A Lactiplantibacillus plantarum strain and its application in preventing Streptococcus pneumoniae respiratory tract infection

Abstract

The present invention provides a Lactiplantibacillus plantarum strain capable of resisting Streptococcus pneumoniae infection in the respiratory tract, with a deposit number of CCTCC NO: M2022359. The Lactiplantibacillus plantarum VHProbi P06 strain provided by the present invention has a strong adhesion effect on nasal epithelial cells and can colonize in the nasal cavity; and can significantly inhibit the adhesion effect of Streptococcus pneumoniae, the main pathogenic microorganism of sinusitis, on nasal epithelial cells; it also has an inhibitory effect on the growth of most other pathogenic microorganisms of sinusitis, including β-hemolytic streptococcus, Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, etc. The Lactiplantibacillus plantarum VHProbi P06 strain provided by the present invention has no toxic effect on the body and has good safety. It can be added to drugs for preventing respiratory diseases or improving respiratory symptoms caused by bacterial infections, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of screening and application of functional probiotics, and specifically relates to a Lactiplantibacillus plantarum capable of resisting Streptococcus pneumoniae respiratory tract infection and its application. Background Art

[0002] Similar to the growth environment of intestinal flora, the human respiratory system, from the nasal cavity to the upper respiratory tract, due to being warm and humid, and having secreted mucus and shed epithelial cells, its environmental conditions are very suitable for the growth of microorganisms, so it also aggregates a wide variety of microorganisms. The microorganisms in the respiratory system include coagulase-negative staphylococci, Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and Pseudomonas aeruginosa, as well as Enterobacteriaceae bacteria such as Escherichia coli and Klebsiella pneumoniae. In the nasal cavity, these microorganisms affect each other and also interact with the respiratory mucosa, forming a sustainable ecosystem. This microbial ecosystem will change in its microbial composition and proportion under the influence of the environment and / or the strength of its own immune ability. In severe cases, it will lead to microecological imbalance in the microhabitat, causing nasal mucosa congestion, swelling, exudation, hyperplasia, etc., thus triggering adverse symptoms such as nasal congestion, nasal itching, and difficulty breathing.

[0003] For the above symptoms in the nasal cavity, traditional treatment methods include drug treatment, surgical treatment, and nasal irrigation treatment. However, these treatment methods only stay at the level of relieving symptoms and reducing pain, and have problems such as repeated attacks, large side effects, and unsatisfactory long-term curative effects. Moreover, these treatment methods will indiscriminately eliminate or kill the bacteria on the nasal mucosa, including both pathogenic bacteria and some beneficial bacteria, which will seriously damage the balance and stability of the nasal microecology.

[0004] In terms of maintaining respiratory health, probiotics colonizing the respiratory mucosa play an indispensable and crucial role. Probiotics act directly on the respiratory mucosa, can adjust the respiratory microecology, and further adjust local or systemic immunity. Summary of the Invention

[0005] The purpose of the present invention is to provide a Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) capable of resisting Streptococcus pneumoniae infection of the respiratory tract and its application. The strain can significantly inhibit the growth and adhesion of Streptococcus pneumoniae, the main pathogenic bacterium in the respiratory tract, and effectively prevent and treat respiratory diseases caused by bacterial infection, with broad application prospects.

[0006] On the one hand, the present invention relates to a Lactiplantibacillus plantarum, which is Lactiplantibacillus plantarum VHProbi P06 ( Lactiplantibacillus plantarumThe Lactiplantibacillus plantarum VHProbi P06 strain was deposited on April 1, 2022 at the China Center for Type Culture Collection of Wuhan University, China, with the deposit number CCTCC NO: M2022359.

[0007] For the Lactiplantibacillus plantarum VHProbi P06 strain, its Riboprinter fingerprint is as Figure 3 shown; its MALDI-TOF ribosomal protein molecular weight map is as Figure 4 shown; its RAPD fingerprint is as Figure 5 shown; its rep-PCR fingerprint is as Figure 6 shown.

[0008] For the Lactiplantibacillus plantarum VHProbi P06 strain, its 16s rDNA sequence is as shown in SEQ ID NO: 1.

[0009] On the one hand, the present invention relates to the application of the Lactiplantibacillus plantarum VHProbi P06 strain in the preparation of products with the function of preventing or treating respiratory diseases.

[0010] The respiratory disease mentioned above is bacterial sinusitis.

[0011] The product mentioned above is a medicine.

[0012] The present invention also relates to a probiotic preparation, which contains any one or several of the viable bacteria cells, dead bacteria cells, intracellular metabolites, and intracellular extracts of the Lactiplantibacillus plantarum VHProbi P06 strain.

[0013] For the Lactiplantibacillus plantarum VHProbi P06 provided by the present invention, both its viable bacteria and fermentation supernatant can significantly inhibit the growth of Streptococcus pneumoniae, the main pathogenic microorganism of sinusitis. The results of one example show that the diameter of the inhibition zone of the fermentation broth containing viable bacteria reaches 16.24 ± 0.08 mm.

[0014] For the Lactiplantibacillus plantarum VHProbi P06 provided by the present invention, it has a strong adhesion effect on nasal epithelial cells and can colonize in the nasal cavity; and it can significantly inhibit the adhesion of Streptococcus pneumoniae, the main pathogenic microorganism of sinusitis, to nasal epithelial cells. The results of one example show that the adhesion inhibition rate of this strain to Streptococcus pneumoniae reaches 71.56%.

[0015] For the Lactiplantibacillus plantarum VHProbi P06 provided by the present invention, its bacterial cells have strong agglutination ability. The self-polymerization rate at 4 h is 19%, and the co-polymerization rate with Streptococcus pneumoniae reaches 21%, providing necessary conditions for its co-aggregation with other microorganisms, especially pathogenic microorganisms, in the nasal cavity environment.

[0016] The Lactiplantibacillus plantarum VHProbi P06 provided by the present invention also has an inhibitory effect on the growth of most other pathogenic microorganisms causing sinusitis, including β-hemolytic streptococcus, Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, etc. Among them, the diameter of the inhibition zone against Streptococcus pyogenes reaches 21.17±0.18 mm.

[0017] The Lactiplantibacillus plantarum VHProbi P06 provided by the present invention has no toxic effect on the body and has good safety. It can be added to medicines for preventing respiratory diseases or improving respiratory symptoms caused by bacterial infections, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the colony map and Gram staining map of strain P06; wherein A is the colony map and B is the Gram staining map;

[0019] Figure 2 It is the API test result map of strain P06;

[0020] Figure 3 It is the Riboprinter fingerprint map of strain P06;

[0021] Figure 4 It is the MALDI-TOF ribosomal protein fingerprint map of strain P06;

[0022] Figure 5 It is the RAPD fingerprint map of strain P06;

[0023] Figure 6 It is the rep-PCR fingerprint map of strain P06;

[0024] Figure 7 It is the photo of the inhibition zone of strain P06 against Streptococcus pneumoniae;

[0025] Figure 8 It is the growth result map of the fermentation supernatant of strain P06 inhibiting Streptococcus pneumoniae;

[0026] Figure 9 It is the macroscopic photo of the co-aggregation of strain P06 and Streptococcus pneumoniae;

[0027] Figure 10 It is the microscopic photo of the co-aggregation of strain P06 and Streptococcus pneumoniae (oil immersion lens, magnified 100×);

[0028] Figure 11 It is the self-aggregation rate of strain P06 and the interactive aggregation rate with Streptococcus pneumoniae;

[0029] Figure 12 It is the adhesion result map of strain P06 to nasopharyngeal carcinoma cells 5-8F;

[0030] Figure 13 Figure showing the inhibitory effect of strain P06 on the adhesion of Streptococcus pneumoniae to nasopharyngeal carcinoma cell line 5-8F;

[0031] Figure 14 Figure showing the inhibitory effect of strain P06 on other respiratory pathogenic bacteria. Detailed implementation manners

[0032] The present invention has screened out a Lactiplantibacillus plantarum strain with strong adhesion and colonization ability, and has significant inhibitory effects on the main respiratory pathogenic bacterium Streptococcus pneumoniae and other pathogenic bacteria, which has very important application value for the prevention and treatment of respiratory diseases caused by bacterial infections.

[0033] The procedures in the isolation process of the Lactiplantibacillus plantarum VHProbi P06 provided by the present invention comply with relevant regulatory requirements. Through polyphasic taxonomic identification, Lactiplantibacillus plantarum VHProbi P06 is a novel Lactiplantibacillus plantarum. The Lactiplantibacillus plantarum VHProbi P32 provided by the present invention has the uses of inhibiting the growth of the main respiratory pathogenic bacterium Streptococcus pneumoniae and killing respiratory pathogenic microorganisms, and has important application value for the prevention and treatment of respiratory diseases.

[0034] The applicant deposited the Lactiplantibacillus plantarum VHProbi P06 strain at the China Center for Type Culture Collection of Wuhan University on April 1, 2022, and its deposit number is CCTCC NO: M2022359.

[0035] The screening method described in the present invention is not limited to that described in the examples, and any known method that can achieve the screening purpose can be used. The screening description in the examples is only an illustration of the present invention, rather than a limitation on the protection scope of the present invention.

[0036] modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all fall within the scope of the present invention.

[0037]

[0038]

[0039] Example 1 Isolation and screening of Lactiplantibacillus plantarum VHProbi P06

[0040] 1.1 Primary screening

[0041] ​​Take 1 g of fresh pickled vegetable fermentation sample, dilute it with sterile normal saline, put it into a sterile sample bag, and mix it evenly with a homogenizer; take 100 μL of the mixed solution and dilute it step by step, then spread it on MRS agar medium (10 g of peptone, 5 g of beef powder, 4 g of yeast powder, 2 g of glucose, 1 ml of Tween 80, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 2 g of ammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15 g of agar powder, 1000 ml of distilled water), and anaerobically culture it at 37 °C for 48 h, and then conduct microscopic examination on the single colonies grown on the plate respectively.

[0042] According to the results of microscopic examination, the applicant screened out a total of 24 potential Lactobacillus strains, which were respectively named P01, P02, ……, P23, P24.

[0043] 2.2 Re-screening

[0044] Prepare 1 L of MRS liquid medium, after autoclaving and cooling, add 3.2 g of porcine gastric mucosa pepsin, shake well to dissolve, and place it in a water bath shaker at 37 °C for 1 h to prepare an acid-resistant medium.

[0045] Inoculate the 24 Lactobacillus strains obtained from the primary screening into the above-mentioned acid-resistant medium respectively according to the inoculation amount of 6%, anaerobically statically culture them at 37 °C for 48 h, and take the fermentation broth for bacterial count.

[0046] The results showed that among the logarithmic values of the viable bacteria counts of the fermentation broths of the 24 screened Lactobacillus strains, the viable bacteria count of strain P06 was the highest after re-screening with the acid-resistant medium, and the logarithmic value was as high as 9.45 Log CFU / mL, indicating that strain P06 had the strongest acid resistance.

[0047] Example 2 Strain Identification

[0048] 2.1 Colony and Bacterial Morphology Identification

[0049] Inoculate strain P06 on MRS agar medium, after anaerobically culturing it at 37 °C for 24 h, it can be seen that the single colony of P06 is milky white, round, opaque, with neat edges, raised surface, smooth and moist, and the colony diameter is about 2 - 3 mm; the bacteria are short rod-shaped under the microscope, with rounded ends, arranged singly, in pairs or in chains, Gram-positive staining, and do not form spores. See the photos of the single colony of P06 and under the optical microscope in Figure 1 .

[0050] 2.2 Carbon Source Metabolism Test

[0051] Use API 50CHL reagent to verify the carbon source metabolism performance of strain P06. API 50CHL reagent can be used to identify the differences of strains at the genus or species level. See the API 50CHL kit instruction manual for the experimental method and result analysis.

[0052] The results are as follows Figure 2 As shown, the carbohydrate metabolic activity of the P06 strain screened by the present invention is basically the same as that of Lactiplantibacillus plantarum.

[0053] 2.3 Molecular biological identification

[0054] 2.3.1 16s rDNA gene sequence analysis

[0055] 1) Genomic DNA extraction

[0056] Operate with reference to the Tiangen Bacterial Genomic DNA Extraction Kit (Catalog No.: DP302).

[0057] 2) 16s rDNA gene amplification

[0058] Primer sequences:

[0059] 27F: AGAGTTTGATCCTGGCTCA;

[0060] 1492R: GGTTACCTTGTTACGACTT.

[0061] The 16s rDNA sequence SEQ ID NO: 1 of the P06 strain was obtained by sequencing, and the specific sequence is as follows:

[0062]

[0063] When SEQ ID NO: 1 was compared in the NCBI database, the result showed that its sequence similarity was the highest with that of Lactiplantibacillus plantarum. Therefore, strain P06 was preliminarily determined to be Lactiplantibacillus plantarum.

[0064] 2.3.2 Riboprinter fingerprint

[0065] A sterile inoculation stick was used to pick up the purified single colony from the agar medium plate and put it into a sample tube containing buffer. It was stirred with a hand-held stirrer to suspend it in the buffer. Then the sample rack was put into a heater for inactivation and then into the Riboprinter system. After the sample went through DNA preparation, membrane transfer, imaging detection and data processing, the bacterial identification result was obtained. The identification result showed that strain P06 was Lactiplantibacillus plantarum. The Riboprinter fingerprint result is shown in Figure 3 .

[0066] 2.3.3 MALDI-TOF-MS detection of ribosomal protein expression of the strain

[0067] Fresh bacterial liquid was inoculated into MRS liquid medium at an inoculation amount of 0.1%. After culturing at 37 °C and 150 rpm for 48 h, the bacterial cells were collected, washed 4 times with sterile water, and the surface moisture was air-dried. Then a small amount of fresh bacterial cells was evenly coated on the target plate in the form of a thin film. 1 μL of lysis solution was added to cover the sample. After air-drying, 1 μL of matrix solution was added to cover the sample. After air-drying, the sample target was put into the mass spectrometer for identification. The co-crystalline film formed by the sample and the matrix was irradiated with laser to ionize the proteins in the sample. The ions were accelerated to fly through the flight tube under the action of an electric field of 10 - 20 KV, and the molecular weights of the proteins were detected according to the different flight times to reach the detector. The protein fingerprint was obtained by using the Autofms 1000 analysis software Autof Analyzer v1.0.

[0068] The main ion peaks of strain P06 were m / z 3936.788, 4747.089, 5188.100, 5738.064, 7875.557, 9494.388, etc. The identification result is as shown in Figure 4 .

[0069] 2.3.4 RAPD and rep-PCR fingerprint identification

[0070] 2.3.4.1 RAPD fingerprint identification

[0071] 1) Primer sequence:

[0072] 5’- GAGGGTGGCGGTTCT-3’;

[0073] 2) RAPD reaction system:

[0074] TaqDNA polymerase (5U / μL) 0.2 μL, 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.

[0075] 3) Electrophoresis

[0076] Prepare a 1.5% agarose gel plate, use DL2000 DNA Marker as the result control, and run the electrophoresis at 100V for 80 minutes. Finally, use the gel imaging system to detect the electrophoresis pattern. Figure 5 shown.

[0077] 2.3.4.2 rep-PCR fingerprint

[0078] 1) Primer sequence: CTACGGCAAGGCGACGCTGACG.

[0079] 2) The reaction system for rep-PCR is:

[0080] Taq DNA polymerase 0.2 μL; 10× Ex Taq DNA Buffer 2 μL; primer (10 uM) 1 μL; dNTPs (2.5 mM) 2 μL; DNA template 2 μL; sterile double-distilled water 12.8 μL.

[0081] 3) Electrophoresis

[0082] DL2000 DNA Marker was used as a control. The amplification results were detected at 100 V and 80 min of electrophoresis. The rep-PCR fingerprint of strain P06 is shown in the figure below. Figure 6 shown.

[0083] 2.3.5 Whole-genome sequencing

[0084] A fresh P06 strain culture was inoculated into 500 mL of MRS broth at a 1% volume ratio. The culture was incubated at 37°C for 20 hours and centrifuged at 8000 rpm for 10 minutes to collect the cells. The cells were sent to a sequencing center, and the complete genome sequence of the strain was obtained. The gene sequences were uploaded to the NCBI gene database, with GenBank accession numbers CP104084-CP104091.

[0085] In summary, combining the colony morphology of the strain, carbon source metabolism, and the identification results of molecular biology, it is determined that the P06 strain screened in the present invention is a new Lactiplantibacillus plantarum, which the applicant names Lactiplantibacillus plantarum VHProbi P06 ( Lactiplantibacillus plantarum VHPribo P06).

[0086] Example 3: Antibacterial test of Lactiplantibacillus plantarum VHProbi P06 against Streptococcus pneumoniae

[0087] 3.1 Strain activation

[0088] Lactiplantibacillus plantarum VHProbi P06: Take the glycerol tube stored frozen, streak it onto an MRS plate, and culture it at 37 °C for 24 - 48 h; after single colonies grow on the plate, pick them under sterile conditions into MRS broth and culture them statically at 37 °C for 24 h.

[0089] Streptococcus pneumoniae ATCC49619: Take the glycerol tube stored frozen, inoculate it into brain heart infusion broth medium (containing 5% fetal bovine serum) at an inoculation amount of 1%, and culture it at 37 °C for 16 - 24 h.

[0090] 3.2 Oxford cup antibacterial test

[0091] Pour the lower layer of the medium. After sterilizing the nutrient agar, pour it into the plate to cover the plate. After the agar solidifies, evenly place 3 sterile Oxford cups on each plate. Pour the upper layer of the medium. After thoroughly mixing the Streptococcus pneumoniae bacterial solution, take 0.2% (v / v) and add it to the brain heart infusion semi-solid medium (containing 5% fetal bovine serum), and take an appropriate amount and evenly spread it on the lower layer of the medium. After the upper layer of the medium solidifies, take out the Oxford cups, and take 100 μl of the mixed fermentation broth of Lactiplantibacillus plantarum VHProbi P06 and add it to the Oxford cup wells. Culture it statically at 37 °C for 24 h, and observe and measure the size of the antibacterial zone.

[0092] The results are as Figure 7 shown. After measurement, the diameter of the antibacterial zone of the Lactiplantibacillus plantarum VHProbi P06 bacterial solution against Streptococcus pneumoniae reaches 16.24 ± 0.08 mm, indicating that the Lactiplantibacillus plantarum VHProbi P06 has a significant inhibitory effect on Streptococcus pneumoniae.

[0093] 3.3 Growth inhibition test of the fermentation supernatant of Lactiplantibacillus plantarum VHProbi P06 against Streptococcus pneumoniae

[0094] 1) Inoculate Lactiplantibacillus plantarum VHProbi P06 into MRS medium at an inoculum amount of 1%, and statically culture at 37 °C for 16 - 24 h. Centrifuge the fermentation broth at 4 °C and 10,000 r / min for 30 min to collect the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the cell-free fermentation supernatant of Lactiplantibacillus plantarum VHProbi P06.

[0095] 2) Add 190 μl of brain heart infusion broth medium supplemented with 5%, 10%, 15%, 20% (v / v) cell-free fermentation supernatant of Lactiplantibacillus plantarum VHProbi P06 into 96-well plates respectively, and then add 10 μl of fresh bacterial suspension of Streptococcus pneumoniae ATCC49619. The brain heart infusion broth medium without adding the cell-free fermentation supernatant of Lactiplantibacillus plantarum VHProbi P06 is used as a control, with 3 parallels in each group. Add 50 μl of sterile paraffin oil to each well to prevent water evaporation during the culture process. Place the 96-well plates in a temperature-controlled microplate reader at 37 °C for 24 h, and measure the OD600 value every 5 min to obtain the growth curve of Streptococcus pneumoniae. The results are as Figure 8 shown.

[0096] It can be seen from the results that compared with the control group, adding the cell-free fermentation supernatant of Lactiplantibacillus plantarum VHProbi P06 can significantly inhibit the growth of the pathogenic bacterium Streptococcus pneumoniae, and with the increase of the addition amount, the inhibitory effect of the fermentation supernatant on Streptococcus pneumoniae is enhanced; when the addition amount of the fermentation supernatant reaches 20% (v / v), Streptococcus pneumoniae stops growing in the brain heart infusion broth medium.

[0097] Example 4 Agglutination and adsorption test of Lactiplantibacillus plantarum VHProbi P06 on Streptococcus pneumoniae

[0098] Take appropriate amounts of activated Lactiplantibacillus plantarum VHProbi P06 and fresh bacterial suspension of Streptococcus pneumoniae, centrifuge at 8000 rpm for 4 min, wash twice with phosphate buffer solution (PBS, pH 7.4), and adjust to OD600 = 4. Add 300 μL of the Lactiplantibacillus plantarum VHProbi P06 bacterial suspension into a 24-well plate, and then add 300 μL of the Streptococcus pneumoniae bacterial suspension as the experimental group; another equal amount of the Lactiplantibacillus plantarum VHProbi P06 bacterial suspension and buffer are mixed as the control group, with 2 parallels set for each control and experimental group. Place the 24-well plate on a microplate constant temperature oscillator, shake and incubate at 400 rpm at room temperature. Microscopically observe and photograph to record the initial well plate state and the well plate state at different times, and observe whether agglutination occurs.

[0099] The macroscopic observation results are as Figure 9As shown, obvious aggregates appeared when Lactobacillus plantarum VHProbi P06 and Streptococcus pneumoniae were combined in the experimental group, while there was no obvious agglutination phenomenon in the control group with only Lactobacillus plantarum VHProbi P06 added; under the microscope, agglutination clumps of Lactobacillus plantarum VHProbi P06 and Streptococcus pneumoniae could be observed in the experimental group ( Figure 10 ).

[0100] Example 5 Aggregation analysis of Lactobacillus plantarum VHProbi P06

[0101] 5.1 Preparation of bacterial suspension

[0102] The activated Lactobacillus plantarum VHProbi P06 was cultured in MRS broth for 18 h, and Streptococcus pneumoniae was cultured in brain heart infusion broth medium at 37 °C for 18 h. After culturing, it was washed 3 times with phosphate buffer solution (PBS, pH 7.4), and the concentration of bacteria was adjusted to make the number of bacteria reach 10 -7 ~10 -8 CFU / mL for standby.

[0103] 5.2 Aggregation analysis

[0104] 20 mL of the bacterial suspension of Lactobacillus plantarum VHProbi P06, the bacterial suspension of Streptococcus pneumoniae, and the equal-volume mixture of the two bacterial suspensions were respectively taken, fully mixed, placed in a constant temperature incubator at 37 °C. The absorbance values at 600 nm of the bacterial suspensions at 0 h, 2 h, 4 h, and 5 h were measured respectively, and the self-aggregation rate and co-aggregation rate were calculated.

[0105] Self-aggregation rate % = [1 - A L / A0] × 100%.

[0106] Co-aggregation rate % = [(A L + A H ) / 2 - A mix / [(A L + A P ) / 2] × 100%.

[0107] A0: Absorbance value of the P06 strain at 0 h;

[0108] A L : Absorbance values of the P06 strain after standing alone for 2, 4, and 5 h;

[0109] A H : Absorbance values of Streptococcus pneumoniae after standing alone for 2, 4, and 5 h;

[0110] A mix : Absorbance values of the mixture of the P06 strain and Streptococcus pneumoniae after standing for 2, 4, and 5 h.

[0111] The results are as Figure 11 shown. The self - polymerization rate of Lactiplantibacillus plantarum VHProbi P06 at 4 h was 19%, and the co - polymerization rate with Streptococcus pneumoniae reached 21%, showing significant effects.

[0112] Example 6 Cytotoxicity test of Lactiplantibacillus plantarum VHProbi P06 against nasopharyngeal carcinoma cells

[0113] 6.1 Preparation of bacterial suspension

[0114] Lactiplantibacillus plantarum VHProbi P06 was cultured in MRS liquid medium until the stationary phase, washed 3 times with phosphate - buffered saline (PBS, pH 7.4), and the concentration of bacteria was adjusted to make the number of bacteria reach 5×10 7 CFU / mL (OD600 absorbance value was about 0.4), and inactivated in a water bath at 70℃ for 20 min for standby.

[0115] 6.2 Cytotoxicity test

[0116] Nasopharyngeal carcinoma cells 5 - 8F were resuscitated and inoculated into a 24 - well culture plate containing cell culture medium with 10% calf serum. The inoculation density was 2×10 5 cells / well, and the cells were cultured for 24 h. The inactivated P06 strain was added to the cells at a ratio of MOI (Multiplicity of Infection) value of 10, and a blank control group without bacteria was set, and the cells were continuously cultured in an incubator for 24 h. MTT solution with a final concentration of 0.3 mg / ml was added to each cell culture well to be detected, and incubated in a carbon dioxide incubator for 3 h. The supernatant was carefully discarded, and 500 μl of DMSO was added to each cell culture well of the 24 - well plate and incubated at 37℃ for 30 min to fully dissolve the purple crystals. The absorbance value at 490 nm was detected.

[0117] From the test results, it can be seen that compared with the control group, Lactiplantibacillus plantarum VHProbi P06 had no significant effect on the proliferation activity of cells, had no cytotoxicity, and had good safety.

[0118] Example 7 Adhesion test of Lactiplantibacillus plantarum VHProbi P06 against nasopharyngeal carcinoma cells

[0119] 7.1 Preparation of bacterial suspension

[0120] Lactiplantibacillus plantarum VHProbi P06 was cultured in MRS liquid medium until the stationary phase, washed 3 times with phosphate - buffered saline (PBS, pH 7.4), resuspended with cell culture medium, and the concentration of bacteria was adjusted to make the number of bacteria reach 1×10 8 CFU / mL for standby.

[0121] 7.2 Cell culture

[0122] Take out nasopharyngeal carcinoma cells 5-8F from the liquid nitrogen tank, resuscitate and passage culture, and expand the number of cultured cells to the required amount. Inoculate 5-8F cells into a six-well culture plate containing 10% calf serum cell culture medium with a cell slide inside. The number of cells plated in each well is about 2×10 6 cells, and place the six-well plate in a carbon dioxide incubator for 24 h.

[0123] 7.3 Adhesion test

[0124] The monolayer of adherent 5-8F single cells in the six-well plate was washed 3 times with PBS buffer, the prepared bacterial suspension of Lactiplantibacillus plantarum VHProbi P06 was added, and it was cultured in a carbon dioxide incubator for 1 h. The cell slide was washed repeatedly 3 times with PBS buffer to remove non-adherent bacteria. It was fixed with absolute methanol for 20 minutes, the cell slide was taken out and dried, and Gram staining was performed. The lactic acid bacteria adhering to 20 random fields of view were observed under an oil immersion microscope at 100× magnification, and a total of 100 cells were observed. Calculate the number of lactic acid bacteria adhering to each cell on average.

[0125] Through statistical analysis, it can be known that the adhesion amount of Lactiplantibacillus plantarum VHProbi P06 to 5-8F cells is 13.10±0.12 CFU / cell, indicating that Lactiplantibacillus plantarum VHProbi P06 can strongly adhere to the surface of nasopharyngeal carcinoma cells and has strong adhesion ability ( Figure 12 )

[0126] Example 8 Adhesion inhibition experiment of Lactiplantibacillus plantarum VHProbi P06 on Streptococcus pneumoniae

[0127] 8.1 Preparation of bacterial suspension

[0128] The fresh bacterial solutions of Lactiplantibacillus plantarum VHProbi P06 and Streptococcus pneumoniae were washed 3 times with phosphate buffer solution (PBS, pH 7.4), resuspended with cell culture medium, and the concentration of bacteria was adjusted to make the number of bacteria reach 1×10 8 CFU / mL for standby.

[0129] 8.2 Adhesion inhibition test

[0130] Wash the adherent 5-8F single cell layer in a 6-well plate 3 times with PBS buffer. Add 1 mL of the above Lactiplantibacillus plantarum VHProbi P06 and Streptococcus pneumoniae bacterial suspensions respectively. The cells without the Lactiplantibacillus plantarum VHProbi P06 bacterial suspension are used as blank controls. Place them in a carbon dioxide incubator and culture for 2 h. Wash the cell slides repeatedly 3 times with PBS buffer to remove the non-adherent bacteria. Fix with absolute methanol for 20 minutes, take out the cell slides and let them dry, perform Gram staining, and examine under an oil immersion microscope at 100× magnification to observe 20 random fields of view, a total of 100 Streptococcus pneumoniae adhering to the cells, and calculate the number of Streptococcus pneumoniae adhering to each cell on average. Compare the changes in the number of Streptococcus pneumoniae adhering under the conditions of the presence and absence of Lactiplantibacillus plantarum VHProbi P06. Taking the adhesion rate of Streptococcus pneumoniae in the group without Lactiplantibacillus plantarum VHProbi P06 as 100%, investigate the reduction of the adhesion rate of Streptococcus pneumoniae when Lactiplantibacillus plantarum VHProbi P06 is present, so as to evaluate the ability of Lactiplantibacillus plantarum VHProbi P06 to inhibit the adhesion of Streptococcus pneumoniae to 5-8F cells.

[0131] The results showed that when not treated with Lactiplantibacillus plantarum VHProbi P06, the adhesion rate of Streptococcus pneumoniae to 5-8F cells was 100%. Lactiplantibacillus plantarum VHProbi P06 could reduce the adhesion rate of Streptococcus pneumoniae to 5-8F cells by 71.56%. Thus, it shows that Lactiplantibacillus plantarum VHProbi P06 can significantly inhibit the adhesion ability of Streptococcus pneumoniae to cells. The results are shown in Figure 13 .

[0132] Example 9 Inhibition of Lactiplantibacillus plantarum VHProbi P06 against other respiratory pathogenic bacteria

[0133] The respiratory pathogenic bacteria used in this example include:

[0134] β-hemolytic streptococcus: CMCC(B)32210, use brain heart infusion broth medium supplemented with 5% (v / v) fetal bovine serum, culture at 37 °C for 16 - 24 h;

[0135] Streptococcus pyogenes: BNCC337110, BNCC185918, use Columbia broth medium supplemented with 5% (v / v) fetal bovine serum, culture at 37 °C for 16 - 24 h;

[0136] Escherichia coli: BNCC337304, BNCC133264, BNCC269342, use nutrient broth medium, culture at 37 °C for 16 - 24 h;

[0137] Staphylococcus aureus: ATCC29213, ATCC25923, ATCC6538, cultured in nutrient broth medium at 37°C for 16 - 24 h.

[0138] Lactiplantibacillus plantarum VHProbi P06 was inoculated into MRS medium at an inoculation amount of 1% and cultured statically at 37°C for 24 h.

[0139] Pour the lower layer of the medium. After sterilizing the nutrient agar, pour it into a petri dish to cover the bottom of the dish. After the agar solidifies, evenly place 3 sterile Oxford cups on each petri dish. Pour the upper layer of the medium. After thoroughly mixing the cultured β - hemolytic streptococcus, Streptococcus pyogenes (a 1:1 volume mixture of 2 strains), Escherichia coli (a 1:1 volume mixture of 3 strains), and Staphylococcus aureus (a 1:1 volume mixture of 3 strains), respectively add 0.2% (v / v) to the corresponding semi - solid medium, and take an appropriate amount and evenly spread it on the lower layer of the medium. After the upper layer of the medium solidifies, remove the Oxford cups, take 100 μl of the well - mixed Lactiplantibacillus plantarum VHProbi P06 bacterial solution and add it to the Oxford cup wells, and culture statically at 37°C for 24 h. Observe and measure the size of the inhibition zone. See the picture of the inhibition zone in Figure 14 , and the size of the inhibition zone is shown in Table 1.

[0140] Table 1: Inhibitory ability of Lactiplantibacillus plantarum VHProbi P06 against 4 respiratory pathogenic bacteria

[0141] Pathogenic bacteria Diameter of inhibition zone / mm β-hemolytic streptococcus 15.16±0.21 Streptococcus pyogenes 21.17±0.18 Escherichia coli 13.72±0.05 Staphylococcus aureus 12.04±0.15

[0142] As can be seen from the above table, Lactiplantibacillus plantarum VHProbi P06 has strong inhibitory effects on β - hemolytic streptococcus, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus.

[0143] In summary, the Lactiplantibacillus plantarum VHProbi P06 described in the present invention has no cytotoxicity, good safety, can significantly inhibit the growth of Streptococcus pneumoniae, the main respiratory pathogenic bacterium, has strong agglutination ability, providing necessary conditions for its co - agglutination with other microorganisms, especially pathogenic microorganisms, in the respiratory environment; this strain has a strong adhesion effect on nasal epithelial cells, providing favorable conditions for its adhesion and colonization in the nasal cavity, and can significantly inhibit the adhesion of Streptococcus pneumoniae, the main respiratory pathogenic microorganism, to nasal epithelial cells; at the same time, this strain has significant inhibitory effects on the growth of respiratory disease - causing pathogenic bacteria such as β - hemolytic streptococcus, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus. The Lactiplantibacillus plantarum VHProbi P06 can be used to prepare products for preventing and treating respiratory diseases, and has broad application prospects.

Claims

1. A Lactiplantibacillus plantarum, characterized in that, The Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) has a deposit number of CCTCC NO: M2022359.

2. Use of Lactiplantibacillus plantarum according to claim 1 in the preparation of a medicament for preventing or treating bacterial rhinosinusitis, wherein the bacteria are Streptococcus pneumoniae, β-hemolytic streptococcus, Streptococcus pyogenes, Escherichia coli or Staphylococcus aureus.

3. A probiotic preparation, characterized in that, The probiotic preparation contains viable cells of Lactiplantibacillus plantarum according to claim 1.

4. The probiotic preparation according to claim 3, wherein The probiotic preparation further contains any one or more of dead cells of Lactiplantibacillus plantarum according to claim 1, intracellular metabolites, and intracellular extracts.

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