Lactobacillus reuteri for improving allergic rhinitis and nasosinusitis as well as product and application of lactobacillus reuteri

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus reuteri for improving allergic rhinitis and nasosinusitis as well as a product and application thereof. The lactobacillus reuteri NHNK-2001 disclosed by the invention is deposited in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2024800. Experiments show that the NHNK-2001 has the functions of inhibiting the growth of nasosinusitis related flora and biofilm formation, flocculating rhinitis pathogenic bacteria, promoting the growth of nasal cavity beneficial bacteria, inhibiting staphylococcus aureus subsp.aureus enterotoxin, degrading histamine, reducing histamine excitation, promoting nasal cavity epithelial cell barrier and reducing nasal cavity epithelial cell inflammation; in addition, a biological membrane formed by the NHNK-2001 thallus can increase the colonization ability to intestinal epithelial cells. The traditional Chinese medicine composition can be used for preparing products for improving allergic rhinitis and nasosinusitis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Lactobacillus reuteri for improving allergic rhinitis and sinusitis, and a product and application thereof. Background Art

[0002] Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mediated by IgE after the body comes into contact with allergens. The diversity of nasal symbiotic flora in AR patients is significantly reduced. Streptococcus salivarius ) is the most abundant commensal bacteria in the nasal cavity of AR patients, aggravating the symptoms of rhinitis. The relative abundance of Propionibacterium acnes, Propionibacterium, and Corynebacterium in the healthy group was significantly higher than that in the AR group. In addition, the researchers found that Staphylococcus aureus subsp. aureus enterotoxin B (SEB) increased the permeability of the nasal mucosa of AR patients in a dose-dependent manner, reduced transepithelial electrical resistance, and destroyed 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 mucosa contain a large amount of histamine. When tissues are damaged, stimulated or produce allergic reactions, histamine is released in large quantities, leading to local tissue edema, airway narrowing, and aggravating respiratory allergy symptoms. Histamine receptors mainly include three subtypes, namely H1R, H2R and H3R, all of which are related to allergic symptoms. In recent years, epidermal growth factor receptor EGFR has been reported to be associated with rhinitis, and its overexpression can cause increased mucus secretion from 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. Common symptoms include nasal congestion, purulent nasal discharge, headache, and loss of smell. It may also cause inflammation of the pharynx, tonsils, and lower nasal cavity, so timely treatment is required. Acute sinusitis is mostly caused by upper nasal infection. The pathogens are mostly pyogenic cocci, such as Streptococcus pneumoniae, Staphylococcus aureus subspecies aureus, Haemophilus influenzae, and Moraxella catarrhalis, while odontogenic infections are mainly caused by oral anaerobic pathogens such as Prevotella. Chronic sinusitis is mostly caused by repeated attacks of acute sinusitis and failure to completely cure it. According to the latest research, the nasal cavity of healthy people is colonized with symbiotic microorganisms. Changes in the nasal flora and damage to the epithelial barrier may be the key factors in inducing acute and chronic sinusitis.

[0005] Beneficial microorganisms can inhibit the growth of pathogens, regulate immune homeostasis and protect the nasal epithelial cell barrier. The active substances of these microorganisms have important application value in various fields. Therefore, it is of great practical significance to provide a microorganism-related product developed using microbial technology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides Lactobacillus reuteri for improving allergic rhinitis and sinusitis, as well as products and applications, which are used to solve the technical problems in the prior art.

[0007] The present invention provides a method for treating Lactobacillus reuteri Limosilactobacillus reuteri ), which is Lactobacillus reuteri ( Limosilactobacillus reuteri )NHNK-2001, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on April 25, 2024, and its preservation number is CCTCC NO: M 2024800 of Lactobacillus reuteri.

[0008] Further, according to the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) prepared by Lactobacillus reuteri ( Limosilactobacillus reuteri ) of live bacteria, inactivated bacteria or fermentation products.

[0009] The present invention also provides the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) or the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) Use of the prepared product in the preparation of products for improving sinusitis.

[0010] Further, the improvement of sinusitis includes at least one of the following a)-d): a) Inhibit the growth of sinusitis-related flora and biofilm formation, inhibit Staphylococcus aureus subspecies aureus ( Staphylococcus aureus subsp. golden ), Streptococcus pneumoniae ( Streptococcus pneumoniae )、Haemophilus influenzae( Haemophilus influenzae salivarius ( Streptococcus salivarius ), Moraxella catarrhalis ( Moraxella catarrhalis ) and Prevotella nigra ( Prevotella nigrescens ) and at least one of growth and biofilm formation; b) Promote the growth of beneficial bacteria in the nasal cavity and promote the growth of Propionibacterium acnes ( Propionibacterium acnes )’s growth; c) Flocculation of sinusitis-related pathogens and Haemophilus influenzae ( Haemophilus influenzae ) and Streptococcus pneumoniae ( Haemophilus influenzae ) at least one of the following: d) Inhibit Staphylococcus aureus enterotoxin and downregulate Staphylococcus aureus enterotoxin B gene oil expression.

[0011] The present invention also proposes the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri) or Lactobacillus reuteri as described above ( Limosilactobacillus reuteri ) Use of the prepared product in the preparation of products for improving allergic rhinitis.

[0012] Further, the improvement of allergic rhinitis includes at least one of the following a)-e): 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 tight junction proteins in 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; e) Bacterial biofilm increases its ability to colonize intestinal epithelial cells.

[0013] Furthermore, the degradation of histamine is the degradation of histamine in culture medium in vitro.

[0014] The present invention also provides a product for improving allergic rhinitis and sinusitis, comprising the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ), or Lactobacillus reuteri as described above ( Limosilactobacillus reuteri ) and acceptable excipients and / or adjuvants.

[0015] The Lactobacillus reuteri disclosed in the present invention ( Limosilactobacillus reuteri ), and its deposit number is CCTCC NO: M 2024800. Experiments have shown that NHNK-2001 has the functions of inhibiting the growth of sinusitis-related bacteria and biofilm formation, flocculating sinusitis-related pathogens, promoting the growth of beneficial nasal bacteria, inhibiting Staphylococcus aureus subspecies aureus enterotoxin, degrading histamine, reducing histamine stimulation, promoting the nasal epithelial cell barrier, and reducing nasal epithelial cell inflammation.

[0016] Biological Deposit Description Lactobacillus reuteri ( Limosilactobacillus reuteri ) NHNK-2001, was deposited in the China Center for Type Culture Collection (CCTCC for short, 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

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The MRS plate colony image and Gram staining image of Lactobacillus reuteri NHNK-2001 in the present invention; Figure 2 This is a graph showing the results of the fermentation product of Lactobacillus reuteri NHNK-2001 of the present invention inhibiting the biofilm formation of sinusitis-related flora; Figure 3 The inactivated bacteria of Lactobacillus reuteri NHNK-2001 in the present invention inhibit the formation of biofilm of sinusitis-related flora; Figure 4 It is a diagram of the flocculation state of Lactobacillus reuteri NHNK-2001, pathogenic bacteria, and Lactobacillus reuteri NHNK-2001 and pathogenic bacteria in the present invention; Figure 5 This is a graph showing the experimental results of histamine degradation by Lactobacillus reuteri NHNK-2001 of the present invention; Figure 6 This is a graph showing the experimental results of the adsorption of live Lactobacillus reuteri NHNK-2001 bacteria and NHNK-2001 biofilm on intestinal epithelial cells Caco-2 in the present invention.

[0019] The embodiments of the present invention will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Furthermore, in the application of the present invention, the Lactobacillus mucosa NHNK-2001 provided by the present invention exists 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.

[0024] 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: Example 1 Isolation of NHNK-2001 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.

[0025] Gram staining microscopy: strain NHNK-2001 is a Gram-positive colony, which appears as a short rod with a round end under a microscope; it grows on an MRS plate and forms white, smooth, round, semi-transparent round colonies with neat edges; it grows evenly in MRS liquid culture medium and becomes a white precipitate after long-term storage. Figure 1 shown.

[0026] Example 2 Nucleic acid identification of NHNK-2001 1. 16S rDNA gene sequence analysis: Pick a single colony in MRS liquid medium, culture at 37℃ overnight, collect the bacteria by centrifugation at 8000 rpm for 1min, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min.

[0027] 2. Results: The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). It was concluded that the NHNK-2001 strain was Lactobacillus reuteri ( Limosilactobacillus reuteri ).

[0028] Example 3 NHNK-2001 inhibits the proliferation of sinusitis-related bacteria 1. Cultivation of sinusitis-related flora Streptococcus pneumoniae ( Streptococcus pneumoniae ) The frozen tube culture liquid of BNCC338425 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v), and cultured at 37℃ for 2 days. After that, the OD value was adjusted to 0. 600 =0.1 to obtain Streptococcus pneumoniae suspension.

[0029] Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. golden ) CGMCC 1.8721 frozen tube bacterial solution was inoculated into BHI medium at 2% (v / v), cultured at 37℃ for 2 days, and then adjusted to OD 600 =0.1 to obtain Staphylococcus aureus subspecies aureus suspension.

[0030] Haemophilus influenzae ( Haemophilus influenzae ) The frozen tube culture liquid of BNCC359479 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v), and cultured at 37℃ for 2 days. After that, the OD was adjusted with TSB medium. 600 =0.1 to obtain Moraxella catarrhalis suspension.

[0031] Streptococcus salivarius ( Streptococcus salivarius ATCC 7073 frozen tube bacterial solution was inoculated into M17 liquid medium at 2% (v / v), and cultured at 42℃ for 2 days. After that, the OD was adjusted with M17 medium. 600 =0.1 to obtain Streptococcus salivarius suspension.

[0032] Moraxella catarrhalis Moraxella catarrhalis) The frozen tube culture solution of BNCC275822 was inoculated into BHI liquid medium at 2% (v / v), and cultured at 37℃ for 2 days. After that, the OD was adjusted with BHI medium. 600 =0.1 to obtain Moraxella catarrhalis suspension.

[0033] Prevotella nigra Prevotella nigrescens ) The frozen tube bacterial solution of BNCC369693 was inoculated into TSB medium containing 5% defibrinated sheep blood at 2% (v / v), and then cultured anaerobically at 37℃ for 2 days and then adjusted to OD with special peptone medium. 600 =0.1 to obtain the black Prevotella suspension.

[0034] 2. Preparation of NHNK-2001 fermentation products NHNK-2001 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v) and cultured at 37°C for 48 h. After that, the OD value was adjusted to 0.04 with MRS. 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product.

[0035] 3. NHNK-2001 fermentation products inhibit the proliferation of sinusitis-related bacteria 160 μL of pathogen suspension was added to each well of a 96-well plate. 40 μL of fermentation product was added to the experimental group, and an equal volume of MRS medium was added to the control group. After culturing at 37°C for 48 hours, the OD 600 The absorbance value at nm. Inhibition rate % = (1-absorbance value of experimental group / absorbance value of control group) * 100%. The results are shown in Table 1: Table 1

[0036] The results showed that the fermentation products of NHNK-2001 could inhibit the growth of Staphylococcus aureus subsp. aureus, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus salivarius, Moraxella catarrhalis and Prevotella nigra, with inhibition rates ranging from 13.66% to 83.14%.

[0037] Example 4 NHNK-2001 promotes the proliferation of Propionibacterium acnes 1. Propionibacterium acnes ( Propionibacterium acnes ) The frozen tube culture solution of Propionibacterium acnes CGMCC 1.5003 was inoculated into a special peptone medium at 2% (v / v). After anaerobic culture at 37°C for 5-6 days, the OD value was adjusted to 0. 600 =0.5 to obtain Propionibacterium acnes suspension.

[0038] 2. Preparation of NHNK-2001 fermentation products The preparation method of NHNK-2001 fermentation product is referred to Example 3.

[0039] 3. NHNK-2001 fermentation products promote the proliferation of Propionibacterium acnes Propionibacterium acnes was added to each well of a 96-well plate at 160 μL. The experimental group was added with 40 μL of fermentation product, and the control group was added with an equal amount of MRS medium. After culturing at 37°C for 48 hours, the OD 600 The absorbance value at nm. Proliferation promotion rate % = (absorbance value of experimental group - absorbance value of control group) / absorbance value of control group * 100%. The results are shown in Table 2: Table 2

[0040] The results showed that the fermentation products of NHNK-2001 could promote the growth of Propionibacterium acnes, with the growth promotion rate being 34.83~38.89%.

[0041] Example 5 NHNK-2001 inhibits sinusitis-associated bacterial biofilm 1. Cultivation of sinusitis-related flora The preparation method of the suspension of Staphylococcus aureus subsp. aureus / Streptococcus pneumoniae / Haemophilus influenzae / Streptococcus salivarius / Mora catarrhalis / Prevotella nigra is as described in Example 3.

[0042] 2. Preparation of NHNK-2001 fermentation products and inactivated bacteria NHNK-2001 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v) and cultured at 37°C for 48 h. After that, the OD value was adjusted to 0.04 with MRS. 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product. Wash the precipitated bacteria three times with sterile PBS, resuspend with sterile PBS and adjust the OD 600 =1.0, sterilize at 121℃ for 15 min to obtain inactivated bacteria.

[0043] 3. NHNK-2001 inhibits sinusitis-related bacterial biofilm 100 μL of sinusitis-related bacterial flora was added to each well of a 96-well plate. 100 μL of NHNK-2001 fermentation product or inactivated bacteria was added to each well of the experimental group, and an equal volume of MRS medium or PBS was added to each well of the control group. Each group had 3 parallels and was then cultured at 37°C for 24 hours.

[0044] After the incubation, 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 minutes. Discard the fixative, add 100 μL of crystal violet to each well and stain at room temperature for 30 minutes. After staining, wash twice with sterile PBS and dry, add 100 μL of anhydrous ethanol to each well, as shown in Figure 2. Figure 2 (fermentation products) and Figure 3 (Inactivated bacteria) as shown, after standing for 1 minute, measure the absorbance at 600nm. Inhibition rate % = (1-experimental group / control group) * 100%.

[0045] The results are shown in Table 3 and Table 4: Table 3

[0046] Table 4

[0047] The results showed that the fermentation products of NHNK-2001 could inhibit the formation of biofilm of sinusitis-related bacteria including Staphylococcus aureus subspecies aureus, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus salivarius, Moraxella catarrhalis and Prevotella nigra, with inhibition rates ranging from 23.50% to 87.04%.

[0048] Example 6 NHNK-2001 Flocculation of Sinusitis-Related Pathogens 1. Cultivation of pathogens related to sinusitis Streptococcus pneumoniae ( Streptococcus pneumoniae ) The frozen tube bacterial solution 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 centrifuged and washed three times with sterile PBS. The cells were resuspended in sterile PBS and the OD was adjusted. 600 =1.0, sterilize at 121℃ for 15 min and set aside.

[0049] Haemophilus influenzae ( Haemophilus influenzae ) The frozen tube bacterial solution 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 centrifuged and washed three times with sterile PBS. The cells were resuspended in sterile PBS and the OD was adjusted. 600 =1.0, sterilize at 121℃ for 15 min and set aside.

[0050] 2. Preparation of NHNK-2001 inactivated bacteria The preparation method of NHNK-2001 inactivated bacteria is shown in Example 5.

[0051] 3. Co-flocculation The pathogenic bacteria solution was mixed with the NHNK-2001 inactivated bacteria solution in a volume ratio of 1:1, and the flocculation condition was observed after standing for 30 minutes.

[0052] 4. Index determination After 30 minutes of reaction, samples of NHNK-2001 alone, pathogens, and the mixed reaction liquid of NHNK-2001 and pathogens were collected. The sampling range was the top 50 μL of the liquid surface. After aspiration, the samples were transferred to a 96-well plate and the absorbance at OD=600nm was measured. At the same time, the flocculation precipitate was stained with Gram stain to observe the flocculation state of the bacteria. Figure 4 shown.

[0053] Calculation formula: Flocculation rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%; Note: Ax: OD measured by NHNK-2001 alone at the reaction time 600 Value; Ay: OD of a single pathogen measured at the reaction time 600 Value; Amix: OD measured at the reaction time after NHNK-2001 and pathogens are mixed 600 Numeric value.

[0054] The results are shown in Table 5: Table 5

[0055] The results showed that NHNK-2001 could flocculate Haemophilus influenzae and Streptococcus pneumoniae, with the flocculation rate ranging from 11.38% to 32.96%.

[0056] Example 7 NHNK-2001 inhibits Staphylococcus aureus subsp. aureus enterotoxin 1. Preparation of NHNK-2001 inactivated bacteria The NHNK-2001 frozen tube bacterial solution was inoculated into MRS medium at 2% and cultured at 37°C for 24 hours. The bacteria were collected by centrifugation at 5000 rpm for 10 minutes, washed with PBS and centrifuged to discard the supernatant. The washed live bacteria were resuspended in PBS and the OD was adjusted. 600 =0.5, sterilize at 121℃ for 15min to obtain inactivated bacterial suspension.

[0057] 2. NHNK-2001 regulates the expression of Staphylococcus aureus subsp. aureus enterotoxin (1) Cultivation and intervention of Staphylococcus aureus subspecies aureus The glycerol culture of Staphylococcus aureus subsp. aureus CGMCC 1.8721 was inoculated into fresh BHI medium at 2% (v / v) and cultured at 37°C with shaking for 24 h. The OD was then adjusted with BHI medium. 600 =0.5. Add the suspension of Staphylococcus aureus subsp. aureus to centrifuge tubes, 1 mL per tube, and add 2 mL of BHI medium. Add 1 mL of NHNK-2001 inactivated bacteria to the experimental group, and add an equal volume of PBS to the control group, and continue to culture at 37°C for 16 hours.

[0058] (2) After the culture, the RNA of Staphylococcus aureus subspecies aureus was extracted using a Gram-positive bacteria RNA extraction kit. After testing the RNA concentration and purity, it was reverse transcribed into cDNA. Using 16S rRNA as the internal reference gene, qPCR fluorescence quantitative technology was used to detect the enterotoxin B gene of Staphylococcus aureus subspecies aureus. oil expression.

[0059] (3) Data processing The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by the method, and the results are shown in Table 6: Table 6

[0060] The results showed that NHNK-2001 inactivated bacteria could inhibit the expression of enterotoxin B gene of Staphylococcus aureus subsp. oil The relative expression multiple was between 0.11 and 0.13.

[0061] Example 8 NHNK-2001 Degradation of Histamine 1. NHNK-2001 Histamine Degradation Experiment NHNK-2001 glycerol bacteria were inoculated at 2% (v / v) in MRS medium containing 500 μg / mL histamine and cultured anaerobically at 37°C for 48 h. The control group was inoculated with an equal volume of MRS medium.

[0062] 2. Reagent preparation ① 50g / L Na2CO3 solution: Dissolve 5g Na2CO3 in pure water and dilute to 100mL with pure water.

[0063] ② 250g / L NaOH solution: Dissolve 25g NaOH in pure water and dilute to 100mL. ③ HCl solution: Add 5 mL of concentrated hydrochloric acid HCl to 55 mL of pure water and mix well.

[0064] ④ Solution A (p-nitroaniline solution): Weigh 0.5g p-nitroaniline, add 5mL HCl to dissolve, dilute to 200mL with pure water, mix well, and prepare before use. Solution B (NaNO2 solution): Weigh 0.5g NaNO2, dissolve and dilute to 100mL with pure water, mix well. Azo reagent: Pipette 5mL of solution A and 40mL of solution B and mix.

[0065] ⑤ 1000 μg mL -1 Histamine phosphate standard solution: accurately weigh 0.025g of histamine dihydrochloride standard, dissolve it in pure water, transfer it into a 25mL volumetric flask, and dilute to the mark with pure water, and mix well. 20μg·mL -1 Histamine phosphate standard solution 1mL 1000μg mL -1 Place the histamine phosphate standard solution in a 50 mL volumetric flask, add pure water to the mark and mix.

[0066] 3. Determination of histamine content 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, which are equivalent to 0, 4, 8, 12, 16, 20 μg of histamine, add water to make up to 1 mL, add 1 mL HCl solution, 3 mL 50 g / L Na2CO3 solution, 3 mL azo reagent, add water to make up to the scale, and measure the OD after leaving for 10 minutes. 480 And draw a standard curve.

[0067] 2. Take 1mL of the sample to be tested, add 1mL of HCl solution, 3mL of 50g / L Na2CO3 solution, and 3mL of azo reagent, add water to the scale, and measure the OD after leaving it for 10 minutes. 480 .like Figure 5 Degradation rate (%) = (1-absorbance value of experimental group / absorbance value of control group)*100.

[0068] The results are shown in Table 7: Table 7

[0069] The results showed that the degradation rate of histamine by NHNK-2001 was between 16.57% and 21.13%.

[0070] Example 9 NHNK-2001 regulates the expression of histamine receptors, barrier and inflammation-related genes in human nasal epithelial cells RPMI-2650 1. Preparation of NHNK-2001 inactivated bacteria NHNK-2001 bacterial solution was inoculated into fresh MRS medium at 2% (v / v), cultured anaerobically at 37°C for 48 h, centrifuged at 5000 rpm for 10 min, the live bacteria precipitated by centrifugation were washed with PBS and resuspended in an appropriate amount, sterilized at 121°C for 15 min, the supernatant was removed after centrifugation, and resuspended with DMEM to adjust OD600=0.5 to obtain an inactivated bacterial suspension.

[0071] 2. NHNK-2001 regulates the expression of histamine receptors, barrier and inflammation-related genes in RPMI-2650 cells (1) Intervention treatment of human nasal epithelial RPMI-2650 cells RPMI-2650 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the cells were 80% confluent, the cells were collected and inoculated in 6-well plates. After overnight culture, the supernatant was discarded. 1 mL of inactivated bacteria and 1 mL of serum-free DMEM medium were added to each well of the experimental group, and the control group was replaced with an equal volume of DMEM, and cultured at 37°C and 5% CO2 for 16 h.

[0072] (2) RNA extraction and fluorescence quantitative PCR After the culture was completed, RIPA lysis buffer was added to lyse the cells, and total RNA was extracted. After testing the RNA concentration and purity, it was reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect histamine receptor related genes H1R、H2R、H3R ; Tight junction protein gene OCLD ; Tumor necrosis factor-α gene TNF-α Epidermal growth factor receptor gene EGFR expression.

[0073] (3) Data processing The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0074] The results are shown in Table 8: Table 8

[0075] The results showed that NHNK-2001 inactivated bacteria down-regulated the histamine receptor gene in human nasal epithelial cells RPMI-2650 H1R、 H2R and H3R down-regulates the expression of tumor necrosis factor-α gene TNF-α and epidermal growth factor receptor gene EGFR The expression of tight junction protein genes was also upregulated OCLDThe expression levels of expressed genes ranged from 0.22 to 1.86 times.

[0076] Example 10 Biofilm formation by NHNK-2001 1. Formation of NHNK-2001 biofilm A single colony of NHNK-2001 was selected and placed in MRS liquid medium, cultured anaerobically at 37°C for 48 h, and then adjusted to OD 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then anaerobic culture was continued at 37 degrees for 24 hours.

[0077] 2. Crystal violet staining After the incubation, discard the supernatant, add 100 μL sterile PBS to each well and wash twice, then add 100 μL 4% paraformaldehyde fixative to each well and fix at room temperature for 30 minutes. Discard the fixative, add 100 μL crystal violet to each well and stain at room temperature for 30 minutes. After staining, wash twice with sterile PBS and dry, add 100 μL anhydrous ethanol to each well, let stand for 1 minute, and measure the absorbance at 600 nm. The results are shown in Table 9: Table 9

[0078] The results showed that NHNK-2001 had a 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0079] Example 10 Determination of the Adhesion Ability of NHNK-2001 to Human Intestinal Epithelial Cells Caco-2 1. Preparation of NHNK-2001 live bacterial suspension Pick a single colony of NHNK-2001 in fresh MRS medium and culture it anaerobically at 37°C for 24 hours. Centrifuge at 5000 rpm for 10 minutes to collect the bacteria, wash twice with sterile PBS, resuspend the bacteria in DMEM medium and adjust the OD 600 =0.5 to obtain a live bacterial suspension.

[0080] 2. Preparation of NHNK-2001 biofilm bacterial suspension A single colony of NHNK-2001 was selected and placed in MRS liquid medium, incubated anaerobically at 37°C for 24 h, and the OD was adjusted to 0. 600=0.2 was added to the bacterial culture dish, and then anaerobically cultured at 37 degrees for 24 hours. After the culture was completed, the upper culture medium was discarded, the bottom biofilm was resuspended after washing twice with PBS, and the bacteria were collected by centrifugation at 5000rpm for 10min. The bacteria were resuspended in DMEM culture medium and the OD was adjusted. 600 =0.5 to obtain biofilm bacterial suspension.

[0081] 3. Determination of the adsorption capacity of NHNK-2001 on Caco-2 cells Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the degree of confluence reached 80%, the cells were digested with trypsin and collected, and inoculated into a 6-well plate containing a slide for overnight culture. After the culture was completed, the supernatant was discarded, and the plate was washed twice with sterile PBS. 1mL of DMEM medium without FBS serum and 1mL of live bacterial suspension or biofilm bacterial suspension were added, and the incubation continued for 2h. After the incubation, it was washed twice with PBS to remove non-adherent bacteria. The slide was fixed in 4% paraformaldehyde for 15 minutes, followed by Gram staining and photography. Figure 6 shown.

[0082] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached 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 metabolism includes at least one of the following a)-d): a) Inhibit the growth of sinusitis-related flora and biofilm formation, inhibit Staphylococcus aureus subspecies 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 at least one of growth and biofilm formation; b) Promote the growth of beneficial bacteria in the nasal cavity and promote the growth of Propionibacterium acnes ( Propionibacterium acnes )’s growth; c) Flocculation of sinusitis-related pathogens and Haemophilus influenzae ( Haemophilus influenzae ) and Streptococcus pneumoniae ( Haemophilus influenzae ) at least one of the following: d) Inhibit Staphylococcus aureus enterotoxin and downregulate Staphylococcus 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)-e): 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 tight junction proteins in 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; e) Bacterial biofilm increases its ability to colonize intestinal epithelial cells.

7. The use according to claim 5, characterized in that: The histamine degradation is the in vitro degradation of histamine in the culture medium.

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