Lactobacillus mucosae Roy and its products and applications for improving allergic rhinitis and sinusitis

Abstract

The present invention relates to the field of microbial technology, and specifically relates to Limosilactobacillus reuteri and products and applications for improving allergic rhinitis and sinusitis. The Limosilactobacillus reuteri NHNK-2001 disclosed in the present invention is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 2024800. Experiments show that NHNK-2001 has the functions of inhibiting the growth and biofilm formation of sinusitis-related flora, flocculating rhinitis pathogens, promoting the growth of beneficial bacteria in the nasal cavity, inhibiting the enterotoxin of Staphylococcus aureus subsp. aureus, degrading histamine, reducing histamine stimulation, promoting the nasal epithelial cell barrier, and reducing nasal epithelial cell inflammation. In addition, the biofilm formed by NHNK-2001 cells can increase the colonization ability of intestinal epithelial cells. It can be used to prepare products for improving allergic rhinitis and sinusitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and particularly relates to Limosilactobacillus reuteri for improving allergic rhinitis and sinusitis, products thereof and applications. Background Art

[0002] Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mediated by IgE after the body contacts allergens. The diversity of the nasal commensal flora in AR patients significantly decreases. Streptococcus salivarius ( Streptococcus salivarius ) is the most abundant commensal bacterium in the nasal cavity of AR patients, aggravating the symptoms of rhinitis. The relative abundances of Propionibacterium acnes, Propionibacterium and Corynebacterium in the healthy group are significantly higher than those in the AR group. In addition, researchers found that Staphylococcus aureus subsp. aureus enterotoxin B (SEB) increases the nasal mucosal permeability of AR patients in a dose-dependent manner, reduces the transepithelial electrical resistance and destroys the integrity of nasal epithelial cells.

[0003] Histamine is an organic nitrogen compound produced by histidine under the action of decarboxylase. Mast cells in the skin, lungs and mucous membranes contain a large amount of histamine. When tissues are damaged, stimulated or have an allergic reaction, histamine is released in large amounts, leading to local tissue edema, narrowing of the airway and aggravating respiratory allergy symptoms. Histamine receptors mainly include three subtypes, namely H1R, H2R and H3R, all of which are related to allergic symptoms. The epidermal growth factor receptor EGFR has been reported to be related to rhinitis in recent years, and its overexpression will cause an increase in mucus secretion of nasal cells. In addition, EGFR inhibitors have been reported to reduce the size of nasal polyps, so they are targets for the treatment of rhinitis or sinusitis.

[0004] Sinusitis is a common sinus infection disease. Common symptoms include nasal congestion, purulent nasal discharge, headache, hyposmia, etc., and it may also cause inflammation in the pharynx, tonsils and lower nasal cavity, so timely treatment is required. Most acute sinusitis is caused by upper nasal cavity infections, and the pathogenic bacteria are mostly pyogenic cocci, such as Streptococcus pneumoniae, Staphylococcus aureus subsp. aureus, Haemophilus influenzae and Moraxella catarrhalis, etc., while odontogenic infections are mainly anaerobic oral pathogenic bacteria such as Prevotella. Chronic sinusitis is mostly caused by repeated attacks of acute sinusitis without complete cure. According to the latest research, the nasal cavity of healthy people has the colonization of commensal microorganisms, and the change of nasal flora and the destruction of the epithelial barrier may be the key factors inducing acute and chronic sinusitis.

[0005] Beneficial microorganisms can inhibit the growth of pathogenic bacteria, regulate immune homeostasis and protect the nasal epithelial cell barrier, and the active substances of these microorganisms have important application values in various fields. Therefore, it has important practical significance to provide a microbial-related product developed by using microbial technology. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides Lactobacillus mucosae ROEMERI and its products and applications for improving allergic rhinitis and sinusitis, aiming to solve the technical problems in the prior art.

[0007] The present invention provides Lactobacillus mucosae ROEMERI ( Limosilactobacillus reuteri ), which is Lactobacillus mucosae ROEMERI ( Limosilactobacillus reuteri ) NHNK-2001, deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, postcode 430072) on April 25, 2024, with the deposit number of CCTCC NO: M 2024800.

[0008] Furthermore, according to the above Lactobacillus mucosae ROEMERI ( Limosilactobacillus reuteri ), the product prepared therefrom is viable bacteria, inactivated bacteria or fermentation products of Lactobacillus mucosae ROEMERI ( Limosilactobacillus reuteri ).

[0009] The present invention also provides the application of the above Lactobacillus mucosae ROEMERI ( Limosilactobacillus reuteri ) or the product prepared therefrom in the preparation of products for improving sinusitis. Limosilactobacillus reuteri

[0010] Furthermore, the improvement of sinusitis includes at least one of the following a)-d):

[0011] a) Inhibiting the growth and biofilm formation of sinusitis-related flora, inhibiting the growth and biofilm formation of at least one of Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. aureus ), Streptococcus pneumoniae ( Streptococcus pneumoniae ), Haemophilus influenzae ( Haemophilus influenzae ), Streptococcus salivarius ( Streptococcus salivarius ), Moraxella catarrhalis ( Moraxella catarrhalis ) and Prevotella nigrescens ( Prevotella nigrescens );

[0012] b) Promoting the growth of beneficial bacteria in the nasal cavity, promoting the growth of Propionibacterium acnes ( Propionibacterium acnes );

[0013] c) Flocculating sinusitis-related pathogenic bacteria, flocculating at least one of Haemophilus influenzae ( Haemophilus influenzae ) and Streptococcus pneumoniae ( Haemophilus influenzae );

[0014] d) Inhibiting the enterotoxin of Staphylococcus aureus subsp. aureus and down-regulating the expression of the enterotoxin B gene of Staphylococcus aureus subsp. aureus seb of.

[0015] The present invention also provides the use of Lactobacillus reuteri as described above ( Limosilactobacillus reuteri ), or a product prepared from Lactobacillus reuteri as described above ( Limosilactobacillus reuteri ) in the preparation of a product for improving allergic rhinitis.

[0016] Furthermore, the improvement of allergic rhinitis includes at least one of the following a)-e):

[0017] a) Degrading histamine;

[0018] b) Reducing histamine stimulation and downregulating the expression of histamine receptors on nasal epithelial cells H1R , H2R and H3R ;

[0019] c) Promoting the nasal epithelial cell barrier and upregulating the expression of tight junction proteins on nasal epithelial cells OCLD ;

[0020] d) Reducing nasal epithelial cell inflammation and regulating the expression of tumor necrosis factor-α gene TNF-α and epidermal growth factor receptor gene EGFR ;

[0021] e) Increasing the colonization ability of the bacterial biofilm on intestinal epithelial cells.

[0022] Furthermore, the degradation of histamine is the degradation of histamine in the in vitro culture medium.

[0023] The present invention also provides a product for improving allergic rhinitis and sinusitis, including Lactobacillus reuteri as described above ( Limosilactobacillus reuteri ), or a product prepared from Lactobacillus reuteri as described above ( Limosilactobacillus reuteri ), and acceptable excipients and / or adjuvants.

[0024] Lactobacillus reuteri disclosed in the present invention ( Limosilactobacillus reuteri ) has a deposit number of CCTCC NO: M 2024800. Experiments show that NHNK-2001 has the functions of inhibiting the growth and biofilm formation of sinusitis-related bacteria, flocculating sinusitis-related pathogenic bacteria, promoting the growth of beneficial nasal bacteria, inhibiting the enterotoxin of Staphylococcus aureus subsp. aureus, degrading histamine, reducing histamine stimulation, promoting the nasal epithelial cell barrier, and reducing nasal epithelial cell inflammation.

[0025] Biological deposit description

[0026] Lactobacillus reuteri ( Limosilactobacillus reuteri)(NHNK-2001) was deposited with the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, postal code 430072) on April 25, 2024, and its deposit number is CCTCC NO: M 2024800. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is a MRS plate colony map and Gram staining map of Lactobacillus mucosae NHNK-2001 in the present invention;

[0029] Figure 2 It is a result map of the inhibitory effect of the fermentation product of Lactobacillus mucosae NHNK-2001 in the present invention on the biofilm formation of sinusitis-related flora;

[0030] Figure 3 It is the inhibition of biofilm formation of sinusitis-related flora by heat-inactivated cells of Lactobacillus mucosae NHNK-2001 in the present invention;

[0031] Figure 4 It is a diagram of the flocculation state of Lactobacillus mucosae NHNK-2001, pathogenic bacteria, and the cells of Lactobacillus mucosae NHNK-2001 and pathogenic bacteria in the present invention;

[0032] Figure 5 It is a result map of the histamine degradation experiment of Lactobacillus mucosae NHNK-2001 in the present invention;

[0033] Figure 6 It is a result map of the experiment on the adsorption of live cells of Lactobacillus mucosae NHNK-2001 and the biofilm of NHNK-2001 to intestinal epithelial cells Caco-2 in the present invention.

[0034] The following will further illustrate the embodiments of the present invention with reference to the drawings. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides Lactobacillus reuteri and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] The Lactobacillus reuteri strain NHNK-2001 of the present invention is derived from the feces of a 2-year-old healthy girl and is identified as Lactobacillus reuteri ( Limosilactobacillus reuteri ). This strain is Gram-positive and appears as a short rod with a round end under a microscope. It grows on MRS plates to form round colonies with smooth, translucent surfaces, white in color, and neat edges. It grows evenly and turbidly in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage. The optimal growth temperature is 37°C. For the specific sequence of 16S rDNA, please refer to the sequence list.

[0037] Lactobacillus reuteri ( Limosilactobacillus reuteri )NHNK-2001, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: April 25, 2024, deposit number: CCTCC NO: CCTCC NO: M 2024800.

[0038] Furthermore, in the application of the present invention, the Lactobacillus mucosa NHNK-2001 provided by the present invention is in the form of sterilized inactivated bacteria, or in the form of fermentation / secretion products (i.e., supernatant), and is not sterilized live bacteria. The derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation products, and inactivated bacteria.

[0039] The reagents and consumables used in the present invention are all common commercially available products. The present invention is further described below in conjunction with the embodiments:

[0040] Example 1 Isolation of NHNK-2001

[0041] The samples were collected from the feces of a healthy 2-year-old girl. After the samples were processed, they were shaken and mixed in physiological saline, and the supernatant was streaked on an MRS solid plate. After anaerobic culture at 37°C for 48 hours, white colonies were picked and repeatedly inoculated and screened until a uniform single colony was obtained, which was named NHNK-2001.

[0042] Gram staining microscopy: The strain NHNK-2001 is a Gram-positive colony, showing short rod-shaped with rounded ends under the microscope; growing on MRS plates, it can form white, smooth and round semi-transparent circular colonies with neat edges; growing uniformly turbid in MRS liquid medium, and the bacteria form white precipitates after standing for a long time. As Figure 1 shown.

[0043] Example 2 Nucleic acid identification of NHNK-2001

[0044] 1. 16S rDNA gene sequence analysis:

[0045] Pick a single colony into MRS liquid medium, culture overnight at 37°C, centrifuge at 8000 rpm for 1 min to collect the bacteria, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used are the universal primers 27F and 1492R for bacterial 16S sequencing, and the PCR amplification system is 20 μL. The PCR amplification program is pre-denaturation at 95°C for 5 min, 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, for 35 cycles; extension at 72°C for 10 min.

[0046] 2. Results:

[0047] After the PCR product was sequenced and compared with the published standard sequences in the GenBank database (BLASTN), it was found that the strain NHNK-2001 was Lactobacillus mucosae ( Limosilactobacillus reuteri ).

[0048] Example 3 Inhibition of the proliferation of sinusitis-related flora by NHNK-2001

[0049] 1. Culture of sinusitis-related flora

[0050] Streptococcus pneumoniae ( Streptococcus pneumoniae ) The frozen stock solution of BNCC338425 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v), cultured with shaking at 37°C for 2 d, and then adjusted to OD 600 = 0.1 to obtain the Streptococcus pneumoniae suspension.

[0051] Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. aureus ) The frozen stock solution of CGMCC 1.8721 was inoculated into BHI medium at 2% (v / v), cultured with shaking at 37°C for 2 d, and then adjusted to OD 600 = 0.1 to obtain the Staphylococcus aureus subsp. aureus suspension.

[0052] Haemophilus influenzae ( Haemophilus influenzae)The bacterial liquid in the cryopreservation tube of BNCC359479 was inoculated into the TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v). After shaking culture at 37 °C for 2 days, the OD was adjusted with the TSB medium 600 = 0.1 to obtain the Moraxella catarrhalis suspension.

[0053] Streptococcus salivarius ( Streptococcus salivarius )The bacterial liquid in the cryopreservation tube of ATCC 7073 was inoculated into the M17 liquid medium at 2% (v / v). After shaking culture at 42 °C for 2 days, the OD was adjusted with the M17 medium 600 = 0.1 to obtain the Streptococcus salivarius suspension.

[0054] Moraxella catarrhalis ( Moraxella catarrhalis )The bacterial liquid in the cryopreservation tube of BNCC275822 was inoculated into the BHI liquid medium at 2% (v / v). After shaking culture at 37 °C for 2 days, the OD was adjusted with the BHI medium 600 = 0.1 to obtain the Moraxella catarrhalis suspension.

[0055] Prevotella nigrescens ( Prevotella nigrescens )The bacterial liquid in the cryopreservation tube of BNCC369693 was inoculated into the TSB medium containing 5% defibrinated sheep blood at 2% (v / v). After anaerobic culture at 37 °C for 2 days, the OD was adjusted with the special peptone medium 600 = 0.1 to obtain the Prevotella nigrescens suspension.

[0056] 2. Preparation of the fermentation product of NHNK-2001

[0057] The glycerol tube bacterial liquid of NHNK-2001 was inoculated into the fresh MRS medium at 2% (v / v). After culturing at 37 °C for 48 h, it was adjusted to OD with MRS 600 = 1.0, centrifuged at 5000 rpm for 10 min, and the supernatant was taken. After filtration through a 0.22 μm filter membrane, the fermentation product was obtained.

[0058] 3. The fermentation product of NHNK-2001 inhibits the proliferation of sinusitis-related bacteria

[0059] The pathogenic bacterial suspension was added to the 96-well plate at 160 μL per well. In the experimental group, 40 μL of the fermentation product was added, and in the control group, an equal volume of MRS medium was added. After culturing at 37 °C for 48 hours, the absorbance value at OD 600 nm was measured. Inhibition rate % = (1 - absorbance value of the experimental group / absorbance value of the control group) * 100%. The results are shown in Table 1 below:

[0060] Table 1

[0061]

[0062] The results showed that the fermentation product of NHNK-2001 could inhibit the growth of Staphylococcus aureus subsp. aureus, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus salivarius, Moraxella catarrhalis, and Prevotella nigrescens, with the inhibition rate ranging from 13.66% to 83.14%.

[0063] Example 4: NHNK-2001 Promotes the Proliferation of Propionibacterium acnes

[0064] 1. Cultivation of Propionibacterium acnes ( Propionibacterium acnes )

[0065] The frozen bacterial liquid of Propionibacterium acnes CGMCC 1.5003 was inoculated into a special peptone medium at 2% (v / v) and anaerobically cultured at 37°C for 5 - 6 days, and then adjusted to OD 600 = 0.5 to obtain the Propionibacterium acnes bacterial suspension.

[0066] 2. Preparation of the fermentation product of NHNK-2001

[0067] The preparation method of the fermentation product of NHNK-2001 refers to Example 3.

[0068] 3. NHNK-2001 fermentation product promotes the proliferation of Propionibacterium acnes

[0069] Propionibacterium acnes was added to a 96-well plate at 160 μL per well. In the experimental group, 40 μL of the fermentation product was added, and in the control group, an equal volume of MRS medium was added. After culturing at 37°C for 48 hours, the absorbance value at OD 600 nm was measured. The proliferation promotion rate (%) = (absorbance value of the experimental group - absorbance value of the control group) / absorbance value of the control group * 100%. The results are shown in Table 2 below:

[0070] Table 2

[0071]

[0072] The results showed that the fermentation product of NHNK-2001 could promote the growth of Propionibacterium acnes, and the proliferation promotion rate was 34.83 - 38.89%.

[0073] Example 5: NHNK-2001 Inhibits the Biofilm of Sinusitis-Related Bacterial Flora

[0074] 1. Cultivation of sinusitis-related bacterial flora

[0075] The preparation method of the bacterial suspension of Staphylococcus aureus subsp. aureus / Streptococcus pneumoniae / Haemophilus influenzae / Streptococcus salivarius / Moraxella catarrhalis / Prevotella nigrescens refers to Example 3.

[0076] 2. Preparation of the fermentation product of NHNK-2001 and inactivated bacteria

[0077] The NHNK-2001 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v). After culturing at 37 °C for 48 h, it was adjusted to OD 600 = 1.0 with MRS. The supernatant was taken after centrifugation at 5000 rpm for 10 min, and the fermentation product was obtained after filtration through a 0.22 μm filter membrane. The precipitated bacteria were washed 3 times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 = 1.0, and sterilized at 121 °C for 15 min to obtain heat-inactivated bacteria.

[0078] 3. NHNK-2001 inhibits the biofilm of sinusitis-related flora

[0079] The sinusitis-related flora bacterial solution was added to a 96-well plate at 100 μL per well. In the experimental group, 100 μL of the NHNK-2001 fermentation product or heat-inactivated bacteria was added to each well, and an equal volume of MRS medium or PBS was added to the control group. There were 3 parallels in each group, and then the culture was continued at 37 °C for 24 hours.

[0080] After the culture was completed, the supernatant was discarded, and each well was washed twice with 100 μL of sterile PBS. Then, 100 μL of 4% paraformaldehyde fixative was added to each well and fixed at room temperature for 30 min. The fixative was discarded, 100 μL of crystal violet was added to each well, and stained at room temperature for 30 min. After the staining was completed, it was washed twice with sterile PBS and air-dried. 100 μL of absolute ethanol was added to each well. As Figure 2 (fermentation product) and Figure 3 (heat-inactivated bacteria) shown, the absorbance at 600 nm was measured after standing for 1 min. Inhibition rate % = (1 - experimental group / control group) * 100%.

[0081] The results are shown in Tables 3 and 4 below:

[0082] Table 3

[0083]

[0084] Table 4

[0085]

[0086] The results showed that the NHNK-2001 fermentation product could inhibit the biofilm formation of Staphylococcus aureus subsp. aureus, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus salivarius, Moraxella catarrhalis, and Prevotella nigrescens, which are related to sinusitis, and the inhibition rate was between 23.50 and 87.04%.

[0087] Example 6 NHNK-2001 flocculates sinusitis-related pathogenic bacteria

[0088] 1. Culture of sinusitis-related pathogenic bacteria

[0089] Streptococcus pneumoniae ( Streptococcus pneumoniae ) The bacterial liquid in the cryopreservation tube of BNCC338425 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v). After shaking culture at 37°C for 2 days, the bacteria were collected by centrifugation and washed 3 times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 = 1.0, sterilized at 121°C for 15 min for standby.

[0090] Haemophilus influenzae ( Haemophilus influenzae ) The bacterial liquid in the cryopreservation tube of BNCC359479 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v). After shaking culture at 37°C for 2 days, the bacteria were collected by centrifugation and washed 3 times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 = 1.0, sterilized at 121°C for 15 min for standby.

[0091] 2. Preparation of inactivated NHNK-2001 bacteria

[0092] The preparation method of inactivated NHNK-2001 bacteria refers to Example 5.

[0093] 3. Co-flocculation

[0094] Mix the pathogenic bacteria liquid and the inactivated NHNK-2001 bacteria liquid according to a volume ratio of 1:1, and observe the flocculation condition after standing for 30 min.

[0095] 4. Index determination

[0096] At 30 min of the reaction, samples of NHNK-2001 alone, pathogenic bacteria, and the mixed reaction solution of NHNK-2001 and pathogenic bacteria were taken. The sampling range was 50 μL from the uppermost layer of the liquid surface. After aspiration, they were transferred to a 96-well plate, and the absorbance at OD = 600 nm was measured. At the same time, the flocculated precipitate was stained with Gram stain and the flocculation state of the bacteria was observed. As Figure 4 shown.

[0097] Calculation formula:

[0098] Flocculation rate (%) = [(Ax + Ay) - 2Amix] / (Ax + Ay) × 100%;

[0099] Note: Ax: OD value of NHNK-2001 alone measured at the reaction time; Ay: OD value of pathogenic bacteria alone measured at the reaction time; Amix: OD value of the mixture of NHNK-2001 and pathogenic bacteria measured at the reaction time. 600 value; Ay: OD value of pathogenic bacteria alone measured at the reaction time 600 value; Amix: OD value of the mixture of NHNK-2001 and pathogenic bacteria measured at the reaction time 600 value.

[0100] The results are shown in Table 5 below:

[0101] Table 5

[0102]

[0103] The results showed that NHNK-2001 could flocculate Haemophilus influenzae and Streptococcus pneumoniae, and the flocculation rate was between 11.38% and 32.96%.

[0104] Example 7 Inhibition of Enterotoxin of Staphylococcus aureus subsp. aureus by NHNK-2001

[0105] 1. Preparation of heat-inactivated NHNK-2001 bacteria

[0106] Inoculate the bacterial liquid in the NHNK-2001 cryopreservation tube into MRS medium at an inoculation rate of 2%, and incubate statically at 37°C for 24 h. Centrifuge at 5000 rpm for 10 min to collect the bacterial cells. After washing with PBS and centrifuging to discard the supernatant, resuspend the washed live bacterial cells with PBS and adjust the OD 600 = 0.5, and sterilize at 121°C for 15 min to obtain a heat-inactivated bacterial suspension.

[0107] 2. Regulation of the expression of enterotoxin of Staphylococcus aureus subsp. aureus by NHNK-2001

[0108] (1) Culture and intervention of Staphylococcus aureus subsp. aureus

[0109] Inoculate the glycerol bacterial liquid of Staphylococcus aureus subsp. aureus CGMCC 1.8721 into fresh BHI medium at an inoculation rate of 2% (v / v), and incubate with shaking at 37°C for 24 h. Then adjust the OD 600 = 0.5. Add the bacterial suspension of Staphylococcus aureus subsp. aureus into centrifuge tubes, 1 mL per tube, and add another 2 mL of BHI medium. Add 1 mL of heat-inactivated NHNK-2001 bacteria to the experimental group, and add an equal volume of PBS to the control group. Continue to culture at 37°C for 16 h.

[0110] (2) After the culture is completed, extract the RNA of Staphylococcus aureus subsp. aureus using a Gram-positive bacteria RNA extraction kit. After detecting the RNA concentration and purity, reverse transcribe it into cDNA. Using 16S rRNA as an internal reference gene, use qPCR fluorescence quantitative technology to detect the expression of the enterotoxin B gene of Staphylococcus aureus subsp. aureus seb .

[0111] (3) Data processing

[0112] The relative expression multiple F of the control group gene = 1. Use the 2 -ΔΔCT method to calculate the F value of each sample. The results are shown in Table 6 below:

[0113] Table 6

[0114]

[0115] The results showed that the heat-inactivated NHNK-2001 could inhibit the expression of the enterotoxin B gene of Staphylococcus aureus subsp. aureus, and the relative expression fold was between 0.11 and 0.13 times. seb

[0116] Example 8 Degradation of histamine by NHNK-2001

[0117] 1. Experiment on the degradation of histamine by NHNK-2001

[0118] Inoculate the glycerol bacteria of NHNK-2001 into MRS medium containing 500 μg / mL histamine at 2% (v / v), and anaerobically culture at 37 °C for 48 h. The control group was inoculated with an equal volume of MRS medium.

[0119] 2. Reagent preparation

[0120] ① 50 g / L Na 2 CO 3 solution: Dissolve 5 g of Na 2 CO 3 in pure water and dilute it to 100 mL with pure water.

[0121] ② 250 g / L NaOH solution: Dissolve 25 g of NaOH in pure water and dilute it to 100 mL.

[0122] ③ HCl solution: Add 5 mL of concentrated hydrochloric acid HCl to 55 mL of pure water and mix well.

[0123] ④ Solution A (p-nitroaniline solution): Weigh 0.5 g of p-nitroaniline, dissolve it in 5 mL of HCl, and then dilute it to 200 mL with pure water and mix well. Prepare it freshly before use. Solution B (NaNO 2 solution): Weigh 0.5 g of NaNO 2 and dissolve it in pure water and dilute it to 100 mL and mix well. Azo reagent: Pipette 5 mL of Solution A and 40 mL of Solution B and mix.

[0124] ⑤ 1000 μg·mL -1 phosphoric acid histamine standard solution: Accurately weigh 0.025 g of histamine dihydrochloride standard product, dissolve it in pure water, transfer it to a 25 mL volumetric flask, and make up the volume to the mark with pure water and mix well. 20 μg·mL -1 phosphoric acid histamine standard working solution Pipette 1 mL of 1000 μg·mL -1 phosphoric acid histamine standard solution into a 50 mL volumetric flask, make up the volume to the mark with pure water and mix well.

[0125] 3. Determination of histamine content

[0126] 1. Pipette 0.0, 0.20, 0.40, 0.60, 0.80, 1.0 mL of 20 μg / mL histamine phosphate standard solution into 10-mL stoppered colorimetric tubes, corresponding to containing 0, 4, 8, 12, 16, 20 μg of histamine. Add water to make the volume up to 1 mL. Then add 1 mL of HCl solution, 3 mL of 50 g / L Na2CO3 solution, 3 mL of azo reagent, add water to make the volume up to the scale, and measure the OD after standing for 10 min 480 And draw a standard curve

[0127] 2. Pipette 1 mL of the sample to be tested, then add 1 mL of HCl solution, 3 mL of 50 g / L Na2CO3 solution, 3 mL of azo reagent, add water to make the volume up to the scale, and measure the OD after standing for 10 min 480 As Figure 5 shown. Degradation rate (%) = (1 - absorbance value of the experimental group / absorbance value of the control group) * 100

[0128] The results are as shown in Table 7 below

[0129] Table 7

[0130]

[0131] The results show that the degradation rate of NHNK-2001 to histamine is between 16.57% and 21.13%

[0132] Example 9 NHNK-2001 regulates the expression of histamine receptor, barrier and inflammation-related genes in human nasal epithelial cells RPMI-2650

[0133] 1. Preparation of inactivated NHNK-2001 bacteria

[0134] Inoculate the NHNK-2001 bacterial solution into fresh MRS medium at 2% (v / v), culture anaerobically at 37 °C for 48 h, centrifuge at 5000 rpm for 10 min. Wash the centrifuged precipitate with PBS and resuspend it in an appropriate amount. Sterilize at 121 °C for 15 min, remove the supernatant after centrifugation, and resuspend it with DMEM to adjust the OD600 to 0.5 to obtain the inactivated bacterial suspension

[0135] 2. NHNK-2001 regulates the expression of histamine receptor, barrier and inflammation-related genes in RPMI-2650 cells

[0136] (1) Intervention treatment of human nasal epithelial cells RPMI-2650 cells

[0137] RPMI-2650 cells are inoculated in DMEM medium containing 10% FBS serum, at 37 °C, 5% CO2 Cultured under the following conditions. When the cell fusion reached 80%, the cells were collected and seeded in a 6-well plate. After overnight culture, the supernatant was discarded. In the experimental group, 1 mL of inactivated bacteria and 1 mL of DMEM medium without serum were added to each well, and in the control group, an equal volume of DMEM was used instead. The cells were cultured at 37 °C and 5% CO 2 for 16 h.

[0138] (2)RNA extraction and fluorescence quantitative PCR

[0139] After the culture was completed, the cells were lysed with RIPA lysis buffer, and total RNA was extracted. After detecting the concentration and purity of the RNA, it was reverse transcribed into cDNA. Using GAPDH as the internal reference gene, the expression of histamine receptor-related genes H1R, H2R, H3R ; tight junction protein genes OCLD ; tumor necrosis factor-α gene TNF-α and epidermal growth factor receptor gene EGFR was detected by qPCR fluorescence quantitative technology.

[0140] (3)Data processing

[0141] The relative expression multiple of the control group gene F = 1, and the F value of each sample was calculated using the 2 -ΔΔCT method.

[0142] The results are shown in Table 8 below:

[0143] Table 8

[0144]

[0145] The results showed that NHNK-2001 inactivated bacteria down-regulated the expression levels of human nasal epithelial cell RPMI-2650 histamine receptor genes H1R, H2R and H3R , down-regulated the expression levels of tumor necrosis factor-α gene TNF-α and epidermal growth factor receptor gene EGFR , and at the same time up-regulated the expression of tight junction protein gene OCLD . The gene expression levels were between 0.22-fold and 1.86-fold.

[0146] Example 10 Formation of biofilm by NHNK-2001

[0147] 1. Formation of NHNK-2001 biofilm

[0148] A single colony of NHNK-2001 was picked into MRS liquid medium and anaerobically cultured at 37 °C for 48 h. It was adjusted to OD with fresh MRS 600= 0.2, add 100 μL of bacterial liquid into each well of a 96-well plate, with 3 parallels in each group, and then continue anaerobic culture at 37 °C for 24 hours.

[0149] 2. Crystal violet staining

[0150] After the culture is completed, discard the supernatant, add 100 μL of sterile PBS to each well and wash twice, then add 100 μL of 4% paraformaldehyde fixative to each well and fix at room temperature for 30 min. Discard the fixative, add 100 μL of crystal violet to each well and stain at room temperature for 30 min. After staining, wash twice with sterile PBS and air dry, add 100 μL of absolute ethanol to each well, let stand for 1 min and then measure the absorbance at 600 nm.

[0151] The results are shown in Table 9 below:

[0152] Table 9

[0153]

[0154] The results show that NHNK-2001 can form biofilms when anaerobically statically cultured in MRS medium at 37 °C for 24 h at a bacterial concentration of OD 600 = 0.2.

[0155] Example 10 Determination of the adhesion ability of NHNK-2001 to human intestinal epithelial cells Caco-2

[0156] 1. Preparation of NHNK-2001 live bacterial suspension

[0157] Pick a single colony of NHNK-2001 into fresh MRS medium and culture anaerobically with shaking at 37 °C for 24 h. Centrifuge at 5000 rpm for 10 min to collect the bacteria, wash twice with sterile PBS, resuspend the bacteria in DMEM medium and adjust OD 600 = 0.5 to obtain the live bacterial suspension.

[0158] 2. Preparation of NHNK-2001 biofilm bacterial suspension

[0159] Pick a single colony of NHNK-2001 into MRS liquid medium and culture anaerobically statically at 37 °C for 24 h, adjust to OD 600 = 0.2 and add it to a bacterial culture dish, then continue anaerobic culture at 37 °C for 24 hours. After the culture is completed, discard the upper layer of the medium, wash twice with PBS and then resuspend the bottom biofilm, centrifuge at 5000 rpm for 10 min to collect the bacteria, resuspend the bacteria in DMEM medium and adjust OD 600 = 0.5 to obtain the biofilm bacterial suspension.

[0160] 3. Determination of the adsorption ability of NHNK-2001 to Caco-2 cells

[0161] Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37 °C and 5% CO 2 conditions. When the confluence reached 80%, the cells were digested with trypsin and collected, and then inoculated into a 6-well plate containing coverslips and cultured overnight. After the culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of DMEM medium without FBS serum and 1 mL of live bacteria suspension or biofilm suspension were added, and the incubation was continued for 2 h. After the incubation, the cells were washed twice with PBS to remove the unadhered bacteria. The coverslips were immersed in 4% paraformaldehyde for 15 min, and then Gram staining was performed and photographed. As Figure 6 shown.

[0162] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A Lactobacillus reuteri strain ( Limosilactobacillus reuteri ), which is Lactobacillus reuteri ( Limosilactobacillus reuteri )NHNK-2001 was deposited in the China Center for Type Culture Collection on April 25, 2024, and its deposit number is CCTCC NO: M 2024800.

2. The Lactobacillus reuteri of claim 1 ( Limosilactobacillus reuteri ) is characterized in that The product is Lactobacillus reuteri ( Limosilactobacillus reuteri ) of live bacteria, inactivated bacteria or fermentation products.

3. The Lactobacillus reuteri of claim 1 ( Limosilactobacillus reuteri ) or the Lactobacillus reuteri of claim 2 ( Limosilactobacillus reuteri ) Use of the prepared product in the preparation of products for improving sinusitis.

4. The use according to claim 3, characterized in that: The improvement of sinusitis includes at least one of the following a)-d): a) Inhibit Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. aureus ), Streptococcus pneumoniae ( Streptococcus pneumoniae )、Haemophilus influenzae( Haemophilus influenzae salivarius ( Streptococcus salivarius ), Moraxella catarrhalis ( Moraxella catarrhalis ) and Prevotella nigra ( Prevotella nigrescens ) and biofilm formation; b) Promote Propionibacterium acnes ( Propionibacterium acnes )’s growth; c) Flocculation of Haemophilus influenzae ( Haemophilus influenzae ) and Streptococcus pneumoniae ( Haemophilus influenzae ) at least one of the following: d) Down-regulation of Staphylococcus aureus subsp. aureus enterotoxin B gene seb expression.

5. The Lactobacillus reuteri of claim 1 ( Limosilactobacillus reuteri ) or the Lactobacillus reuteri of claim 2 ( Limosilactobacillus reuteri ) Use of the prepared product in the preparation of products for improving allergic rhinitis.

6. The use according to claim 5, characterized in that: The improvement of allergic rhinitis includes at least one of the following a)-d): a) Degradation of histamine; b) Reduce histamine stimulation and downregulate histamine receptors in nasal epithelial cells H1R , H2R and H3R Expression; c) Promote the nasal epithelial cell barrier and upregulate the tight junction proteins of nasal epithelial cells OCLD Expression; d) Reduce inflammation of nasal epithelial cells and regulate tumor necrosis factor-α gene TNF-α and epidermal growth factor receptor gene EGFR expression.

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