Lactobacillus rhamnosus with effect of preventing helicobacter pylori infection and application of lactobacillus rhamnosus

Abstract

The invention discloses lactobacillus rhamnosus with an effect of preventing helicobacter pylori infection and application of the lactobacillus rhamnosus, and belongs to the technical field of biological medicines and microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 21, 2025, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 34273. The JGSLR02 is a lactobacillus rhamnosus strain with excellent probiotic characteristics, and especially has excellent helicobacter pylori antagonism potential in vitro. A helicobacter pylori infected C57BL / 6 mouse model proves that the rhamnosus lactobacillus has the capability of preventing occurrence and development of mouse helicobacter pylori infection, and can antagonize helicobacter pylori in vivo, prevent helicobacter pylori infection, relieve inflammatory response and promote gastric mucosa repair.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and microbiology, and in particular to a strain of Lactobacillus rhamnosus that has the effect of preventing Helicobacter pylori infection and its application. Background Technology

[0002] Probiotics have gained widespread attention in the prevention and treatment of chronic diseases due to their safety, effectiveness, and healthy properties. Currently, the use of probiotics and their fermented products is an emerging alternative for the prevention and adjunctive treatment of Helicobacter pylori infection, which can improve the eradication rate of Helicobacter pylori, balance the normal gut flora, and reduce antibiotic-related side effects.

[0003] Helicobacter pylori ( Helicobacter pylori Helicobacter pylori (H. pylori) is a Gram-negative pathogen that colonizes the human gastric mucosa, with a global infection rate exceeding 50%. It is a Group 1 carcinogen for chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Although antibiotics currently developed can eradicate H. pylori infection, they face increasingly serious problems such as drug resistance and treatment side effects, including diarrhea, nausea, and intestinal flora imbalance. There is an urgent need to develop safe and effective alternative or adjuvant treatment strategies.

[0004] Probiotics and their fermentation products have attracted widespread attention due to their potential in regulating the gut microbiota, enhancing the mucosal barrier, and modulating immunity. Existing research has shown that some functional probiotics can exert adjuvant anti-infection effects through mechanisms such as competitively inhibiting Helicobacter pylori adhesion, secreting antimicrobial substances, or modulating the host's immune response, and are characterized by safety, high efficacy, no toxic side effects, and easy availability. Meanwhile, there is a significant market demand for probiotic strains capable of preventing and treating Helicobacter pylori infection, necessitating the development of probiotics and their fermentation products with excellent Helicobacter pylori prevention and treatment capabilities to fill this gap. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Lactobacillus rhamnosus* with preventive efficacy against *Helicobacter pylori* infection and its applications, thereby addressing the problems existing in the prior art. This invention screens a strain of *Lactobacillus rhamnosus* with excellent probiotic properties from animal intestines, particularly its superior potential for antagonizing *Helicobacter pylori* in vitro. A mouse model of *Helicobacter pylori* infection demonstrates that the *Lactobacillus rhamnosus* strain of this invention has the ability to prevent the occurrence and development of *Helicobacter pylori* infection in mice, and can significantly antagonize *Helicobacter pylori* in vivo, prevent *Helicobacter pylori* infection, alleviate inflammatory responses, and promote gastric mucosal repair.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosusJGSLR02, this strain was deposited on April 21, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 34273.

[0007] The second technical solution of the present invention is a microbial inoculant, comprising the Lactobacillus rhamnosus JGSLR02.

[0008] The third technical solution of the present invention is the application of Lactobacillus rhamnosus JGSLR02 or the microbial agent in the preparation of drugs for the prevention and / or treatment of Helicobacter pylori infection.

[0009] The fourth technical solution of the present invention is a drug for preventing and / or treating Helicobacter pylori infection, comprising the Lactobacillus rhamnosus JGSLR02 or the microbial agent.

[0010] The fifth technical solution of the present invention is the application of Lactobacillus rhamnosus JGSLR02 or the microbial agent in the preparation of antibacterial agents.

[0011] The sixth technical solution of the present invention is an antibacterial agent, comprising the Lactobacillus rhamnosus JGSLR02 or the microbial agent.

[0012] The seventh technical solution of the present invention is a composition containing Lactobacillus rhamnosus JGSLR02, wherein the composition is in the form of, but is not limited to, health products, drugs, dietary supplements, intestinal nutrition preparations or therapeutic agents.

[0013] Based on the above technical solution, the present invention has the following technical effects: (1) In this invention, Lactobacillus rhamnosus JGSLR02 has excellent acid and bile salt resistance.

[0014] (2) The metabolites of Lactobacillus rhamnosus JGSLR02 in this invention have a significant inhibitory effect on common pathogenic bacteria in the intestine and Helicobacter pylori.

[0015] (3) In this invention, Lactobacillus rhamnosus JGSLR02 can significantly inhibit the urease activity of Helicobacter pylori.

[0016] (4) The Lactobacillus rhamnosus JGSLR02 in this invention has excellent antioxidant capacity.

[0017] (5) In this invention, Lactobacillus rhamnosus JGSLR02 has excellent adhesion properties to gastric mucin and human gastric adenocarcinoma (AGS) cells.

[0018] (6) In this invention, Lactobacillus rhamnosus JGSLR02 has the effect of preventing or treating Helicobacter pylori infection. After mice with Helicobacter pylori infection model ingested Lactobacillus rhamnosus JGSLR02, the activity of Helicobacter pylori and urease in the gastric antrum of mice was reduced, the inflammation level of the gastric mucosa of mice was significantly reduced, the damage to gastric mucosal tissue and apoptosis of gastric epithelial cells in mice were relieved, and the repair of gastric mucosa in mice was promoted. Moreover, this beneficial effect is dose-related. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus JGSLR02.

[0020] Figure 2 Image of Gram-stained Lactobacillus rhamnosus JGSLR02.

[0021] Figure 3 This image shows Helicobacter pylori after treatment with probiotic supernatant, stained with NucGreen / EthD-III fluorescence - double staining of live / dead cells.

[0022] Figure 4 The effect of Lactobacillus rhamnosus JGSLR02 on the content of Helicobacter pylori in the gastric tissue of mice.

[0023] Figure 5 The effect of Lactobacillus rhamnosus JGSLR02 on urease activity in the gastric mucosa of mice.

[0024] Figure 6 The effects of Lactobacillus rhamnosus JGSLR02 on the histopathology of gastric antrum tissue in the pylorus region of mice.

[0025] Figure 7 The effects of Lactobacillus rhamnosus JGSLR02 on MPO activity and cytokines in mouse gastric tissue were investigated. A represents myeloperoxidase (MPO) activity, B represents interleukin IL-1β, C represents interleukin IL-6, and D represents the anti-inflammatory factor IL-10.

[0026] Figure 8 The effect of Lactobacillus rhamnosus JGSLR02 on SOD in mouse gastric tissue.

[0027] Figure 9 The effect of Lactobacillus rhamnosus JGSLR02 on apoptosis of mouse gastric epithelial cells.

[0028] Figure 10 The effect of Lactobacillus rhamnosus JGSLR02 on gastric mucosal epithelial repair proteins in mice. Detailed Implementation

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0030] This invention provides a strain of Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus JGSLR02, this strain was deposited on April 21, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 34273.

[0031] This invention also provides a microbial inoculant, including the Lactobacillus rhamnosus JGSLR02.

[0032] This invention also provides the use of the Lactobacillus rhamnosus JGSLR02 or the microbial agent in the preparation of medicaments for the prevention and / or treatment of Helicobacter pylori infection.

[0033] This invention also provides a drug for preventing and / or treating Helicobacter pylori infection, including the Lactobacillus rhamnosus JGSLR02 or the microbial agent.

[0034] This invention also provides the application of the Lactobacillus rhamnosus JGSLR02 or the microbial agent in the preparation of antibacterial agents.

[0035] In some specific implementations, the antibacterial agent is used to inhibit Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Helicobacter pylori.

[0036] This invention also provides an antibacterial agent, including the Lactobacillus rhamnosus JGSLR02 or the microbial agent.

[0037] In some specific implementations, the antibacterial agent is used to inhibit Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Helicobacter pylori.

[0038] The Lactobacillus rhamnosus JGSLR02 disclosed in this invention has the following properties: (1) Acid resistance: The survival rate after treatment in a pH 2.5 environment for 3 h was 90.85%; (2) Bile salt tolerance: The survival rate was as high as 93.03% after treatment in an environment with a bile salt concentration of 0.3% for 4 hours; (3) The scavenging rates of DPPH free radicals and hydroxyl free radicals were 49.88% and 58.35%, respectively, and the reducing power was 36.98%; (4) It has strong inhibitory properties against three common foodborne pathogens: Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium. (5) The inhibition zones of the three Helicobacter pylori indicator strains all exceeded 20 mm, which could significantly inhibit the growth of Helicobacter pylori and the inhibition rate of urease activity of Helicobacter pylori reached 82.60%; (6) The adhesion rate to gastric mucin reached 19.58%, and the adhesion rate to human gastric adenocarcinoma (AGS) cells reached 12.68%; (7) Prevention and treatment effects on Helicobacter pylori-infected mouse models: ① Reduce the activity of Helicobacter pylori in the gastric antrum of a mouse model of Helicobacter pylori infection; ② Reduce the activity of Helicobacter pylori urease in a mouse model of Helicobacter pylori infection; ③ Alleviate gastric mucosal tissue damage in Helicobacter pylori-infected mouse models; ④ Reduce the level of inflammation in the gastric mucosa of mice with Helicobacter pylori infection; ⑤ Reducing gastric epithelial cell apoptosis in Helicobacter pylori-infected mouse models; ⑥ Promotes the repair of gastric mucosa in Helicobacter pylori-infected mouse models.

[0039] Preparation method of MRS broth medium: Weigh 48.3g of MRS medium powder, add it to 1000mL of distilled water, heat to boiling to dissolve, dispense into containers, autoclave at 121℃ for 20 minutes, remove after sterilization and cool to room temperature to obtain MRS broth medium.

[0040] Preparation method of MRS solid culture medium: Weigh 63.3g of MRS culture medium powder, add it to 1000mL of distilled water, heat to boiling to dissolve, dispense into portions, autoclave at 121℃ for 20 minutes, after sterilization, remove and cool to 55℃, pour into petri dishes (15 mL / dish, 3-5 mm thick) to obtain MRS solid culture medium.

[0041] Preparation method of LB liquid culture medium: Weigh 25g of LB liquid culture medium powder and 1000mL of distilled water into a beaker, mix them, and then place the mixed solution in a sterilizer for sterilization for 20 minutes. After sterilization, remove it and cool it to room temperature to obtain LB liquid culture medium.

[0042] Preparation method of LB solid culture medium: Weigh 40g of LB solid culture medium powder and 1000mL of distilled water into a beaker, mix them, and then put the mixed solution into a sterilizer for sterilization for 20 minutes. After sterilization, take it out and cool it to 55℃, then pour it into a petri dish (15mL / dish, 3-5mm thick) to obtain LB solid culture medium.

[0043] CAB Campylobacter agar medium: Weigh 26.5 g of solid medium, dissolve in distilled water, and bring the volume to 500 mL. Place in an autoclave and sterilize at 121 °C for 15 min. Cool to approximately 45 °C, add 5.0% sterile defibrinated sheep blood and 1.0% mixed antibiotics to prepare solid plates, and store at 4 °C.

[0044] BHI brain and heart extract liquid culture medium was dissolved in an appropriate amount of distilled water (DW), and the volume was adjusted to 500 mL. It was then autoclaved at 121 °C for 15 min and stored at 4 °C.

[0045] The 16S rRNA sequence of *Lactobacillus rhamnosus* JGSLR02 described in this invention is as follows (SEQ ID NO.1):

[0046] Example 1: Isolation, screening and identification of Lactobacillus rhamnosus JGSLR02 Obtaining Lactobacillus rhamnosus JGSLR02 in this invention: Fecal samples were collected from healthy infants aged 0-24 months in the Nanchang area. A sterile sampler was used to collect samples from the inner wall of the infant's feces. Samples were sent to the laboratory within 4 hours for subsequent experiments. Samples were serially diluted with physiological saline (10T). -1 10 -2 10 -3 10 -4 10 -5 Afterwards, 100 μL of the serially diluted solution was spread onto MRS solid medium supplemented with 0.04% (w / v) bromocresol purple. The medium was incubated at room temperature for 8 min to allow the bacterial culture to be absorbed. The petri dishes were then inverted and incubated at 37 ℃ for 48 h. Colonies showing a yellow color ring around their periphery were selected. Strains of different morphologies and sizes were streaked twice, repeating this process 2-3 times until pure single colonies with consistent morphology were isolated and purified. Further Gram staining was performed. The specific steps were as follows: A drop of distilled water was placed on a glass slide, and a single colony prepared using an inoculation loop was placed in the water drop. After the water evaporated, the slide was placed over a flame to fix the single colony. A drop of crystal violet staining solution was then added to the single colony, and the slide was gently shaken. After the crystal violet staining covered the single colony, it was held for 1 minute and then rinsed with water. A drop of iodine solution was then added to the single colony, and the slide was gently shaken. After the iodine solution covered the single colony, it was held for 1 minute and then rinsed with water. 95% ethanol was then added to decolorize the single colony. After no more purple liquid was removed, it was rinsed with water. Frucrine solution was added, and the slide was held for 30 seconds and then rinsed with water. Finally, the liquid on the surface of the slide was blotted dry with absorbent paper, and the single colony (Gram-positive bacteria) was observed under an oil immersion microscope. Single colonies with a purple color (Gram-positive bacteria) were retained. Colonies of suspected strains purified by streak plating were treated with Gram staining, and morphological observation was performed simultaneously. The results are shown in [Figure number missing]. Figure 1 and Figure 2 As shown.

[0047] from Figure 1 The colonies in the culture medium were observed to be milky white, with a smooth surface, and easily detached. Gram staining revealed that the strain was Gram-positive, possessed a capsule, and lacked flagella. Figure 2 .

[0048] Single colonies were inoculated into MRS broth medium to obtain bacterial suspensions (10... 8(CFU / mL) Using a pipette, 0.5 mL of bacterial suspension and 0.5 mL of 20% sterile glycerol were mixed evenly in a sterilized bacterial culture tube and stored at -80°C. Subsequently, the strain's acid and bile salt tolerance, antioxidant capacity, and inhibition of common pathogens and Helicobacter pylori were measured to obtain a strain with good performance. The selected strain was inoculated into MRS broth medium and cultured at 37°C for 24 hours. DNA was then extracted using a bacterial genomic DNA extraction kit.

[0049] The extracted DNA was subjected to PCR amplification using 1 μL of 10 μM upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 2), 1 μL of 10 μM downstream primer 1495R (5'-CTACGGCTACCTTGTTAGA-3', SEQ ID NO. 3), 12.5 μL of 2×Taq plus Buffer, and 1 μL of template DNA. The volume was brought to 25 μL with sterile dd H2O. Sterile ultrapure water was used as a negative control instead of template DNA.

[0050] The amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 29 cycles, with a final extension at 72℃ for 5 min.

[0051] The target DNA fragment was recovered and subjected to 16S rRNA sequencing. The sequencing results were compared with BLAST, and the strain was identified as *Lactobacillus*. Lacticaseibacillus ), 16S rDNA homology analysis results showed that it was similar to *Lactobacillus rhamnosus* (known in the GeneBank database) Lacticaseibacillus rhamnosus The homology between the strain and the isolated *Lactobacillus rhamnosus* reached 100%. The strain was named JGSLR02 and deposited. The deposit information is as follows: Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus JGSLR02 was deposited on April 21, 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. 34273.

[0052] Example 2: Acid and bile salt tolerance of Lactobacillus rhamnosus JGSLR02 Lactobacillus rhamnosus JGSLR02 was inoculated into MRS broth medium and cultured at 37 ℃ for 24 hours to obtain bacterial fermentation broth (bacterial suspension). The bacterial suspension was centrifuged at 8000 g for 5 min (4 ℃), the supernatant was discarded and the bacterial cells were collected. The cells were washed twice with sterile PBS and then resuspended in MRS liquid medium at pH 2.5. The suspension was incubated at 37 ℃ for 3 h, and 1 mL of the bacterial suspension was taken at 0, 1, 2, and 3 h respectively and serially diluted 10-fold with 0.9% physiological saline (10... -1 -10 -8 Then, an appropriate concentration was spread onto MRS agar plates and anaerobically incubated at 35°C for 72 hours. After incubation, the viable cell count was determined, with three replicates for each sample. Simultaneously, the acid tolerance of *Lactobacillus rhamnosus* CICC 6144 was determined under the same conditions.

[0053] JGSLR02 bacterial cells were collected and washed according to the above method. The bacterial cells were then resuspended in MRS liquid medium (pH 8.0) containing 0.3% ox bile salts and incubated at 37°C for 3 hours. At 0, 2, and 4 hours, 1 mL of the bacterial suspension was taken and serially diluted 10-fold with 0.9% physiological saline. -1 -10 -7 Then, an appropriate concentration was spread onto MRS agar plates and anaerobically incubated at 35°C for 72 hours. After incubation, the viable bacterial count was determined, with three replicates for each sample. Simultaneously, the bile salt tolerance of *Lactobacillus rhamnosus* CICC 6144 was determined under the same conditions. The survival rate of lactic acid bacteria was calculated using the following formula: Strain survival rate (%) = N1 / N0 × 100%; In the formula, N1 is the number of surviving bacteria after incubation; N0 is the initial number of surviving bacteria.

[0054] Table 1. Acid tolerance of Lactobacillus rhamnosus

[0055] Table 2. Bile salt tolerance of Lactobacillus rhamnosus

[0056] As shown in Table 1, the survival rate of *Lactobacillus rhamnosus* JGSLR02 remained above 90% after treatment in a pH 2.5 environment for 3 h; as shown in Table 2, the survival rate reached over 93% after treatment in an environment with a bile salt concentration of 0.3% for 4 h. The experimental results indicate that *Lactobacillus rhamnosus* JGSLR02 possesses strong acid and bile salt tolerance.

[0057] Example 3 Antioxidant properties of Lactobacillus rhamnosus JGSLR02 Example 1: Lactobacillus rhamnosus JGSLR02 was obtained through streak plating purification, morphological identification, and molecular identification. The antioxidant capacity of strain JGSLR02 was determined. The specific method is as follows: Lactobacillus rhamnosus was inoculated into MRS broth medium and cultured at 37°C for 24 h to achieve a strain concentration of 10. 9 At CFU / mL, the bacterial cells were collected by centrifugation at 5000g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 2 mL of physiological saline. This was the sample to be tested. The antioxidant capacity of the strain was determined by 2,2-dip Henyl-1-picrylhydrazyl (DPPH) scavenging rate, hydroxyl radical scavenging rate, and reducing power.

[0058] 1. Experiment on scavenging DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radicals 2 mL of bacterial suspension was mixed with 2 mL of DPPH methanol solution (0.2 mM) and incubated at room temperature in the dark for 30 min to obtain a mixture, which was designated as the sample group. 2 mL of physiological saline was used instead of the bacterial suspension, and the same conditions were applied as the sample group to obtain a mixture, which was designated as the control group. 2 mL of ethanol was used instead of the DPPH methanol solution, and the same conditions were applied as the sample group to obtain a mixture, which was designated as the blank group. 2 mL of vitamin C solution (0.8 mg / mL) was used instead of the bacterial suspension, and the same conditions were applied as the sample group to obtain a mixture, which was designated as the positive control group. The absorbance at 517 nm was measured for the sample group, control group, blank group, and positive control group, respectively. The DPPH free radical scavenging rate was calculated as follows: ; In the formula, A1 is the absorbance of the sample group or positive control group; A2 is the absorbance of the control group; and A0 is the absorbance of the blank group.

[0059] The DPPH free radical scavenging rate was calculated as the average of three results. The results are shown in Table 3. The DPPH scavenging rate of strain *Lactobacillus rhamnosus* JGSLR02 was 49.88%, which is similar to the 50.61% scavenging rate of vitamin C (0.8 mg / mL). The DPPH scavenging rate of strain CICC 6144 was 39.70%. Therefore, *Lactobacillus rhamnosus* JGSLR02 exhibits a strong ability to scavenge DPPH free radicals.

[0060] 2. Hydroxyl radical scavenging test 1 mL of bacterial suspension was mixed with 1 mL of salicylic acid, 1 mL of ferrous sulfate (FeSO4), and 1 mL of hydrogen peroxide and incubated at 37 °C for 15 min to obtain a mixture, denoted as A1. 1 mL of pure water was used instead of hydrogen peroxide, and the treatment was the same as A1, yielding a mixture denoted as A2. 1 mL of physiological saline was used instead of bacterial suspension, and the treatment was the same as A1, yielding a mixture denoted as A0. 1 mL of Vc solution was used instead of bacterial suspension, and the treatment was the same as the sample group, yielding a mixture denoted as the positive control group. The absorbance at 510 nm was measured for each of the above groups. The concentration of FeSO4 solution was 0.005 mol / L, the concentration of Vc solution was 0.8 mg / mL, and the hydroxyl radical scavenging rate was calculated as follows: ; The hydroxyl radical scavenging rate was the average of three results. The results are shown in Table 3. The hydroxyl radical scavenging rate of strain JGSLR02 was 58.35%, which was better than that of strain Vc and strain CICC 6144, indicating that Lactobacillus rhamnosus JGSLR02 has a strong hydroxyl radical scavenging ability.

[0061] 3. Determination of reducing power Take 0.5 mL of bacterial suspension and add it to a mixture of 0.5 mL of 0.2 mol / L PBS solution (pH 6.6) and 0.5 mL of 1% potassium ferricyanide solution. Mix well, incubate at 50 ℃ for 20 min, cool rapidly, add 0.5 mL of 10% trichloroacetic acid, centrifuge at 5000 g for 5 min, take the supernatant and mix with 1 mL of distilled water and 1 mL of 0.1% trichloroacetic acid. After standing for 10 min, sample group A1 is obtained. Following the above preparation method, replace the bacterial suspension with PBS buffer to prepare the blank control group A0, and replace the bacterial suspension with Vc solution to prepare the positive control group. Measure the absorbance values ​​of the sample group, positive control group, and blank control group at 700 nm and calculate the reducing power as follows: ; The reducing ability was calculated as the average of three results. The results are shown in Table 3. The reducing ability of strain JGSLR02 was 36.98%, which was not significantly different from the reducing ability of vitamin C, indicating that *Lactobacillus rhamnosus* JGSLR02 has a strong reducing ability.

[0062] Table 3 Antioxidant activity of strains

[0063] The above experimental results show that Lactobacillus rhamnosus JGSLR02 has acid and bile salt resistance and strong antioxidant activity, and can be used to prepare food or health products with anti-aging and antioxidant effects.

[0064] Example 4: Inhibitory ability of Lactobacillus rhamnosus JGSLR02 against common pathogenic bacteria and Helicobacter pylori The antibacterial activity of fermentation supernatant of Lactobacillus rhamnosus JGSLR02 against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Helicobacter pylori was determined by the perforation method.

[0065] Inhibition ability against *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella typhimurium* was determined: *Lactobacillus rhamnosus* JGSLR02 and *Lactobacillus rhamnosus* CICC 6144 were passaged twice and inoculated into fresh MRS medium at a 2% inoculum. The medium was incubated at 37℃ for 24 h, centrifuged (4℃, 7000×g, 10 min), and the supernatant was filtered through a 0.22 μm filter for later use. Indicator bacteria *Escherichia coli* (CMCC 44350), *Staphylococcus aureus* (CMCC 26003), and *Salmonella typhimurium* (ATCC 13311) were inoculated into LB liquid medium to prepare 10... 8 CFU / mL indicator bacterial suspensions were prepared and inoculated into sterilized semi-solid LB agar medium (10). 7 -10 8 After shaking well, the solution (CFU / mL) was poured into sterile petri dishes. Once the culture medium had cooled and solidified, three wells were punched into each LB agar plate using a 6 mm diameter punch. 200 μL of the prepared *Lactobacillus rhamnosus* JGSLR0 fermentation supernatant was added to each well, and the plate was incubated at 37 °C for 12–16 h. The diameter of the inhibition zone was measured using calipers. Simultaneously, the inhibitory effect of *Lactobacillus rhamnosus* CICC 6144 on pathogenic bacteria was determined using the same method.

[0066] Inhibition ability against Helicobacter pylori: Lactobacillus rhamnosus JGSLR02 and Lactobacillus rhamnosus CICC 6144 were passaged twice and inoculated into fresh MRS medium at a 2% inoculum. The medium was incubated at 37℃ for 24 h, centrifuged for 10 min (4℃, 7000×g), and the supernatant was filtered through a 0.22 μm filter membrane for later use. Using three internationally common standard strains of Helicobacter pylori—26695, SS1, and ATCC43504—as indicator bacteria, the indicator bacteria, Helicobacter pylori SS1, ATCC26695, and ATCC43504, frozen at -80 °C, were streaked onto CAB blood agar plates and incubated under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) at 37 °C for 48 h. After two generations of activation, the bacteria were spread onto CAB agar plates and incubated for 48 h. The bacterial cells were then resuspended in BHI liquid medium to prepare a Helicobacter pylori bacterial suspension. For liquid culture, the Helicobacter pylori bacterial suspension was inoculated at 5% in BHI liquid medium supplemented with 10% fetal bovine serum and cultured overnight under microaerophilic conditions with shaking. Finally, *Helicobacter pylori* was washed and resuspended in PBS (0.01 mol / L, pH 7.2-7.4). The optical density (OD) of the resuspended solution was adjusted to 0.50 ± 0.05 at 600 nm to prepare the *Helicobacter pylori* resuspension. The in vitro inhibition test of the strain against *Helicobacter pylori* was determined using a perforation method. First, 100 μL of *Helicobacter pylori* resuspension was spread on CAB agar medium. Then, 100 μL of bacterial suspension was injected into 6 mm agar wells perforated with a sterile stainless steel perforator. The plates were incubated in a tri-gas incubator for 48 h, and the diameter of the inhibition zone was measured using calipers.

[0067] Table 4. Inhibitory effect of lactic acid bacteria on common pathogenic bacteria and Helicobacter pylori (mm)

[0068] As shown in Table 4, the fermentation supernatant of *Lactobacillus rhamnosus* JGSLR02 exhibited strong inhibitory effects against three common foodborne pathogens: *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella typhimurium*. Furthermore, the inhibitory effect on *Helicobacter pylori* determined by this invention showed that *Lactobacillus rhamnosus* JGSLR02 formed inhibition zones exceeding 20 mm against all three *Helicobacter pylori* indicator strains, significantly inhibiting the growth of *Helicobacter pylori*. This indicates that *Lactobacillus rhamnosus* JGSLR02 possesses a strong inhibitory effect on *Helicobacter pylori*.

[0069] Co-culture analysis with Helicobacter pylori: A co-culture system of Helicobacter pylori with supernatant or bacterial suspension of Lactobacillus rhamnosus was established using microbial co-culture technology. First, Helicobacter pylori ATCC26695 was resuspended in Brucella broth, and supernatant or bacterial suspension of Lactobacillus rhamnosus JGSLR02 and Lactobacillus rhamnosus CICC6144 were added, respectively, and cultured under microaerophilic conditions at 37°C. During co-culture, the survival count of Helicobacter pylori ATCC26695 was assessed at regular time intervals, and the survival count was determined by culturing on CAB agar. Simultaneously, Helicobacter pylori was resuspended in supernatant of Lactobacillus rhamnosus JGSLR02 and Lactobacillus rhamnosus CICC6144, respectively. Subsequently, the supernatant was placed at 37°C to induce Helicobacter pylori into a VBNC (viable but unculturable) state. After 24 h, the number of culturable cells in the induction solution was determined by plate counting. The VBNC state transition of Helicobacter pylori was detected using a LIVE / DEAD staining kit. First, NucGreen / EthD-III was added to an appropriate amount of cell suspension. The cell suspension was then gently mixed and stained in the dark for 15 min. Finally, 10 μL of the mixture was dropped onto a glass slide covered with an 18 mm square coverslip and observed using a Leica DM3000B fluorescence microscope.

[0070] Table 5. Changes in the survival count of Helicobacter pylori during co-culture with probiotic supernatant and probiotic suspension.

[0071] Table 5 shows that after 24 h of co-culturing with Helicobacter pylori, the fermentation supernatant of Lactobacillus rhamnosus JGSLR02 reduced the number of Helicobacter pylori to 1.95 × 10⁻⁶. 2 CFU / mL, superior to *Lactobacillus rhamnosus* CICC6144. For probiotic cell resuspensions, *Lactobacillus rhamnosus* JGSLR02 cell resuspension reduced *Helicobacter pylori* counts to 3.63 × 10⁻⁶. 3 This indicates that *Lactobacillus rhamnosus* JGSLR02 has a strong ability to inhibit *Helicobacter pylori*. The results of the LIVE / DEAD staining assay are as follows: Figure 3 As shown, after being stressed in the supernatant of Lactobacillus rhamnosus JGSLR02 for 24 h, a large number of Helicobacter pylori cells died (red), and the effect was better than that of Lactobacillus rhamnosus CICC6144.

[0072] Example 5: Inhibitory effect of Lactobacillus rhamnosus JGSLR02 on Helicobacter pylori urease activity Method for determining the inhibition of Helicobacter pylori urease activity: Helicobacter pylori bacterial suspension and Lactobacillus rhamnosus JGSLR02 strain resuspension were prepared according to the method in Example 4. 50 μL of each suspension was added to a 96-well plate and incubated at 37 °C under microaerophilic conditions for 24 h. Then, 150 μL of urea-phenol red solution (20% urea and 0.012% phenol, pH 6.5) was added to the wells to initiate the urease biochemical reaction. Color change was observed after 30 min, and absorbance was measured at 561 nm. A separate culture of Helicobacter pylori was used as a control. The same method was used to determine the inhibitory effect of Lactobacillus rhamnosus CICC 6144 on Helicobacter pylori urease activity. The urease activity inhibition was calculated as follows: ; In the formula, A 0 OD of the control group after 24 hours 561 value, A t The OD of each experimental group after 24 hours 561 value.

[0073] The inhibition results of the strain on Helicobacter pylori urease activity are shown in Table 6. Lactobacillus rhamnosus JGSLR02 inhibited the urease activity of Helicobacter pylori by 82.60%, which was better than strain CICC 6144. The results indicate that Lactobacillus rhamnosus JGSLR02, which can inhibit the urease activity of Helicobacter pylori, has important potential in preventing pathogen infection.

[0074] Example 6: Adhesion properties of Lactobacillus rhamnosus JGSLR02 to mucin and human gastric adenocarcinoma (AGS) cells Method for determining the adhesion properties of gastric mucin type II: A resuspension of *Lactobacillus rhamnosus* strain JGSLR02 was prepared according to the method described in Example 4. Porcine gastric mucin type II was spread onto a 6-well cell plate. 1 mL of *Lactobacillus rhamnosus* JGSLR02 cell suspension was added to each well coated with mucin, and the plate was incubated at 37 °C for 4 h. Each well was then thoroughly washed with PBS to remove unadhered lactic acid bacteria cells. 1 mL of 0.05% Triton X-100 solution was then added to each well and reacted for 4 h to remove adhered lactic acid bacteria cells (i.e., N...). adhered Then, the lysate was collected, spread on MRS agar medium, and incubated for 48 h. Plate counting was used to determine the amount of mucin adhering to the protein (N). initial ) and adhered to mucin (i.e. N) adhered The number of lactic acid bacteria was measured. The adhesion properties of *Lactobacillus rhamnosus* CICC 6144 to gastric mucin were also determined using the same method.

[0075] Methods for determining the adhesion properties of human gastric adenocarcinoma (AGS) cells: AGS cells were placed in 6-well plates and cultured at 37 °C for 48 h until a complete monolayer of cells was formed. Then, 1 mL of Lactobacillus rhamnosus JGSLR02 resuspension was added and the cells were cultured for 4 h. Adhering bacterial colonies were removed with trypsin, and the adhesion properties were measured by plate counting. The same method was used to determine the adhesion properties of Lactobacillus rhamnosus CICC 6144 to gastric mucin.

[0076] The adhesion properties of *Lactobacillus rhamnosus* to mucin and AGS cells are expressed as adhesion rate: ; In the formula, N adhered It is the number of colonies adhering to mucin or AGS cells, N initial It is the initial number of bacterial colonies added.

[0077] Table 6. Urease inhibition properties of the strain, adhesion properties to mucin and human gastric adenocarcinoma (AGS) cells.

[0078] As shown in Table 6, the adhesion rates of Lactobacillus rhamnosus JGSLR02 to gastric mucin and AGS cells were 19.58% and 12.68%, respectively, which were better than those of strain CICC 6144. This indicates that Lactobacillus rhamnosus JGSLR02 has good adhesion properties to the gastric mucosa and has the potential to reduce Helicobacter pylori adhesion.

[0079] Methods for determining inflammatory factors in Helicobacter pylori-infected human gastric adenocarcinoma (AGS) cells: AGS cells were cultured in microplates until a complete monolayer was formed. Then, AGS cells were co-infected with Helicobacter pylori 26695 and Lactobacillus rhamnosus JGSLR02 at an infection ratio of 1:100 and cultured for 12 h. Helicobacter pylori infection alone served as a positive control, and untreated AGS cells served as a negative control. The concentration of IL-8 in the cell supernatant was calculated using an interleukin-8 (IL-8) ELISA kit (Solepro Beijing) according to the standard curve, and the corresponding OD value was calculated according to the manufacturer's instructions. Simultaneously, the same method was used to determine inflammatory factors in AGS cells co-infected with Helicobacter pylori 26695 and Lactobacillus rhamnosus CICC 6144.

[0080] Table 7. Interleukin-8 (IL-8) levels in Helicobacter pylori-infected AGS cells treated with Lactobacillus rhamnosus

[0081] Table 7 shows that Helicobacter pylori 26695 increased IL-8 secretion in gastric AGS cells to 108.33 pg / mL, while Lactobacillus rhamnosus JGSLR02 and CICC 6144 reduced IL-8 secretion levels in AGS cells stimulated by Helicobacter pylori, with secretion levels of 72.14 and 82.50 pg / L, respectively. This indicates that Lactobacillus rhamnosus JGSLR02 has a better anti-inflammatory effect.

[0082] Example 7: The preventive and therapeutic effects of Lactobacillus rhamnosus JGSLR02 on Helicobacter pylori-infected mice. Seventy-eight male C57BL / 6 mice aged 4-6 weeks (20-24 grams) were randomly divided into six groups of 13 mice each. The groups were: blank control group, model group, positive control group, low-dose Lactobacillus rhamnosus group, medium-dose Lactobacillus rhamnosus group, and high-dose Lactobacillus rhamnosus group.

[0083] One week after acclimatization, all mice (n=8 per group) except for the control group (replaced with saline) received Helicobacter pylori solution (10... 8 Mice were administered CFU / mL, 20 μL / g via gavage to establish infection patterns. This was repeated every other day for four weeks. After this period, 2 mice were euthanized and infection was assessed. Four mice were randomly selected for dissection, and the infection was then analyzed. Gastric tissue was plated, and a Helicobacter pylori infection concentration greater than 6.0 Lg (CFU / mL) in the model group indicated successful modeling.

[0084] During the above experimental period: the Lactobacillus rhamnosus group began daily gavage administration of different doses (high dose: 1×10) after the fourth week. 9 CFU / mL; Medium dose: 1×10 8 CFU / mL; Low dose: 1×10 7 Resuspend 200 μL of Lactobacillus rhamnosus JGSLR02 bacterial suspension in PBS (CFU / mL) until the end of the experiment.

[0085] The positive control group received a mixture of antibiotics, 0.125 μg / mL amoxicillin and 0.5 μg / mL metronidazole, by gavage daily for 14 days starting from week 4. Mice in the model group and the normal group were administered 200 μL of PBS by gavage daily.

[0086] Mice were kept in an environment of 23-25 ​​℃ and 55-60% humidity with 12-hour alternating light. Mice were observed daily and weighed and recorded weekly. After drug administration was discontinued, mice were euthanized by cervical dislocation at week 9. Serum samples were collected from retroorbital blood by centrifugation (2000g, 10 min) and stored at -80 ℃. The stomach was removed, dissected along the greater and lesser curvatures, rinsed with sterile PBS, and the gastric body and antrum were collected and preserved separately. The number of Helicobacter pylori, urease, pathological findings, inflammatory factors, and immunohistochemical indicators in the stomach were measured. The results are as follows: (1) Effects of Lactobacillus rhamnosus JGSLR02 on the activity of Helicobacter pylori and urease in the gastric antrum of mice infected with Helicobacter pylori The results are as follows Figure 4 As shown, the model group exhibited the highest Helicobacter pylori colonization activity at 6.87 Lg (CFU / mL), while the low-dose and medium-dose groups showed colonization activities of 5.78 and 4.8 Lg (CFU / mL), respectively. The high-dose group and the positive control group showed colonization activities of only 1.94 and 0.45 Lg (CFU / mL), respectively. Furthermore, the urease content in mouse gastric tissue was determined by adding a urease indicator to the tissue homogenate and measuring the absorbance of the resulting color at a wavelength of 561 nm. Figure 5 The absorbance of urease in the model group was 3.995, while that in the positive control group was 0.63. The absorbances of the low-dose, medium-dose, and high-dose groups were 3.08, 2.24, and 1.90, respectively. Therefore, *Lactobacillus rhamnosus* JGSLR02 can reduce the activity of *Helicobacter pylori* and urease in mice infected with *Helicobacter pylori*, and has a certain preventive and alleviating effect on *Helicobacter pylori* infection.

[0087] (2) Effects of Lactobacillus rhamnosus JGSLR02 on gastric tissue pathology in Helicobacter pylori-infected mice Pathological examination of a mouse model of Helicobacter pylori infection: Gastric tissue was fixed in 4% paraformaldehyde buffer (pH 7.4) for 24 h. It was first embedded in paraffin, then micromechanically sectioned, dewaxed with xylene, and stained with hematoxylin and eosin (H&E). Finally, the stained sections were observed under an optical microscope. Representative images were randomly selected from the sections. To assess inflammation and mucosal damage, inflammatory cells in the lamina propria were counted on the H&E-stained sections, and regional observations were performed on the entire mucosal layer.

[0088] Pathological results of mouse gastric antrum tissue as follows Figure 6As shown in the figure, the gastric mucosa of mice in the control group was clear, with intact epithelium, tightly packed mucosal glands, normal morphology, and no obvious inflammatory response. In the model group, the gastric mucosal epithelial cells and some glandular cells were damaged, vacuolation was observed in the lamina propria, and a large number of neutrophils infiltrated the epithelial and lamina propria layers. Compared with the model group, the gastric mucosa structure in the low-dose and medium-dose groups was clearer and more intact, with a relatively tighter arrangement, a significant reduction in neutrophils in the lamina propria, and the disappearance of neutrophil hemorrhage in the lamina propria and muscularis mucosae; however, slight lymphocyte infiltration was still present in the gastric tissue pathological sections. Furthermore, some erythrocytes appeared in the muscularis mucosa of the positive control group, indicating hemorrhage and ulceration; however, the gastric mucosal tissue damage in the high-dose group recovered after 4 weeks of treatment. Therefore, *Lactobacillus rhamnosus* JGSLR02 can alleviate gastric tissue lesions in mice infected with *Helicobacter pylori*, prevent further lesions of the gastric mucosa, and has a certain preventive effect against *Helicobacter pylori* infection.

[0089] (3) Effects of Lactobacillus rhamnosus JGSLR02 on gastric mucosal inflammation in Helicobacter pylori-infected mice According to the manufacturer's instructions, serum IL-1β, IL-6, and IL-10 levels were determined using an ELISA kit. Serum frozen at -80 °C was thawed to room temperature. The diluted serum was incubated with the ELISA test solution in a microplate reader, and the OD value was immediately measured at 450 nm. The activities of gastric mucosal myeloperoxidase (MPO) and superoxide dismutase (SOD) were detected using a commercial kit: gastric tissue was homogenized with PBS, centrifuged (4000 g, 10 min), and the cell supernatant was collected. MPO and SOD were measured separately using the kit, and the absorbance was measured at OD450 using a microplate reader.

[0090] The degree of neutrophil infiltration can be measured by the activity of myeloperoxidase (MPO). MPO can catalyze the synthesis of various cytotoxic oxidants. Increased gastric mucosal damage further promotes MPO release. After Helicobacter pylori colonizes the host, its self-expressed urease and neutrophil activation proteins can act as antigens, mediating neutrophil chemotaxis. Subsequently, neutrophils infiltrate the gastric mucosa and exert mucosal immunity by secreting cytokines and releasing molecules such as MPO and reactive oxygen species. Figure 7As shown, the level of myeloperoxidase (MPO) in the gastric mucosa of the model group mice was higher than that in other groups, while the MPO level was lowest in the positive control group. Treatment with different doses of *Lactobacillus rhamnosus* JGSLR02 reduced MPO activity in the gastric mucosa of mice to varying degrees, with the high-dose group showing a greater effect than other dose groups. Furthermore, intervention with *Lactobacillus rhamnosus* JGSLR02 effectively reduced the levels of pro-inflammatory cytokines such as interleukin IL-1β and IL-6, and increased the level of the anti-inflammatory factor IL-10. These results indicate that *Lactobacillus rhamnosus* JGSLR02 can alleviate the inflammatory level of the gastric mucosa in *Helicobacter pylori*-infected mouse model, thereby exerting an anti-inflammatory effect to alleviate the occurrence and development of *Helicobacter pylori* infection.

[0091] Superoxide dismutase (SOD) is an important antioxidant that protects cells from oxidative damage by scavenging superoxide anion radicals induced by free radicals. Exogenous SOD can effectively alleviate acute gastric mucosal damage caused by various factors. For example... Figure 8 As shown, intervention with Lactobacillus rhamnosus JGSLR02 can increase the level of superoxide dismutase (SOD) in the gastric mucosa of mice, indicating that Lactobacillus rhamnosus JGSLR02 has an antioxidant stress effect.

[0092] (4) The repair effect of Lactobacillus rhamnosus JGSLR02 on gastric mucosa of Helicobacter pylori-infected mice Assessment of gastric epithelial cell apoptosis: Paraffin sections were stained with Ki67 and β-catenin heterologous double-label immunofluorescence. After incubation with primary antibody, Alexa Fluor 488-labeled goat anti-rabbit IgG and CY3-labeled goat anti-rabbit IgG were used as secondary antibodies. Assessment of gastric mucosal repair: Paraffin sections were stained with Occludin and ZO-1 homologous double-label immunofluorescence. After incubation with primary antibody, HRP-labeled goat anti-rabbit IgG and CY3-labeled goat anti-rabbit IgG were used as secondary antibodies. All double staining was enhanced using a tyramine signal amplification system (TSA). After staining, cell nuclei were counterstained with DAPI (4',6-diamidindo-2-phenylindole), and finally, images were observed and captured at the appropriate wavelengths under a fluorescence microscope.

[0093] Inflammation-induced oxidative stress, mediated by reactive oxygen species, can induce changes in mitochondrial membrane potential and permeability. This not only promotes the translocation of apoptosis-related proteins on the mitochondrial membrane but also releases apoptosis factors into the cytoplasm, thereby activating the mitochondrial apoptosis pathway. Ki-67 is a cell proliferation-specific nuclear antigen; β-catenin is a core molecule regulating proliferation and differentiation in the classical Wnt signaling pathway. Therefore, immunofluorescence was used to detect the expression of β-catenin and the proliferating cell nuclear antigen Ki-67 in mouse gastric mucosa. Immunofluorescence staining images of mouse gastric tissue are shown below. Figure 9 The results showed that different dose groups could resist apoptosis to varying degrees, while the number of gastric epithelial cells in the high-dose group JGSLR02 was close to that in the control group, indicating that high-dose Lactobacillus rhamnosus JGSLR02 has good anti-apoptotic ability.

[0094] Zona occludens 1 (ZO-1), a tight junction protein, is an important regulator of the banded region of intercellular tight junctions, playing a crucial role in tight junction formation. Occludin-1 is a membrane-integrated protein that forms and regulates the permeability barrier surrounding tight junctions. ZO-1 protein can interact with Occludin-1 protein to form a complex mediating intercellular tight junctions and signal transduction. Figure 10 As shown, the expression of ZO-1 and Occludin-1 proteins in the gastric tissue of mice treated with medium- and high doses of *Lactobacillus rhamnosus* JGSLR02 was upregulated, and the protein levels tended to normalize. These results indicate that *Lactobacillus rhamnosus* JGSLR02 can restore gastric mucosa damaged by *Helicobacter pylori* by inhibiting gastric epithelial cell apoptosis and promote the expression of gastric mucosal repair proteins in mouse gastric tissue.

[0095] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus JGSLR02, characterized in that, The strain was deposited in China General Microbiological Culture Collection Center on April 21, 2025, the deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 34273.

2. A microbial inoculant, characterized in that, Lacticaseibacillus rhamnosus JGSLR02 of claim 1.

3. Use of the Lacticaseibacillus rhamnosus JGSLR02 of claim 1 or the microbial inoculant of claim 2 in the preparation of a medicament for preventing and / or treating Helicobacter pylori infection.

4. A medicament for preventing and / or treating Helicobacter pylori infection, characterized by, Lacticaseibacillus rhamnosus JGSLR02 of claim 1 or the microbial inoculant of claim 2.

5. Use of the Lacticaseibacillus rhamnosus JGSLR02 of claim 1 or the microbial inoculant of claim 2 in the preparation of a bacteriostatic agent.

6. Use according to claim 5, characterized in that, The bacteriostatic agent is used for inhibition of Escherichia coli, Salmonella typhi, Staphylococcus aureus and Helicobacter pylori.

7. A bacteriostatic agent, characterized in that, Lacticaseibacillus rhamnosus JGSLR02 of claim 1 or the microbial inoculant of claim 2.

8. The bacteriostatic agent of claim 7, wherein, The bacteriostatic agent is used for inhibition of Escherichia coli, Salmonella typhi, Staphylococcus aureus and Helicobacter pylori.

Images