Bifidobacterium longum bn-bb01 and its use in improving helicobacter pylori infection

Abstract

The present application relates to the field of microorganisms, discloses a strain of Bifidobacterium longum BN-BB01 and its use in improving Helicobacter pylori infection. The Bifidobacterium longum BN-BB01 provided by the present application has a preservation number of CGMCC No.34432, can inhibit urease activity and inhibit the colonization and reproduction of Helicobacter pylori, can also regulate intestinal metabolism, thereby increasing the content of beneficial fatty acids in the intestinal tract and significantly reducing the content of rancid fatty acids. In addition, the complete viable body of the Bifidobacterium longum has tissue remodeling ability and can specifically reverse gastric mucosal fibrosis caused by Helicobacter pylori infection, thereby providing a new treatment approach for solving the intestinal barrier and gastric mucosal structure damage caused by Helicobacter pylori infection.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of Bifidobacterium longum BN-BB01 and its use in improving Helicobacter pylori infection. Background Technology

[0002] Helicobacter pylori (Hp) infection is a triggering factor for chronic active gastritis, peptic ulcers, and even gastric cancer. Hp infection not only directly damages gastric tissue but also disrupts the homeostasis of the gastrointestinal axis, leading to dysbiosis of the downstream intestinal flora. This results in the abnormal accumulation of putrefactive fatty acids (branched short-chain fatty acids, especially isobutyric acid) and a severe deficiency of beneficial fatty acids (straight-chain short-chain fatty acids, especially butyric acid). Furthermore, the persistent inflammatory storm triggered by Hp infection activates the gastric mucosa's inherent fibroblasts, leading to excessive collagen deposition and ultimately irreversible pathological fibrosis, causing atrophic gastritis and even gastric cancer.

[0003] While commonly used clinical methods for treating Helicobacter pylori infection, such as the "quadruple therapy" of antibiotics, can effectively kill the bacteria and achieve a good Hp eradication effect, patients still face long-term problems such as gastrointestinal discomfort and recurrent inflammation after successfully eradicating Hp, thus the treatment effect is not ideal.

[0004] Therefore, there is an urgent need to develop new drug formulations that have better therapeutic and alleviating effects on diseases and symptoms caused by Helicobacter pylori infection. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned problems in the prior art and provide a strain of Bifidobacterium longum BN-BB01 and its use in improving Helicobacter pylori infection. The Bifidobacterium longum provided by this invention not only effectively inhibits the colonization and reproduction of Helicobacter pylori, but also has the effect of repairing gastrointestinal mucosal damage caused by Helicobacter pylori infection.

[0006] The first aspect of this invention provides a strain of Bifidobacterium longum ( Bifidobacterium longum The preservation number of this Bifidobacterium longum is CGMCC No. 34432.

[0007] A second aspect of the present invention provides a microbial agent containing the *Bifidobacterium longum* and / or the metabolites of the *Bifidobacterium longum* described in the first aspect.

[0008] A third aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect.

[0009] The fourth aspect of the present invention provides the use of the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention and / or treatment of diseases or symptoms caused by Helicobacter pylori infection.

[0010] The fifth aspect of the present invention provides a method for inhibiting the reproduction of Helicobacter pylori in vitro, the method comprising contacting the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect, with Helicobacter pylori cultured in vitro.

[0011] The sixth aspect of the present invention provides a method for inhibiting the colonization of Helicobacter pylori in an in vitro culture, the method comprising contacting the in vitro culture with the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect, thereby inhibiting the colonization of Helicobacter pylori in the in vitro culture.

[0012] The beneficial effects obtained by the present invention through the above technical solution include at least the following: (1) The Bifidobacterium longum provided by the present invention is a probiotic isolated from human fecal samples. It can easily colonize in the body and exert its effects. When used as a probiotic product, it can have good in vivo stability.

[0013] (2) The Bifidobacterium longum provided by the present invention can inhibit urease activity and inhibit Hp colonization and reproduction, and has the potential to be used as a probiotic drug or preparation for anti-Helicobacter pylori infection.

[0014] (3) The *Bifidobacterium longum* provided by this invention can regulate the metabolism of digestive tract cells, promote the increase of beneficial fatty acid content in the intestine (for example, experimental verification shows that the *Bifidobacterium longum* provided by this invention can increase butyric acid content to an ultra-high level of 165.69 μmol / g), and significantly reduce the content of putrefactive fatty acids in the intestine. Moreover, the intact live *Bifidobacterium longum* of this invention exhibits tissue remodeling ability and can specifically reverse digestive tract damage such as gastric mucosal fibrosis induced by *H. pylori* infection. Thus, this strain provides a solution to the problems of intestinal barrier and gastric mucosal structural damage after *H. pylori* infection, and the difficulty of existing treatments in addressing these damages. Attached Figure Description

[0015] Figure 1 This is a colony morphology diagram of Bifidobacterium longum BN-BB01 obtained in Example 1.

[0016] Figure 2 This is a graph showing the in vitro inhibition results of different BN-BB01 strain preparations against Helicobacter pylori in Example 2.

[0017] Figure 3These are cell morphology diagrams of the control group and the model group in the adhesion inhibition experiment of Example 4.

[0018] Figure 4 This is a cell morphology diagram of the BN-BB01 strain preparation group in the adhesion inhibition experiment of Example 4.

[0019] Figure 5 These are cell morphology diagrams of the control group and the model group in the invasion inhibition experiment of Example 4.

[0020] Figure 6 This is a cell morphology diagram of the BN-BB01 strain preparation group in the invasion inhibition experiment of Example 4.

[0021] Figure 7 This is a diagram showing the HE staining results of the gastric mucosa of mice treated with different BN-BB01 strain preparations in Example 5.

[0022] Figure 8 This is a graph showing the results of detecting the inflammatory infiltration area of ​​the gastric mucosa in mice treated with different BN-BB01 strain preparations in Example 5.

[0023] Figure 9 This is a diagram showing the Masson staining results of the gastric mucosa of mice treated with different BN-BB01 strain preparations in Example 5.

[0024] Figure 10 This is a graph showing the relative collagen area of ​​the gastric mucosa of mice treated with different BN-BB01 strain preparations in Example 5.

[0025] Biological Preservation The strain provided by this invention is classified and named Bifidobacterium longum. Bifidobacterium longum It was deposited on May 6, 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. 34432. Detailed Implementation

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] Through long-term research, the inventors discovered that while commonly used clinical methods for treating Helicobacter pylori infection, such as the "quadruple therapy" of antibiotics, can effectively kill the bacteria, they only focus on the eradication of Hp and neglect the treatment of conditions such as gastric mucosal fibrosis and mucosal barrier damage caused by Hp infection. As a result, patients still face long-term problems such as stomach discomfort and recurrent inflammation after successfully eradicating Helicobacter pylori, requiring further treatment to relieve symptoms.

[0028] After conducting in-depth research on numerous existing probiotic anti-Hp infection preparations, the inventors of this invention discovered that existing anti-Hp microbial preparations not only suffer from significant deficiencies in butyrate conversion efficiency, effective concentration, and generation rate in the intestine, but also cause an increase in putrefactive fatty acid content. Although they possess Hp-inhibiting efficacy, they cannot meet the rapid, high-dose metabolic demands required for intestinal mucosal repair under severe Hp infection conditions, resulting in unsatisfactory treatment effects similar to antibiotic therapy. Through long-term research and screening, the inventors of this invention accidentally obtained a strain of Bifidobacterium longum from the feces of long-lived elderly individuals that can simultaneously increase beneficial fatty acids and decrease putrefactive fatty acids, named BN-BB01. This strain not only increases beneficial fatty acids and reduces putrefactive fatty acids but also inhibits Helicobacter pylori proliferation and colonization in the body, effectively inhibits urease activity, regulates intestinal microecological balance, and repairs damaged gastric mucosal barriers, potentially providing a new treatment approach for combating Hp infection and improving related conditions.

[0029] Based on the above findings, the first aspect of the present invention provides a strain of Bifidobacterium longum (… Bifidobacterium longum The preservation number of this Bifidobacterium longum is CGMCC No. 34432.

[0030] In this invention, "BN-BB01", "Bifidobacterium longum BN-BB01", and "Bifidobacterium longum provided by this invention" have the same meaning, all referring to Bifidobacterium longum with accession number CGMCC No. 34432. Unless otherwise specified, "Bifidobacterium longum" in this invention also refers to Bifidobacterium longum with accession number CGMCC No. 34432.

[0031] A second aspect of the present invention provides a microbial agent containing the *Bifidobacterium longum* and / or the metabolites of the *Bifidobacterium longum* described in the first aspect.

[0032] According to some specific embodiments of the present invention, the metabolites may include active substances produced by *Bifidobacterium longum* through fermentation, including intracellular metabolites and / or extracellular metabolites. Specific forms of the metabolites may include, but are not limited to, fermentation supernatant (mainly containing intracellular metabolites, obtained by centrifugation to remove bacterial cells after fermentation) and bacterial lysis supernatant (including intracellular metabolites and / or extracellular metabolites, obtained by centrifugation after bacterial fermentation and cell lysis, which can simultaneously enrich intracellularly released active ingredients and extracellularly secreted active substances). The metabolites can be obtained from the *Bifidobacterium longum* fermentation system using conventional processes such as centrifugation, filtration, concentration, and freeze-drying.

[0033] According to some specific embodiments of the present invention, the fermentation supernatant may further include the supernatant obtained by centrifuging to remove the bacterial cells after fermentation of the strain without pH adjustment, and / or the supernatant obtained by centrifuging to remove the bacterial cells after fermentation of the strain and further adjusting the pH to neutral (in this invention, it is referred to as "neutral supernatant", with a pH of 6.8-7.2, such as 6.8, 6.85, 6.9, 6.95, 7, 7.05, 7.1, 7.15, 7.2, or any value between the above values ​​or any range of any two values).

[0034] According to some specific embodiments of the present invention, the viable count of Bifidobacterium longum in the bacterial agent can be 10. 2 -10 10 CFU / mL. 10 2 -10 10 CFU / mL refers to a concentration of 10 effective viable Bifidobacterium longum in the bacterial agent. 2 -10 10 CFU / mL level, for example, 1×10 2 CFU / mL, 5×10 2 CFU / mL, 8×10 2 CFU / mL, 9.9×10 2 CFU / mL were all within 10 2 CFU / mL level. That is, in this invention, the effective viable count of Bifidobacterium longum in the bacterial agent is greater than or equal to 1 × 10⁻⁶. 2 CFU / mL to less than 1×10 11 CFU / mL.

[0035] For example, the viable count of Bifidobacterium longum in the bacterial agent can be 1 × 10⁻⁶. 2 CFU / mL, 5×10 2 CFU / mL, 9×10 2 CFU / mL, 1×10 3 CFU / mL, 5×10 3 CFU / mL, 9×103 CFU / mL、1×10 4 CFU / mL、5×10 4 CFU / mL、9×10 4 CFU / mL、1×10 5 CFU / mL、5×10 5 CFU / mL、9×10 5 CFU / mL、1×10 6 CFU / mL、5×10 6 CFU / mL、9×10 6 CFU / mL、1×10 7 CFU / mL、2×10 7 CFU / mL、3×10 7 CFU / mL、4×10 7 CFU / mL、5×10 7 CFU / mL、6×10 7 CFU / mL、7×10 7 CFU / mL、8×10 7 CFU / mL、9×10 7 CFU / mL、1×10 8 CFU / mL、2×10 8 CFU / mL、3×10 8 CFU / mL、4×10 8 CFU / mL、5×10 8 CFU / mL、6×10 8 CFU / mL、7×10 8 CFU / mL、8×10 8 CFU / mL、9×10 8 CFU / mL、1×10 9 CFU / mL、2×10 9 CFU / mL、3×10 9 CFU / mL、4×10 9 CFU / mL、5×10 9 CFU / mL、6×10 9 CFU / mL、7×10 9 CFU / mL、8×10 9 CFU / mL、9×10 9 CFU / mL、1×10 10 CFU / mL、5×10 10 CFU / mL、9×10 10 CFU / mL、9.9×10 10CFU / mL, or any value between the above values ​​or a range consisting of any two values.

[0036] According to some preferred embodiments of the present invention, the method for preparing the microbial agent or metabolite (or preparation containing the metabolite) may include at least one of the following: (1) Bacterial suspension: BN-BB01 was inoculated into MRS liquid medium and cultured anaerobically at 35-39℃ until the logarithmic or stationary growth phase. The viable cell concentration of the fermentation broth was adjusted to 10. 7- 10 9 CFU / mL is sufficient; Alternatively, the fermentation broth can be filtered or centrifuged to collect the cells, and then resuspended in MRS medium to a viable cell concentration of 10⁻⁶. 7- 10 9 CFU / mL; (2) Supernatant: BN-BB01 is inoculated in MRS liquid medium and cultured anaerobically at 35-39℃ until the logarithmic or stationary growth phase. The cells are removed by filtration or centrifugation, and the remaining liquid is the supernatant. (3) Neutral supernatant: Adjust the pH of the supernatant obtained by preparation method (2) to 6.8-7.2; (4) Lysis supernatant: BN-BB01 is inoculated in MRS liquid medium and cultured anaerobically at 35-39℃ until the logarithmic or stable growth phase. The cells are lysed, and then centrifuged or filtered to remove cell fragments. The remaining liquid is the lysis supernatant.

[0037] Preferably, the preparation methods (2)-(4) above may further include sterilizing the collected supernatant, neutral supernatant and lysed supernatant. Sterilization is preferably performed by filtration (e.g., using a 0.22 μm filter membrane).

[0038] According to some preferred embodiments of the present invention, in the above preparation method (4), the lysis of bacterial cells can be carried out by grinding and shaking with glass beads, preferably with a glass bead diameter of 0.05-0.15 mm and a shaking time of 25-35 s.

[0039] The Bifidobacterium longum and its metabolites provided by the present invention are active or effective against damage or effects caused by Helicobacter pylori infection. Based on the above findings, the third aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition includes the Bifidobacterium longum described in the first aspect or the bacterial agent described in the second aspect.

[0040] According to some preferred embodiments of the present invention, the pharmaceutical composition may further contain pharmaceutically acceptable excipients. Any excipient commonly used in pharmaceutical preparation in the art is suitable for the present invention, such as excipients, preservatives, stabilizers, buffers, etc.

[0041] According to some specific embodiments of the present invention, the pharmaceutical composition may be provided in any dosage form, including liquid, semi-solid, and solid.

[0042] According to some specific embodiments of the present invention, the formulation may be a liquid formulation or a solid formulation; specifically, the supernatant / lysis supernatant may be concentrated, lyophilized or spray-dried to form a dry powder; the bacterial suspension may be lyophilized in the presence of a protectant to form a live bacterial powder; the dry powder may be further prepared into solid formulations such as granules, pills, capsules, tablets, or formulated with a pharmaceutically acceptable carrier into ointments, gels, sprays or liquid formulations.

[0043] Through extensive research, the inventors discovered that the *Bifidobacterium longum* provided by this invention can prevent and / or treat diseases or symptoms caused by *Helicobacter pylori* infection by inhibiting the reproduction and / or colonization process of *Helicobacter pylori*. Specifically, the *Bifidobacterium longum* provided by this invention can not only directly inhibit the growth and reproduction of *Helicobacter pylori*, but also co-agglutinate with *Helicobacter pylori* that has not successfully colonized, thereby reducing its contact with tissue cells by encapsulating *Helicobacter pylori*, thus reducing its adhesion and colonization probability and promoting its excretion with gastrointestinal contents; moreover, the *Bifidobacterium longum* provided by this invention can not only prevent *Helicobacter pylori* from colonizing by competitively occupying adhesion sites on the surface of tissue cells (competitive mode) when *Helicobacter pylori* has not yet successfully infected or colonized, but also displace *Helicobacter pylori* that has already adhered to / invaded tissue cells when tissue cells have been colonized (displacement mode), and preventively reduce the probability of *Helicobacter pylori* infection by pre-adhering to tissue cells (rejection mode), thus reducing the *Helicobacter pylori* load in the body in three ways.

[0044] Specifically, BN-BB01 can not only inhibit the adhesion of Helicobacter pylori to tissue cells and thus inhibit its colonization, but also inhibit its invasion of tissue cells. Adhesion refers to the process by which Helicobacter pylori binds to specific receptors on the surface of tissue cells, thereby attaching itself to the cell surface. Invasion refers to the process by which Helicobacter pylori, after adhering to the surface of tissue cells, breaks through surface barriers (such as cell membranes and mucosal layers), enters the tissue cells or intercellular spaces, further establishes colonization, and may cause cell damage.

[0045] Therefore, the discovery of BN-BB01 provides a basis for the prevention and / or treatment of diseases or conditions caused by Helicobacter pylori infection.

[0046] Based on this, the fourth aspect of the present invention provides the use of the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention and / or treatment of diseases or symptoms caused by Helicobacter pylori infection.

[0047] According to some preferred embodiments of the present invention, the diseases or symptoms caused by Helicobacter pylori infection may include primary symptoms related to Helicobacter pylori infection, such as chronic atrophic gastritis, duodenal ulcer, and precancerous lesions of the stomach caused by Helicobacter pylori infection, or secondary symptoms that occur or are aggravated by long-term inflammatory stimulation or microecological metabolic disorders after Helicobacter pylori infection, such as chronic inflammation of the gastric mucosa and irritable bowel syndrome.

[0048] According to some preferred embodiments of the present invention, the disease or symptom caused by Helicobacter pylori infection may include damage to the digestive tract mucosa. The digestive tract may include the upper and / or lower digestive tract, for example, at least one of the oral cavity, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, colon, and rectum). According to a preferred embodiment of the present invention, the digestive tract is at least one of the stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, colon, rectum), preferably the stomach (i.e., the digestive tract mucosa is the gastric mucosa).

[0049] According to some preferred embodiments of the present invention, the gastrointestinal mucosal injury includes inflammatory changes and / or fibrotic lesions (including fibrotic lesions caused by collagen deposition).

[0050] In further research, the inventors also found that the application of BN-BB01 can improve intestinal metabolism, specifically by increasing the content of beneficial fatty acids (such as C2-C5 saturated straight-chain fatty acids) in the intestine and inhibiting the production of putrefactive fatty acids (such as C4-C5 saturated branched-chain fatty acids).

[0051] Based on this, according to some preferred embodiments of the present invention, the drug is used to increase the content of C2-C5 saturated straight-chain fatty acids in the intestine and / or decrease the content of C4-C5 saturated branched-chain fatty acids in the intestine.

[0052] Furthermore, the present invention also provides the use of the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a medicament for increasing the content of C2-C5 saturated straight-chain fatty acids and / or decreasing the content of C4-C5 saturated branched-chain fatty acids in the intestine.

[0053] In this invention, C2-C5 saturated straight-chain short-chain fatty acids are fatty acids produced by the fermentation of dietary fiber (carbohydrates) in the intestine, and are referred to in the art as beneficial short-chain fatty acids. Exemplarily, C2-C5 saturated straight-chain short-chain fatty acids may include one or more of acetic acid, propionic acid, butyric acid, and valeric acid. In this invention, C4-C5 saturated branched-chain fatty acids are fatty acids produced by the fermentation of dietary fiber (carbohydrates) in the intestine, and are different from C2-C5 saturated straight-chain short-chain fatty acids, and are referred to in the art as putrefactive short-chain fatty acids. Exemplarily, C4-C5 branched-chain fatty acids may include isobutyric acid and / or isovaleric acid.

[0054] In further research, the inventors also discovered that BN-BB01 has an inhibitory effect on urease activity, thereby weakening the ability of Helicobacter pylori to colonize by producing and releasing urease, and treating or alleviating diseases or symptoms caused by changes in the digestive tract microenvironment due to urease.

[0055] Based on this, according to some preferred embodiments of the present invention, the pharmaceutical composition is used to inhibit urease activity.

[0056] According to some specific embodiments of the present invention, the urease (mainly) is derived from Helicobacter pylori. Urease activity is a key factor in the colonization of Helicobacter pylori in the stomach and the induction of gastric mucosal damage. Specifically, the pharmaceutical composition can inhibit the catalytic activity of Helicobacter pylori urease through its contained bacterial cells and / or metabolites (such as fermentation supernatant, lysis supernatant), blocking the pathway by which Helicobacter pylori utilizes urea decomposition to produce ammonia to neutralize gastric acid and create a survival microenvironment, thereby weakening the colonization ability of Helicobacter pylori and reducing its pathogenic risk.

[0057] Furthermore, the present invention also provides the use of the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a medicament for inhibiting urease activity (also referred to as a "urease inhibitor").

[0058] The present invention further provides a method for in vitro inhibition of urease activity, the method comprising contacting the Bifidobacterium longum (including its cells and / or metabolites) described in the first aspect with urease, or contacting the bacterial agent described in the second aspect with urease, or contacting the pharmaceutical composition described in the third aspect with urease.

[0059] In some preferred embodiments, the method may include contacting the *Bifidobacterium longum* (cells and / or metabolites) described in the first aspect with *Helicobacter pylori*, or contacting the bacterial agent described in the second aspect with *Helicobacter pylori*, or contacting the pharmaceutical composition described in the third aspect with *Helicobacter pylori*.

[0060] According to some preferred embodiments of the present invention, the method for inhibiting urease activity in vitro may further include contacting *Bifidobacterium longum* (bacterial cells and / or metabolites) with a sample containing *Helicobacter pylori*. Preferably, the sample containing *Helicobacter pylori* may include a pure culture of *Helicobacter pylori*, gastric mucosal tissue (homogene) infected with *Helicobacter pylori*, or a clinical gastric tissue biopsy sample infected with *Helicobacter pylori*. The method provided by the present invention can inhibit urease activity in the sample, thereby reducing the dependence of in vitro experiments on *Helicobacter pylori* load and facilitating related in vitro studies under conditions of low *Helicobacter pylori* content.

[0061] The fifth aspect of the present invention provides a method for inhibiting the reproduction of Helicobacter pylori in vitro, the method comprising contacting the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect with Helicobacter pylori cultured in vitro.

[0062] The sixth aspect of the present invention provides a method for inhibiting the colonization of Helicobacter pylori in an in vitro culture, the method comprising contacting the in vitro culture with the Bifidobacterium longum described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect, thereby inhibiting the colonization of Helicobacter pylori in the in vitro culture.

[0063] According to some specific embodiments of the present invention, the in vitro culture may include cells or organoids. Any cell or organoid capable of maintaining growth or function in an artificially simulated biological environment and capable of being infected by Helicobacter pylori can be used to inhibit the colonization of Helicobacter pylori in the culture using the Bifidobacterium longum described in the first aspect, the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0064] Preferably, the in vitro culture is obtained by in vitro culture of cells or tissues derived from the digestive tract.

[0065] This invention further provides the application of the *Bifidobacterium longum* described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect, in constructing in vitro and in vivo experimental models of *Helicobacter pylori* infection. Exemplarily, the *Bifidobacterium longum* (such as its cells and / or metabolites) provided by this invention can intervene in the infection process in organs or tissues infected with *Helicobacter pylori*. By regulating the intestinal fatty acid metabolism profile (promoting the restoration of short-chain fatty acid composition in a favorable direction) and promoting mucosal barrier repair, it enables the *Helicobacter pylori* infection model to present a stable and controllable pathological state. Simultaneously, it can be used as an intervention group reagent to evaluate the therapeutic effects of different regimens on *Helicobacter pylori* infection.

[0066] The present invention further provides a method for constructing an in vivo or in vitro experimental model of Helicobacter pylori infection, the method comprising contacting the Bifidobacterium longum (including its cells and / or metabolites) described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect, with cells or tissues infected with Helicobacter pylori.

[0067] In some preferred embodiments, the experimental model is an in vitro model. That is, the cells or cell cultures infected with Helicobacter pylori are cultured in vitro (such as two-dimensional cell cultures, and three-dimensional cultures such as organoids).

[0068] In some preferred embodiments, the experimental model is an in vivo model. That is, the animals infected with Helicobacter pylori are experimental animals.

[0069] The method provided by the present invention applies the present invention's Bifidobacterium longum, bacterial agent, or pharmaceutical composition to the cells or cell cultures and experimental animals infected with Helicobacter pylori, simulating the state of cells, tissues, and organisms during the treatment and recovery period after infection. This method can be used for mechanism studies related to Helicobacter pylori infection and treatment, drug action mechanism studies, and can also be used as a positive control in the drug screening process.

[0070] The present invention will be further described below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0071] In the following examples, unless otherwise specified, all reagents and materials used were commercially available products purchased from reputable chemical or biological reagent / material suppliers, and all reagents were of analytical grade or met the purity requirements of the experiment.

[0072] Example 1 This embodiment illustrates the isolation, activation, identification, and preparation process of various strains of Bifidobacterium longum BN-BB01.

[0073] Strain isolation, activation, and identification: The inventors isolated a bacterium from a fecal sample obtained from a centenarian in Bama County, Guangxi Province, and named it BN-BB01. Figure 1 As shown, BN-BB01 colonies are milky white to light cream-colored, nearly round, with neat edges, a smooth and slightly glossy surface, and are distinctly raised, with the center being even higher, giving them an overall convex to conical (or cushion-like) shape. After 16S rRNA gene PCR amplification and sequencing, followed by sequence alignment and molecular biological and physiological / biochemical tests (acid and bile salt tolerance), BN-BB01 was comprehensively confirmed as *Bifidobacterium longum*. Bifidobacterium longum ).

[0074] Preparation of bacterial suspension: Strain BN-BB01 was inoculated into MRS liquid medium and anaerobically cultured at 37℃ until the logarithmic growth phase or stationary phase. The fermentation broth (viable cell concentration 1×10⁻⁶) was then vortexed and mixed. 8 After centrifuging at 12000g for 10 min at 4℃, the supernatant was discarded, and the bacterial cell pellet was collected. The bacterial cells were resuspended in sterile PBS, washed twice, and finally resuspended in sterile PBS to obtain a bacterial suspension (viable cell concentration of 1×10⁻⁶ CFU / mL). 8 (CFU / mL).

[0075] Preparation of the supernatant: BN-BB01 strain was inoculated into MRS liquid medium and anaerobic cultured at 37℃ until the logarithmic growth phase or stationary phase. The fermentation broth (viable cell concentration 1×10⁻⁶) was then vortexed and mixed. 8 After centrifuging at 12000g for 10 min at 4℃, the precipitate was discarded, and the resulting liquid was filtered through a 0.22μm sterile filter membrane to obtain the supernatant.

[0076] Preparation of neutral supernatant: The pH of the supernatant was adjusted dropwise to 7±0.2 with sterile NaOH aqueous solution (concentration 1mol / L), and then filtered through a 0.22μm sterile filter membrane to obtain neutral supernatant.

[0077] Preparation of lysis supernatant: The bacterial suspension was shaken with 0.1 mm glass beads at 5000 rpm for 30 s, then centrifuged at 12000 g for 10 min at 4 °C. The collected supernatant was filtered through a 0.22 μm sterile filter membrane to obtain the lysis supernatant.

[0078] Example 2 This embodiment illustrates the in vitro inhibitory effect of Bifidobacterium longum BN-BB01 on Helicobacter pylori.

[0079] In this embodiment, the microaerophilic culture environment is formed by placing the culture in a sealed container and adding a 2.5L microaerophilic gas generating bag (purchased from Mitsubishi Corporation, Japan, gas ratio: oxygen 9±3% by volume, carbon dioxide 6±2% by volume, and the remainder nitrogen).

[0080] Activation process of Helicobacter pylori (Hp, ATCC43504): The frozen Hp culture was removed from -80℃ and thawed. 20 μL of the culture was spread onto a 2:1 (w / w) agar plate containing 10% fetal bovine serum (FBS). After inoculation, the plate was placed in a sealed container and incubated at 37℃ under a microaerophilic environment until typical small, translucent / grayish-white colonies appeared (approximately 3-5 days). Sterile PBS was added to the plate, and Hp was scraped off and evenly spread onto a fresh plate of the same formulation to obtain the first generation of activated Hp. The first generation of activated Hp was then inoculated onto freshly prepared solid culture medium of the same formulation and incubated until typical small, translucent / grayish-white colonies appeared (approximately 3-5 days), yielding the second generation of activated Helicobacter pylori. The concentration of Hp in the activated culture was adjusted to 1 × 10⁻⁶ using sterile PBS. 8 CFU / mL was plated onto fresh Columbia agar plates containing a selective antibiotic for Helicobacter pylori (Solepro, catalog number LA7471, 1 mL antibiotin / 100 mL culture medium). The plates were immediately placed in Oxford cups, and 100 μL each of the various forms of BN-BB01 strain preparations (supernatant, neutral supernatant, bacterial suspension, and lysis supernatant) prepared in Example 1 were added. The plates were then placed in a sealed container and incubated in a microaerophilic environment at 4°C for 1 hour, followed by incubation at 37°C for 72 hours. The diameter of the inhibition zone was measured, and the ratio of the area of ​​a single well in the inhibition zone to the area of ​​the culture dish (single-well area ratio) was calculated. Measurements were repeated three times. The results of the in vitro antibacterial experiment are as follows: Figure 2 As shown in the figure (the positions of the inhibition zones of different bacterial preparations are drawn with circles), the specific diameters of the inhibition zones and the percentage of the area per well are shown in Table 1.

[0081] Percentage of area per well (%) = Area of ​​inhibition zone of bacterial strain / Area of ​​petri dish × 100 Table 1

[0082] Note: In Table 1, different letters indicate that the differences between groups are statistically significant. p <0.05; the same letter indicates that the difference between groups is not statistically significant ( p >0.05).

[0083] The results showed that all forms of BN-BB01 strain preparations exhibited significant antibacterial activity, with the neutral supernatant showing the best performance. Using the area of ​​the inhibition zone as a parameter to characterize the amount of secreted non-acid-dependent specific antibacterial factors, the amount of non-acid-dependent specific antibacterial factors secreted in the neutral supernatant was nearly 47% higher than that in the lysed supernatant and nearly 43% higher than that in the supernatant. This indicates that strain BN-BB01 can exert antibacterial effects in different microenvironments of the gastric mucus layer: it can fight bacteria not only in acidic gastric mucosa environments but also in neutral microenvironments of the gastric mucus layer (such as the side of the mucus layer near the gastric mucosal epithelial cells). Furthermore, because Helicobacter pylori can decompose urea to produce ammonia through urease, and ammonia neutralizes gastric acid to form a protective microenvironment, the antibacterial effect of the neutral supernatant further proves that BN-BB01 can effectively eliminate Helicobacter pylori even under non-acidic conditions.

[0084] In the Oxford cup inhibition zone experiment, although the bacterial suspension showed no inhibition zone formation, this was mainly because the bacterial suspension was mainly composed of intact live bacterial cells, which had limited diffusion ability in the agar medium and could not fully contact Helicobacter pylori. The effect of Bifidobacterium longum BN-BB01 on inhibiting the reproduction of Helicobacter pylori still needs further confirmation.

[0085] Example 3 This example illustrates the effect of Bifidobacterium longum BN-BB01 on the aggregation of Helicobacter pylori.

[0086] (1) Self-aggregation BN-BB01 or Hp bacterial suspension was adjusted to OD500 with PBS 600 =0.5±0.05 to obtain the test liquid, place it in a 37℃ static incubator, and measure the OD of the upper layer of the test liquid before incubation. 600 Absorbance value A0 and OD values ​​at different incubation times 600 Absorbance A t The self-agglomeration rate of BN-BB01 or Hp was calculated according to the following formula (I). The results of the self-agglomeration rate over time are shown in Table 2.

[0087] Self-agglomeration rate (%) = (A0 - A) t ) / A0×100 formula (I) (2) Coagulation BN-BB01 bacterial suspension was mixed with an equal volume of Hp bacterial suspension to obtain a mixed bacterial suspension. The mixture was thoroughly shaken for 5 min and then incubated at 37°C. The OD values ​​of the initial supernatant of the test liquid and the Hp supernatant were measured. 600 Absorbance B x and B y and OD values ​​of mixed bacterial cultures at different times 600 Absorbance B tThe coagulation rate of BN-BB01 and Hp was calculated according to the following formula (II). The results of the coagulation rate over time are shown in Table 2.

[0088] Coagulation rate (%) = (1-2B) t / (B) x +B y (II) × 100 Table 2

[0089] The results are shown in Table 2. Regarding autoagglutination, the autoagglutination rate of BN-BB01 was higher than that of Helicobacter pylori, indicating that BN-BB01 cells are more likely to form aggregates than Hp, which may facilitate better colonization of the gastrointestinal tract and prolong the duration of its probiotic function. Regarding coagulation, the coagulation rate of BN-BB01 and Hp increased significantly over time, significantly increasing after 16 hours, reaching its highest level at 24 hours, and significantly exceeding the autoagglutination rate of Hp. This result suggests that BN-BB01 possesses the surface characteristic of recognizing and binding to Hp. Under prolonged coexistence conditions, live BN-BB01 cells may physically disrupt the dispersion of Hp and form coprecipitates, which is the basis for the bacterial suspension group blocking Hp colonization through a physical repulsion mechanism.

[0090] In summary, BN-BB01 can prevent Hp colonization through coagulation and has the potential to be used as a probiotic for the treatment and eradication of Helicobacter pylori.

[0091] Example 4 This example illustrates the inhibitory effect of Bifidobacterium longum BN-BB01 on the proliferation and colonization of Helicobacter pylori (Hp) in vitro.

[0092] GES-1 gastric epithelial cells (purchased from Wuhan Pronosei Life Science Technology Co., Ltd., hereinafter referred to as gastric epithelial cells) were used at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 1:1 in 96-well plates and cultured at 37°C and 5% CO2 until 80-90% confluence. The culture medium was then discarded, and the cells were washed twice with serum-free Dalberg modified Eagle medium (DMEM). Subsequently, the cells were treated in three parallel groups (each group was tested in duplicate). The culture conditions were 37°C and 5% CO2. (1) Competition mode: 100 μL each of Hp bacterial culture (MOI=100) and BN-BB01 strain preparation in different forms were added to the samples and cultured for 4 hours. (2) Exclusion mode: First, add 100 μL of different forms of BN-BB01 bacterial preparations, incubate for 2 hours, wash with PBS, and then add 100 μL of Hp bacterial solution for co-culture for 2 hours. (3) Replacement mode: First, add 100 μL of Hp bacterial solution and incubate for 2 hours. After washing with PBS, add 100 μL of different forms of BN-BB01 bacterial preparations and continue incubation for 2 hours.

[0093] In each of the modes (1)-(3), a control group without Hp bacterial solution and BN-BB01 and a model group with only Hp were set up.

[0094] After culture, a cell adhesion inhibition experiment was performed, with one set of parallel experiments used to test the adhesion inhibition rate. Another set of parallel experiments underwent gentamicin post-treatment to perform a cell invasion inhibition experiment, followed by testing the invasion inhibition rate.

[0095] Post-treatment with gentamicin: Wash cells three times with PBS to remove suspended bacteria, add culture medium containing 100 μg / mL gentamicin, and incubate at 37°C for 2 hours to specifically kill extracellular bacteria that have not yet invaded, and use this to test the invasion inhibition rate.

[0096] Adhesion / invasion inhibition rate test procedure: After each reaction mode was completed, the culture medium was aspirated, and the cells were gently washed 5 times with PBS to thoroughly remove unadhesed bacteria. 40 g / L paraformaldehyde was added for fixation at room temperature for 15 minutes, and the cells were then air-dried. Next, 100 μL of 0.1 g / L crystal violet solution was added to each well for staining at room temperature for 15 minutes. After washing away any excess stain and air-drying, 33% (v / v) glacial acetic acid was added and the cells were shaken for 10 minutes (until completely dissolved). The absorbance (OD) values ​​at 550 nm of the model group and the treatment group were measured using a microplate reader. 模型组 and OD 处理组 .

[0097] Adhesion / Invasion Inhibition Rate (%) = [(OD 模型组 –OD 处理组 ) / OD 模型组 ]×100 For cell adhesion inhibition experiments: Cell morphology diagrams of the control group and the model group are shown below. Figure 3 As shown, where, Figure 3 (A) is a cell morphology diagram of the control group, showing the basic morphology of gastric epithelial cells in the absence of Hp. Figure 3 (B) shows the cell morphology of the model group. The white arrows point to a large number of dark red *Helicobacter pylori* (Hp) cells aggregated and adhered to the cell membrane surface and edges. Numerous short, rod-shaped or dot-shaped particles (*Helicobacter pylori*) stained red are tightly adhered and aggregated on the surface and intercellular spaces of gastric epithelial cells. Compared to the control group with a clean background, this confirms the successful construction of the *Hp* in vitro adhesion model, and *Hp* exhibits extremely strong adhesion properties.

[0098] Figure 4(A)-(C) show the cell morphology of the supernatant, bacterial suspension, and lysate supernatant in the competition mode, respectively. The blue arrows indicate BN-BB01 cells (which are slender rod-shaped). Compared with the model group, all three experimental groups treated with different forms of BN-BB01 bacterial strains showed significant anti-adhesion effects. The number of red Helicobacter pylori adhering to the surface of gastric epithelial cells was significantly reduced compared with the model group, and the morphology of gastric epithelial cells remained intact, with no large bacterial clumps observed. Specifically: exist Figure 4 In (B), suspected BN-BB01 bacteria were observed to coexist with Helicobacter pylori around gastric epithelial cells. This phenomenon suggests that live BN-BB01 bacteria can physically block the contact between Helicobacter pylori and host cells through steric hindrance or competitive binding sites. Figure 4 (A) and Figure 4 (C) indicates that not only do live BN-BB01 cells have anti-adhesion activity, but their secreted metabolites and intracellular active substances also have the function of destroying or blocking the adhesion of Helicobacter pylori.

[0099] For the invasion inhibition experiment: Cell morphology diagrams of the control group and the model group are shown below. Figure 5 As shown, where, Figure 5 (A) is a cell morphology diagram of the control group, showing the basic morphology of gastric epithelial cells in the absence of Helicobacter pylori. Figure 5 (B) is a cell morphology diagram of the model group. As shown in the figure, the gastric epithelial cells exhibit characteristics of severe infection. A large number of dark red Helicobacter pylori clumps are visible on and around the surface of the gastric epithelial cells, and some cells show stress-induced changes such as nuclear pyknosis, deep cytoplasmic staining, and cell shrinkage.

[0100] Figure 6 (A)-(C) are, in order, diagrams of cell morphology in the supernatant, bacterial suspension, and lysis supernatant treatments in the competition mode. Figure 6 As shown, all three experimental groups treated with different forms of BN-BB01 bacterial strain preparations exhibited significant anti-invasion effects: Figure 6 (A) and Figure 6 In (C), the field of view is extremely clear, the gastric epithelial cells have intact morphology and structure, close to the control group level, and almost no dark red Helicobacter pylori particles are seen in the field of view, indicating that the extracellular metabolites and intracellular active ingredients of BN-BB01 can effectively block the internalization process of Hp, preventing Hp from entering or colonizing the host cells. Figure 6(B) indicates that although scattered Hp and rod-shaped probiotic cells can be seen around the gastric epithelial cells, the degree of Hp aggregation and the amount of invasion into the gastric epithelial cells are significantly reduced compared with the model group. This suggests that the live bacteria play an anti-infection role through spatial hindrance or competitive exclusion mechanisms. Moreover, the deep-stained clumps that appear in some severely infected cells are a pathological manifestation of cell nuclear solidification.

[0101] Table 3

[0102] Note: In Table 3, different letters indicate that the differences between groups are statistically significant. p <0.05; the same letter indicates that the difference between groups is not statistically significant ( p >0.05).

[0103] In adhesion experiments, BN-BB01 exhibited a significant anti-adhesion mechanism primarily based on repulsion, particularly evident in the bacterial suspension treatment group: the inhibition rate of the bacterial suspension in the "repulsion mode" (40.26%) was significantly higher than that in the competition and displacement modes (25.97% and 26.53%, respectively), indicating that BN-BB01 can better block *Helicobacter pylori* (Hp) adhesion by pre-occupying adhesion sites to form a steric barrier, thereby achieving optimal blocking effects. Meanwhile, the supernatant showed the highest inhibitory activity in both the "repulsion" and "displacement" modes (46.01% and 44.53%, respectively), suggesting that the secretory products of BN-BB01 possess both preventative blocking potential and the ability to intervene in already adhered *Hp*, demonstrating its dual pharmacological potential for both infection prevention and colonization clearance.

[0104] In the invasion assay, BN-BB01 exhibited a significant "cell- and intracellular component-dominated" characteristic in blocking *Helicobacter pylori* (Hp) invasion of host cells: the bacterial suspension group showed high invasion inhibition rates (72.51% and 69.08%) in both "competitive mode" and "rejection mode," demonstrating that BN-BB01 can construct an efficient defense line at the source through a dual mechanism of physical barrier and receptor competition. Furthermore, the lysate supernatant group also showed comparable high inhibitory activity (72%) to the bacterial suspension in "rejection mode," indicating that the bacterial cells contain a specific anti-invasion factor that, after pre-acting on the host cell, can significantly reduce the endocytosis efficiency of *Hp*.

[0105] Combining microscopic morphological observation and quantitative analysis of adhesion inhibition rate, the lysate supernatant group showed high inhibitory activity comparable to the bacterial suspension under "rejection mode," indicating that the bacterial cells contain a specific anti-invasion factor that can pre-act on host cells to reduce the endocytosis efficiency of *Helicobacter pylori* (Hp), with a stronger anti-invasion effect than the fermentation supernatant. This result suggests that to block the deep invasion of *Hp* into gastric epithelial cells and protect host cells, it is necessary to rely not only on secreted metabolites but also on bacterial cell structural components.

[0106] Example 5 This embodiment illustrates the effects of Bifidobacterium longum BN-BB01 on the colonization of Helicobacter pylori, fatty acid metabolism, and digestive tract tissue characteristics in vivo.

[0107] Eight-week-old male SPF-grade Balb / C mice (n=10 per group, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., catalog number 8000002242) were selected and acclimatized for 7 days before model initiation. Days 8-21 were the infection modeling period. Except for the control group, the other mice were fasted for 24 hours and then administered 0.2 mL of NaHCO3 solution (0.2 mol / L) by gavage to neutralize gastric acid. Thirty minutes later, they were administered 0.2 mL of *Helicobacter pylori* suspension (concentration 5 × 10⁻⁶). 8 The fasting and gavage treatments were cycled every two days (i.e., one day without fasting, one day with gavage, and one treatment cycle was two days), lasting for two weeks. Days 22-28 were the Hp colonization period, during which mice ate water and feed normally without any additional treatment. Days 29-70 were the intervention treatment period, during which the model mice were randomly divided into the model group, the mixed antibiotic group (the mixed antibiotic was a compound solution of 0.025 μg / mL omeprazole, 0.125 μg / mL amoxicillin and 0.5 μg / mL metronidazole), the supernatant group, the bacterial suspension group, and the lysis supernatant group (the dosage of mixed antibiotic, supernatant, bacterial suspension and lysis supernatant were all 0.2 mL / mouse / day), and they ate water and feed normally.

[0108] 1. Detection of short-chain fatty acids (SCFAs) in colon contents The contents of SCFAs such as acetic acid, propionic acid, and butyric acid in colonic contents were determined by gas chromatography-mass spectrometry (GC-MS). The results are shown in Table 4, with units of μmol / g (i.e., the molar content of the corresponding fatty acids per gram of colonic contents).

[0109] Table 4

[0110] Note: In Table 4, different letters indicate that the differences between groups are statistically significant. p <0.05; the same letter indicates that the difference between groups is not statistically significant (p >0.05).

[0111] Results from the model and control groups showed that *H. pylori* infection led to a deficiency of beneficial fatty acids (acetic acid and butyric acid) and an abnormal accumulation of putrefactive fatty acids (isovaleric acid and isobutyric acid) in the mouse intestine. Intervention results showed that the supernatant exhibited superior butyric acid-promoting properties, significantly higher than the normal control group. It is speculated that the supernatant of BN-BB01 is rich in bioactive factors that promote butyric acid bacteria proliferation, and by increasing butyric acid levels, it helps repair the damaged intestinal epithelial barrier. In contrast, the bacterial suspension showed a significant advantage in inhibiting putrefactive metabolism, significantly reducing the concentrations of isovaleric acid and isobutyric acid in the intestine and effectively reversing the enhanced protein fermentation induced by *H. pylori*.

[0112] 2. Examination of digestive tract tissue characteristics Gastric tissue acquisition and fixation process: The stomach was longitudinally cut along the greater curvature. The gastric cavity was gently rinsed with PBS to remove chyme / mucus, avoiding vigorous scraping of the mucosa. The gastric tissue was laid flat on a sterile operating table. First, a 2mm wide section of pyloric tissue was cut from near the pyloric ring (to detect the relative urease activity of the gastric tissue supernatant). The remaining gastric tissue was divided into two parts longitudinally (cardia-pyloric axis): one half was used for fixation, laid flat with the mucosal side facing up and fixed in 40g / L paraformaldehyde; the other half was used for cryopreservation, the surface liquid was aspirated, and the tissue was placed in a pre-cryopreservation tube, flash-frozen in liquid nitrogen, and then transferred to... Store at 80℃ for subsequent molecular or metabolic-related assays. After fixation, gastric tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then prepared into 4-5 μm serial sections.

[0113] HE staining: The sections were routinely stained with hematoxylin and eosin (H&E) and observed under a light microscope to observe the infiltration of inflammatory cells, epithelial shedding, and structural integrity of the gastric mucosa.

[0114] Masson staining: Sections were treated with Masson's trichrome staining method, and collagen fiber deposition was observed under a microscope (collagen fibers appear blue, and muscle fibers appear red). ImageJ software was used for semi-quantitative analysis of the positive area of ​​collagen fibers to assess the degree of gastric mucosal fibrosis.

[0115] The results and analysis of the above tests are as follows: The results of HE staining and Masson staining are as follows: Figure 7 and Figure 9 As shown, where, Figure 7 and Figure 9 In the diagram, A, B, C, D, E, and F represent the staining results of the control group, model group, mixed antibiotic group, supernatant group, bacterial suspension group, and lysis supernatant group, respectively.

[0116] ImageJ was used to perform color separation on HE images to obtain the Hematoxylin channel. Within the tissue region of interest (ROI), nuclear-staining positive areas were segmented using a threshold and their areas were measured. The inflammatory infiltration area was calculated using the following formula, and the results are as follows: Figure 8 As shown in Table 5; Inflammatory infiltration area (%) = Nuclear staining positive area / Tissue area × 100 The area of ​​collagen-positive regions within the tissue ROI of the Masson image was obtained by color thresholding, and the relative collagen area (CVF) was calculated using the following formula. The results are as follows. Figure 10 As shown in Table 5.

[0117] Relative collagen area (%) = Collagen-positive area / Tissue area × 100 (1) Results of HE staining of cell morphology Figure 7 (A) shows that the gastric mucosa in the control group was structurally intact, with a continuous and dense surface epithelium, regularly arranged and clearly defined glands, and uniform glandular morphology; no obvious mucosal shedding, erosion, or necrosis was observed. There was very little inflammatory cell infiltration in the lamina propria, and the muscularis mucosae was continuous, intact, and well-preserved, suggesting that the gastric mucosa was in a normal physiological state.

[0118] Figure 7 (B) shows that the gastric mucosal structure in the model group was significantly damaged, with obvious degeneration, necrosis, and shedding of the superficial epithelial cells, resulting in an uneven mucosal surface and local defects (erosions). Large areas of mucosal shedding and focal erosions were visible. The glands were disordered, with some glands deformed, atrophied, or even broken. The lamina propria was significantly thickened, with extensive infiltration of inflammatory cells, and local congestion and edema were observed. These changes suggest that Helicobacter pylori successfully colonized and adhered to the gastric mucosal surface, inducing significant mucosal barrier disruption and inflammatory response.

[0119] Figure 7 (C) shows that, compared with the model group, the degree of gastric mucosal damage was significantly reduced in the mixed antibiotic group. Surface epithelial continuity was significantly improved, the area of ​​mucosal shedding was significantly reduced, glandular structure was more regular with only mild disordered arrangement, and the degree of inflammatory cell infiltration was significantly decreased. This indicates that mixed antibiotic intervention can effectively reduce Helicobacter pylori-related mucosal damage.

[0120] Figure 7 (D) shows that the gastric mucosa in the supernatant group had a more intact overall structure, with significantly better epithelial continuity than the model group, and only a small amount of focal mucosal shedding was observed; the glands were arranged in a basically regular manner, and the infiltration of inflammatory cells in the lamina propria was reduced. Combined with the results of the in vitro adhesion inhibition experiment, this suggests that this treatment can effectively reduce the adhesion and invasion ability of Helicobacter pylori to the gastric mucosa, thereby alleviating the tissue damage induced by it.

[0121] Figure 7(E) shows that the gastric mucosal morphology of the bacterial suspension group was further improved, with a continuous and dense surface epithelium. Although slight roughness was occasionally observed, almost no obvious mucosal sloughing or erosion foci were observed. The glandular structure was intact and its arrangement was close to that of the control group. The degree of inflammatory cell infiltration was low, suggesting that this strain / treatment has a stronger effect in protecting the gastric mucosal structure. Combined with the results of the in vitro adhesion inhibition experiment, it is suggested that its mucosal protective effect may be related to blocking the Hp adhesion process.

[0122] Figure 7 (F) shows that the gastric mucosal structure in the lysate group had neater cleft edges, less inflammatory cell infiltration in the interstitium, and more obvious recovery. The epithelial and glandular structures were intact and clear, and there were very few inflammatory cells in the lamina propria. However, the area of ​​inflammatory infiltration was still higher than that in the mixed antibiotic group, and the tissue morphology was close to that of the control group. The results indicate that this treatment has significant advantages in preventing Helicobacter pylori adhesion and maintaining the integrity of the gastric mucosal barrier.

[0123] (2) Masson staining results of cell morphology Figure 9 (A) shows that the collagen fibers in the gastric mucosa and submucosa of the control group were evenly and continuously distributed, with only a small amount of blue-stained collagen and no abnormal deposition, indicating no obvious tissue remodeling or fibrosis.

[0124] Figure 9 (B) shows that the model group had a large amount of blue-stained collagen fiber deposits in the gastric mucosa and submucosa, which were distributed in a cord-like or clump-like manner and had a disordered structure. Some areas highly overlapped with the mucosal shedding and gland destruction areas, suggesting that the matrix remodeling (fibrosis) and abnormal tissue repair response occurred under the background of chronic inflammation induced by the continuous adhesion stimulation of Helicobacter pylori.

[0125] Figure 9 (C) shows that the degree of collagen deposition was significantly reduced in the mixed antibiotic group, the blue staining area was reduced compared with the model group, and the fiber distribution was relatively regular, suggesting that the intervention can effectively reduce the inflammatory fibrotic response associated with Helicobacter pylori.

[0126] Figure 9 (D) shows that collagen fiber deposition in the supernatant group was not significantly improved. Compared with the model group, a large number of blue-stained collagen fibers were still visible in the submucosa and glandular stroma, with a relatively disordered structure. This suggests that although extracellular metabolites alone can inhibit acute inflammation, they are insufficient to reverse the pathological matrix remodeling (fibrosis) induced by chronic infection.

[0127] Figure 9 (E) shows that the area of ​​collagen stained in blue decreased in the bacterial suspension group compared with the supernatant group, and the fiber arrangement was regular and similar to that of the control group, demonstrating the specific advantage of intact live bacteria in blocking the fibrosis process.

[0128] Figure 9 (F) shows that the acute inflammatory damage of the mucosa in the lysate group was alleviated compared with the model group. However, obvious blue-stained collagen fiber deposition was still visible in the submucosa and glandular stroma, and the fiber arrangement was slightly disordered in some areas, not fully restored to a dense and orderly physiological state. This indicates that although the intracellular lysate component alone has advantages in anti-inflammatory effects, its efficacy in reversing pathological matrix remodeling (fibrosis) induced by chronic infection is relatively limited, and it cannot achieve deep repair of tissue structure like intact live bacteria.

[0129] (3) Results of inflammatory infiltration and relative collagen area Table 5

[0130] Note: In Table 5, different letters indicate that the differences between groups are statistically significant. p <0.05; the same letter indicates that the difference between groups is not statistically significant ( p >0.05).

[0131] Figure 8 The results in Table 5, showing the inflammatory infiltration area, confirm that the infection led to a widespread inflammatory response and spread of inflammation in the gastric mucosa of the model group mice. Compared with the model group, the lysate supernatant group and the bacterial suspension group showed more significant anti-inflammatory effects, with a significantly reduced infiltration area, showing a significant difference from the model group. However, the reduction in infiltration area in the supernatant group was relatively smaller, with no statistically significant difference from the model group, indicating that its efficacy in reducing the inflammatory area was weaker. This result suggests that the BN-BB01 lysate supernatant group and the bacterial suspension group are superior to the supernatant group in reducing deep inflammatory infiltration of the gastric mucosa. That is, the effect of bacterial structural components in regulating the local immune microenvironment to alleviate pathological inflammatory infiltration in local tissues is better than that of secreted metabolites.

[0132] Figure 10 The results of the relative collagen area in gastric tissue showed a significant increase in collagen deposition in the model group, suggesting that infection-related abnormal tissue repair and matrix remodeling processes were activated. Different forms of bacterial metabolites all exhibited positive intervention effects, especially the bacterial suspension group, which showed a significant intervention effect, reducing the collagen deposition area to approximately 14%. The intact live bacteria preparation had a more prominent advantage in alleviating abnormal collagen deposition / anti-fibrosis, successfully reversing pathological tissue remodeling. This result indicates that reversing gastric mucosal fibrosis depends not only on killing bacteria but also, and more importantly, on the physical occupation and colonization of intact live bacteria on the gastric mucosal surface. The physical barrier constructed by live bacteria can effectively block fibrosis signaling pathways, with better effects than metabolites alone.

[0133] In summary, after treatment, based on the quantitative results of the inflammatory infiltration area, the degree of gastric mucosal inflammation, from mild to severe, was ranked as follows: control group, mixed antibiotic group, lysate supernatant group, bacterial suspension group, supernatant group, and model group; based on the relative collagen area, the degree of gastric mucosal fibrosis, from mild to severe, was ranked as follows: control group, mixed antibiotic group, bacterial suspension group, supernatant group, lysate supernatant group, and model group.

[0134] Combined HE and Masson staining results showed that Helicobacter pylori infection significantly disrupts the gastric mucosal barrier, inducing mucosal shedding, glandular structural disorder, inflammatory cell infiltration, and abnormal collagen deposition. Different treatments with BN-BB01 improved these pathological changes to varying degrees, with the most effective treatment group significantly reducing mucosal shedding and fibrosis, and promoting the restoration of gastric mucosal structure to near-normal conditions. These results suggest that BN-BB01-related interventions may exert a synergistic mucosal protective effect through multiple pathways and are related to reducing infection-related mucosal damage and abnormal repair processes. Combined with the aforementioned in vitro adhesion / invasion inhibition results, its protective effect is closely related to inhibiting the adhesion and colonization of Helicobacter pylori on the gastric mucosal surface, thereby indirectly reducing bacterial-mediated inflammatory damage and promoting tissue remodeling.

[0135] Example 6 This example illustrates the in vitro inhibitory effect of Bifidobacterium longum BN-BB01 on urease activity.

[0136] Urease indicator formulation: 0.9% by weight NaCl, 20 mmol / L urea, 14 μg / mL phenol red. Adjust pH to 6.8 (the color change point of phenol red) with HCl. Fresh *Helicobacter pylori* (Hp) was washed twice with PBS and then prepared to a concentration of 1 × 10⁻⁶ on Brain Heart Infusion Agar (BHI) medium. 7 CFU / mL *H. pylori* bacterial suspension was added to each well of a 96-well plate. 40 μL of *H. pylori* and 10 μL of the BN-BB01 strain preparation prepared in Example 1 were added and mixed thoroughly. The plate was then incubated at 37°C under microaerophilic conditions (the apparatus and environment were the same as in Example 2) for 48 h. Then, 150 μL of urease indicator was added to each well. After color change, the plate was shaken to mix thoroughly and the absorbance (OD) at 550 nm was immediately measured using a microplate reader. 样品 Additionally, a blank control group was set up in which urease indicator was added only to 96 wells (the absorbance OD was measured). 空白 ) and the model group in which the strain preparation was replaced with BHI (absorbance OD was measured). 模型 The urease inhibition rate was calculated using the following formula. The results of the urease inhibition rate calculation are shown in Table 6.

[0137] Relative activity of urease (%) = (OD) 样品 -OD 空白 ) / (OD 模型-OD 空白 ) × 100% Table 6

[0138] Note: In Table 6, different letters indicate that the differences between groups are statistically significant. p <0.05; the same letter indicates that the difference between groups is not statistically significant ( p >0.05).

[0139] The neutral supernatant group exhibited significant urease inhibitory activity, indicating that the active component of this strain that inhibits *Helicobacter pylori* urease can do so independently of acidic organic acids and may also possess specific extracellular metabolites adapted to neutral environments. This characteristic enables it to penetrate the gastric acid barrier and exert a sustained inhibitory effect deep within the gastric mucus layer (near a neutral environment) where *Helicobacter pylori* colonizes, thereby depriving *Helicobacter pylori* of its acid-adaptive survival ability at its source.

[0140] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Bifidobacterium longum ( Bifidobacterium longum ), characterized in that, The preservation number of this Bifidobacterium longum is CGMCC No. 34432.

2. A microbial agent, characterized in that, The inoculum contains the Bifidobacterium longum as described in claim 1 and / or the fermentation broth of the Bifidobacterium longum as described in claim 1; The fermentation broth includes at least one of the following: Supernatant: Bifidobacterium longum CGMCC No.34432 was inoculated into MRS liquid medium and cultured anaerobically at 35-39℃ until the logarithmic or stationary growth phase. The bacterial cells were removed by filtration or centrifugation, and the remaining liquid was the supernatant. Neutral supernatant: Adjust the pH of the supernatant to 6.8-7.2; Lysis supernatant: Bifidobacterium longum CGMCC No.34432 was inoculated into MRS liquid medium and cultured anaerobically at 35-39℃ to the logarithmic or stationary growth phase. The bacterial cells were lysed, and then centrifuged or filtered to remove bacterial fragments. The remaining liquid portion is the lysis supernatant.

3. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the Bifidobacterium longum as described in claim 1, or the bacterial agent as described in claim 2.

4. The use of the Bifidobacterium longum of claim 1, or the bacterial agent of claim 2, or the pharmaceutical composition of claim 3 in the preparation of a medicament for the prevention and / or treatment of diseases or symptoms caused by Helicobacter pylori infection.

5. The application according to claim 4, wherein, The disease or condition mentioned includes damage to the digestive tract mucosa caused by Helicobacter pylori infection.

6. A method for inhibiting the proliferation of Helicobacter pylori in vitro, characterized in that, The method includes contacting the Bifidobacterium longum of claim 1, or the bacterial agent of claim 2, or the pharmaceutical composition of claim 3 with Helicobacter pylori cultured in vitro.

7. A method for inhibiting the colonization of Helicobacter pylori in in vitro cultures, characterized in that, The method includes contacting the in vitro culture with the Bifidobacterium longum of claim 1, or the bacterial agent of claim 2, or the pharmaceutical composition of claim 3, thereby inhibiting the colonization of Helicobacter pylori in the in vitro culture.

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