Polysaccharide prepared from a lactobacillus reuteri ccfm1453 for promoting gastric mucosa repair

Abstract

This invention discloses a polysaccharide prepared from *Lactobacillus reuteri* CCFM1453 that promotes gastric mucosal repair, belonging to the fields of microbial technology and pharmaceutical technology. *Lactobacillus reuteri* CCFM1453 is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No:65608. The mitigation of gastric mucosal damage by *Lactobacillus reuteri* CCFM1453 is manifested in: improving gastric mucosal tissue morphology; significantly reducing the levels of gastric mucosal damage markers PGE2 and TFF3; reducing the levels of inflammatory factors such as IL-6 and IL-8; increasing the levels of anti-inflammatory factors such as IL-10; and protecting the gastric mucosa and alleviating inflammation by regulating key signaling pathways such as STAT3 and SOCS3.

Description

Technical Field

[0001] This invention relates to a polysaccharide prepared from a strain of Lactobacillus reuteri CCFM1453 that promotes gastric mucosal repair, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their antipyretic, analgesic, and anti-inflammatory effects by inhibiting cyclooxygenase activity and reducing prostaglandin synthesis. However, they frequently cause gastric mucosal damage, which is usually related to the drug's influence on the gastric mucosal barrier, including interference with gastric mucosal protective mechanisms such as reducing mucus secretion and increasing gastric acid secretion, thus making the gastric mucosa more susceptible to injury. Gastritis, as a typical manifestation of gastric mucosal inflammation, is closely related to the imbalance of mucosal protective mechanisms, activation of pro-inflammatory factors, and exacerbation of oxidative stress. Pathologically, persistent inflammation is prone to progress to gastric ulcers, bleeding, or perforation, severely disrupting gastric homeostasis. Functionally, gastric acid secretion disorders and impaired mucosal repair can lead to malabsorption of nutrients, thereby inducing systemic complications such as anemia and metabolic disorders. In addition, chronic gastritis is closely related to precancerous lesions such as intestinal metaplasia and dysplasia, significantly increasing the risk of gastric cancer transformation.

[0003] The challenge of treating gastric mucosal damage caused by nonsteroidal anti-inflammatory drugs (NSAIDs) lies in minimizing side effects on the gastric mucosa while maintaining drug efficacy. This may require the development of novel drug delivery systems or the combined use of gastric mucosal protectants. Probiotic extracellular polysaccharides (EPPs) exhibit good biocompatibility and typically do not react with drugs, making them a promising option for adjunctive treatment of NSAID-induced gastritis. Screening for probiotic EPPs with gastritis-relieving effects and developing corresponding probiotic EPP products has profound scientific significance and substantial practical value. This research area can provide new strategies for treating gastric mucosal damage, especially NSAID-induced damage, while simultaneously driving the innovation and development of postbiotic products, thus having an undeniable impact on promoting human health and improving quality of life. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of extracellular polysaccharides of Limosilactobacillus reuteri CCFM1453 in products that alleviate gastric mucosal damage.

[0005] This invention provides a strain of *Limosilactobacillus reuteri* CCFM1453, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 11, 2024, with accession number GDMCC NO: 65608.

[0006] The Limosilactobacillus reuteri CCFM1453 cells appear as slightly irregular, round-ended curved bacteria under a microscope. When inoculated onto MRS medium, the colonies are white, moist and smooth, with neat edges.

[0007] The *Limosilactobacillus reuteri* CCFM1453 is a Gram-positive, facultatively anaerobic, thermophilic bacterium with an optimal growth temperature of 35–40°C and an optimal growth pH of 6.0–7.0.

[0008] This invention also provides a method for preparing extracellular polysaccharides from *Lactobacillus reuteri* CCFM1453, comprising the following steps: *Lactobacillus reuteri* CCFM1453 is inoculated into a fermentation medium and cultured to obtain a culture broth. The culture supernatant is collected, and four volumes of anhydrous ethanol are added to precipitate the extracellular polysaccharides. The mixture is placed at room temperature for 24 hours, and then centrifuged at 8000 rpm for 10 minutes to separate the crude polysaccharides. The crude polysaccharides are completely dissolved in deionized water at a concentration of 10 mg / ml, and trichloroacetic acid is added to bring the final concentration to 4% (m / v). The mixture is placed at 4°C for 12 hours, centrifuged at 6000 rpm for 15 minutes at 4°C, and the supernatant is collected. The supernatant is then transferred to a dialysis bag (1000 Da) and dialyzed at 4°C for 48 hours, with the water changed every 12 hours. The polysaccharide content in the solution is determined using the phenol-sulfuric acid method. The dialyzed polysaccharides are then freeze-dried.

[0009] In one embodiment, the fermentation conditions are: fermentation temperature 25-40℃, fermentation time 10-30h; preferably, fermentation temperature 37℃, fermentation time 18-24h.

[0010] In one embodiment, the fermentation involves inoculating a seed culture of *Lactobacillus reuteri* CCFM1453 into a culture medium; the inoculation amount is 1-5% (v / v), preferably 2% (v / v).

[0011] In one embodiment, the culture medium contains 10-12 g / L glucose, 5.0-7.0 g / L yeast extract, 9.0-11.0 g / L peptone, 2-3 g / L anhydrous sodium acetate, 2-2.5 g / L diammonium citrate, 2-2.6 g / L dipotassium hydrogen phosphate, 0.05-0.25 g / L manganese sulfate monohydrate, 0.1-0.5 g / L magnesium sulfate heptahydrate, and 1.0-1.5 mL / L Tween-80. In another embodiment, the concentration of *Lactobacillus reuteri* CCFM1453 cells in the culture medium is not less than 5.0 × 10⁻⁶. 7 CFU / mL.

[0012] The present invention also provides a fermentation composition obtained by the above method.

[0013] The present invention also provides a product containing the above-mentioned *Lactobacillus reuteri* CCFM1453 or the above-mentioned extracellular polysaccharide.

[0014] In one embodiment, the product comprises food or health supplements.

[0015] In one embodiment, the product comprises a pharmaceutical or cosmetic product.

[0016] In one embodiment, the food also includes conventional auxiliary ingredients.

[0017] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0018] In one embodiment, the health product further includes conventional excipients, which include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0019] In one embodiment, the drug further comprises a drug carrier and / or pharmaceutical excipients.

[0020] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0021] In one embodiment, the pharmaceutical excipient comprises at least one of the following: solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesion agent, integrator, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, encapsulating agent, humectant, absorbent, diluent, flocculant and anti-flocculation agent, filter aid, or release inhibitor.

[0022] In one embodiment of the present invention, the cosmetic comprises the above-described composition, matrix raw materials and / or conventional excipients.

[0023] In one embodiment of the present invention, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.

[0024] In one embodiment of the present invention, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0025] The present invention also provides the application of the above-mentioned *Lactobacillus reuteri* CCFM1453 or the above-mentioned extracellular polysaccharide in the preparation of a medicine for relieving gastritis and promoting gastric mucosal repair.

[0026] In one embodiment, the dosage of the extracellular polysaccharide of *Limosilactobacillus reuteri* CCFM1453 in the drug is not less than 5 mg / kg body weight.

[0027] Beneficial effects:

[0028] The extracellular polysaccharide of *Limosilactobacillus reuteri* CCFM1453 of this invention, when taken orally after oral administration, has the ability to alleviate gastritis and gastric mucosal damage in the host, specifically manifested in:

[0029] (1) Relief of gastric mucosal injury: Reduces the content of PGE2 in gastric tissue and decreases the inflammatory response of the gastric mucosa. Reduces the overexpression of TFF3 in the stomach of mice with gastric mucosal injury and promotes gastric mucosal repair.

[0030] (2) Anti-inflammatory mechanism: It significantly reduced the level of inflammatory factor IL-6 in the gastric tissue of mice, alleviating the inflammatory response caused by gastric mucosal injury; it significantly increased the expression of IL-10, inhibiting excessive inflammatory response; it significantly reduced the level of COX-2 in the serum of mice with gastric mucosal injury, thereby inhibiting the activation of inflammatory pathways; it reduced the expression of STAT3, regulated the expression of SOCS3, alleviated gastric inflammation and apoptosis signaling pathways, and alleviated gastric mucosal injury; it inhibited the expression of MyD88, IKBα, and AKT, intervened in the TLR4 / MyD88 / NF-κB signaling pathway, and blocked the cascade reaction of inflammatory factors; it downregulated the expression of TRPV4, alleviated cell damage caused by calcium overload, and protected the integrity of gastric mucosa; it increased the expression of Bcl2, reduced apoptosis, and protected gastric mucosal cells.

[0031] Preservation of biological materials:

[0032] A strain of *Limosilactobacillus reuteri* (CCFM1453), taxonomically named *Limosilactobacillus reuteri*, was deposited on December 11, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 65608), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0033] Figure 1 Effects of different probiotic extracellular polysaccharides on the proliferation of RAW264.7 cells.

[0034] Figure 2 Effects of different probiotic extracellular polysaccharides on the repair of LPS-induced expression of IL-1β mRNA, IL-6 mRNA, IL-10 mRNA, and TNF-α mRNA in RAW264.7 cells.

[0035] Figure 3 Flowchart of mouse experiments.

[0036] Figure 4 Effects of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453 on the levels of PGE2 and TFF3, markers of gastric mucosal injury in mice.

[0037] Figure 5 Effects of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453 on the levels of inflammatory markers IL-6, IL-8, IL-10, and COX-2 in mice with gastritis.

[0038] Figure 6 The effects of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453 on alleviating gastric mucosal damage through different pathways.

[0039] Figure 7 Pathological section of Lactobacillus reuteri CCFM1453 extracellular polysaccharide promoting gastric mucosal repair.

[0040] "*" indicates a statistically significant difference from the Model group (P<0.05), "**" indicates a statistically significant difference from the Model group (P<0.01), "***" indicates an extremely statistically significant difference from the Model group (P<0.001), and "****" indicates an extremely statistically significant difference from the Model group (P<0.0001). Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments.

[0042] The mouse macrophages (RAW264.7) used in the following examples were purchased from the Shanghai Cell Bank.

[0043] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0044] The *Lactobacillus reuteri* CCFM1453, *Lactobacillus reuteri* RC14, *Lactobacillus reuteri* 45B1, *Lactobacillus reuteri* 12M3, and *Lactobacillus reuteri* 215 strains involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0045] The culture media involved in the following examples are as follows:

[0046] MRS liquid culture medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, and Tween-80 1 mL / L, pH 6.2–6.4.

[0047] MRS solid medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, and agar 20.0 g / L, pH 6.2–6.4.

[0048] MRS simplified liquid culture medium: glucose 10 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, anhydrous sodium acetate 2 g / L, diammonium citrate 2 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.05 g / L, magnesium sulfate heptahydrate 0.1 g / L, and Tween-80 1 mL / L, pH 6.2–6.4.

[0049] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content 10000U / mL, streptomycin concentration 10mg / mL).

[0050] Example 1: Cell resuscitation and culture

[0051] First, the frozen mouse macrophages (RAW264.7) were taken out and rapidly thawed in a 37°C water bath. Then, they were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in an appropriate volume of cell culture medium. The cells were then placed in a culture dish and cultured in a 37°C incubator containing 5% CO2. When the cells regained viability and reached 70%–80% confluence after 1–2 days, they were passaged.

[0052] Example 2: Screening and extraction of extracellular polysaccharides from *Lactobacillus reuteri* CCFM1453

[0053] (1) Screening of Lactobacillus reuteri CCFM1453

[0054] The samples were derived from cow's milk. After pretreatment, the samples were stored in 20% glycerol at -80°C. After thawing, the samples were mixed, and 0.5 mL of the sample was added to 4.5 mL of physiological saline. Serial dilutions were performed using physiological saline, and the appropriate serial dilutions were plated onto MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus reuteri* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium for enrichment and preserved in 30% glycerol to obtain the strain. The genome of the strain was extracted, and 16S rDNA was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The 16S rDNA sequencing results, confirmed by NCBI sequence alignment, identified *Lactobacillus reuteri* as *Limosilactobacillus reuteri*, named *Limosilactobacillus reuteri* CCFM1453, and deposited at the Guangdong Provincial Microbial Culture Collection Center on December 11, 2024, with accession number GDMCC No: 65608.

[0055] (2) Extraction of extracellular polysaccharides from *Lactobacillus reuteri* CCFM1453, *Lactobacillus reuteri* RC14, *Lactobacillus reuteri* 45B1, *Lactobacillus reuteri* 12M3, and *Lactobacillus reuteri* 215

[0056] 1) Recover Lactobacillus reuteri CCFM1453 by streaking from the preservation tube, and incubate it in a water-jacketed incubator at 37°C for 24-48 hours on MRS solid medium to obtain single colonies; pick a single colony and inoculate it into MRS liquid medium, and incubate it at 37°C for 12-18 hours to obtain culture medium 1;

[0057] 2) Inoculate culture solution 1 into MRS liquid medium at an inoculation rate of 2% (v / v) and incubate at 37°C for 12 h to obtain seed culture;

[0058] 3) Inoculate the seed culture at 2-5% (v / v) into MRS simplified liquid medium for expansion culture, and culture at 37℃ for 18-24h to obtain bacterial culture a.

[0059] 4) Centrifuge bacterial culture a at 8000 rpm for 30 min to obtain the supernatant. Add 4 volumes of anhydrous ethanol to precipitate the extracellular polysaccharides. Incubate at room temperature for 24 h, then centrifuge at 8000 rpm for 10 min to separate the crude polysaccharides. Dissolve the crude polysaccharides completely in deionized water at a concentration of 10 mg / ml. Add trichloroacetic acid to a final concentration of 4% (m / v) and incubate at 4℃ for 12 h. Centrifuge at 6000 rpm for 15 min at 4℃ and collect the supernatant. Transfer the supernatant to a dialysis bag (1000 Da) and dialyze at 4℃ for 48 h, changing the water every 12 h. Determine the polysaccharide content in the solution using the phenol-sulfuric acid method. Freeze-dry the dialyzed polysaccharides.

[0060] Example 3: Effect of extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 on the proliferation of mouse macrophages RAW264.7

[0061] The specific steps are as follows:

[0062] (1) Take 100 μL of logarithmic growth phase mouse macrophages RAW264.7 and administer at 7×10⁻⁶ mg / L. 3 Cells were seeded at a concentration of 100 cells / well in a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 24 hours to allow the cells to adhere, blank control group, control group, and post-genetic treatment group were set up.

[0063] The blank group consisted of only cell culture medium and no mouse macrophages RAW264.7.

[0064] The control group consisted of cell culture medium and mouse macrophages RAW264.7, but not probiotic extracellular polysaccharides.

[0065] The probiotic extracellular polysaccharide treatment group consisted of cell culture medium containing different extracellular polysaccharides obtained in Example 2 and mouse macrophages RAW264.7.

[0066] Preparation of cell culture medium containing probiotic extracellular polysaccharides: The lyophilized extracellular polysaccharides were resuspended in cell culture medium (the amount of resuspended extracellular polysaccharides was the same as that fermented to a concentration of 5.0 × 10⁻⁶). 7 The amount of extracellular polysaccharides extracted from bacterial cultures at CFU / mL is equivalent to that extracted from probiotic extracellular polysaccharides, thus obtaining a cell culture medium containing probiotic extracellular polysaccharides.

[0067] (2) The above well plates were incubated in an incubator at 37°C for 24 hours. After incubation, 10 μL of CCK8 solution was added to each well and incubated for 2 hours. The absorbance (OD) at 450 nm was then measured.

[0068] Cell viability is calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0069] The effects of probiotic extracellular polysaccharides on cell proliferation, such as Figure 1 As shown, compared with the control group, the cell proliferation rates of *Lactobacillus reuteri* CCFM1453, *Lactobacillus reuteri* RC14, *Lactobacillus reuteri* 45B1, *Lactobacillus reuteri* 12M3, and *Lactobacillus reuteri* 215 with added extracellular polysaccharides were 90.75%, 92.24%, 87.92%, 91.70%, and 80.46%, respectively.

[0070] According to the ISO 10993-5:2009 toxicity grading evaluation method, cell viability greater than 70% can be considered non-toxic. The above results indicate that the mouse macrophage RAW264.7 cells at the above-mentioned extracellular polysaccharide concentrations showed high viability (above 90%). Considering its non-cytotoxicity, an inactivated bacterial concentration of 5.0 × 10⁻⁶ cells could be selected. 7 CFU / mL is a suitable postbiotic concentration for subsequent cell experiments.

[0071] Example 4: Effect of extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 on the repair of inflammatory factor mRNA expression levels in RAW264.7 cells after LPS intervention.

[0072] The specific steps are as follows:

[0073] (1) RAW264.7 cells were cultured at 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / mL in 6-well plates and cultured overnight until cell adhesion was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. Control group 1 and model group 1 were set up.

[0074] Control group 1 was given 2 mL of standard cell culture medium;

[0075] Add 2 mL of ordinary culture medium containing 1 μg / mL LPS to modeling agent group 1.

[0076] (2) The above-mentioned well plates were incubated in an incubator at 37°C for 24 hours. After incubation, the old culture medium of the control group and the modeling agent group was discarded, and the plates were washed three times with PBS. The control group, model group and treatment group were set up:

[0077] The control group consisted of control group 1, which had 2 mL of ordinary cell culture medium added after the medium was changed;

[0078] The model group consisted of the modeling agent group 1, which was changed and then 2 mL of ordinary cell culture medium was added.

[0079] The treatment groups were as follows: After changing the medium, 2 mL of culture medium containing Lactobacillus reuteri extracellular polysaccharide was added to the modeling agent group 1.

[0080] The preparation method of extracellular polysaccharides from *Lactobacillus reuteri* was as described in Example 2. Extracellular polysaccharides from *Lactobacillus reuteri* CCFM1453, *Lactobacillus reuteri* RC14, *Lactobacillus reuteri* 45B1, *Lactobacillus reuteri* 12M3, and *Lactobacillus reuteri* 215 were resuspended in 2 mL of cell culture medium, respectively, until the amount of resuspended extracellular polysaccharides was equal to the concentration obtained after fermentation at 5.0 × 10⁻⁶. 7 The amount of extracellular polysaccharide prepared from bacterial suspensions of CFU / mL was equivalent to that of Lactobacillus reuteri, thus obtaining a culture medium containing extracellular polysaccharide of Lactobacillus reuteri.

[0081] (3) Each sample in the above-mentioned well plate was in triplicate and incubated at 37℃ for 24 h. The culture supernatant was discarded, and each well was washed three times rapidly with PBS. 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HSF cells was detected by real-time quantitative PCR. The expression levels of IL-1β mRNA, IL-6 mRNA, IL-10 mRNA, and TNF-α mRNA were calculated using the 2-ΔΔCt formula, with β-actin as the internal control. The primers are described in Table 1 below. The results are as follows: Figure 2 As shown.

[0082] Table 1: Primer sequences

[0083]

[0084] The results show that, by Figure 2 It was found that the expression level of IL-1β mRNA in the control group was approximately 1, while the expression level in the model group increased to 128.78 after LPS intervention. The extracellular polysaccharide extracted from *Lactobacillus reuteri* CCFM1453 significantly reduced the expression level of IL-1β mRNA in RAW264.7 cells to 42.12. However, the extracellular polysaccharides of other *Lactobacillus reuteri* strains, such as RC14, 45B1, 12M3, and 215, did not have a significant effect on downregulating the increased IL-1β mRNA expression induced by modeling as the extracellular polysaccharide of CCFM1453.

[0085] The expression level of IL-6 mRNA in the control group was approximately 1, while the expression level in the model group increased to 48.64 after LPS intervention. Extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 significantly reduced the expression level of IL-6 mRNA in RAW264.7 cells to 32.66. However, the extracellular polysaccharides of other *Lactobacillus reuteri* strains, such as RC14, 45B1, 12M3, and 215, did not have a significant effect on downregulating the increased IL-6 mRNA expression induced by modeling as the extracellular polysaccharide of CCFM1453. Some of these polysaccharides even caused an increase in IL-6 mRNA expression after intervention.

[0086] The expression level of IL-10 mRNA in the control group was approximately 1, while the expression level in the model group increased to 3.29 after LPS intervention. The extracellular polysaccharide extracted from *Lactobacillus reuteri* CCFM1453 significantly increased the expression level of IL-10 mRNA in RAW264.7 cells to 6.30. However, the extracellular polysaccharides of other *Lactobacillus reuteri* strains, such as RC14, 45B1, 12M3, and 215, did not have a significant effect on increasing IL-10 mRNA expression induced by modeling as the extracellular polysaccharide of CCFM1453.

[0087] The expression level of TNF-α mRNA in the control group was approximately 1, while the expression level in the model group increased to 2.03 after LPS intervention. The extracellular polysaccharide extracted from *Lactobacillus reuteri* CCFM1453 significantly increased the expression level of TNF-α mRNA in RAW264.7 cells to 0.43. However, the extracellular polysaccharides of other *Lactobacillus reuteri* strains, such as RC14, 45B1, 12M3, and 215, did not have a significant effect on alleviating the increased TNF-α mRNA expression caused by modeling as the extracellular polysaccharide of CCFM1453.

[0088] This indicates that the extracellular polysaccharide extracted from *Lactobacillus reuteri* CCFM1453 reduces the expression of IL-1β mRNA, IL-6 mRNA, and TNF-α mRNA in RAW264.7 cells under LPS intervention, while increasing the expression of IL-10 mRNA. This demonstrates that the extracellular polysaccharide from *Lactobacillus reuteri* CCFM1453 has an anti-inflammatory effect on RAW264.7 macrophages.

[0089] Example 5: Effects of extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 on the levels of PGE2 and TFF3 in gastric tissue of mice with gastric mucosal injury.

[0090] The specific steps are as follows:

[0091] Preparation of extracellular polysaccharides for gavage: Following the method in Example 2, colonies were collected at a count of 1 × 10⁻⁶. 9 The supernatant from the fermentation broth of CFU was used to extract crude polysaccharides, remove proteins, dialyze, and freeze-dry to obtain Lactobacillus reuteri extracellular polysaccharide powder.

[0092] Experimental grouping: Thirty-five healthy male BALB / c mice aged 8 weeks were randomly divided into 7 cages with 5 mice per cage. The 7 cages were as follows: 1 cage for pseudo-sterile control group (p-Control), 1 cage for pseudo-sterile model group (p-Model), 1 cage for pseudo-sterile Lactobacillus reuteri CCFM1453 extracellular polysaccharide group (p-CCFM1453eps), 1 cage for control group (Control), 1 cage for model group (Model), 1 cage for Lactobacillus reuteri CCFM1453 extracellular polysaccharide group (CCFM1453eps), and 1 cage for Yangshen group (p-pc).

[0093] Experimental procedure:

[0094] (1) After one week of adaptation, except for the control group, the other groups were administered acetylsalicylic acid (0.2 mL 50 mg / mL acetylsalicylic acid) by gavage at a daily dose of 400 mg / kg to induce chronic gastritis in BALB / c mice for 7 days (7-14 days).

[0095] (2) From day 13 to day 25, all pseudo-sterile groups were given a pseudo-sterile mouse model by gavage with a quadruple antibiotic regimen. The quadruple antibiotic regimen consisted of: vancomycin 100 mg / kg; neomycin 200 mg / kg; ampicillin 200 mg / kg; and metronidazole 200 mg / kg. The gavage dose was 0.2 mL.

[0096] (3) From day 15 to day 25, the p-CCFM1453eps group and the CCFM1453eps group were administered extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453 by gavage at a daily dose of 50 mg / kg (0.2 mL 6.25 mg / mL); the control group and the model group were administered the same volume of physiological saline by gavage as controls; the Yangshen group was administered aluminum magnesium carbonate by gavage at a daily dose of 30 mg / kg until the end of the experiment (day 25). All groups had free access to water and food, and the experimental procedure was as follows. Figure 3 As shown.

[0097] After the experiment, the mice were euthanized, and their stomach tissue was collected, preserved in paraformaldehyde, and stained with H&E. The results are as follows: Figure 7 As shown in the figure. Compared to the control group, the model group showed superficial erosion, loose and edematous mucosa and lamina propria, a few inflammatory infiltrations, and loose and edematous lamina propria. After repair with CCFM1453eps, the mucosal barrier damage in the pseudo-germ-free mouse group and the normal mouse group improved, the degree of edema was reduced, and the infiltration of inflammatory cells was significantly reduced.

[0098] After the experiment, mice were euthanized, and blood was collected from the eyeballs. After standing for 40 minutes, the blood was centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected for ELISA detection. Skin tissue from the back was cut and homogenized with PBS at a weight-to-volume ratio of 1:10. This homogenate was centrifuged at 3000 rpm for 20 minutes, and the skin supernatant was collected. The PGE2 content in the mouse stomach tissue was detected using an ELISA kit. The results are as follows: Figure 4 As shown:

[0099] Compared with the p-Control group (3.28 pg / mg), the PGE2 content in the gastric tissue of the p-Model group was significantly increased to 11.70 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the content of PGE2, a gastritis marker, in the gastric tissue of mice compared with p-Model, decreasing it to 4.04 pg / mg. Compared with the Control group (5.17 pg / mg), the PGE2 content in the gastric tissue of the Model group was significantly increased to 9.37 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the content of PGE2, a gastritis marker, in the gastric tissue of mice compared with Model, decreasing it to 3.69 pg / mg.

[0100] The TFF3 content in mouse gastric tissue was detected using an ELISA kit, and the results are as follows: Figure 4 As shown:

[0101] Compared with the p-Control group (29.18 pg / mg), the TEE3 content in the gastric tissue of the p-Model group was significantly increased to 87.42 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the content of the gastritis marker TFF3 in the gastric tissue of mice compared with p-Model, reducing it to 50.59 pg / mg, which was more effective than magnesium aluminum carbonate. Compared with the Control group (58.53 pg / mg), the TFF3 content in the gastric tissue of the Model group was significantly increased to 92.58 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the content of the gastritis marker TFF3 in the gastric tissue of mice compared with Model, reducing it to 87.56 pg / mg.

[0102] The results show that the extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 has the ability to alleviate the accumulation of PGE2 and TFF3 in the gastric tissue of mice with gastric mucosal damage. Compared with the model group, the improvement is significant, and the improvement effect is better than that of the drug aluminum magnesium carbonate.

[0103] Example 6: Effects of extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 on the levels of inflammatory markers in gastric tissue and serum of mice with gastric mucosal injury.

[0104] The animal experimental design and gavage groups involved in the following examples are the same as in Example 5. The levels of IL-6, IL-8, IL-10, and COX-2 in the gastric tissue and serum of mice with gastric mucosal injury were detected using an ELISA kit from Nanjing Senbega Pharmaceutical Co., Ltd. Figure 5 As shown.

[0105] (1) IL-6 content in gastric tissue: Compared with the p-Control group (2.37 pg / mg), the IL-6 content in the gastric mucosa of the p-Model group was significantly increased to 7.12 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the IL-6 content in the gastric tissue of mice to 3.53 pg / mg. Compared with the Control group (3.54 pg / mg), the IL-6 content in the gastric tissue of the Model group was significantly increased to 7.86 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the IL-6 content in the gastric tissue of mice compared with the model group to 4.35 pg / mg.

[0106] (2) IL-8 content in gastric tissue: Compared with the p-Control group (1.82 pg / mg), the IL-8 content in the gastric mucosa of the p-Model group increased significantly to 6.36 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the IL-8 content in the gastric tissue of mice to 4.72 pg / mg. Compared with the Control group (3.06 pg / mg), the IL-8 content in the gastric tissue of the Model group increased significantly to 7.16 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the IL-8 content in the gastric tissue of mice compared with the model group to 5.67 pg / mg.

[0107] (3) IL-10 content in gastric tissue: Compared with the p-Control group (40.10 pg / mg), the IL-10 content in the gastric mucosa of the p-Model group was significantly reduced to 7.08 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly increased the IL-10 content in the gastric tissue of mice, increasing to 36.53 pg / mg. Compared with the Control group (53.00 pg / mg), the IL-10 content in the gastric tissue of the Model group was significantly reduced to 37.55 pg / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly increased the IL-10 content in the gastric tissue of mice compared with the model group, increasing to 43.24 pg / mg.

[0108] (4) Serum COX-2 content: Compared with the p-Control group (3.01 ng / mg), the serum COX-2 content in the p-Model group increased significantly to 4.85 ng / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the serum COX-2 content in mice to 2.70 ng / mg. Compared with the Control group (3.58 ng / mg), the serum COX-2 content in the Model group increased significantly to 7.23 ng / mg. Oral administration of extracellular polysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the serum COX-2 content in mice compared with the model group to 4.98 ng / mg.

[0109] The results of relevant biochemical indicators in animal serum showed that extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 could reduce the levels of inflammatory factors IL-6, IL-8, IL-10, and COX-2 in the gastric tissue and serum of mice with gastric mucosal damage, thus alleviating inflammation and combating gastric mucosal damage. In observing the differences between pseudo-germ-free mice and normal mice, the intervention effect was found to be better in the pseudo-germ-free mouse group. This may be because antibiotics increase gastrointestinal permeability, which may alter the absorption pattern of polysaccharides, making them easier to absorb.

[0110] Example 7: Effects of extracellular polysaccharides extracted from *Lactobacillus reuteri* CCFM1453 on the expression of key target genes in gastric tissue of mice with gastric mucosal injury.

[0111] The animal experimental design, gavage groups, and RNA extraction and detection methods used in the following examples are the same as in Example 5. Primers for key genes STAT3, SOCS3, TRPV4, MyD88, AKT, and IKBα in the gastric tissue of mice with gastric mucosal injury are described in Table 2 below. Gene expression results are shown in... Figure 6 .

[0112] Table 2: Primer sequences

[0113]

[0114] (1) STAT3: STAT3 is a member of the Stat family of cytoplasmic transcription factors and is involved in many biological processes, including cell proliferation, survival, differentiation, and angiogenesis, as well as many pathological processes, including inflammation, tumors, and immune responses. The expression level of STAT3 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 4.09 in the p-Model group and 10.42 in the Model group due to the influence of modeling. However, after gavage administration of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of STAT3 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.48 and 1.11, respectively.

[0115] (2) SOCS3: SOCS3 belongs to the SOCS protein family and is involved in the regulation of inflammatory cytokines, insulin resistance, and glucose metabolism. Downregulation of SOCS3 reduces the severity of intestinal inflammation. Due to the influence of modeling, the expression level of SOCS3 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 4.22 in the p-Model group and 2.22 in the Model group. However, after gavage administration of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of SOCS3 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 0.99 and 0.80, respectively.

[0116] (3) TRPV4: TRPV4 is a widely expressed non-selective cation channel that plays an important role in various physiological and pathological processes. Due to the influence of modeling, the expression level of TRPV4 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 4.73 in the p-Model group and 11.50 in the Model group. However, after gavage administration of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of TRPV4 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.11 and 1.09, respectively.

[0117] (4) AKT: As one of the serine / threonine protein kinases, AKT plays a key role in the treatment of inflammatory gastritis. Due to the influence of modeling, the expression level of AKT mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 7.40 in the p-Model group and 8.68 in the Model group. However, after gavage administration of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of AKT mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.28 and 4.13, respectively.

[0118] (5) MyD88: MyD88 is a key molecule in the TLR4 / MyD88 / NF-κB signaling pathway, which plays an important role in the development of gastric mucosal injury. Due to the influence of modeling, the expression level of MyD88 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 3.13 in the p-Model group and 2.12 in the Model group. However, after gavage administration of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of MyD88 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.50 and 1.32, respectively.

[0119] (6) IKBα: The role of IKBα in gastric mucosal injury is mainly related to its regulation of the NF-κB signaling pathway. Due to the influence of modeling, the expression level of IKBα mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 2.55 in the p-Model group and 4.01 in the Model group. However, after gavage administration of extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of IKBα mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 0.91 and 0.65, respectively.

[0120] Extracellular polysaccharides extracted from Lactobacillus reuteri CCFM1453 can inhibit gastric mucosal damage through the JAK / STAT3 signaling pathway, PI3K / AKT signaling pathway, NF-κB signaling pathway, and MAPK signaling pathway. These signaling pathways play important roles in cell proliferation, differentiation, immune regulation, and inflammatory responses.

[0121] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1453, the Lactobacillus reuteri CCFM1453, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:65608.

2. A method for preparing extracellular polysaccharides from *Lactobacillus reuteri* CCFM1453, characterized in that, The Lactobacillus reuteri CCFM1453 of claim 1 was fermented in a culture medium, and the fermentation supernatant was collected, purified by alcohol precipitation. The fermentation process involves inoculating a seed culture of *Lactobacillus reuteri* CCFM1453 into a culture medium and culturing it at 25-40°C for at least 10-30 hours to obtain a fermentation broth. The concentration of *Lactobacillus reuteri* CCFM1453 in the fermentation broth is not less than 5.0 × 10⁻⁶. 7 CFU / mL; The culture medium contains 10-12 g / L glucose, 5.0-7.0 g / L yeast extract, 9.0-11.0 g / L peptone, 2-3 g / L anhydrous sodium acetate, 2-2.5 g / L diammonium citrate, 2-2.6 g / L dipotassium hydrogen phosphate, 0.05-0.25 g / L manganese sulfate monohydrate, 0.1-0.5 g / L magnesium sulfate heptahydrate, and 1.0-1.5 mL / L Tween-80.

3. The extracellular polysaccharide obtained by the method of claim 2.

4. A medicine containing the extracellular polysaccharide of claim 3.

5. The medicine as described in claim 4, characterized in that, The drug also includes a drug carrier and / or pharmaceutical excipients; the pharmaceutical excipients include excipients and additives.

6. The medicine as described in claim 5, characterized in that, The dosage forms of the medicine include tablets, granules, capsules, or liquid preparations.

7. The use of the extracellular polysaccharide according to claim 3 in the preparation of a medicament for relieving chronic gastritis caused by acetylsalicylic acid.

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