Application of rhodomonas mucilaginosa exopolysaccharide in preparation of medicine for relieving and / or treating allergic rhinitis

Abstract

The invention discloses an application of mucorhomonas exopolysaccharide in preparation of a medicine for relieving and / or treating allergic rhinitis, and the mucorhomonas exopolysaccharide is obtained by performing sterilization, deproteinization, absolute ethyl alcohol precipitation, DEAE ion exchange resin and gel column separation and purification on fermentation liquor of the mucorhomonas DL-1. The rhodomonas mucilaginosa DL-1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number of the rhodomonas mucilaginosa DL-1 is CGMCC (China General Microbiological Culture Collection Center) No.25967. The rhodomonas mucilaginosa DL-1 extracellular polysaccharide disclosed by the invention has a relieving and / or treating effect on OVA induced allergic rhinitis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of Roseomonas mucilaginosa exopolysaccharide in preparing a medicine for alleviating and / or treating allergic rhinitis. Background Art

[0002] Inflammation is a mainly defensive immune response of the host to stimulation by inflammatory factors. Various cells in the body are recruited into the immune microenvironment to produce cytokines to eliminate the invasion of external factors and restore the health of the body. However, persistent inflammation can cause damage to the body's organs and systemic responses, and plays an important role in the occurrence and development of many complex diseases.

[0003] Allergic rhinitis is an upper respiratory tract disease. Foreign studies have shown that 10% to 25% of the general population suffers from allergic rhinitis worldwide. It is a global health problem that affects about 500 million people worldwide and has a huge impact on public health. Although the pathology and treatment mechanisms of allergic rhinitis have been widely studied, many aspects of allergic rhinitis remain unclear and require further research. Allergic rhinitis is a chronic inflammatory disease dominated by Th2-type immune response, and its pathological characteristics include significant changes in mast cells and goblet cells. Specifically, the aggregation of mast cells in allergic rhinitis is one of its main characteristics. Mast cells release a variety of inflammatory mediators through degranulation, which are involved in the pathophysiological process of allergic rhinitis and are key sensitizing cells for pathological changes in the nasal mucosa. Therefore, mast cells and goblet cells play an important role in allergic rhinitis. Mast cells participate in allergic reactions by releasing inflammatory mediators, while the proliferation of goblet cells reflects the pathological changes in the nasal mucosa. The two work together to promote the development and persistence of allergic rhinitis.

[0004] In most cases, antihistamines and intranasal corticosteroids can relieve allergic rhinitis symptoms, and common new therapies include highly purified allergen extracts and purified or recombinant allergens, allergens, peptides and new adjuvants for specific allergen immunotherapy, and specific monoclonal antibodies (biological agents) for blocking the action of IgE, IL-4 / IL-5 and / or their receptors. Despite many studies on the treatment of allergic rhinitis, the drugs used for the treatment of allergic rhinitis are still limited.

[0005] Ovalbumin (OVA) is the main method for modeling allergic rhinitis in mice, and aluminum hydroxide is an important adjuvant for preparing allergic rhinitis mouse models. The process of allergic rhinitis is that after the body comes into contact with allergens, antigen presenting cells absorb the information of allergens, causing the body to initiate an adaptive immune response. With the help of adjuvants, intraperitoneal injection of OVA can induce systemic sensitization in mice, and then allergen stimulation in the nose can form a local allergic inflammatory response.

[0006] Polysaccharides are high-molecular carbohydrates composed of more than 10 monosaccharides linked in the form of glycosidic bonds. Bioactive polysaccharides have the effects of immunomodulation, anti-tumor, hypoglycemic, anti-inflammatory and regulating the intestinal microenvironment. The development and use of suitable bioactive polysaccharides is expected to provide a new treatment for allergic rhinitis. Summary of the invention

[0007] Purpose of the invention: The present invention provides a new application of Roseomonas mucilaginosa exopolysaccharide in the preparation of a medicament for treating allergic rhinitis.

[0008] The mucilaginous roseomonas exopolysaccharide is obtained by the following steps: inoculating the activated mucilaginous roseomonas into a fermentation medium for cultivation, sterilizing, deproteinizing, and precipitating the obtained fermentation broth with anhydrous ethanol, separating the obtained broth with DEAE centrifugal exchange resin, collecting 0.2-0.5 mol / L NaCl eluted components and freeze-drying them, and then separating them with a gel column, setting the flow rate to 0.4-0.6 mL / min, and collecting the effluent for 9-15 minutes to obtain the exopolysaccharide with a weight average molecular weight of 3000-15000 Da, wherein the deposit number of the mucilaginous roseomonas is CGMCC No.25967.

[0009] The mucus roseomonas (Roseomonas mucosa), strain number DL-1, China General Microbiological Culture Collection Center, preservation time is October 26, 2022, preservation number is CGMCCNo.25967, preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, zip code 100101. This strain is a skin commensal bacteria strain screened by the inventor from healthy human skin in November 2021.

[0010] Specifically, the fermentation broth of Roseomonas mucilaginosa is prepared by the following method: inoculating the Roseomonas mucilaginosa into R2A culture medium for activation at 25-37° C. for 12-24 hours, transferring the activated Roseomonas mucilaginosa into fermentation medium, and culturing at 30-37° C. for 24-36 hours.

[0011] Among them, the components of R2A culture medium are: yeast extract powder 0.5g / L, peptone 0.5g / L, casein hydrolysate 0.5g / L, glucose 0.5g / L, soluble starch 0.5g / L, dipotassium hydrogen phosphate 0.3g / L, anhydrous magnesium sulfate 0.024g / L, sodium pyruvate 0.3g / L, agar 15.0g / L, pH 7.2.

[0012] Preferably, the fermentation medium is formulated as follows: carbon source 10-100 g / L, nitrogen source 1-30 g / L, inorganic salt 0.01-50 g / L, pH 5.0-9.0, and the solvent is water.

[0013] The carbon source is any one or more combinations of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol, starch and molasses, preferably any one or more combinations of sucrose, glucose, lactose, citric acid and starch; the nitrogen source is any one or more combinations of yeast extract, beef extract, peptone, yeast extract, corn steep liquor, soybean cake powder, cottonseed cake powder, urea, (NH4)2SO4, NH4Cl, (NH4)2HPO4 and NH4NO3, preferably any one or more combinations of peptone, yeast extract, corn steep liquor, (NH4)2SO4, NH4Cl, (NH4)2HPO4 and NH4NO3; the inorganic salt is any one or more combinations of sodium chloride, sulfate, phosphate, dihydrogen phosphate, dihydrogen phosphate and hydrochloride.

[0014] In a preferred embodiment, tryptone 15.0-18.0 g / L, soy peptone or soy papain hydrolysate 2.0-4.0 g / L, sodium chloride 4.0-6.0 g / L, dipotassium hydrogen phosphate 2.0-3.0 g / L, glucose 2.0-3.0 g / L, pH 7.2-7.3, more preferably, the fermentation medium components are tryptone 17.0 g / L, soy peptone or soy papain hydrolysate 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.3.

[0015] In a specific embodiment, the exopolysaccharide of Roseomonas mucilaginosa is prepared by the following method: inoculating Roseomonas mucilaginosa into 200 mL of fermentation medium and culturing at 32° C. for 24 h, sterilizing by centrifugation at 3000-5000 rpm for 10 min, adding 1 / 2 volume of sevage reagent to remove protein 3 times, adding 1 volume of anhydrous ethanol and incubating at 4° C. overnight to obtain a crude sugar precipitate of exopolysaccharide, re-dissolving with deionized water, separating with DEAE-52, collecting 0.2M NaCl eluted components for freeze-drying, and then separating with a gel column at a flow rate of 0.4-0.6 mL / min, collecting the effluent for 3-9 min to obtain an exopolysaccharide with a weight average molecular weight of 50-90 kDa, and collecting the effluent for 9-15 min to obtain an exopolysaccharide with a weight average molecular weight of 3000-15000 Da, wherein the deposit number of the Roseomonas mucilaginosa is CGMCC No. 25967.

[0016] Preferably, the gel column is a Superdex 75prep grade gel column, and the 0.2-0.5 mol / L NaCl elution fraction is collected and freeze-dried, and then dissolved in pure water and separated using the gel column, and the eluent is pure water.

[0017] The monosaccharide composition of the extracellular polysaccharide with a weight average molecular weight of 3000-15000Da includes glucose, mannose and rhamnose, wherein the molar ratio of the above monosaccharides is 30-60:30-80:1-10.

[0018] The monosaccharide composition of the extracellular polysaccharide with a weight average molecular weight of 50-90 kDa includes mannose, rhamnose and galactose, and the molar ratio of mannose, rhamnose and galactose is 40-60:10-25:10-25.

[0019] The invention provides the effect of Roseomonas mucilaginosa exopolysaccharide in alleviating allergic rhinitis.

[0020] Wherein, the Roseomonas mucilaginosa exopolysaccharide alleviates OVA-induced allergic rhinitis.

[0021] Specifically, the exopolysaccharide of Roseomonas mucilaginosa reduces the number of nasal scratching, the increase of inflammatory cells in the nasal mucosa, the increase of goblet cells and the increase of mast cells induced by OVA. At the same time, the exopolysaccharide of Roseomonas mucilaginosa inhibits the increase of IL-4, IL-13, IL-6 and IFN-γ in serum induced by OVA.

[0022] Beneficial effects: (1) The present invention prepares high molecular weight exopolysaccharides and low molecular weight exopolysaccharides of Roseomonas mucosa DL-1 by a fermentation separation method, and verifies that both polysaccharides have the effect of alleviating OVA-induced allergic dermatitis. Specifically, Roseomonas mucosa exopolysaccharides can reduce the number of nasal scratching, increase in inflammatory cells in the nasal mucosa, increase in goblet cells and mast cells induced by OVA, and at the same time alleviate the increased levels of IL-4, IL-6 and IFN-γ in the serum of OVA-induced BALB / c allergic rhinitis mice, providing a new means for the treatment of allergic rhinitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the ion chromatogram of the standard sample;

[0024] Figure 2 Ion chromatograms of low molecular weight exopolysaccharide (A) and high molecular weight exopolysaccharide (B) samples of Roseomonas mucilaginosa DL-1;

[0025] Figure 3 Scattering spectra of low molecular weight exopolysaccharide (A) and high molecular weight exopolysaccharide samples of Roseomonas mucilaginosa DL-1 (B);

[0026] Figure 4 Figure 2 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on the pathological changes of OVA-induced BALB / c allergic rhinitis mice;

[0027] Figure 5 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on scratching in OVA-induced BALB / c allergic rhinitis mice;

[0028] Figure 6 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on nasal mucosal goblet cells in OVA-induced BALB / c allergic rhinitis mice;

[0029] Figure 7 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on mast cells in the nasal mucosa of BALB / c mice with OVA-induced allergic rhinitis;

[0030] Figure 8 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on IL-4, IL-13, IL-6 and IFN-γ in the serum of OVA-induced BALB / c allergic rhinitis mice. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0032] The detailed information of Roseomonas mucilaginosa DL-1 used in the following examples is disclosed in CN116103205A.

[0033] In the text, Roseomonas mucilaginosa DL-1 exopolysaccharide and Roseomonas mucilaginosa exopolysaccharide have the same meaning, and Roseomonas mucilaginosa DL-1 and Roseomonas mucilaginosa have the same meaning.

[0034] Example 1 Preparation of Roseomonas mucilaginosa DL-1 exopolysaccharide.

[0035] 1) Cultivation of Roseomonas mucilaginosa DL-1

[0036] The mucilaginous Roseomonas DL-1 stored on a slant at 4°C was activated and inoculated on a seed culture medium (i.e., activation culture medium) at an activation temperature of 32°C for 16 hours. 10% of the seed fermentation liquid was inoculated into the fermentation medium at a rotation speed of 200 rpm and cultured at 32°C for 24 hours to obtain a fermentation liquid. The number of live bacteria in the fermentation liquid could reach 5×10 9 CFU / mL and above.

[0037] The activation culture medium is R2A culture medium, and the culture medium components are: 0.5 g / L yeast extract powder, 0.5 g / L peptone, 0.5 g / L casein hydrolyzate, 0.5 g / L glucose, 0.5 g / L soluble starch, 0.3 g / L dipotassium hydrogen phosphate, 0.024 g / L anhydrous magnesium sulfate, 0.3 g / L sodium pyruvate, 15.0 g / L agar, and a pH value of 7.2.

[0038] The fermentation medium 1 comprises 17.0 g / L tryptone, 3.0 g / L soy peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH value of 7.3.

[0039] 2) Preparation of exopolysaccharide from Roseomonas mucilaginosa DL-1

[0040] The fermentation broth obtained above was centrifuged (3000-4000rpm, 10min) to sterilize, and 1 volume of the fermentation broth was added to 2 volumes of sevag reagent (chloroform: n-butanol = 4:1), and repeated 2-3 times to remove protein, and then 1 volume of anhydrous ethanol was added, and centrifuged overnight at 4°C (3000-4000rpm, 10min) to obtain crude exopolysaccharide precipitate, which was dried and re-dissolved in deionized water. About 5-6g of crude exopolysaccharide was determined by sulfuric acid-phenol method for DEAE centrifugal exchange resin separation and purification, and the 0.2M NaCl elution fraction was collected and freeze-dried, dissolved in pure water, and the concentration was 50mg / ml, and then passed through Superdex75prep grade gel column (26mm×1000mm, Cytiva, USA), and high molecular weight exopolysaccharides (3-9min) and low molecular weight exopolysaccharides (9-15min) were separated according to the elution time, and their monosaccharide composition and molecular weight were determined.

[0041] The method for determining the monosaccharide of extracellular polysaccharide is as follows: Thermo ICS5000 ion chromatography system (ICS5000, ThermoFisher Scientific, USA) uses an electrochemical detector to analyze and detect the monosaccharide components of the mixed standard and sample. After accurately weighing the standard products required for this project, add water to make a 10 mg / mL standard solution mother solution single standard, then take an appropriate amount of the standard mother solution single standard and mix it to make a standard mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL, and prepare the series of standards required for the machine according to the following concentration gradient.

[0042] Table 1. Monosaccharide standard gradient concentration information

[0043]

[0044]

[0045] *Standard products mainly come from Sigma

[0046] Using Dionex TM C Allergic RhinitisboPac TMPA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2MNaAc), flow rate 0.5ml / min; column temperature was 30℃; elution gradient: 0min A phase / B phase / C phase (95:5:0, V / V), 26min A phase / B phase / C phase (85:5:10, V / V), 42min A phase / B phase / C phase (85:5:10, V / V), 42.1min A phase / B phase / C phase (60:0:40, V / V), 52minA phase / B phase / C phase (60:40:0, V / V), 52.1min A phase / B phase / C phase (95:5:0, V / V), 60min A phase / B phase / C phase (95:5:0, V / V).

[0047] The results of standard determination are as follows Figure 1 As shown in Figure 1, all 13 standards are single peaks. Among them, the sample chromatogram of low molecular weight extracellular polysaccharide is as follows Figure 2 As shown in A, based on the standard curve of standard retention time and sample concentration, the main monosaccharide composition of low molecular weight extracellular polysaccharide was determined to be glucose: mannose: rhamnose = 46.00: 52.15: 1.84. The results of high molecular weight polysaccharide are shown in Figure 2 As shown in B, it was determined that the high molecular weight exopolysaccharide monosaccharides mainly include mannose, rhamnose and glucose, which are composed of a molar ratio of 44.71:22.16:21.24.

[0048] The molecular weight of the obtained polysaccharide was further detected. The method for determining the molecular weight of extracellular polysaccharide is as follows: the chromatographic system used is a gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system is U3000 (Thermo, USA), the differential detector is Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector is DAWN HELEOSⅡ (Wyatt technology, CA, USA). Gel exclusion chromatography columns (Ohpak SB-805HQ (300×8mm), OhpakSB-804HQ (300×8mm) and Ohpak SB-803HQ (300×8mm) were connected in series. The column temperature was 45°C, the injection volume was 100μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.4mL / min, and the elution gradient was: isocratic, 100min. The results are as follows. Figure 3 As shown in A, the weight average molecular weight of the low molecular weight Roseomonas mucilaginosa exopolysaccharide EPS is 11.7 kDa. Figure 3As shown in B, the weight average molecular weight of the high molecular weight Roseomonas mucilaginosa exopolysaccharide EPS is 97.2 kDa.

[0049] Example 2 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on nasal tissue of OVA-induced BALB / c allergic rhinitis mice.

[0050] Housing environment: SPF grade, mice: BALB / c female mice, 8 weeks old, weighing 18-20g. The preparation of the OVA-induced allergic rhinitis model includes the following steps: (1) Systemic basal sensitization: On days 0, 3, 6, 9, 12 and 15, intraperitoneal injection of 200μL saline containing 100μg OVA (Sigma-Aldrich, USA) and 2mg aluminum hydroxide; (2) Nasal reinforcement stimulation: On days 21-30, intranasal treatment with 10μL saline containing 200μg OVA. After one week of stable feeding, the purchased mice were randomly divided into 4 groups, with 6 biological replicates for each treatment, for a total of 24 mice. The drug administration was unified as local administration, with drug administration in the morning and modeling in the afternoon every day. The treatment methods of each group are as follows:

[0051] Treatment 1 (HC), no treatment, i.e., the healthy control group;

[0052] Treatment 2 (Vehicle), OVA modeling, 10 μL saline nasal drops, which is the model group;

[0053] Treatment 3 (DLEPS-H), 10 μL of physiological saline containing 0.5 wt % of the high molecular weight Roseomonas mucosa exopolysaccharide prepared in Example 1 was administered nasally from the 21st day of OVA modeling, which was the high molecular weight polysaccharide treatment group.

[0054] Treatment 4 (DLEPS-L), 10 μL of physiological water nasal drops containing 0.5 wt % of the low molecular weight Roseomonas mucosa extracellular polysaccharide prepared in Example 1 were administered from the 21st day of OVA modeling, which was the low molecular weight polysaccharide treatment group.

[0055] The other daily management of the four treatments remained the same. After the experiment, the mice were sacrificed to obtain tissue and serum samples. The pathological analysis of the nasal mucosa of the mice was performed. The results are as follows Figure 4 As shown in the figure, the nasal mucosa structure of mice in the blank group was intact, no obvious inflammatory cell infiltration was observed under the mucosa, and no obvious hyperplasia and edema were observed in the interstitium; the nasal mucosal epithelium of mice in the model group was significantly damaged, the cilia were disordered, a large number of neutrophils infiltrated under the mucosa, hyperplasia and edema appeared in the interstitium, and small blood vessels were dilated and hyperplastic. The low-molecular-weight and high-molecular-weight exopolysaccharide treatment groups showed significant improvement compared with the model group, with reduced infiltration of inflammatory cells in the nasal mucosa of mice and reduced thickness of epidermal cells.

[0056] Example 3 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on the frequency of nasal itching and scratching in OVA-induced BALB / c allergic rhinitis mice.

[0057] Nasal drip provocation was performed 15 days after OVA induction. 24 hours before sacrifice, half an hour of activity images of each mouse were recorded in a non-disturbing space, and the number of scratching times was calculated in the middle time period. The statistical results are shown in the figure. Figure 5 As shown in the results, the exopolysaccharide of Roseomonas mucilaginosa DL-1 had a certain effect on alleviating OVA-induced nasal itching in mice, and both the low-molecular-weight and high-molecular-weight exopolysaccharide treatment groups were statistically significant compared with the model group. The difference between the low-molecular-weight exopolysaccharide treatment group and the model group was more significant, P≤0.001; the difference between the high-molecular-weight exopolysaccharide treatment group and the model group was statistically significant P≤0.01.

[0058] Example 4 Effect of Roseomonas mucilaginosa DL-1 exopolysaccharide on the number of goblet cells in the nasal mucosa of BALB / c mice induced by OVA.

[0059] The goblet cell staining steps are as follows:

[0060] (1) Place the mouse nasal mucosa paraffin sections in an oven at 60°C for 1 to 2 h;

[0061] (2) Paraffin sections were dewaxed with conventional xylene and ethanol until hydrated;

[0062] (3) Oxidation with 0.5% periodic acid aqueous solution for 5 minutes.

[0063] (4) Rinse with distilled water for more than 5 minutes;

[0064] (5) Schiff's reagent acts for 10-30 minutes;

[0065] (6) Wash three times with sulfurous acid solution, each time for 1.5-2 minutes.

[0066] (7) Wash with running water for 15 minutes;

[0067] (8) Lightly stain the nuclei with Harry's hematoxylin for 1-2 minutes (too dark may indicate differentiation);

[0068] (9) Rinse with running water;

[0069] (10) Dehydrate, clear, and seal the slides as usual.

[0070] Under the microscope, goblet cells appear reddish purple, and the nucleus appears blue. Figure 6 As shown, OVA can induce the proliferation of goblet cells in the nasal mucosa of BALB / c mice. The order of proliferation is model group, high molecular weight exopolysaccharide group and low molecular weight exopolysaccharide group. The exopolysaccharide treatment groups all have the effect of significantly reducing OVA-induced goblet cell proliferation in the nasal mucosa of BALB / c mice.

[0071] Example 5 Effect of Roseomonas mucilaginosa DL-1 exopolysaccharide on mast cell infiltration in the nasal mucosa of OVA-induced BALB / c allergic rhinitis mice.

[0072] The heads of the sacrificed mice were decalcified and then embedded in paraffin and sliced. Mast cells were stained with toluidine blue. The staining method was as follows: (1) The tissue was fixed in neutral formaldehyde solution or formaldehyde ethanol solution; (2) The tissue sections were dewaxed; (3) The sections were placed in toluidine blue solution (0.5 g toluidine blue was diluted to 100 ml with distilled water) and stained for 30 min. (4) The sections were rinsed with water; (5) The sections were differentiated with glacial acetic acid solution (0.5 ml glacial acetic acid was diluted to 100 ml with distilled water) until the nuclei and particles were clear (under a microscope); (6) The sections were rinsed with water and dried with cold air; (7) The sections were sealed with xylene or transparent neutral gum.

[0073] The results are as follows Figure 7 As shown, mast cell granules are red-purple and nuclei are blue. OVA treatment can significantly induce mast cell infiltration in the nasal mucosa of mice. Compared with the model group, the mucoid Roseomonas DL-1 exopolysaccharide treatment group can alleviate OVA-induced mast cell infiltration in mice, and there are significant differences in microscopic observation. Among them, the low molecular weight exopolysaccharide treatment group has a better effect than the high molecular weight exopolysaccharide treatment group.

[0074] Example 6 Effects of Roseomonas mucilaginosa DL-1 exopolysaccharide on IL-4, IL-13, IL-6 and IFN-γ in the serum of OVA-induced BALB / c allergic rhinitis-like mice.

[0075] The specific experimental steps are as follows:

[0076] ① Before the experiment begins, the collected specimens and various reagents in the kit need to be placed at room temperature for 30 minutes.

[0077] ② Divide the ELISA plate into three parts: blank well, standard well, and sample well, and add sample diluent, standard and sample to be tested in turn, 100μL each. When adding samples, try not to touch the well wall, add to the bottom of the ELISA plate, shake gently to mix, and avoid bubbles. The sample addition time is within 10 minutes. Then cover the ELISA plate with a film and place it in an electric constant temperature water bath for incubation at 37°C for 120 minutes.

[0078] ③ During the incubation process, prepare the working solution of detection solution A. After the incubation is completed, discard the solution and spin dry, and do not wash the ELISA plate. Add 100 μL of the working solution of detection solution A to each well of the ELISA plate, shake to mix, cover the ELISA plate with film, and place it in the electric constant temperature water bath for incubation again at 37°C for 60 minutes.

[0079] ④During the second incubation, dilute the concentrated washing solution to 20 times with distilled water. Shake off the liquid in the wells, add about 350μL of the prepared washing solution to each well, soak for 1-2 minutes, and then shake off the liquid in the ELISA plate. Repeat this washing step 3 times.

[0080] ⑤ Prepare the detection solution B working solution. After washing, add the detection solution B working solution to the ELISA plate wells, about 100 μl per well, shake to mix, cover the ELISA plate with a film, and place it in the electric constant temperature water bath for incubation again at 37°C for 60 minutes.

[0081] ⑥After the second incubation, discard the liquid and spin dry, then wash 5 times in total. The washing method is the same as step ④.

[0082] ⑦ Add 90 μL of substrate solution to each well of the ELISA plate, cover with a film and incubate at 37°C in the dark for about 15 minutes. When a clear gradient appears in the standard well, the color development is finished.

[0083] ⑧ Add 50 μL of stop solution to each well to stop the reaction. Add the stop solution in the order of adding the substrate solution. When the color of the liquid in the well of the ELISA plate immediately changes from blue to yellow, the reaction is terminated.

[0084] ⑨After the reaction is terminated, the ELISA plate is immediately placed in the ELISA reader, with the blank well as the control, the standard value is adjusted to 0, the wavelength is set to 450nm, and the OD value of each well is measured. Then, a standard curve is established according to the instructions, a standard equation is generated, and the absorbance value in each well is substituted into the equation to calculate the concentration of IL-4, IL-13, IL-6 and IFN-γ in each sample.

[0085] The results are as follows Figure 8 As shown in the results, the exopolysaccharide of Roseomonas mucilaginosa DL-1 inhibited the increase of IL-4, IL-13, IL-6 and IFN-γ in the serum of mice with allergic rhinitis induced by OVA. The low molecular weight and high molecular weight exopolysaccharide treatment groups were statistically significant compared with the model group, but the low molecular weight DLEPS could not significantly inhibit the increase of IL-13 levels in serum induced by OVA, which may also be caused by the differences between the groups.

[0086] In summary, the present invention provides a preparation method and application of Roseomonas mucosa DL-1 exopolysaccharide of skin commensal bacteria that can relieve allergic rhinitis. The OVA-induced BALB / c allergic rhinitis animal model verified the alleviating effect of Roseomonas mucosa DL-1 exopolysaccharide on the number of scratching, inflammatory factor accumulation and nasal mucosal damage in mice with allergic rhinitis. Therefore, Roseomonas mucosa DL-1 exopolysaccharide has a certain application prospect in the treatment of allergic rhinitis.

[0087] The present invention provides a method and idea for alleviating allergic rhinitis. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. Use of Roseomonas mucilaginosa exopolysaccharide in the preparation of a medicament for relieving and / or treating allergic rhinitis, wherein: The Roseomonas mucilaginosa exopolysaccharide is obtained by the following steps: inoculating the activated Roseomonas mucilaginosa into a fermentation medium for cultivation, sterilizing, deproteinizing, and anhydrous ethanol precipitation of the obtained fermentation broth, separating and purifying it by using DEAE centrifugal exchange resin, collecting 0.2-0.5 mol / L NaCl elution components and freeze-drying them, and then separating them by using a gel column with a flow rate set to 0.4-0.6 mL / min, collecting the effluent for 9-15 minutes to obtain the exopolysaccharide with a weight average molecular weight of 3000-15000 Da, wherein the deposit number of the Roseomonas mucilaginosa is CGMCC No. 25967.

2. The use according to claim 1, characterized in that: The gel column is a Superdex 75 prep grade gel column. The 0.2-0.5 mol / L NaCl eluted fraction is collected and freeze-dried, then dissolved in pure water and separated using the gel column. The eluent is pure water.

3. The use according to claim 1, characterized in that: The monosaccharide composition of the mucilaginous roseomonas exopolysaccharide includes glucose, mannose and rhamnose, wherein the molar ratio of the above monosaccharides is 30-60: 30-80: 1-10.

4. Use of Roseomonas mucilaginosa exopolysaccharide in the preparation of a medicament for alleviating and / or treating allergic rhinitis, wherein: The Roseomonas mucilaginosa DL-1 extracellular polysaccharide is obtained by the following steps: inoculating the activated Roseomonas mucilaginosa into a fermentation medium for cultivation, sterilizing, deproteinizing, and anhydrous ethanol precipitation of the obtained fermentation broth, separating the broth by using DEAE centrifugal exchange resin, collecting 0.2-0.5 mol / L NaCl elution components and freeze-drying them, and then separating them by using a gel column with a flow rate set to 0.4-0.6 mL / min, collecting effluent for 3-9 min, and obtaining an extracellular polysaccharide with a weight average molecular weight of 50-90 kDa, wherein the deposit number of the Roseomonas mucilaginosa is CGMCC No. 25967.

5. The use according to claim 4, characterized in that: The gel column is a Superdex 75 prep grade gel column. The 0.2-0.5 mol / L NaCl eluted fraction is collected and freeze-dried, then dissolved in pure water and separated using the gel column. The eluent is pure water.

6. The use according to claim 4, characterized in that: The monosaccharide composition of the mucilaginous roseomonas exopolysaccharide includes mannose, rhamnose and galactose, and the molar ratio of mannose, rhamnose and galactose is 40-60: 10-25: 10-25.

7. The use according to any one of claims 1 to 6, characterized in that: The fermentation medium comprises: 15.0-18.0 g / L tryptone, 2.0-4.0 g / L soy peptone or soy papain hydrolysate, 4.0-6.0 g / L sodium chloride, 2.0-3.0 g / L dipotassium hydrogen phosphate, 2.0-3.0 g / L glucose, and a pH value of 7.2-7.3, preferably, 17.0 g / L tryptone, 3.0 g / L soy peptone or soy papain hydrolysate, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH value of 7.

3.

8. The use according to claim 1 or 4, characterized in that: The Roseomonas mucilaginosa exopolysaccharide alleviates OVA-induced allergic rhinitis.

9. The use according to claim 1 or 4, characterized in that: The Roseomonas mucilaginosa exopolysaccharide reduces the frequency of nasal scratching, increase in inflammatory cells of the nasal mucosa, increase in goblet cells and increase in mast cells induced by OVA.

10. The application according to any one of claims 1 or 3, wherein: The Roseomonas mucilaginosa exopolysaccharide inhibits the increase of IL-4, IL-13, IL-6 and IFN-γ in serum induced by OVA.

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