Lactobacillus rhamnosus with efficacy of inhibiting pathogenic bacteria of respiratory tract infection and application thereof

Abstract

The present application relates to the functional microorganism screening and application technical field, specifically provide a new lactobacillus rhamnosus and its application, the preservation number of the strain is CCTCC NO: M2022361.The live bacteria and fermentation supernatant of the lactobacillus rhamnosus can significantly inhibit the growth of the main pathogenic microorganism of sinusitis haemophilus influenzae, and have strong adhesion to nasal epithelial cells.The lactobacillus rhamnosus provided in the present application has no toxic effect on the body, good safety, can be added in the medicine, is used for preventing respiratory diseases or improving bacterial sinusitis symptoms, has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of probiotic screening and application technology, specifically relating to a strain of Lactobacillus rhamnosus that can resist Haemophilus influenzae respiratory tract infection and its application. Background Technology

[0002] Sinusitis is a general term for inflammatory diseases of the sinus mucosa, a common otolaryngological condition. Its main symptoms include nasal congestion, purulent nasal discharge, and headache, often accompanied by a decreased or lost sense of smell. Sinusitis is divided into acute and chronic sinusitis. Acute sinusitis has a higher incidence and can occur in all populations, especially children and the elderly, whose overall immune systems are lower. Studies have shown that Haemophilus influenzae is one of the main pathogens of acute sinusitis. In addition, pyogenic cocci, Escherichia coli, and anaerobic bacteria may also cause acute sinusitis. Chronic sinusitis often develops from repeated episodes of acute sinusitis; the overall prevalence of chronic sinusitis in my country is 8%.

[0003] Current treatment practices require adequate doses and courses of antibiotics for patients with acute sinusitis. However, excessive antibiotic use can easily lead to antibiotic resistance. Furthermore, patients should be aware of potential adverse reactions such as gastrointestinal issues, allergic reactions, nervous system reactions, and abnormal liver and kidney function before taking any antibiotics. Other treatment methods, such as oral traditional Chinese medicine, can cause numerous adverse reactions with long-term use, including gastric mucosal cell hyperplasia or chronic atrophic gastritis. Therefore, finding natural and safe novel antibacterial agents is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a novel strain of *Lactobacillus rhamnosus* and its applications. This strain was isolated from fecal samples of healthy adults, and both its live bacteria and fermentation supernatant exhibit inhibitory effects on the growth of pathogenic microorganisms causing sinusitis. Therefore, the *Lactobacillus rhamnosus* strain of this invention can be used to prepare products for the prevention and elimination of pathogenic microorganisms causing sinusitis, and has significant application value in the prevention and treatment of respiratory diseases such as sinusitis.

[0005] The Lactobacillus rhamnosus provided in this invention, named Lactobacillus rhamnosus VHProbi F20, was deposited at the China Center for Type Culture Collection on April 1, 2022, with accession number CCTCC NO: M2022361, and the depositary address is Wuhan University, Wuhan, China.

[0006] The MALDI-TOF molecular weight profile of Lactobacillus rhamnosus strain VHProbi F20 provided by this invention is as follows: Figure 3 As shown; its Riboprinter fingerprint spectrum is as follows. Figure 4 As shown; its RAPD fingerprint spectrum is as follows. Figure 5 As shown; rep-PCR fingerprint pattern as shown Figure 6 As shown.

[0007] The present invention also provides an application of Lactobacillus rhamnosus strain VHProbi F20 in the preparation of articles for the prevention or treatment of respiratory diseases;

[0008] The respiratory disease described, as a specific example, is sinusitis caused by pathogenic microorganisms;

[0009] As a specific example, the pathogenic microorganisms are Haemophilus influenzae, β-hemolytic streptococci, pyogenic streptococci, Escherichia coli, or Staphylococcus aureus.

[0010] The product in question is a pharmaceutical product.

[0011] In another aspect, the present invention provides a probiotic preparation comprising at least one of the following: live bacteria, inactivated bacterial cells, extracellular metabolites, or intracellular extracts of the *Lactobacillus rhamnosus* strain VHProbi F20.

[0012] The live bacteria of Lactobacillus rhamnosus VHProbi F20 and the fermentation supernatant provided by this invention can significantly inhibit the growth of Haemophilus influenzae, the main pathogenic microorganism of sinusitis. In one specific embodiment, the diameter of the inhibition zone of the fermentation broth containing live Lactobacillus rhamnosus VHProbi F20 reached 20.12±0.15mm, and when the amount of fermentation supernatant added was 15%, the growth inhibition rate against Haemophilus influenzae was as high as 43.16%.

[0013] The Lactobacillus rhamnosus strain VHProbi F20 provided by this invention has a strong adhesion effect on nasal epithelial cells, providing favorable conditions for their adhesion and colonization in the nasal cavity; and can significantly inhibit the adhesion of Haemophilus influenzae, with an adhesion inhibition rate of 59.15%.

[0014] The Lactobacillus rhamnosus VHProbi F20 provided by this invention has a strong agglutination ability, which provides the necessary conditions for its co-agglutination with other microorganisms, especially pathogenic microorganisms, in the nasal cavity environment.

[0015] The Lactobacillus rhamnosus VHProbi F20 provided by this invention has an inhibitory effect on the growth of pathogenic microorganisms causing sinusitis.

[0016] The Lactobacillus rhamnosus VHProbi F20 provided by this invention has no toxic effects on the body, good safety, and can be added to medicines for the prevention of respiratory diseases or the improvement of symptoms of bacterial sinusitis, showing broad application prospects. Attached Figure Description

[0017] Figure 1 The images show colony diagrams and Gram staining results for strain F20; where A is the colony diagram and B is the Gram staining result.

[0018] Figure 2 The image shows the API test results for strain F20.

[0019] Figure 3 MALDI-TOF ribosomal protein fingerprint of strain F20;

[0020] Figure 4 Riboprinter fingerprint of strain F20;

[0021] Figure 5 RAPD fingerprint of strain F20;

[0022] Figure 6 The rep-PCR fingerprint of strain F20;

[0023] Figure 7 Photograph of the inhibition zone of strain F20 against Haemophilus influenzae;

[0024] Figure 8 This is a photograph of strain F20 co-agglutinated with Haemophilus influenzae;

[0025] Figure 9 Microscopic images of coagulation of strain F20 with Haemophilus influenzae (oil immersion, 100× magnification).

[0026] Figure 10 The graph shows the self-aggregation rate and the cross-aggregation rate with Haemophilus influenzae for strain F20.

[0027] Figure 11 This is a diagram showing the cytotoxicity of strain F20 against nasopharyngeal carcinoma cells 5-8F.

[0028] Figure 12 Adhesion diagram of strain F20 on nasopharyngeal carcinoma cells 5-8F;

[0029] Figure 13 The graph shows the adhesion inhibition of strain F20 on nasopharyngeal carcinoma cells 5-8F by Haemophilus influenzae.

[0030] Figure 14 A diagram showing the inhibition of Haemophilus influenzae growth by the fermentation supernatant of strain F20;

[0031] Figure 15 This diagram illustrates the inhibitory effect of strain F20 on other sinusitis pathogens. Detailed Implementation

[0032] This invention screened a strain of Lactobacillus rhamnosus that has a significant inhibitory effect on Haemophilus influenzae and other pathogens causing bacterial sinusitis, which has very important application value for the treatment of bacterial sinusitis.

[0033] The screening process for Lactobacillus rhamnosus VHProbi F20 provided by this invention is carried out in accordance with regulatory requirements at every step. Polyphasic taxonomic identification has confirmed that Lactobacillus rhamnosus VHProbi F20 is a novel strain of Lactobacillus rhamnosus.

[0034] On April 1, 2022, the applicant deposited Lactobacillus rhamnosus VHProbi F20 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2022361. The depositary address is Wuhan University, Wuhan, China.

[0035] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0036] The present invention will now be described in detail with reference to specific embodiments.

[0037] Example 1: Isolation and screening of Lactobacillus rhamnosus VHProbi F20

[0038] 1.1 Initial screening

[0039] Prepare MRS agar medium, adjust pH to 6.2-6.5, and autoclave at 121℃ for 15 min.

[0040] A 1g fecal sample from a healthy adult (the sampling process complied with ethical standards for biosampling) was taken, diluted with sterile saline, and placed in a sterile sample bag. The sample was then homogenized using a homogenizer. 100μL of the homogenate was serially diluted, spread onto MRS agar, and anaerobically incubated at 37°C for 48 hours. Single colonies were observed under a microscope after growth. Based on the microscopic examination results, the applicant identified 31 potential lactobacilli strains, named F01, F02, ..., F20, F21, F22, ..., F30, and F31.

[0041] 2.2 Secondary screening

[0042] Prepare 1L of MRS liquid culture medium, autoclave at 121℃ for 15min, and after cooling, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to prepare an acid-resistant culture medium. Inoculate the 31 selected lactobacillus strains at a 6% inoculum into the above acid-resistant culture medium, and anaerobically culture at 37℃ for 48h. Collect the fermentation broth for bacterial count.

[0043] The results showed that among the 31 Lactobacillus strains obtained from the initial screening, strain F20 had the highest viable count after rescreening on an acid-resistant medium, with a log value as high as 9.65 Log CFU / mL, indicating that strain F20 had the strongest acid resistance.

[0044] Example 2: Strain Identification

[0045] 2.1 Identification of colony and cell morphology

[0046] F20 strain was inoculated onto MRS agar medium and anaerobically cultured at 37°C for 24 hours. Single F20 colonies were observed to be white, moist, smooth, round, and opaque, with a diameter of approximately 0.5–2 mm. Under a microscope, the bacteria appeared as short rods, occurring singly, in pairs, or in chains. They did not form spores, were Gram-positive, non-motile, and lacked flagella. Figure 1 shows the single F20 colony and its culture state under an optical microscope.

[0047] 2.2 Carbon source metabolism experiment

[0048] The carbon source metabolism performance of strain F20 was verified using API 50CHL reagent. API 50CHL reagent can be used to identify differences in strains at the genus or species level. For experimental methods and result analysis, please refer to the API 50CHL kit instructions. Analysis showed that strain F20 had an ID value of 99.8% with *Lactobacillus rhamnosus*, indicating identical carbohydrate metabolic activity. The API test results are shown below. Figure 2 .

[0049] 2.3 Molecular biological identification

[0050] 2.3.1 16S rDNA gene sequence analysis

[0051] 1) Genomic DNA extraction

[0052] Follow the instructions in the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302).

[0053] 2) 16S rDNA gene amplification

[0054] Primer sequences:

[0055] 27F: 5′-AGAGTTTTGATCCTGGCTCA-3′;

[0056] 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0057] The 16S rDNA sequence of strain F20 was obtained by sequencing and is SEQ ID NO:1, as follows:

[0058]

[0059] The 16S rDNA sequence of SEQ ID NO:1 was compared with the NCBI database, and strain F20 was identified as Lactobacillus rhamnosus.

[0060] 2.3.2 MALDI-TOF-MS detection of ribosomal protein expression in strains

[0061] Fresh bacterial culture was inoculated into MRS liquid medium at an inoculum rate of 0.1%. After incubation at 37°C and 150 rpm for 48 hours, the bacterial cells were collected, washed four times with sterile water, and air-dried. A small amount of fresh bacterial cells was then uniformly coated onto a target plate in the form of a thin film. 1 μL of lysis buffer was added to cover the sample, and after air-drying, 1 μL of matrix solution was added to cover the sample again. After air-drying, the sample target was placed in a mass spectrometer for identification. The co-crystallized film formed by the sample and matrix was irradiated with a laser, causing the proteins in the sample to ionize. The ions were accelerated through the flight tube under the action of an electric field of 10-20 kV, and the molecular weight of the protein was determined based on the different flight times to the detector. Protein fingerprints were obtained using Autofms 1000 Analyzer v1.0 software. The main ion peaks of strain F20 were: m / z 3419.341, 4696.587, 5348.637, 5914.392, 6842.358, and 9392.936, etc. The identification results are as follows: Figure 3 As shown.

[0062] 2.3.3 Riboprinter fingerprint spectrum

[0063] A single purified colony was picked up from an agar plate using a sampling stick and placed into a sample tube containing buffer. The colony was then stirred with a hand stirrer to suspend it in the buffer. After inactivation in a heater, the sample was placed into the Riboprinter system. Following DNA preparation, transfer, imaging, and data processing, the Riboprinter fingerprint of strain F20 was obtained. Figure 4 ).

[0064] 2.3.4 Identification using RAPD and rep-PCR fingerprinting

[0065] 2.3.4.1 RAPD fingerprint identification

[0066] 1) Primer sequence: M13(5′- GAGGGTGGCGGTTCT-3′);

[0067] 2) The RAPD reaction system is shown in Table 1.

[0068] Table 1: RAPD Reaction System

[0069] Reactive components volume Taq DNA polymerase (5 U / μL) 0.2 μL <![CDATA[10× Buffer (containing Mg 2+ )]]> 2 μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 0.8 μL DNA template 2 μL sterile double-distilled water 14 μL Total volume 20 μL

[0070] 3) Electrophoresis

[0071] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a control. Electrophoresis was performed at a constant voltage of 100V for 80 min, and the electrophoresis pattern was finally detected using a gel imaging system. The RAPD fingerprint of strain F20 is shown below. Figure 5 As shown.

[0072] 2.3.4.2 rep-PCR fingerprinting

[0073] 1) Primer sequence: 5′-CTACGGCAAGGCGACGCTGACG-3′.

[0074] 2) The reaction system for rep-PCR is shown in Table 2.

[0075] Table 2: Reaction system of rep-PCR

[0076] Reactive components volume r Taq DNA polymerase 0.2μL 10×Ex Taq DNA Buffer 2μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 2μL DNA template 2μL sterile double-distilled water 12.8 μL

[0077] 3) Electrophoresis

[0078] The DL2000 DNA Marker was used as a result control. Amplification results were detected at 100 V for 80 min. The rep-PCR fingerprint of strain F20 is shown below. Figure 6 As shown.

[0079] 2.3.5 Whole genome sequencing

[0080] Fresh F20 bacterial culture was inoculated into 500 mL of MRS broth at a volume ratio of 1%, incubated at 37°C for 20 h, centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were sent to a sequencing center to obtain the complete genome sequence of the bacterium. The genome sequence was uploaded to the NCBI GenBank database, with GenBank accession numbers CP094952-CP094953.

[0081] In summary, based on the colony morphology, carbon source metabolism, and molecular biological identification results of strain F20, the applicant has determined that strain F20 is a novel Lactobacillus rhamnosus and named it Lactobacillus rhamnosus VHProbi F20.

[0082] Example 3: Antibacterial test of Lactobacillus rhamnosus VHProbi F20 against Haemophilus influenzae.

[0083] 3.1 Culture medium preparation

[0084] MRS culture medium and MRS broth: Qingdao Haibo Biotechnology;

[0085] Brain and heart extract chocolate liquid culture medium: Brain and heart extract broth, autoclave at 121℃ for 15 min, cool to about 50℃, add 7% sterile defibrinated sheep blood and mix well, heat in an 80℃ water bath for about 10 min while shaking continuously until the culture medium turns brown (chocolate color), cool to room temperature and store at 4℃ for later use.

[0086] Brain and heart infusion chocolate agar medium: After autoclaving brain and heart infusion agar, add 7% sterile defibrinated sheep blood, heat in an 80℃ water bath for 10 minutes until the medium turns brown, cool to 45~50℃ and pour into sterile Petri dishes for later use.

[0087] 3.2 Strain activation

[0088] Lactobacillus rhamnosus VHProbi F20: Take a frozen glycerol tube, streak it onto an MRS plate, and incubate at 37°C for 24-48 hours; after single bacteria have grown on the plate, pick them under aseptic conditions and transfer them to MRS broth, and incubate at 37°C for 24 hours.

[0089] Haemophilus influenzae ATCC49766: Take a frozen glycerol tube, streak it onto brain heart and heart infusion chocolate agar medium, and incubate at 37°C and 5% CO2 for 24 hours; after single bacteria have grown on the plate, aseptically transfer it to brain heart and heart infusion chocolate liquid medium and incubate at 37°C and 5% CO2 for 18-24 hours.

[0090] 3.3 Antibacterial test

[0091] Spread the lower layer of culture medium, sterilizing the nutrient agar and pouring it into the agar plate until it is full. After the agar solidifies, spread the upper layer of culture medium, evenly spreading the brain heart and heart infusion chocolate agar medium on top. After the upper layer of culture medium solidifies, dilute the cultured Haemophilus influenzae and spread an appropriate dilution onto the surface of the medium. Punch wells and add 100 μL of the mixed lactic acid bacteria fermentation broth to each well. Incubate at 37°C and 5% CO2 for 24 hours, then measure the size of the inhibition zone.

[0092] Measurements showed that *Lactobacillus rhamnosus* strain VHProbi F20 had a significant inhibitory effect on *Haemophilus influenzae*, with an inhibition zone diameter of 20.12 ± 0.15 mm. The results are as follows: Figure 7 As shown.

[0093] Example 4: Agglutination and adsorption effect of Lactobacillus rhamnosus VHProbi F20 on Haemophilus influenzae

[0094] Take an appropriate amount of activated Lactobacillus rhamnosus VHProbi F20 and fresh Haemophilus influenzae bacterial suspension, centrifuge at 8000 rpm for 4 min, wash twice with phosphate buffer solution (PBS, pH 7.4), and adjust to OD600=4 to obtain bacterial suspension.

[0095] Add 300 μL of *Lactobacillus rhamnosus* VHProbi F20 bacterial suspension to a 24-well plate, followed by 300 μL of *Haemophilus influenzae* bacterial suspension as the experimental group. Separately, mix an equal volume of *Lactobacillus rhamnosus* VHProbi F20 bacterial suspension with buffer as the control group. Each control and experimental group was replicated in two places. The 24-well plates were placed in a microplate shaker at 400 rpm at room temperature and incubated with shaking. The initial and subsequent plate conditions were observed and photographed under a microscope to record the plate's state, noting any aggregation.

[0096] Macroscopic observation results such as Figure 8 As shown, in the experimental group, *Lactobacillus rhamnosus* VHProbi F20 and *Haemophilus influenzae* showed obvious agglutination, while no agglutination was observed in the control group where *Lactobacillus rhamnosus* VHProbi F20 was added alone. Microscopic observation revealed agglutinated clumps of *Lactobacillus rhamnosus* VHProbi F20 and *Haemophilus influenzae* in the experimental group, such as... Figure 9 As shown.

[0097] The above results show that the Lactobacillus rhamnosus VHProbi F20 provided by the present invention has a significant agglutination and adsorption effect on Haemophilus influenzae, achieving unexpected technical results.

[0098] Example 5 Aggregation analysis of Lactobacillus rhamnosus VHProbi F20

[0099] 5.1 Preparation of bacterial suspension

[0100] Activated *Lactobacillus rhamnosus* VHProbi F20 was cultured in MRS broth for 18 hours, and *Haemophilus influenzae* was cultured in brain heart extract chocolate liquid medium at 37°C and 5% CO2 for 18 hours. After culturing, the bacteria were washed three times with phosphate-buffered saline (PBS, pH 7.4) to adjust the bacterial concentration to achieve a count of 10⁻⁶. -7 ~10 -8 CFU / mL, for later use.

[0101] 5.2 Cluster Analysis

[0102] Equal volumes of 20 mL of Lactobacillus rhamnosus VHProbi F20 bacterial suspension, Haemophilus influenzae bacterial suspension, and both bacterial suspensions were mixed thoroughly and placed in a 37℃ incubator. The absorbance at 600 nm of the bacterial suspensions at 0 h, 2 h, 4 h, and 5 h were measured, and the self-polymerization rate and copolymerization rate were calculated.

[0103] Self-polymerization rate % = [1-A] L / A0]×100%.

[0104] Copolymerization rate % = [(A L +A H ) / 2-A mix ] / [( A L + A P ) / 2]×100%.

[0105] A0: Absorbance value of Lactobacillus rhamnosus VHProbi F200h;

[0106] A L : Absorbance values ​​of Lactobacillus rhamnosus VHProbi F20 after standing alone for 2, 4, and 5 hours;

[0107] A H : Absorbance values ​​of Haemophilus influenzae after standing alone for 2, 4, and 5 hours;

[0108] A mix The absorbance values ​​of Lactobacillus rhamnosus VHProbi F20 and Haemophilus influenzae after standing for 2, 4 and 5 hours.

[0109] The results are as follows Figure 10 As shown, the self-polymerization effect of Lactobacillus rhamnosus VHProbi F20 and its copolymerization effect with Haemophilus influenzae continuously increased with time. At 5 hours, the self-polymerization rate of this strain reached 23%, and the copolymerization rate with Haemophilus influenzae reached 26%, achieving unexpected results.

[0110] Example 6: Cytotoxicity test of Lactobacillus rhamnosus VHProbi F20 against nasopharyngeal carcinoma cells.

[0111] 6.1 Preparation of bacterial suspension

[0112] Lactobacillus rhamnosus VHProbi F20 was cultured in MRS liquid medium to the stationary phase, washed three times with phosphate-buffered saline (PBS, pH 7.4), and the bacterial concentration was adjusted to reach 5 × 10⁻⁶. 7 CFU / mL (OD600 absorbance value approximately 0.4), inactivated in a 70℃ water bath for 20 min for later use.

[0113] 6.2 Cytotoxicity test

[0114] Nasopharyngeal carcinoma cells at 5-8F were resuscitated and seeded into 24-well culture plates containing 10% fetal bovine serum at a seeding density of 2 × 10⁻⁶ cells / well. 5 Cells / well, cultured for 24 h. Inactivated strain F20 was added to the cells at an MOI (Multiplicity of Infection) of 10, and a blank control group without bacterial infection was set up. Cells were cultured for another 24 h. MTT solution was added to each well to a final concentration of 0.3 mg / ml, and the cells were incubated in a CO2 incubator for 3 h. The supernatant was carefully discarded, and 500 μL of DMSO was added to each well of a 24-well plate. The cells were incubated at 37°C for 30 min to fully dissolve the purple crystals. The absorbance was measured at 490 nm.

[0115] Test results as follows Figure 11 As shown, compared with the control group, Lactobacillus rhamnosus VHProbi F20 had no significant effect on the proliferation activity of nasopharyngeal carcinoma 5-8F cells, showed no cytotoxicity, and had good safety.

[0116] Example 7 Adhesion test of Lactobacillus rhamnosus VHProbi F20 on nasopharyngeal carcinoma cells

[0117] 7.1 Preparation of bacterial suspension

[0118] Lactobacillus rhamnosus VHProbi F20 was cultured in MRS liquid medium to the stationary phase, washed three times with phosphate-buffered saline (PBS, pH 7.4), resuspended in cell culture medium, and the bacterial concentration was adjusted to reach 1 × 10⁻⁶ cells / year. 8 CFU / mL, for later use.

[0119] 7.2 Culture of nasopharyngeal carcinoma 5-8F cells

[0120] Nasopharyngeal carcinoma cells (5-8F) were retrieved from a liquid nitrogen tank, revived, and passaged to expand the cell number to the required level. The 5-8F cells were then seeded into six-well plates containing 10% fetal bovine serum-containing cell culture medium and built-in cell spreaders, with approximately 2 × 10⁶ cells per well. 6 Cells, place the six-well plate in a carbon dioxide incubator for 24 hours.

[0121] 7.3 Adhesion Test

[0122] Nasopharyngeal carcinoma 5-8F monocell layers that had adhered to the wells of a six-well plate were washed three times with PBS buffer, and then a *Lactobacillus rhamnosus* VHProbi F20 bacterial suspension prepared above was added. The plates were then incubated in a CO2 incubator for 1 hour. Cell slides were washed three times repeatedly with PBS buffer to remove unadhered bacteria. The slides were fixed with anhydrous methanol for 20 minutes, air-dried, and Gram-stained. Twenty random fields (100 cells) were examined under a 100x oil immersion microscope to observe the lactic acid bacteria adhering to each cell, and the average number of lactic acid bacteria adhering to each cell was calculated.

[0123] Statistical analysis showed that *Lactobacillus rhamnosus* VHProbi F20 adhered to nasopharyngeal carcinoma cells at a rate of 13.35 ± 0.71 CFU / cell, indicating that this strain has a strong adhesion ability to nasopharyngeal carcinoma cells. Figure 12 As shown.

[0124] Example 8: Adhesion inhibition experiment of Lactobacillus rhamnosus VHProbi F20 against Haemophilus influenzae.

[0125] 8.1 Preparation of bacterial suspension

[0126] Fresh bacterial cultures of *Lactobacillus rhamnosus* VHProbi F20 and *Haemophilus influenzae* were washed three times with phosphate-buffered saline (PBS, pH 7.4), resuspended in cell culture medium, and the bacterial concentration was adjusted to achieve a count of 1 × 10⁻⁶. 8 CFU / mL, for later use.

[0127] 8.2 Adhesion Inhibition Test

[0128] Nasopharyngeal carcinoma 5-8F monocell layers that had adhered to the wells of a six-well plate were washed three times with PBS buffer. 1 mL of *Lactobacillus rhamnosus* VHProbi F20 and *Haemophilus influenzae* bacterial suspensions were added to each well, respectively. Cells without *Lactobacillus rhamnosus* VHProbi F20 suspension served as a blank control. The plates were incubated in a CO2 incubator for 2 hours. Cell slides were washed three times with PBS buffer to remove unadhered bacteria. The slides were fixed with anhydrous methanol for 20 minutes, air-dried, and Gram-stained. *Haemophilus influenzae* adhering to 100 cells in 20 random fields were observed under a 100x oil immersion microscope. The average number of *Haemophilus influenzae* bacteria adhering to each cell was calculated. The changes in the number of Haemophilus influenzae adhering to cells were compared under the conditions of presence and absence of Lactobacillus rhamnosus VHProbi F20. The adhesion rate of Haemophilus influenzae in the group without Lactobacillus rhamnosus VHProbi F20 was taken as 100%, and the inhibition rate of Lactobacillus rhamnosus VHProbi F20 on Haemophilus influenzae adhering cells was calculated.

[0129] Adhesion inhibition rate (%) = (Adhesion amount of Haemophilus influenzae in the control group - Adhesion amount of Haemophilus influenzae in the experimental group) / Adhesion amount of Haemophilus influenzae in the control group × 100%.

[0130] The results showed that *Lactobacillus rhamnosus* VHProbi F20 significantly reduced the adhesion of *Haemophilus influenzae* to nasopharyngeal carcinoma cells, with an adhesion inhibition rate of 59.15%. Figure 13 As shown.

[0131] Example 9: Inhibitory effect of Lactobacillus rhamnosus VHProbi F20 on the growth of Haemophilus influenzae.

[0132] 1) Haemophilus influenzae ATCC49766: Inoculate into brain heart extract chocolate liquid medium and incubate at 37°C and 5% CO2 for 16-24 hours.

[0133] Lactobacillus rhamnosus VHProbi F20 was inoculated into MRS medium at a 1% inoculum and cultured statically at 37°C for 16–24 h. The fermentation broth was centrifuged at 10,000 r / min for 30 min at 4°C to collect the supernatant, which was then filtered through a 0.22 μm microporous membrane to obtain cell-free fermentation supernatant of Lactobacillus rhamnosus VHProbi F20.

[0134] 2) Fresh Haemophilus influenzae ATCC49766 bacterial culture was inoculated at a 1% inoculum into brain and heart extract chocolate liquid medium supplemented with 5%, 10%, and 15% (v / v) cell-free fermentation supernatant of strain F20, respectively. Brain and heart extract chocolate liquid medium without the addition of Lactobacillus rhamnosus VHProbi F20 cell-free fermentation supernatant served as a control. The medium was incubated at 37°C and 5% CO2 for 24 h. The growth of Haemophilus influenzae was measured at a wavelength of 600 nm.

[0135] The results are as follows Figure 14 As shown, compared with the control group, the addition of cell-free fermentation supernatant of Lactobacillus rhamnosus VHProbi F20 significantly inhibited the growth of pathogenic Haemophilus influenzae. Moreover, with the increase of the addition amount, the inhibitory effect of Lactobacillus rhamnosus VHProbi F20 fermentation supernatant on Haemophilus influenzae was enhanced. When the addition amount of fermentation supernatant was 15%, the inhibition rate of Haemophilus influenzae reached 43.16%, achieving an unexpected effect.

[0136] Example 10: Inhibitory effect of Lactobacillus rhamnosus VHProbi F20 on other sinusitis pathogens

[0137] The sinusitis pathogens used in this example include:

[0138] β-hemolytic streptococci: CMCC(B)32210, cultured in brain heart infusion broth medium supplemented with 5% (v / v) fetal bovine serum at 37°C for 16-24 h;

[0139] Streptococcus pyogenes: BNCC337110, BNCC185918, cultured in Columbia broth medium supplemented with 5% (v / v) fetal bovine serum at 37°C for 16-24 hours;

[0140] Escherichia coli: BNCC337304, BNCC133264, BNCC269342, cultured in nutrient broth at 37°C for 16-24 hours;

[0141] Staphylococcus aureus: ATCC29213, ATCC25923, ATCC6538, cultured in nutrient broth at 37°C for 16-24 hours.

[0142] Lactobacillus rhamnosus VHProbi F20 was inoculated into MRS medium at a rate of 1% and incubated at 37°C for 24 hours.

[0143] For the lower culture medium layer, sterilize the nutrient agar and pour it into the plates, covering the entire plate. After the agar solidifies, evenly place three sterile Oxford cups on each plate. For the upper culture medium layer, thoroughly mix the cultured β-hemolytic streptococci, pyogenic streptococci (two strains mixed in equal volumes), Escherichia coli (three strains mixed in equal volumes), and Staphylococcus aureus (three strains mixed in equal volumes). Take 0.2% (v / v) of each culture and add it to the corresponding semi-solid medium. Take an appropriate amount and evenly spread it onto the lower culture medium layer. After the upper culture medium solidifies, remove the Oxford cups and add 100 μL of the mixed Lactobacillus rhamnosus VHProbi F20 bacterial suspension to each well. Incubate at 37°C for 24 hours, observe and measure the size of the inhibition zone. See the image for the inhibition zone. Figure 15 The size of the inhibition zone is shown in Table 3.

[0144] Table 3: Antibacterial activity of Lactobacillus rhamnosus VHProbi F20 against four sinusitis pathogens

[0145] Pathogenic bacteria Antibacterial zone diameter / mm β-hemolytic streptococci 14.14±0.09 Streptococcus pyogenes 21.06±0.24 Escherichia coli 14.67±0.15 Staphylococcus aureus 11.54±0.08

[0146] from Figure 15 As shown in Table 3, Lactobacillus rhamnosus VHProbi F20 has a strong inhibitory effect on β-hemolytic streptococci, pyogenic streptococci, Escherichia coli and Staphylococcus aureus.

[0147] In summary, the *Lactaseibacillus rhamnosus* VHProbi F20 strain provided by this invention has the ability to inhibit and kill the growth of pathogenic microorganisms causing sinusitis, both in its live cells and fermentation supernatant. The bacteria possess strong agglutination ability, providing the necessary conditions for co-agglutination with other microorganisms, especially pathogenic microorganisms, in the nasal cavity environment. It also exhibits strong adhesion to nasal epithelial cells, providing favorable conditions for adhesion and colonization in the nasal cavity, and significantly inhibits the adhesion of *Haemophilus influenzae*, the main pathogenic microorganism of sinusitis. Furthermore, it significantly inhibits the growth of most other pathogenic microorganisms of sinusitis, such as β-hemolytic streptococci, *Streptococcus pyogenes*, *Escherichia coli*, and *Staphylococcus aureus*. It is non-cytotoxic, has good safety profile, and broad application prospects. It can be used as an additive in nasal cleaning products and nasal treatment drugs, and has significant application value in preventing respiratory diseases and improving symptoms of bacterial sinusitis.

Claims

1. A type of Lactobacillus rhamnosus, characterized in that, The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The accession number of the object is CCTCC NO: M2022361.

2. The use of *Lactobacillus rhamnosus* as described in claim 1 in the preparation of products for the prevention or treatment of respiratory diseases; wherein the respiratory disease is sinusitis caused by pathogenic microorganisms; wherein the pathogenic microorganisms are any one or more of *Haemophilus influenzae*, *Streptococcus pyogenes*, *Escherichia coli*, or *Staphylococcus aureus*; and wherein the product is a pharmaceutical product.

3. A probiotic preparation, characterized in that, The probiotic preparation contains live Lactobacillus rhamnosus as described in claim 1.

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