 2 to 6, characterized in that, The products include pharmaceuticals.

8. The application according to claim 6 or 7, characterized in that, The anti-Helicobacter pylori treatment includes relieving gastritis and / or systemic inflammation caused by Helicobacter pylori infection.

9. The application according to claim 8, characterized in that, The anti-Helicobacter pylori treatment includes at least one of inhibiting urease activity, reducing the content of pro-inflammatory factors, and reducing the content of immunoglobulin G.

10. A product for treating Helicobacter pylori, characterized in that, The product includes a bacterial agent and excipients containing *Lactobacillus reuteri* PFL-2501 or a derivative of *Lactobacillus reuteri* PFL-2501; the preservation number of *Lactobacillus reuteri* PFL-2501 is CGMCC No. 35503.

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