Leuconostoc mesenteroides strain for relieving allergic rhinitis as well as separation method, probiotic preparation and application of leuconostoc mesenteroides strain

Abstract

The invention discloses a leuconostoc mesenteroides strain for relieving allergic rhinitis as well as a separation method, a probiotic preparation and application of the leuconostoc mesenteroides strain. The leuconostoc mesenteroides strain is leuconostoc mesenteroides YMJ2024LM-1, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation date is December 31, 2024, and the preservation number is CGMCC No.33257. The invention further discloses a preparation method of the leuconostoc mesenteroides strain for relieving allergic rhinitis. Experiments show that the leuconostoc mesenteroides YMJ2024LM-1 has a good effect of improving the allergic rhinitis, can relieve symptoms of the allergic rhinitis of mice, reduce the IgE content and the proinflammatory cytokine level in serum of the mice with the allergic rhinitis and repair damaged nasal mucosa of the mice with the allergic rhinitis, can be used for preparing products for preventing and / or treating the allergic rhinitis, and has a good application prospect. And the method has good value and application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a mesenteric leuconostoc strain for allergic rhinitis relief, an isolation method thereof, a probiotic preparation and application thereof. Background Art

[0002] Allergic rhinitis is a common upper respiratory tract disease, which is mainly manifested by inflammation of the nasal mucosa. Symptoms include nasal congestion, nasal itching, sneezing, and watery nasal discharge. Allergic rhinitis is an inflammatory reaction caused by an imbalance of Th1 and Th2 immune responses caused by the in vitro environmental factors acting on the body through an IgE-mediated type I hypersensitivity reaction. Among them, the function of Th1 cells is mainly to secrete IFN-γ, promote cellular immunity, and inhibit the overactivation of Th2 cells; while the function of Th2 cells is mainly to secrete cytokines such as IL-4, IL-5, and IL-13, which promote humoral immunity and allergic reactions. That is, Th2-type cytokines are sensitizing cytokines, and their increased expression indicates that the immune system has abnormally activated Th2-type immune responses. Among them, the increased expression of IL-4 can promote B cells to produce IgE antibodies, and the increased expression of IL-5 can promote the proliferation and differentiation of eosinophils. IL-13 has a highly homologous amino acid sequence with IL-4, and its increased expression can also promote the production of IgE. At the same time, the expression activity of inflammatory factors in patients with allergic rhinitis will also increase significantly, including IL-6, IL-1β, IL-33 and TSLP; in addition, studies have shown that allergic rhinitis is also accompanied by changes in the expression activity of regulatory cytokines, including IL-10 and TGF-β; therefore, allergic rhinitis is often accompanied by pathological changes in nasal tissue, such as congestion, swelling, epithelial damage, inflammatory infiltration (such as eosinophils), etc.

[0003] At present, the treatment of allergic rhinitis mainly relies on drugs such as antihistamines (such as loratadine, cetirizine), glucocorticoids and decongestants, but these drugs may have side effects such as dry mouth, drowsiness, insomnia, and long-term use has limited effect. In recent years, probiotics have gradually become a research hotspot for the treatment of inflammatory diseases due to their regulation of the immune system and anti-inflammatory effects. For example, the Chinese patent document CN109512854B discloses a probiotic composition for the prevention and treatment of allergic rhinitis and a nasal preparation and preparation method based thereon, and reports that Lactobacillus acidophilus and Bifidobacterium can more quickly and directly exert an immunomodulatory effect on the nasal mucosa, by promoting the increased secretion of interleukin 12 (IL-12) and interferon γ, regulating Th1 type immune response and inhibiting immunoglobulin IgE, and improving the allergic phenomenon of excessive Th2 type immune response; Chinese patent application document CN119074903A discloses a biological combination preparation for allergic rhinitis and its preparation method, reporting that Lactobacillus rhamnosus and Lactobacillus reuteri Bacillus can regulate the immune system to reduce allergic reactions and relieve the symptoms of allergic rhinitis; another Chinese patent document CN111647526B discloses plant lactobacillus SD-H9, its probiotic agent and its application. The probiotic agent provided in the report can effectively relieve the nasal symptoms of rats with allergic rhinitis, reduce the levels of histamine, OVA-specific IgE and IL-4 in the serum of rats with rhinitis, and increase the IFN-γ content in the serum of rats, which helps to relieve allergic reactions and induce correct immune responses, thereby changing the Th2 cell bias that promotes allergic reactions, and exerting a therapeutic effect on allergic rhinitis by regulating the host immune function, which can effectively improve the symptoms of rhinitis.

[0004] Leuconostoc mesenteroides is an important species of the genus Leuconostoc in lactic acid bacteria, with high acid production, antioxidant capacity and antagonism to pathogenic bacteria. Existing studies have shown that Leuconostoc mesenteroides has the effects of regulating intestinal flora, enhancing immunity and anti-inflammatory, but there are no reports on its research in the treatment and relief of allergic rhinitis. Summary of the invention

[0005] In view of the above problems, the present invention provides a strain of Leuconostoc mesentericus for alleviating allergic rhinitis, its isolation method, probiotic preparation and application, which can effectively relieve rhinitis symptoms by regulating the immune system and inhibiting inflammatory response. The specific technical scheme is as follows:

[0006] One of the purposes of the present invention is to provide a mesenteric Leuconostoc strain for relieving allergic rhinitis, the strain is Leuconostoc mesenteroides YMJ2024LM-1 (Leuconostoc mesenteroides YMJ2024LM-1), the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, the preservation date is December 31, 2024, and the preservation number is CGMCC No.33257.

[0007] The aforementioned mesenteric Leuconostoc strain used to relieve allergic rhinitis can grow well under anaerobic conditions. The colonies are spherical, bean-shaped or short rod-shaped, arranged in pairs or in short chains, non-motile, with a smooth surface, milky white, and does not produce any pigment. It can grow in an environment of pH ≤ 5.

[0008] The aforementioned Leuconostoc mesenteroides strain used to relieve allergic rhinitis has a genome size of 1,987,684 bp and a GC content of 37.64%; the dDDH and ANI values ​​between it and the model strain of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 are 94.90% and 99.29%, respectively.

[0009] The second object of the present invention is to provide a method for isolating the aforementioned Leuconostoc mesentericus strain for allergic rhinitis, comprising the following steps:

[0010] 1) Take fermented kimchi from Hubei as sample, take 100 μL of kimchi extract, add it to 900 μL sterile PBS EP tube, and dilute the sample in sequence until the concentration of sample extract is 10 -6 times;

[0011] 2) Take 100 μL of samples of different dilutions and spread them on MRS medium and culture them in an anaerobic incubator at 37°C for 48 h;

[0012] 3) Use a sterile inoculation loop to pick colonies with different morphological characteristics on the culture plate, transfer them to new MRS solid medium for further purification, continue anaerobic culture at 37°C for 48 hours, and passage for 3 times to obtain purified strains;

[0013] 4) The purified strains were cultured in MRS liquid culture medium at pH=3.0 to screen the probiotic strains with acid resistance, which are the Leuconostoc mesentericus strains for alleviating allergic rhinitis.

[0014] The third object of the present invention is to provide a use of the aforementioned Leuconostoc mesentericus strain in the preparation of a product for treating, preventing or improving allergic rhinitis.

[0015] As a preferred technical solution, the product prepared using the aforementioned Leuconostoc mesentericus strain can reduce serum IgE concentration and / or nasal mucosal pro-inflammatory cytokine concentration.

[0016] As a preferred technical solution, the product prepared using the aforementioned mesenteric Leuconostoc strain can inhibit the expression of nasal mucosal allergenic cytokines.

[0017] As a preferred technical solution, the product prepared using the aforementioned Leuconostoc mesentericus strain can alleviate the pathological changes in nasal tissue induced by allergic rhinitis.

[0018] The fourth object of the present invention is to provide a probiotic preparation for relieving allergic rhinitis, which is made from the aforementioned Mesenteric Leuconostoc strain for relieving allergic rhinitis, or contains the aforementioned Mesenteric Leuconostoc strain for relieving allergic rhinitis as an active ingredient.

[0019] As a preferred technical solution, the aforementioned probiotic preparation for relieving allergic rhinitis includes any form of capsules, lyophilized powder, suspension or tablets.

[0020] As a preferred technical solution, in the aforementioned probiotic preparation for relieving allergic rhinitis, the concentration of Leuconostoc mesenteroides is 1×10^8 to 1×10^11 CFU / mL.

[0021] Beneficial effects of the present invention:

[0022] 1) The Leuconostoc mesenteroides YMJ2024LM-1 provided by the present invention can effectively relieve rhinitis symptoms by regulating the immune system and inhibiting inflammatory responses. Animal experiments have shown that Leuconostoc mesenteroides YMJ2024LM-1 can effectively relieve the symptoms of allergic rhinitis model mice and reduce the serum IgE concentration of allergic rhinitis model mice; after 7 days of YMJ2024LM-1 intervention, the IgE concentration in the serum of allergic rhinitis model mice was significantly reduced to 0.68 times the original level (295.94 / 436.62); oral administration of the YMJ2024LM-1 strain can inhibit the relative expression level of the sensitizing cytokine IL-4, making it close to the normal expression, and also has an inhibitory trend on the expression of IL-5 and IL-13.

[0023] 2) The intestinal mesenteroides YMJ2024LM-1 provided by the present invention has an inhibitory effect on the expression activity of the inflammatory cytokine IL-6, has an inhibitory trend on the expression of IL-1β, and can inhibit the secretion level of the proinflammatory cytokine IL-33, and shows a tendency to inhibit the expression of the regulatory cytokine IL-10.

[0024] 3) Pathological analysis showed that oral administration of Leuconostoc mesenteroides YMJ2024LM-1 provided by the present invention can also alleviate the pathological changes in nasal tissue induced by allergic rhinitis.

[0025] 4) The Leuconostoc mesenteroides YMJ2024LM-1 provided by the present invention can be used to prepare foods, health products or medicines for relieving allergic rhinitis, and has good economic value and excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of oral administration of YMJ2024LM-1 on the number of nose scratching in mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the number of nose scratching in mice, *** indicates p<0.001);

[0027] Figure 2 The effect of oral administration of YMJ2024LM-1 on serum IgE levels in mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the serum IgE concentration, * indicates p<0.05, ** indicates p<0.01);

[0028] Figure 3 The effect of oral administration of YMJ2024LM-1 on the expression activity of IL-4, a pro-allergic cytokine, in the nasal tissue of mice with allergic rhinitis induced by OVA (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the transcription level of IL-4 in the nasal tissue, and * indicates p<0.05);

[0029] Figure 4 The effect of oral administration of YMJ2024LM-1 on the concentration of IL-5, a sensitizing cytokine, in the nasal tissue of mice with allergic rhinitis induced by OVA (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the concentration level of IL-5 in the nasal tissue, ns means no statistical difference);

[0030] Figure 5 The effect of oral administration of YMJ2024LM-1 on the concentration of IL-13, a sensitizing cytokine, in the nasal tissue of mice with allergic rhinitis induced by OVA (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the concentration level of IL-13 in the nasal tissue, ns means no statistical difference);

[0031] Figure 6 The effect of oral administration of YMJ2024LM-1 on the expression activity of inflammatory cytokine IL-6 in nasal tissue of mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the transcription level of IL-6 in nasal tissue, ** indicates p<0.01, *** indicates p<0.001);

[0032] Figure 7 The effect of oral administration of YMJ2024LM-1 on the expression activity of inflammatory cytokine IL-1β in nasal tissue of mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the transcription level of IL-1β in nasal tissue, * indicates p<0.05, ns indicates no statistical difference);

[0033] Figure 8 The effect of oral administration of YMJ2024LM-1 on the expression activity of regulatory cytokine IL-10 in nasal tissue of mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the transcription level of IL-10 in nasal tissue, * indicates p<0.05, **** indicates p<0.0001, ns indicates no statistical difference);

[0034] Fig. 9 The effect of oral administration of YMJ2024LM-1 on the expression activity of regulatory cytokine TGF-β in nasal tissue of mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the transcription level of TGF-β in nasal tissue, ns means no statistical difference);

[0035] Fig.10 The effect of oral administration of YMJ2024LM-1 on the concentration of inflammatory cytokine IL-33 in nasal tissue of mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the concentration level of IL-33 in nasal tissue, * indicates p<0.05, ** indicates p<0.01);

[0036] Fig.11 The effect of oral administration of YMJ2024LM-1 on the concentration of inflammatory cytokine TSLP in the nasal tissue of mice with OVA-induced allergic rhinitis (in the figure: the horizontal axis is the different animal treatment groups, and the vertical axis is the concentration level of TSLP in the nasal tissue);

[0037] Fig.12 The pathological tissue characteristics of nasal mucosa of mice in the normal group;

[0038] Fig.13 The pathological tissue characteristics of nasal mucosa in mice with OVA-induced allergic rhinitis;

[0039] Fig.14 To show the pathological characteristics of nasal mucosa in mice with OVA-induced allergic rhinitis after oral administration of YMJ2024LM-1;

[0040] Fig.15 The nasal mucosal pathological tissue characteristic scores of mice in different treatment groups (in the figure: the horizontal axis is the different animal treatment groups, the vertical axis is the pathological tissue characteristic score, ** indicates p < 0.01);

[0041] Fig.16 This is a draft genome information of Leuconostoc mesenteroides YMJ2024LM-1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings. These embodiments are only exemplary and do not constitute any limitation on the scope of protection defined in the claims of the present invention.

[0043] Example 1 Isolation, preservation and identification of Leuconostoc mesenteroides YMJ2024LM-1

[0044] 1.1 Isolation of YMJ2024LM-1 strain

[0045] (1) MRS liquid medium: peptone 10 g / L, beef powder 10 g / L, yeast powder 5 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 3 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween-80 1 g / L, pH 6.0.

[0046] (2) MRS solid medium: peptone 10 g / L, beef powder 10 g / L, yeast powder 5 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 3 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween-80 1 g / L, agar 15 g / L, pH 6.0.

[0047] (3) Using fermented kimchi from Hubei as the sample, 100 μL of kimchi extract was taken and added to a 900 μL sterile PBS EP tube. The sample was diluted in sequence until the concentration of the sample extract was 10 -6 times; 100 μL of samples with different dilutions were spread on MRS medium and cultured in an anaerobic incubator at 37°C for 48 h;

[0048] (4) Use a sterile inoculation loop to pick colonies with different morphological characteristics on the culture plate, transfer them to new MRS solid medium for further purification, continue anaerobic culture at 37°C for 48 h, and subculture three times to obtain purified strains;

[0049] (5) The purified strain was cultured in MRS liquid medium at pH = 3.0, and a probiotic strain with acid resistance was screened, namely, Leuconostoc mesenteroides YMJ2024LM-1, which was used in subsequent experiments and stored frozen at the same time.

[0050] 1.2 Bacterial strain preservation

[0051] Use BHI medium containing 30% glycerol as the bacterial preservation solution for frozen preservation of bacterial strains. The specific operation method is as follows: after the bacteria are transferred to the solid culture medium twice in succession, add 2mL of high-pressure sterilized bacterial preservation solution to the culture plate; use an L rod to scrape and spread to make the colonies fully dissolved in the bacterial preservation solution, transfer the bacterial solution to a pre-sterilized bacterial preservation tube, and about 1.5mL of the bacterial solution can be drawn out. After mixing, store it at -80℃.

[0052] 1.3 Observation of colony appearance and bacterial morphology

[0053] The mesenteric Leuconostoc YMJ2024LM-1 prepared in this example is a Gram-positive bacterium that grows well under anaerobic conditions. The colonies are spherical, bean-shaped or short rod-shaped, some of which are arranged in pairs or in short chains. They are non-motile, have a smooth surface, are milky white, do not produce any pigment, and can grow in an environment with a pH of ≤5.

[0054] 1.4 Extraction of total bacterial DNA

[0055] YMJ2024LM-1 colony was inoculated on MRS medium and cultured anaerobically at 37°C overnight. The whole genomic DNA of bacteria was extracted according to the instructions of Genomic DNA Purification Kit (Promega).

[0056] 1.5 Bacterial taxonomic identification

[0057] The genome of YMJ2024LM-1 was sequenced by the second generation of bacterial whole genome sequencing to obtain the draft genome sequence, and the bacterial taxonomy was identified using GTDB-Tk software. After that, the average nucleotide identity (Average Nucleotide Identity, ANI; ANI < 95%, https: / / www.ezbiocloud.net / tools / ani) and genomic hybridization (digital DNA-DNA hybridization, dDDH; dDDH < 70%, http: / / ggdc.dsmz.de) analysis was performed by comparison with the model strain to further identify the strain. Both dDDH and ANI can be calculated and analyzed using the online analysis platform.

[0058] 1.6 Genome correlation analysis

[0059] (1) Basic characteristics of the genome

[0060] The sequencing draft results are as follows Fig.16 The results showed that the genome size of YMJ2024LM-1 strain was 1,987,684 bp and the GC content was 37.64%.

[0061] (2) dDDH and ANI

[0062] The dDDH and ANI values ​​of the type strains of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and YMJ2024LM-1 were compared. The results showed that the dDDH and ANI values ​​between the type strains ATCC 8293 and YMJ2024LM-1 were 94.90% and 99.29%, respectively, supporting that the two strains were the same bacterial species.

[0063] The preservation information of this strain is as follows: the strain preservation number is CGMCC NO.33257, the preservation classification name is Leuconostoc mesenteroidesYMJ2024LM-1 strain, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation date is December 31, 2024.

[0064] Example 2 Effect of Leuconostoc mesenteroides YMJ2024LM-1 on OVA-induced allergic rhinitis

[0065] 2.1 Preparation of YMJ2024LM-1 bacterial suspension

[0066] The mesenteric Leuconostoc YMJ2024LM-1 strain was streaked on MRS solid culture medium and cultured anaerobically at 37°C for 48 hours to obtain a single colony; a single colony was picked and inoculated into fresh MRS liquid culture medium and cultured anaerobically at 37°C for 18 hours to obtain an activated solution; the activated solution was inoculated into MRS liquid culture medium at an inoculum amount of 10% (v / v), and cultured anaerobically at 37°C for 18 hours to obtain a bacterial solution; the bacterial solution was centrifuged at 3000g for 15 minutes to obtain mesenteric Leuconostoc cells, which were washed with 0.9% (m / v) saline and then resuspended in 0.9% saline to a bacterial concentration of 1×10^10 CFU / mL (2×10^9 CFU / mouse, 200μL for oral gavage) to obtain a YMJ2024LM-1 bacterial suspension.

[0067] 2.2 Experimental process

[0068] Twenty-four SPF BABL / c female mice aged 6 to 8 weeks (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) with a body weight range of 16 to 20 g were selected.

[0069] According to the experimental requirements, the mice were divided into four groups: blank group, model group, YMJ2024LM-1 probiotic intervention group and loratadine positive control group, with 6 mice in each group.

[0070] After the mice adapted for one week, the mice in the model group, probiotic intervention group and positive control group were intraperitoneally injected with 200 μL of ovalbumin (OVA) suspension (concentration of 1 mg / mL, solvent: normal saline) on days 0, 7 and 14 of the experiment to sensitize the mice.

[0071] Starting from the 14th day of the experiment, the mice in the YMJ2024LM-1 probiotic intervention group were gavaged with 200 μL of YMJ2024LM-1 bacterial suspension with a bacterial concentration of 1×10^10 CFU / mL (prepared by resuspending in 0.9% saline) every day, the mice in the positive control group were gavaged with loratadine (each mouse was given 15.15 mg / kg according to body weight), and the mice in the blank group and the model group were gavaged with an equal amount of 0.9% saline.

[0072] Starting from the 21st day of the experiment, the model group, YMJ2024LM-1 probiotic intervention group and positive control group were given 500 μg OVA intranasally daily to create an allergic rhinitis mouse model.

[0073] Starting from the 27th day of the experiment, the symptoms of allergic rhinitis (including the number of nose scratching and sneezing) of mice in all groups were recorded within ten minutes after nasal drops.

[0074] The 28th day was the end point of the experiment. All mice were anesthetized and blood was collected from their eyeballs, and then euthanized. The collected mouse serum was analyzed for IgE content, and the mouse nasal mucosal tissue was collected for pro-inflammatory cytokine content, pro-allergenic cytokine expression, regulatory cytokine expression, and tissue pathology section evaluation.

[0075] 2.3 Detection methods

[0076] 2.3.1 Serum IgE concentration was detected using a commercial kit according to the instructions. The detection kit was purchased from Thermo Fisher Scientific (Cat. No. 88-50460-88).

[0077] 2.3.2 Detection of expression and secretion of inflammatory cytokines and allergenic cytokines in nasal mucosa.

[0078] The nasal mucosal tissue was placed in a grinding tube containing 1 mL of Trizol for cryo-grinding. After grinding, 200 μL of chloroform was added to the tube, shaken and mixed, and placed on ice for 20 min. Then centrifuged at 12000 rpm for 20 min at 4 °C, and the upper aqueous phase was collected. 500 μL of isopropanol was added, inverted and mixed, incubated at room temperature for 10 min, and centrifuged again at 4 °C and 12000 rpm for 20 min to discard the supernatant. The resulting white precipitate was washed with 75% ethanol to obtain RNA, dissolved in 100 μL of enzyme-free water, and the RNA concentration was determined.

[0079] According to the instructions of the kit, 40 μL of reverse transcription reaction system was prepared, 500 ng of RNA was added to each tube, and reverse transcription reaction was performed: 37°C for 15 minutes, followed by 85°C for 5 minutes to obtain cDNA. According to the instructions of the kit, 20 μL of quantitative PCR reaction system (containing 2 μL of cDNA template) was prepared for quantitative PCR detection. The reaction conditions were: 95°C pre-denaturation for 30 seconds, followed by 40 cycles, each cycle including 95°C denaturation for 3 seconds and 60°C annealing for 30 seconds, and fluorescence signals were collected at the end of each cycle. β-actin was used as the internal reference gene, and the relative expression of the target gene was calculated by the 2-ΔΔCt method.

[0080] Table 1. Primer sequences used for PCR

[0081]

[0082] The secretion level of allergenic cytokine (IL-5) in nasal mucosal tissue was detected using a commercial kit according to the instructions. The detection kit was purchased from Thermo Fisher Scientific (Cat. No. 88-7054-88).

[0083] The secretion level of sensitizing cytokine (IL-13) in nasal mucosal tissue was detected using a commercial kit according to the instructions. The detection kit was purchased from Thermo Fisher Scientific (Cat. No. 88-7137-22).

[0084] The secretion level of pro-inflammatory cytokines (IL-33) in nasal mucosal tissue was detected using a commercial kit according to the instructions. The detection kit was purchased from Thermo Fisher Scientific (Cat. No. 88-7333-22).

[0085] The expression level of pro-inflammatory cytokine (TSLP) in nasal mucosal tissue was detected using a commercial kit according to the instructions. The detection kit was purchased from Beijing Thermo Fisher Scientific Co., Ltd. (Cat. No. 88-7490-22).

[0086] 2.3.3 Pathological examination of nasal tissue: The mouse nasal tissue was sealed with 4% paraformaldehyde for 24 h in advance; the preparation of pathological sections, HE staining and section scanning were all completed by Beijing Anheyuan Biotechnology Co., Ltd.

[0087] 2.4 Statistical methods:

[0088] All data were statistically analyzed using GraphPad Prism 10.4.1 software. Data are presented as mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance and LSD test (equal variance) or Dunnett's test (unequal variance) as multiple comparisons. Data that did not conform to normal distribution were logarithmically transformed or analyzed by nonparametric tests.

[0089] 2.5 Experimental Results

[0090] 1) Number of nose scratches

[0091] The number of nose scratching is one of the important indicators for evaluating the symptoms of rhinitis in mice. The more times, the more severe the allergic rhinitis symptoms of the mice. Figure 1 As shown in the figure, compared with the blank group (about 1.7 times), the number of nose scratching in the model group mice increased significantly (increased to 6.3 times), while the number of nose scratching in the YMJ2024LM-1 probiotic intervention group mice decreased significantly compared with the model group (decreased to about 3.3 times), close to the number of nose scratching in the loratadine treatment group mice (about 2.7 times). The above results show that YMJ2024LM-1 can alleviate the increase in the number of nose scratching in mice caused by OVA, and YMJ2024LM-1 can significantly alleviate the symptoms of allergic rhinitis in mice caused by OVA.

[0092] 2) Serum IgE level

[0093] Allergic rhinitis is mainly caused by the immune system's overreaction to allergens, which leads to increased levels of immunoglobulin E (IgE). Blood tests of most patients show increased IgE values. Previous studies have confirmed that serum IgE levels are significantly increased in the OVA-induced mouse allergic rhinitis model. In the experiment of this example, Figure 2 As shown in the figure, compared with the blank group, the serum IgE level of the OVA-sensitized model group mice increased significantly (about 2 times). The serum IgE content of the mice in the YMJ2024LM-1 probiotic intervention group was significantly reduced (down 32%), which was similar to the effect of the loratadine treatment group (down 37%), proving that YMJ2024LM-1 has the potential to treat allergic rhinitis.

[0094] 3) Sensitizing cytokines

[0095] IL-4, IL-5, and IL-13 in the interleukin (IL) family have been shown to be positively correlated with the severity of allergic rhinitis as sensitizing cytokines. Figures 3 to 5 As shown, compared with the blank group, the expression level of IL-4 in the nasal mucosa of the model group was significantly increased, while the expression of IL-4 was significantly inhibited in the mice in the YMJ2024LM-1 probiotic intervention group, and its inhibitory effect was similar to that in the loratadine treatment group, suggesting that YMJ2024LM-1 can relieve the symptoms of allergic rhinitis by regulating the pro-sensitizing cytokine IL-4 in the nasal mucosal tissue.

[0096] 4) Pro-inflammatory factors

[0097] IL-6, IL-1β, IL-33 and TSLP in the interleukin (IL) family have been shown to be positively correlated with inflammation in vivo. IL-10 and TGF-β play an important immunomodulatory role in allergic rhinitis, reducing allergic reactions by inhibiting the production of proinflammatory cytokines, promoting immune tolerance and regulating the function of Treg cells. In the experiment of this example, Figures 6 to 9 As shown in the figure, after OVA stimulation, the expression of IL-6 and IL-1β in the nasal mucosa of mice increased significantly, and the secretion of IL-33 also increased (by about 1.3 times). In the mice in the YMJ2024LM-1 probiotic intervention group, the expression level of IL-6 decreased (by about 57%), and the secretion of IL-33 was also inhibited (by 19%), indicating that YMJ2024LM-1 can inhibit inflammation in mice and has a positive effect on the treatment of allergic rhinitis.

[0098] 5) Regulatory cytokines

[0099] For regulatory cytokines, such as Figure 10 to Figure 11 As shown in the figure, the expression of IL-10 in the nasal mucosa of mice increased after OVA stimulation, while TGF-β had no effect. In the mice in the YMJ2024LM-1 probiotic intervention group, the expression of IL-10 showed a downward trend. Although the effect was not as good as that in the loratadine treatment group, it also suggested that YMJ2024LM-1 had a certain effect in inhibiting inflammation through the IL-10 pathway.

[0100] 6) Pathological sections of nasal mucosa tissue

[0101] Compared with normal mice, the pathological sections of the nasal mucosa of mice with allergic rhinitis showed congestion, swelling, epithelial damage, and inflammatory infiltration (such as eosinophils). Figure 12 to Figure 14 As shown in the figure, the nasal mucosal surface of the mice in the control group was intact, and there was no mucosal congestion or damage. However, the mucosal layer of the model group was significantly thickened, and the mucosal surface was irregular, with epithelial damage and inflammatory infiltration. The nasal mucosal tissue of the mice in the YMJ2024LM-1 probiotic intervention group was significantly restored, and epithelial damage and inflammatory infiltration were alleviated. Through scoring statistical analysis, such as Fig.15 As shown, strain YMJ2024LM-1 intervention can significantly reduce the nasal mucosal tissue pathological score of OVA-induced mice.

[0102] In summary, oral administration of Mesenteric Leuconostoc YMJ2024LM-1 can reduce the increase of serum IgE in OVA-induced allergic rhinitis mice, alleviate the expression and secretion of allergic cytokines and inflammatory cytokines in allergic rhinitis mice, and alleviate the nasal mucosal tissue damage caused by allergic rhinitis, which is of great significance for the prevention and auxiliary treatment of allergic rhinitis. It also reveals that Mesenteric Leuconostoc YMJ2024LM-1 can be used to prepare foods, health products or medicines for allergic rhinitis, etc., and has good economic value and excellent application prospects.

[0103] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view. In addition, it should be understood that although this specification is described in accordance with the implementation mode, it does not include only one technical solution. This narrative mode of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A mesenteric Leuconostoc strain for allergic rhinitis, characterized in that: The strain is Leuconostoc mesenteroides YMJ2024LM-1, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation date is December 31, 2024, and the preservation number is CGMCC No.33257.

2. The Leuconostoc mesentericum strain for allergic rhinitis relief according to claim 1, characterized in that: The strain can grow well under anaerobic conditions. The colonies are spherical, bean-shaped or short rod-shaped, arranged in pairs or short chains, non-motile, with a smooth surface, milky white, and no pigment. It can grow in an environment with a pH of ≤5.

3. The Leuconostoc mesentericum strain for allergic rhinitis relief according to claim 1, characterized in that: The genome size of this strain is 1,987,684 bp, and the GC content is 37.64%. The dDDH and ANI values ​​of this strain compared with the type strain of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 are 94.90% and 99.29%, respectively.

4. A method for isolating the Leuconostoc mesentericum strain for allergic rhinitis according to any one of claims 1 to 3, characterized in that: The steps include: 1) Take fermented kimchi from Hubei as sample, take 100 μL of kimchi extract, add it to 900 μL sterile PBS EP tube, and dilute the sample in sequence until the concentration of sample extract is 10 -6 times; 2) Take 100 μL of samples of different dilutions and spread them on MRS medium and culture them in an anaerobic incubator at 37°C for 48 hours; 3) Use a sterile inoculation loop to pick colonies with different morphological characteristics on the culture plate, transfer them to new MRS solid medium for further purification, continue anaerobic culture at 37°C for 48 h, and subculture for 3 times to obtain purified strains; 4) The purified strains were cultured in MRS liquid culture medium at pH=3.0 to screen probiotic strains with acid resistance, which are the Leuconostoc mesentericus strains for alleviating allergic rhinitis.

5. Use of the Leuconostoc mesentericum strain according to any one of claims 1 to 3 in the preparation of a product for treating, preventing or improving allergic rhinitis.

6. The use according to claim 5, characterized in that: The product is able to reduce serum IgE concentrations and / or nasal mucosal pro-inflammatory cytokine concentrations.

7. The use according to claim 5, characterized in that: This product can inhibit the expression of nasal mucosal allergenic cytokines.

8. The use according to claim 5, characterized in that: This product can alleviate the pathological changes of nasal tissue induced by allergic rhinitis.

9. A probiotic preparation for allergic rhinitis, characterized in that: The probiotic preparation is made from the Leuconostoc mesentericum strain for relieving allergic rhinitis as described in any one of claims 1 to 3, or contains the Leuconostoc mesentericum strain for relieving allergic rhinitis as described in any one of claims 1 to 3 as an effective ingredient.

10. The probiotic preparation for allergic rhinitis according to claim 9, characterized in that: The dosage form of the probiotic preparation includes any one of capsules, lyophilized powder, suspension or tablet.

11. The probiotic preparation for relieving allergic rhinitis according to claim 9 or 10, characterized in that: The concentration of Leuconostoc mesenteroides in the probiotic preparation is 1×10^8 to 10×10^11 CFU / mL.

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