Bifidobacterium longum subsp. Longum strain for inhibiting helicobacter pylori and preparation method thereof

Abstract

The invention discloses a bifidobacterium longum subsp. Longum strain for inhibiting helicobacter pylori and a preparation method, the strain is bifidobacterium longum subsp. Longum Bi-PG-14, the classification name of the strain is Bifidobacterium longum subsp. Longum, the preservation number is CGMCC No.31862, the preservation time is September 4, 2024, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC No.31862, the preservation number is CGMCC No.31862, the preservation number is CGMCC No.31862, the preservation number is CGMCC No.31862, the preservation number is CGMCC No.31862, the preservation number is CGMCC No.31862, and the preservation number is CGMCC No.31862. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bifidobacterium longum subsp. Longum Bi-PG-14 is separated from faeces of healthy babies, has the function of inhibiting helicobacter pylori and has a certain antibacterial effect on pathogenic bacteria such as staphylococcus aureus, bacillus subtilis and escherichia coli, so that the strain with the function of inhibiting the helicobacter pylori is screened out, and the strain can be used for preparing a medicine for preventing and treating the pathogenic bacteria such as the staphylococcus aureus, the bacillus subtilis and the escherichia coli. The method is of great significance to improvement of body health, and provides high-quality strain resources for the probiotic industry.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori and its preparation method. Background Technology

[0002] Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric epithelium and is one of the pathogenic factors for gastric diseases such as chronic gastritis and peptic ulcers. Currently, the main treatments for eradicating Helicobacter pylori are triple and quadruple therapy. However, due to the overuse of antibiotics, Helicobacter pylori resistance is constantly increasing, which greatly affects the efficacy of treatments. Therefore, it is particularly important to find safe treatments with fewer side effects.

[0003] Probiotics are important beneficial bacteria in the human and animal body. They can regulate intestinal balance, alleviate colitis symptoms, enhance immunity, and exert antibacterial and anti-inflammatory effects while improving the gut microbiota. When these microorganisms enter the human body, they may temporarily reside in the sites occupied by Helicobacter pylori, thereby inhibiting the infection of uncolonized Helicobacter pylori. Therefore, it is feasible to treat Helicobacter pylori infection using oral probiotics.

[0004] Existing strains of Bifidobacterium longum are unable to inhibit Helicobacter pylori in the human stomach. When using antibiotics to treat Helicobacter pylori, it is easy to lead to increased drug resistance in Helicobacter pylori, which affects the treatment effect. Summary of the Invention

[0005] The purpose of this invention is to provide a long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori and its preparation method. This strain has the advantages of strong resistance to acid, alkali, and bile salts, strong resistance to simulated gastrointestinal fluid, and strong inhibition of Helicobacter pylori. It solves the problem that existing long subspecies of Bifidobacterium longum strains are difficult to inhibit Helicobacter pylori in the human stomach, and that the use of antibiotics to treat Helicobacter pylori can easily lead to increased drug resistance of Helicobacter pylori, thus affecting the treatment effect.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a strain of Bifidobacterium longum subsp. Longum that inhibits Helicobacter pylori. This strain is Bi-PG-14, classified as Bifidobacterium longum subsp. Longum, with accession number CGMCC No. 31862, accession date of September 4, 2024, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] A method for preparing a long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori includes the following steps:

[0008] S1. Dilute fecal samples with physiological saline and spread them on modified MRS solid medium plates containing 0.05 g / L cysteine ​​hydrochloride and 0.05 g / L mupirocin lithium. Incubate at 37°C for 48 h in anaerobically. Select single colonies with different morphologies and sizes from the plates and streak them multiple times on the modified MRS solid medium for purification until they are identified as single colonies. Select single colonies for microscopic examination, physiological and biochemical analysis, and 16S RNA homology comparison. Preserve the isolated strains in glycerol.

[0009] S2. Fully activate and expand the Bifidobacterium longum subsp. Bi-PG-14, which has been cryopreserved with glycerol;

[0010] S3. Inoculate the fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial suspension onto Columbia blood agar medium and perform a hemolysis test.

[0011] S4. The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different pH values ​​and cultured at 37℃ for 24 h to conduct acid resistance tests on the strain.

[0012] S5. The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different salt concentrations and cultured at 37℃ for 24 h to conduct salt tolerance test of the strain.

[0013] S6. The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different bile salt concentrations and cultured at 37℃ for 24 h to conduct a bile salt tolerance test of the strain.

[0014] S7. Centrifuge the fully activated and expanded Bifidobacterium longum subsp. longum Bi-PG-14 bacterial solution, collect the bacterial sludge, and prepare a bacterial suspension with physiological saline for naked bacterial simulated gastrointestinal test.

[0015] S8. After activating the indicator bacteria, they were inoculated into LB liquid medium. After activating Bifidobacterium longum subsp. longum Bi-PG-14, they were inoculated into MRS liquid medium. After culturing at 37℃ for 24h, the antibacterial test of Bifidobacterium longum subsp. longum Bi-PG-14 was carried out.

[0016] S9. The agar diffusion method was used to measure the antibacterial activity of the strain against Helicobacter pylori.

[0017] S10. The urease activity of the strain was determined by colorimetric method to measure the urease activity of Bi-PG-14 subsp. longum.

[0018] In a preferred embodiment of the method for preparing the Bifidobacterium longum subsp. longum strain that inhibits Helicobacter pylori according to the present invention, the fecal sample in S1 is infant feces, and the Bifidobacterium longum subsp. longum strain Bi-PG-14 is isolated from the infant feces.

[0019] In a preferred embodiment of the method for preparing the Bifidobacterium longum subsp. longum strain that inhibits Helicobacter pylori according to the present invention, when the Bifidobacterium longum subsp. longum Bi-PG-14 in S2 is activated, it is inoculated into sterile MRS liquid culture medium containing 0.5 g / L L-cysteine, and first cultured anaerobically at 37°C for 24 h, and then cultured anaerobically at 37°C for another 24 h to fully activate the Bifidobacterium longum subsp. longum Bi-PG-14.

[0020] In a preferred embodiment of the method for preparing the Bifidobacterium longum subsp. longum strain that inhibits Helicobacter pylori according to the present invention, in step S3, when the Bifidobacterium longum subsp. longum Bi-PG-14 bacterial suspension is inoculated onto Columbia blood agar medium, it is incubated in a constant temperature incubator at 37°C for 24 hours, and then the hemolysis phenomenon around the colony is observed.

[0021] In a preferred embodiment of the method for preparing the *Bifidobacterium longum* subsp. *longum* strain that inhibits *Helicobacter pylori* according to the present invention, during the acid resistance test of the strain in S4, the pH value of the MRS medium is adjusted with HCl at a concentration of 4.0 mol / L, and multiple MRS mediums with different pH values ​​are prepared, namely 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0. The strain is inoculated into the medium with different pH values, and after being cultured at 37°C for 24 h, the OD600 nm value of the bacterial suspension is measured, its survival rate is calculated, and the acid resistance ability of *Bifidobacterium longum* subsp. *longum* Bi-PG-14 is determined.

[0022] In a preferred embodiment of the method for preparing the *Bifidobacterium longum* subsp. *longum* strain that inhibits *Helicobacter pylori* according to the present invention, during the salt tolerance test of the strain in S5, the strain is inoculated into MRS medium containing salt concentrations of 1, 2, 3, 4, and 5 g / 100 mL at a 3% inoculation rate. After culturing at 37°C for 24 h, the OD600 nm value of the bacterial suspension is measured. The OD value of the strain in MRS medium without added salt is used as a control. The survival rate is calculated, and the effect of salt on the activity of *Bifidobacterium longum* subsp. *longum* Bi-PG-14 is determined.

[0023] In a preferred embodiment of the method for preparing the *Bifidobacterium longum* subsp. *longum* strain that inhibits *Helicobacter pylori* according to the present invention, during the bile salt tolerance test in step S6, the strain is inoculated at a 3% inoculum into MRS medium containing bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively. After culturing at 37°C for 24 hours, the OD600 nm value of the bacterial suspension is measured. The OD value of the strain in MRS medium without bile salt is used as a control. The survival rate is calculated, and the effect of bile salt on the Bi-PG-14 activity of *Bifidobacterium longum* subsp. *longum* is determined.

[0024] In a preferred method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to the present invention, during the simulated gastrointestinal test of the naked bacteria in step S7, the strain is activated and passaged twice in MRS medium at 37°C. After centrifugation, 1.0 g of bacterial sludge is added to 5.0 mL of artificial gastric fluid. After treatment at 37°C for 3 hours, the precipitate is collected under aseptic conditions. According to the ratio of 1.0 g bacterial sludge to 5.0 mL physiological saline, sterile physiological saline is added, mixed thoroughly, diluted, and spread. Colony counting is performed. Then, 1.0 g of artificial gastric fluid is taken... After treatment with the simulated gastrointestinal tract, the bacterial sludge was added to 5.0 mL of artificial bile and treated at 37°C for 20 min. After centrifugation at 8000 r / min for 10 min, the bacterial sludge was collected and colony counted. Then, 1.0 g of the bacterial sludge treated with artificial bile was added to 5.0 mL of artificial intestinal fluid and treated at 37°C for 4 or 8 h respectively. After centrifugation at 8000 r / min for 10 min, the bacterial sludge was collected and colony counted. The colony count in MRS medium without simulated gastrointestinal tract treatment was used as a control to calculate the survival rate of the strain.

[0025] In a preferred embodiment of the method for preparing the *Bifidobacterium longum* subsp. *longum* strain that inhibits *Helicobacter pylori* according to the present invention, during the antibacterial test of *Bifidobacterium longum* subsp. *longum* Bi-PG-14 in S8, the Oxford cup agar diffusion method is used. *Staphylococcus aureus*, *Bacillus subtilis*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Listeria monocytogenes*, and *Salmonella* are used as indicator bacteria for plate spreading. Physiological saline is used as a blank control. Four Oxford cups are evenly placed on each plate. 200 μL of *Bifidobacterium longum* subsp. *longum* Bi-PG-14 bacterial suspension is added to three Oxford cups, and physiological saline is added to one Oxford cup. After incubation at 37°C for 24 h, the size of the inhibition zone is measured with calipers.

[0026] In a preferred embodiment of the method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to the present invention, when measuring the antibacterial activity of the strain against *Helicobacter pylori* using the agar diffusion method in step S9, a 0.05 mg / mL metronidazole solution is used as a positive control, and MRS liquid culture medium is used as a blank control. 100 μL of *Helicobacter pylori* bacterial suspension is spread on a Columbia blood agar plate without antibiotics, and 100 μL of the test liquid is added to each well. The test liquids are lactic acid bacteria bacterial suspension, positive control, and blank control, respectively. The plates with fermentation broth are placed in a microaerophilic environment at 37°C for 72-96 h. After the culture is completed, the diameter of the inhibition zone is measured with vernier calipers.

[0027] In a preferred embodiment of the method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to the present invention, when determining the urease activity of the strain by colorimetry in step S10, 40 μL of *Helicobacter pylori* bacterial suspension is mixed with 10 μL of bacterial suspension, and 10 μL of sterile liquid culture medium is used as a control. The mixture is added to a clean, sterile 96-well plate and incubated at 37°C in a microaerophilic environment for 48 h. The incubated mixture is then removed, and 150 mL of urease reagent is added to each well. The color change is observed and the OD550 value is measured. The urease reagent includes 0.9% NaCl, 20 mmol / L urea, and 4 μg / mL phenol red. The pH is then adjusted to 6.8 with HCl.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The Bi-PG-14 subspecies of Bifidobacterium longum of the present invention was isolated from the feces of healthy infants. This strain has the function of inhibiting Helicobacter pylori and has a certain antibacterial effect on pathogenic bacteria such as Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Therefore, screening out strains with the function of inhibiting Helicobacter pylori is of great significance for improving the body's health and provides high-quality strain resources for the probiotic industry.

[0030] 2. The Bifidobacterium long subspecies Bi-PG-14 prepared by this invention has good resistance to acid, salt, bile salt and simulated gastrointestinal fluid. When it enters the human body from the human body, it can reach the intestine with a high survival rate.

[0031] 3. The Bi-PG-14 subsp. longissimus prepared in this invention has the function of inhibiting Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella. The diameters of the inhibition zones are 18.46±0.04, 22.80±0.05, 25.66±0.05, 16.06±0.10, 21.46±0.05 and 20.46±0.04, respectively, showing strong antibacterial ability.

[0032] 4. The Bi-PG-14 subspecies of Bifidobacterium longum provided by the present invention has the function of inhibiting Helicobacter pylori, with an inhibition zone diameter of 23.06±0.14 and an inhibition rate of 88.71%, demonstrating a significant ability to inhibit Helicobacter pylori. Attached Figure Description

[0033] Figure 1 Image showing the colony morphology and Gram staining results of Bi-PG-14 plate of Bifidobacterium longum subsp. longum;

[0034] Figure 2 The image shows the results of the hemolysis test for Bi-PG-14 subsp. longibracte.

[0035] Figure 3 A graph showing the acid resistance of Bifidobacterium longum subsp. Bi-PG-14;

[0036] Figure 4 Diagram showing the salt tolerance of Bifidobacterium longum subsp. Bi-PG-14;

[0037] Figure 5 A diagram showing the bile salt tolerance of Bifidobacterium longum subsp. Bi-PG-14;

[0038] Figure 6 A graph showing the tolerance of Bifidobacterium longum subsp. longum Bi-PG-14 to simulated gastrointestinal fluid.

[0039] Figure 7 Figure 1 shows the results of the test on the inhibition of pathogenic bacteria by the supernatant of fermentation broth of Bifidobacterium longum subsp. longum Bi-PG-14.

[0040] Figure 8 Figure 1 shows the results of the experiment on the inhibition of Helicobacter pylori by fermentation broth of Bi-PG-14 subsp. longibrachium.

[0041] Figure 9 Graph showing the antibacterial activity test data of Bi-PG-14 subsp. longum;

[0042] Figure 10 The graph shows the inhibition rate and urease activity data of Bi-PG-14 of Bifidobacterium longum subsp. longum against Helicobacter pylori.

[0043] Figure 11 This is a comparison chart showing the ability of other probiotic strains to inhibit Helicobacter pylori. Detailed Implementation

[0044] Example 1

[0045] A strain of Bifidobacterium longum subsp. Longum that inhibits Helicobacter pylori was discovered. The strain is Bi-PG-14, classified as Bifidobacterium longum subsp. Longum, with accession number CGMCC No. 31862, deposited on September 4, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] This strain was isolated and purified from the feces of healthy infants. The strain has a strong ability to inhibit Helicobacter pylori, with an inhibition zone diameter of 23.06 mm or more. In addition, the strain has strong resistance to acid, salt, bile salts and artificial simulated gastrointestinal fluid, and possesses the characteristics of probiotics.

[0047] Example 2

[0048] A method for preparing a long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori includes the following steps:

[0049] First, 1g of fecal sample was diluted with 9mL of sterile physiological saline. After shaking and mixing, it was further diluted 10-fold with sterile physiological saline. Different dilutions were then spread on modified MRS solid medium plates containing 0.05g / L cysteine ​​hydrochloride and 0.05g / L mupirocin lithium salt and cultured anaerobically at 37℃ for 48h.

[0050] Furthermore, the fecal sample was infant feces, and the long subspecies of Bifidobacterium longum Bi-PG-14 strain was isolated from the infant feces.

[0051] Furthermore, single colonies of different morphologies and sizes were picked from the plates and purified by streaking multiple times on modified MRS solid medium until they were identified as single colonies. Single colonies were then selected for microscopic examination, physiological and biochemical analysis, and 16S RNA homology comparison. The isolated strains were then cryopreserved in glycerol.

[0052] Furthermore, during microscopic examination, single colonies were picked and inoculated into MRS broth medium containing 0.5 g / L L-cysteine. After culturing for 24 hours, Gram staining was performed, and the bacterial solution was smeared. After Gram staining, the cell morphology and arrangement were observed under an optical microscope. Strains that conformed to the typical morphology of Bifidobacterium were selected by microscopic observation, and the colony morphology was photographed and recorded.

[0053] Furthermore, during physiological and biochemical tests, hydrogen peroxide test, indole test, and carbohydrate utilization physiological and biochemical tests are used to identify and screen bacterial strains.

[0054] Further reference Figure 1The isolated and purified strain is milky white with a raised center and a smooth, moist surface. It has a diameter of 1.02 ± 0.02 mm. The strain is Gram-positive, and the cells are short rod-shaped and arranged in pairs. Its physicochemical characteristics are negative for hydrogen peroxide and positive for indole reaction. It is positive for aesculin hydrolysis test and can ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0055] Furthermore, during 16SRNA homology comparison, the bacterial strain was expanded and cultured, the bacterial solution was centrifuged to obtain bacterial cells, and sequencing was performed. The sequencing was completed by Nanjing Paisennong Gene Technology Co., Ltd. The rDNA sequence of Bifidobacterium longum subsp. Bi-PG-14 is as follows:

[0056]

[0057] Example 3

[0058] Activation of Bifidobacterium longum subspecies Bi-PG-14 includes the following steps:

[0059] First, the Bifidobacterium longum subsp. Bi-PG-14, which was cryopreserved with glycerol, was fully activated and expanded. When Bifidobacterium longum subsp. Bi-PG-14 was activated, it was inoculated into sterile MRS liquid medium containing 0.5 g / L L-cysteine. It was first cultured anaerobically at 37°C for 24 h, and then cultured anaerobically at 37°C for another 24 h to fully activate Bifidobacterium longum subsp. Bi-PG-14.

[0060] Example 4

[0061] The hemolysis test for Bi-PG-14, a subspecies of Bifidobacterium longum, includes the following steps:

[0062] The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated onto Columbia blood agar medium for hemolysis test.

[0063] Furthermore, when the Bi-PG-14 subsp. longum bacterial suspension was inoculated onto Columbia blood agar medium, it was incubated in a constant temperature incubator at 37°C for 24 hours, and then the presence of hemolysis around the colonies was observed.

[0064] Furthermore, the presence of a translucent hemolysis ring on the plate indicates α-hemolysis, while the presence of a clearly defined, completely transparent hemolysis ring indicates β-hemolysis. The absence of a hemolysis ring indicates γ-hemolysis, i.e., no hemolysis.

[0065] refer to Figure 2 The results show the detection results of Bifidobacterium longum subsp. Bi-PG-14, with Staphylococcus aureus as the control strain (left image). Bifidobacterium longum subsp. Bi-PG-14 (right image) does not hemolyze and is a safe strain.

[0066] Example 5

[0067] When performing acid resistance testing on Bi-PG-14, a strain of Bifidobacterium longum subsp. longum, the following steps are included:

[0068] The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different pH values ​​and cultured at 37℃ for 24 h to conduct acid resistance tests on the strain.

[0069] Furthermore, during the acid tolerance test of the strain, the pH of the MRS medium was adjusted with 4.0 mol / L HCl, and multiple MRS media with different pH values ​​were prepared: 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0. The strain was inoculated into the media with different pH values ​​and cultured at 37°C for 24 h. The OD600nm value of the bacterial suspension was measured, with three replicates. The OD value of the MRS medium (pH 6.0) was used as a control. The survival rate was calculated to determine the acid tolerance of Bifidobacterium longum subsp. Bi-PG-14.

[0070] That is, survival rate = Nt / N0

[0071] In the formula: Nt is the OD value of MRS medium with pH values ​​of 1.0-5.0;

[0072] N0 is the OD value of the MRS medium at pH 6.0.

[0073] refer to Figure 3 ,from Figure 3 It can be seen that *Bifidobacterium longum* subsp. *bi-PG-14* exhibits poor tolerance at pH 1.0, with a survival rate of only 4.54% after 24 hours of treatment. However, it demonstrates strong tolerance at pH 4.0, with a survival rate as high as 63.46% after 24 hours of treatment. This indicates that the bacterium has a strong ability to tolerate acidic conditions.

[0074] Example 6

[0075] When performing a salt tolerance test on Bi-PG-14, a strain of Bifidobacterium longum subsp. longum, the following steps are included:

[0076] The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different salt concentrations and cultured at 37℃ for 24 h to conduct salt tolerance tests on the strain.

[0077] Furthermore, during the salt tolerance test, the strain was inoculated at a 3% inoculum into MRS medium containing salt concentrations of 1, 2, 3, 4, and 5 g / 100 mL, respectively. After culturing at 37°C for 24 h, the OD600nm value of the bacterial suspension was measured. The OD value of the strain in MRS medium without salt was used as a control. The survival rate was calculated, and the effect of salt on the activity of Bifidobacterium longum subsp. longum Bi-PG-14 was determined.

[0078] Furthermore, three replicates were set up, with the OD value of MRS medium as a control, and the survival rate was calculated, i.e., survival rate = Nt / N0;

[0079] In the formula: Nt is the OD value of MRS medium with sodium chloride concentrations of 1.0-5.0 g / mL;

[0080] N0 is the OD value of MRS medium when the sodium chloride concentration is 0 g / mL.

[0081] refer to Figure 4 The salt tolerance of the strains was tested by selecting sodium chloride concentrations ranging from 1.0 to 5.0 g / mL. Figure 4 It can be seen that the survival rate of Bifidobacterium longum subspecies Bi-PG-14 gradually decreased with increasing salt concentration, but after culturing for 24 hours at a salt concentration of 5 g / mL, its survival rate was still 41.27%, indicating that the bacterium has a strong ability to tolerate salt conditions.

[0082] Example 7

[0083] When performing a bile salt tolerance test on Bi-PG-14, a strain of *Bifidobacterium longum* subsp. *longum*, the following steps are included:

[0084] The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different bile salt concentrations and cultured at 37℃ for 24 h to conduct bile salt tolerance tests on the strain.

[0085] Furthermore, in the bile salt tolerance test, the strain was inoculated at a 3% inoculum into MRS medium containing bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively. After incubation at 37°C for 24 h, the OD600nm value of the bacterial suspension was measured. The OD value of the strain in MRS medium without bile salt was used as a control. The survival rate was calculated, and the effect of bile salt on the activity of Bifidobacterium longum subsp. longum Bi-PG-14 was determined.

[0086] Furthermore, three replicates were set up, with the OD value of MRS medium without bile salts as a control, and the survival rate of the strain was calculated, i.e., survival rate = Nt / N0;

[0087] In the formula: Nt is the number of viable bacteria in MRS culture medium containing different concentrations of bile salts;

[0088] N0 represents the number of viable bacteria in MRS medium without bile salts.

[0089] refer to Figure 5 In this study, five different concentrations of bile salts (0.1%, 0.3%, 0.5%, 0.7%, and 0.9%) were used to treat *Bifidobacterium longum* subsp. *bifidobacterium* Bi-PG-14 strain for 24 hours (with the strain cultured without bile salts as a control). Figure 5 It can be seen that as the bile salt concentration increases, the survival rate of the strain gradually decreases. After 24 hours of treatment with 0.5% bile salt, the survival rate is still 33.97%, indicating that Bifidobacterium longum subsp. Bi-PG-14 has good bile salt tolerance.

[0090] Example 8

[0091] A naked microbial gastrointestinal assay was performed on strain Bi-PG-14 of *Bifidobacterium longum* subsp. *longum*, including the following steps:

[0092] The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was centrifuged, the bacterial sludge was collected, and it was prepared into a bacterial suspension with physiological saline for naked bacterial simulated gastrointestinal experiment.

[0093] Furthermore, in the simulated gastrointestinal tract test of naked bacteria, the strain was activated and passaged twice in MRS medium at 37°C. After centrifugation, 1.0g of bacterial sludge was added to 5.0mL of simulated gastric fluid and treated at 37°C for 3 hours. Under aseptic conditions, the precipitate was collected, and sterile physiological saline was added according to the ratio of 1.0g bacterial sludge to 5.0mL physiological saline. The mixture was diluted, spread, and colony counts were performed. Then, 1.0g of bacterial sludge treated with simulated gastric fluid was added to 5.0mL of simulated bile and treated at 37°C for 20 minutes. After centrifugation at 8000r / min for 10 minutes, the bacterial sludge was collected and colony counts were performed. Then, 1.0g of bacterial sludge treated with simulated bile was added to 5.0mL of simulated intestinal fluid and treated at 37°C for 4 or 8 hours respectively. After centrifugation at 8000r / min for 10 minutes, the bacterial sludge was collected and colony counts were performed. The colony count in MRS medium without simulated gastrointestinal tract treatment was used as a control to calculate the survival rate of the strain.

[0094] Furthermore, the survival rate of artificial gastrointestinal fluid = Nt / N0

[0095] In the formula: Nt is the number of viable bacteria after artificial treatment with simulated gastric juice, bile, and intestinal juice;

[0096] N0 represents the number of viable bacteria before treatment.

[0097] refer to Figure 6 ,Depend on Figure 6 It can be seen that after 3 hours of treatment with gastric juice, the survival rate of Bifidobacterium longum subsp. Bi-PG-14 was 72.68%, with a viable count of 14.42±0.20lg(cfu / g). After 20 minutes of treatment with bile, the survival rate was 68.38%, with a viable count of 13.57±0.31lg(cfu / g). After 4 hours of treatment with intestinal juice, the survival rate was still 60.20%, with a viable count of 11.94±0.15lg(cfu / g). This shows that Bifidobacterium longum subsp. Bi-PG-14 has a strong ability to tolerate artificial simulated gastrointestinal juice.

[0098] Example 9

[0099] An antibacterial test was performed on strain Bi-PG-14 of *Bifidobacterium longum* subsp. *longum*, including the following steps:

[0100] The indicator bacteria were activated and inoculated into LB liquid medium, and Bifidobacterium longum subsp. longum Bi-PG-14 was activated and inoculated into MRS liquid medium. After being cultured at 37℃ for 24 hours, the antibacterial test of Bifidobacterium longum subsp. longum Bi-PG-14 was carried out.

[0101] Furthermore, in the antibacterial test of Bifidobacterium longum subsp. longum Bi-PG-14, the Oxford cup agar diffusion method was used. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella were used as indicator bacteria for plate spreading. Physiological saline was used as a blank control. Four Oxford cups were evenly placed on each plate. 200 μL of Bifidobacterium longum subsp. longum Bi-PG-14 bacterial suspension was added to three Oxford cups, and physiological saline was added to one Oxford cup. After incubation at 37°C for 24 h, the size of the inhibition zone was measured with vernier calipers.

[0102] from Figure 7 and Figure 9 It can be seen that inhibition zones appeared in all the petri dishes, and the diameter of the inhibition zones all reached more than 16.06 μm, indicating that Bifidobacterium longum subsp. longum Bi-PG-14 has an inhibitory effect on the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella typhimurium. The diameters of the inhibition zones were 18.46±0.04, 22.80±0.05, 25.66±0.05, 16.06±0.10, 21.46±0.05 and 20.46±0.04, respectively, indicating that Bifidobacterium longum subsp. longum Bi-PG-14 has a significant antibacterial effect and strong antibacterial ability.

[0103] Example 10

[0104] The antibacterial activity test of Bi-PG-14, a strain of Bifidobacterium longum subsp. longum, against Helicobacter pylori included the following steps:

[0105] The antibacterial activity of the strain against Helicobacter pylori was measured using the agar diffusion method.

[0106] Furthermore, when measuring the antibacterial activity of the strain against Helicobacter pylori using the agar diffusion method, a 0.05 mg / mL metronidazole solution was used as a positive control, and MRS liquid medium was used as a blank control. 100 μL of Helicobacter pylori suspension was spread on an antibiotic-free Columbia blood agar plate, and 100 μL of the test liquid was added to each well. The test liquids were lactic acid bacteria suspension, positive control, and blank control, respectively. The plates with fermentation broth were placed in a microaerophilic environment at 37°C for 72-96 h. After the culture was completed, the diameter of the inhibition zone was measured with vernier calipers.

[0107] The formula for calculating the inhibition rate of lactic acid bacteria against Helicobacter pylori is as follows:

[0108] Helicobacter pylori inhibition rate (%) = Rx / Ry × 100

[0109] In the formula: Rx represents the diameter of the inhibition zone of lactic acid bacteria against Helicobacter pylori, and Ry represents the diameter of the inhibition zone of the positive control against Helicobacter pylori.

[0110] from Figure 8 and Figure 10 As can be seen, inhibition zones appeared in the petri dish, and the diameter of the inhibition zones was measured to be over 23.06 mm. The inhibition rate of Helicobacter pylori was 88.71%, indicating that Bifidobacterium longum subsp. Bi-PG-14 has a strong inhibitory effect on the growth of Helicobacter pylori.

[0111] The results of comparisons with other probiotic strains on their inhibitory effects on Helicobacter pylori are listed below. Figure 11 ,Depend on Figure 11 It can be seen that the inhibitory effect of Bifidobacterium longum subspecies Bi-PG-14 on Helicobacter pylori is significantly higher than that of other common probiotic strains, indicating that Bifidobacterium longum subspecies Bi-PG-14 is an excellent strain with strong inhibitory function against Helicobacter pylori.

[0112] Example 11

[0113] The determination of urease activity in Bi-PG-14 subsp. longum includes the following steps:

[0114] The urease activity of the strain was determined by colorimetry to measure the urease activity of Bi-PG-14 subsp. longum.

[0115] Furthermore, when determining the urease activity of the strain using a colorimetric method, 40 μL of Helicobacter pylori bacterial suspension was mixed with 10 μL of bacterial suspension, with 10 μL of sterile liquid culture medium as a control. The mixture was added to a clean, sterile 96-well plate and incubated at 37°C in a microaerophilic environment for 48 h. After incubation, 150 mL of urease reagent was added to each well, and the color change was observed and the OD value was measured. 550 The urease reagent consists of 0.9% NaCl, 20 mmol / L urea, and 4 μg / mL phenol red, and is then adjusted to pH 6.8 with HCl.

[0116] Furthermore, a key characteristic of Helicobacter pylori is its ability to produce highly active urease. Infection with Helicobacter pylori stimulates the body's immune system to produce urease antibodies. The activity of Helicobacter pylori can be determined through a urease test. Figure 10It can be seen that when *Bifidobacterium longum* subsp. *longum* Bi-PG-14 was co-cultured with *Helicobacter pylori*, the urease activity of *Helicobacter pylori* showed a decreasing trend compared with the group without added lactic acid bacteria, indicating that *Bifidobacterium longum* subsp. *longum* Bi-PG-14 has an inhibitory effect on urease activity.

[0117] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori, characterized in that: The strain is Bi-PG-14, a subsp. longum of Bifidobacterium longum. Its classification name is Bifidobacterium longum subsp. Longum, its accession number is CGMCC No. 31862, its accession date is September 4, 2024, its depositary is the China General Microbiological Culture Collection Center, and its deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for preparing a long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori, applicable to the long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori as described in claim 1, characterized in that, Includes the following steps: S1. Dilute fecal samples with physiological saline and spread them on modified MRS solid medium plates containing 0.05 g / L cysteine ​​hydrochloride and 0.05 g / L mupirocin lithium. Incubate at 37°C for 48 h in anaerobically. Select single colonies with different morphologies and sizes from the plates and streak them multiple times on the modified MRS solid medium for purification until they are identified as single colonies. Select single colonies for microscopic examination, physiological and biochemical analysis, and 16S RNA homology comparison. Preserve the isolated strains in glycerol. S2. Fully activate and expand the Bifidobacterium longum subsp. Bi-PG-14, which has been cryopreserved with glycerol; S3. Inoculate the fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial suspension onto Columbia blood agar medium and perform a hemolysis test. S4. The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different pH values ​​and cultured at 37℃ for 24 h to conduct acid resistance tests on the strain. S5. The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different salt concentrations and cultured at 37℃ for 24 h to conduct salt tolerance test of the strain. S6. The fully activated and expanded Bifidobacterium longum subsp. Bi-PG-14 bacterial culture was inoculated into MRS medium with different bile salt concentrations and cultured at 37℃ for 24 h to conduct a bile salt tolerance test of the strain. S7. Centrifuge the fully activated and expanded Bifidobacterium longum subsp. longum Bi-PG-14 bacterial solution, collect the bacterial sludge, and prepare a bacterial suspension with physiological saline for naked bacterial simulated gastrointestinal test. S8. After activating the indicator bacteria, they were inoculated into LB liquid medium. After activating Bifidobacterium longum subsp. longum Bi-PG-14, they were inoculated into MRS liquid medium. After culturing at 37℃ for 24h, the antibacterial test of Bifidobacterium longum subsp. longum Bi-PG-14 was carried out. S9. The agar diffusion method was used to measure the antibacterial activity of the strain against Helicobacter pylori. S10. The urease activity of the strain was determined by colorimetric method to measure the urease activity of Bi-PG-14 subsp. longum.

3. The method for preparing the long subspecies of Bifidobacterium longum strain that inhibits Helicobacter pylori according to claim 2, characterized in that: The fecal sample in S1 is infant feces, and the Bifidobacterium longum subsp. Bi-PG-14 strain was isolated from the infant feces.

4. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 3, characterized in that: When activating Bifidobacterium longum subsp. Bi-PG-14 in S2, it is inoculated into sterile MRS liquid culture medium containing 0.5 g / L L-cysteine. It is first cultured anaerobicly at 37°C for 24 h, and then cultured anaerobicly at 37°C for another 24 h to fully activate Bifidobacterium longum subsp. Bi-PG-14.

5. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 4, characterized in that: In step S3, when the Bi-PG-14 subsp. longissimus bacterial culture was inoculated onto Columbia blood agar medium, it was incubated at 37°C for 24 hours, and then the presence of hemolysis around the colonies was observed.

6. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 5, characterized in that: In the acid resistance test of the strain in S4, the pH value of the MRS medium was adjusted with HCl at a concentration of 4.0 mol / L, and multiple MRS mediums with different pH values ​​were prepared, namely 1.0, 2.0, 3.0, 4.0, 5.0 and 6.

0. The strain was inoculated into the medium with different pH values, and after being cultured at 37°C for 24 h, the OD600nm value of the bacterial suspension was measured, its survival rate was calculated, and the acid resistance ability of Bifidobacterium longum subsp. longum Bi-PG-14 was determined.

7. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 6, characterized in that: In the S5 test, the strain was inoculated into MRS medium containing salt concentrations of 1, 2, 3, 4, and 5 g / 100 mL at a 3% inoculation rate. After culturing at 37°C for 24 h, the OD600nm value of the bacterial suspension was measured. The OD value of the strain in MRS medium without salt was used as a control. The survival rate was calculated, and the effect of salt on the activity of Bifidobacterium longum subsp. longum Bi-PG-14 was determined.

8. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 7, characterized in that: In the bile salt tolerance test of the strain in S6, the strain was inoculated at a 3% inoculum into MRS medium containing bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively. After incubation at 37°C for 24 h, the OD600nm value of the bacterial suspension was measured. The OD value of the strain in MRS medium without bile salt was used as a control. The survival rate was calculated, and the effect of bile salt on the activity of Bifidobacterium longum subsp. longum Bi-PG-14 was determined.

9. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 8, characterized in that: In the S7 test for simulating the gastrointestinal tract with naked bacteria, the strain was activated and passaged twice in MRS medium at 37°C. 1.0g of bacterial sludge was centrifuged and added to 5.0mL of artificial gastric fluid. After treatment at 37°C for 3 hours, the precipitate was collected under aseptic conditions. Sterile physiological saline was added according to a ratio of 1.0g bacterial sludge to 5.0mL physiological saline, mixed thoroughly, diluted, spread, and colony counted. Then, 1.0g of bacterial sludge treated with artificial gastric fluid was added to 5.0mL of artificial bile. After treatment at 37°C for 20 minutes, the mixture was centrifuged at 8000r / min for 10 minutes. The bacterial sludge was collected and colony counted. Then, 1.0g of bacterial sludge treated with artificial bile was added to 5.0mL of artificial intestinal fluid. After treatment at 37°C for 4 or 8 hours respectively, the mixture was centrifuged at 8000r / min for 10 minutes. The bacterial sludge was collected and colony counted. The colony count in MRS medium without simulated gastrointestinal treatment was used as a control to calculate the strain survival rate.

10. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 9, characterized in that: In the S8 test for the inhibition of Bifidobacterium longum subsp. longum Bi-PG-14, the Oxford cup agar diffusion method was used. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella were used as indicator bacteria for plate spreading. Physiological saline was used as a blank control. Four Oxford cups were evenly placed on each plate. 200 μL of Bifidobacterium longum subsp. longum Bi-PG-14 bacterial suspension was added to three Oxford cups, and physiological saline was added to one Oxford cup. After incubation at 37°C for 24 h, the size of the inhibition zone was measured with vernier calipers.

11. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 10, characterized in that: In step S9, when measuring the antibacterial activity of the strain against Helicobacter pylori using the agar diffusion method, a 0.05 mg / mL metronidazole solution was used as a positive control, and MRS liquid medium was used as a blank control. 100 μL of Helicobacter pylori suspension was spread on a Columbia blood agar plate without antibiotics, and 100 μL of the test liquid was added to each well. The test liquids were lactic acid bacteria suspension, positive control, and blank control, respectively. The plates with fermentation broth were placed in a microaerophilic environment at 37°C for 72-96 h. After the culture was completed, the diameter of the inhibition zone was measured with vernier calipers.

12. The method for preparing the *Bifidobacterium longum* subspecies strain that inhibits *Helicobacter pylori* according to claim 11, characterized in that: In step S10, when determining the urease activity of the strain using a colorimetric method, 40 μL of Helicobacter pylori bacterial suspension was mixed with 10 μL of bacterial suspension, and 10 μL of sterile liquid culture medium was used as a control. The mixture was added to a clean, sterile 96-well plate and incubated at 37°C in a microaerophilic environment for 48 h. The incubated mixture was then removed, and 150 mL of urease reagent was added to each well. The color change was observed, and the OD550 value was measured. The urease reagent consisted of 0.9% NaCl, 20 mmol / L urea, and 4 μg / mL phenol red. The pH was then adjusted to 6.8 with HCl.

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