Application of Weissella cibaria X-5 and its exopolysaccharide in preventing and treating obstructive pulmonary diseases

Abstract

The present invention belongs to the field of biomedical technology, and discloses Weissella cibaria X-5 and its application. The Weissella cibaria X-5 has a deposit number of CCTCC NO: M 20232333 and is deposited in the China Center for Type Culture Collection. The Weissella cibaria X-5 can secrete extracellular polysaccharide, and the secreted extracellular polysaccharide can significantly improve the lung function of mice with chronic obstructive pulmonary disease. It is found through observation of lung tissue sections that it can also significantly improve the level of pulmonary cell infiltration in chronic obstructive pulmonary disease, has no effect on the body weight and growth status of mice, and causes no damage to important organs, with high safety. In addition, cell experiments show that the extracellular polysaccharide can also significantly improve the expression of genes related to the MHS inflammatory response of macrophages, and has no effect on the viability of macrophages MHS. Therefore, the Weissella cibaria X-5 of the present invention and the extracellular polysaccharide secreted by it can be used to prepare drugs for alleviating and / or treating chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of Weissella cibaria X-5 and its exopolysaccharide in the prevention and treatment of obstructive pulmonary diseases. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a disease that seriously affects the respiratory system function, characterized by persistent airflow limitation, mainly caused by chronic bronchitis and / or emphysema. This disease can not only cause symptoms such as dyspnea and cough in patients, but may also further develop into pulmonary heart disease and respiratory failure. Although its exact cause is not yet clear, factors such as smoking, air pollution and genetics are considered to be one of the main inducing factors. COPD progresses slowly, the condition is prone to recurrence and difficult to reverse, so timely diagnosis and effective treatment are crucial. According to epidemiological investigation and statistics, the incidence, prevalence and mortality of COPD are increasing year by year, and this trend exists not only in different regions and races, but also among different populations. Globally, in the population over 40 years old, the incidence of COPD has reached as high as 9%-10%. The earliest symptoms of COPD often resemble ordinary coughs, so its diagnosis requires the support of pulmonary function test results.

[0003] At present, the treatment of chronic obstructive pulmonary disease (COPD) involves a variety of methods, including drug treatment, oxygen therapy, rehabilitation training, supportive treatment and in some cases surgical treatment such as lung transplantation. Drug treatment usually includes the use of drugs such as bronchodilators, steroids and antibiotics to relieve symptoms, improve lung function and prevent acute exacerbation. Oxygen therapy can be used to treat hypoxemia and improve the quality of life of patients. Rehabilitation training helps patients better cope with the disease and improve the quality of life through measures such as physical exercise, nutritional guidance and psychological support. However, although the existing treatment methods can relieve symptoms and delay the progression of the disease to a certain extent, the treatment of COPD still faces many problems. First, the effect of drug treatment is limited, it cannot completely reverse the decline of lung function, and long-term use may lead to drug resistance and side effects. Second, although surgical treatments such as lung transplantation are effective in some cases, their application is limited due to the shortage of donors and rejection after surgery. In addition, the high treatment cost has also become one of the important challenges faced by patients and the medical system. Therefore, it is urgent to strengthen the research on the effective prevention and treatment of COPD in order to provide new strategies for its clinical treatment.

[0004] Exopolysaccharides produced by lactic acid bacteria are natural macromolecules that exhibit excellent biosafety and biocompatibility in their biological properties. Exopolysaccharides produced by lactic acid bacteria have the potential to modulate the immune system and can be used as immunomodulators to regulate innate and adaptive immunity. At the cellular level, exopolysaccharides produced by lactic acid bacteria have a positive impact on the viability and proliferation of various immune cell types, including macrophages, dendritic cells, and lymphocytes. In addition, exopolysaccharides produced by lactic acid bacteria can also induce the production of cytokines, thereby enhancing the defensive function of the host immune system and reducing the inflammatory response. The inflammatory response is often the main manifestation in the initial stage of obstructive lung disease. Currently, the research on exopolysaccharides mainly focuses on Lactobacillus, but the exopolysaccharide production of Lactobacillus is relatively low, and its specific mechanism in obstructive lung disease has not been elucidated, which limits the widespread application of exopolysaccharides produced by lactic acid bacteria in the treatment of obstructive lung disease. Therefore, exploring lactic acid bacteria with high exopolysaccharide production and deeply investigating the role and mechanism of exopolysaccharides in the prevention and treatment of obstructive lung disease are of great significance for its application in the prevention and treatment of obstructive lung disease.

[0005] Weissella cibaria, as a member of the lactic acid bacteria family, is widely distributed in the natural environment, mainly present in saliva, breast milk, the human gastrointestinal tract, and traditional fermented foods, and has an important ecological status. Its exopolysaccharide production is relatively high, and it is mainly used as a thickener, stabilizer, and gelling agent in the fields of food processing, cosmetics, and pharmaceuticals. Research has shown that exopolysaccharides produced by Weissella cibaria have antioxidant, antibacterial, promoting the growth of lactic acid bacteria and bifidobacteria, and immunomodulatory effects. However, there is currently no report on Weissella cibaria and its exopolysaccharides in obstructive lung disease, especially the research on the prevention and treatment of obstructive lung disease and its related mechanisms has not been reported. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of Weissella cibaria X-5 and its exopolysaccharides in the prevention and treatment of obstructive lung disease.

[0007] To achieve the purpose of the invention, the technical solutions adopted by the present invention are as follows:

[0008] In the first aspect of the present invention, a Weissella cibaria is provided, and the nucleic acid sequence of the Weissella cibaria contains the sequence shown in Sequence 1 (SEQ ID NO.1).

[0009] According to the above-mentioned Weissella cibaria, preferably, the Weissella cibaria is Weissella cibaria X-5. The preservation number of Weissella cibaria X-5 is CCTCC NO: M20232333, the preservation institution is the China Center for Type Culture Collection (CCTCC), and the address of the preservation institution is the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is November 27, 2023.

[0010] The screening and identification process of the above-mentioned Weissella cibaria X-5 is as follows: Sterile PBS is fully shaken and resuspended with the fermented pickled Chinese cabbage, the supernatant is taken, and serially diluted to 10 -5 . This dilution is added to the MRS liquid medium for lactic acid bacteria growth, and statically cultured at 37°C for 48 h. The bacterial liquid is aspirated and serially diluted to 10 -5 . Then, the dilution is aspirated and spread on the MRS solid medium, and statically cultured at 37°C until obvious single colonies grow. The single colonies are picked into the MRS liquid medium, and after statically culturing at 37°C for 24 h, 0.5 ml of the bacterial liquid is taken, centrifuged at 5000 g for 5 min at 4°C, and after removing the supernatant, the bacterial precipitate is resuspended with sterile PBS. The 16S rDNA sequence of the bacteria is amplified using primers (27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R: 5’-TACGGCTACCTTGTTACGACTT-3’), and the amplified sequence is sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing result of 16S rDNA is input into the BLAST database of NCBI for identification. Finally, according to the homologous sequence analysis, the isolated strain is confirmed to be Weissella cibaria, named Weissella cibaria X-5, and this strain is preserved in the China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCC NO: M 20232334. The 16S rDNA sequence of Weissella cibaria X-5 is shown as Sequence 1 (SEQ ID NO.1).

[0011] The nucleotide sequence of Sequence 1 is:

[0012]

[0013] In the second aspect of the present invention, there is provided the use of Weissella esculenta or a culture containing Weissella esculenta described in the first aspect above in the preparation of a product for preventing, alleviating or / and treating obstructive pulmonary diseases.

[0014] In the third aspect of the present invention, there is provided an extracellular polysaccharide secreted by Weissella esculenta described in the first aspect above.

[0015] According to the above extracellular polysaccharide, preferably, the extracellular polysaccharide is obtained by fermenting and culturing Weissella esculenta X-5 and separating and purifying the fermentation culture solution.

[0016] According to the above extracellular polysaccharide, preferably, the preparation method of the extracellular polysaccharide is as follows: inoculating Weissella esculenta X-5 described in the first aspect above into a medium for fermentation culture to obtain a culture solution, removing the thalli and removing proteins from the culture solution to obtain a supernatant; performing alcohol precipitation treatment on the supernatant, collecting the precipitate, and subjecting the precipitate to dialysis for impurity removal and freeze-drying treatment to obtain the extracellular polysaccharide.

[0017] According to the above extracellular polysaccharide, preferably, the medium is MRS medium or mMRS medium (modified MRS medium).

[0018] According to the above extracellular polysaccharide, preferably, the composition of the mMRS medium is: dipotassium hydrogen phosphate 2 g / L, beef extract 10 g / L, diammonium citrate 5 g / L, sucrose 100 g / L, peptone 10 g / L, magnesium sulfate 0.2 g / L, anhydrous sodium acetate 5 g / L, manganese sulfate 0.05 g / L, Tween 80 1 ml / L, yeast extract 5 g / L, and the balance is water.

[0019] According to the above extracellular polysaccharide, preferably, the culture temperature for fermenting Weissella esculenta X-5 is 30 °C to 37 °C. More preferably, the culture temperature is 37 °C.

[0020] According to the above extracellular polysaccharide, preferably, the dialysis is carried out in distilled water, and the dialysis cut-off molecular weight is 8000 to 14000 Da.

[0021] According to the above extracellular polysaccharide, preferably, the alcohol precipitation treatment is to add absolute ethanol to the supernatant, mix evenly and stand at 4 °C for 12 h to 18 h.

[0022] According to the above extracellular polysaccharide, preferably, the specific operation for protein removal is as follows: Add trichloroacetic acid to the culture solution after removing the bacterial cells, mix evenly to obtain a mixed solution, place the mixed solution at 4°C and let it stand for 12 h to 18 h, and then centrifuge to remove the precipitate. More preferably, the dosage of trichloroacetic acid is such that the mass-volume percentage concentration of trichloroacetic acid in the mixed solution is 4%.

[0023] According to the above extracellular polysaccharide, preferably, the specific operation for bacterial cell removal is as follows: Centrifuge the culture solution to remove the bacterial cells. More preferably, the centrifugation conditions are: centrifuge at 12,000 g at 4°C for 30 min.

[0024] The fourth aspect of the present invention provides the application of the extracellular polysaccharide described in the third aspect above in the preparation of products for preventing, alleviating or / and treating obstructive lung diseases.

[0025] According to the above application, preferably, the obstructive lung disease is chronic obstructive pulmonary disease.

[0026] The fifth aspect of the present invention provides a drug for preventing, treating and / or alleviating obstructive lung diseases. The drug contains an active ingredient, and the active ingredient is Weissella cibaria described in the first aspect above or a culture containing Weissella cibaria or the extracellular polysaccharide described in the second aspect above.

[0027] According to the above drug, preferably, when the active ingredient is Weissella cibaria described in the first aspect above or a culture containing Weissella cibaria, Weissella cibaria can be live bacterial cells or dead bacterial cells.

[0028] According to the above drug, preferably, the drug is administered by oral administration, gavage administration or parenteral administration. More preferably, the parenteral administration method is intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intranasal administration or rectal administration.

[0029] According to the above drug, preferably, the drug further contains pharmaceutically acceptable excipients. More preferably, the excipients include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants or flavoring agents, etc.

[0030] Compared with the prior art, the positive and beneficial effects achieved by the present invention:

[0031] (1) The Weissella cibaria X-5 (W. cibaria X-5) screened in the present invention can secrete extracellular polysaccharide, and the secreted extracellular polysaccharide can significantly improve the lung function of mice with chronic obstructive pulmonary disease. It is found through observation of lung tissue sections that it can also significantly improve the level of pulmonary cell infiltration in chronic obstructive pulmonary disease and can effectively relieve the state of obstructive pulmonary disease. Moreover, cell experiments show that the extracellular polysaccharide can also significantly improve the expression of genes related to the inflammatory response of macrophage MHS and has no effect on the viability of macrophage MHS. Therefore, the Weissella cibaria X-5 of the present invention and the extracellular polysaccharide secreted by it can be used to prepare drugs for relieving and / or treating chronic obstructive pulmonary disease.

[0032] (2) The Weissella cibaria X-5 of the present invention is screened from fermented pickled Chinese cabbage, and its metabolite extracellular polysaccharide is also a natural component produced during the metabolism of lactic acid bacteria. This extracellular polysaccharide has no effect on the body weight and growth status of mice, causes no damage to the important organs of mice, and has high safety. From the perspective of clinical application, compared with other existing drugs for treating obstructive pulmonary disease, the extracellular polysaccharide of Weissella cibaria X-5 is safer. Description of the Drawings

[0033] Figure 1 Results of the screening of the fermentation medium of W. cibaria X-5 and the extraction of extracellular polysaccharide; among them, A is a photo of the extracellular polysaccharide secretion of W. cibaria X-5 on an MRS solid culture plate; B is a photo of the extracellular polysaccharide secretion of W. cibaria X-5 on an mMRS solid culture plate; C is a photo of the freeze-dried sample of the extracellular polysaccharide;

[0034] Figure 2 SEM result diagram of the extracellular polysaccharide of Weissella cibaria X-5;

[0035] Figure 3 UV spectrum scanning analysis result diagram of the extracellular polysaccharide of Weissella cibaria X-5;

[0036] Figure 4 IR spectrum scanning analysis result diagram of the extracellular polysaccharide of Weissella cibaria X-5;

[0037] Figure 5 HPLC analysis result diagram of the extracellular polysaccharide of Weissella cibaria X-5;

[0038] Figure 6 Weight changes (g) of mice in each group during the 0-8th week (model establishment period); among them, Control represents the Control group. Compared with the Control group: * P < 0.05;

[0039] Figure 7Weight changes (g) of mice in each group during the 8th - 16th weeks (treatment period); where Control represents the Control group, Model represents the Model group, extracellular polysaccharide represents the extracellular polysaccharide treatment group, and aminophylline represents the aminophylline treatment group; n = 8, compared with the Control group: * P < 0.05; compared with the Model group: ▲ P < 0.05; compared with the extracellular polysaccharide treatment group: # P < 0.05;

[0040] Figure 8 Changes in pulmonary function of mice in each group at the 16th week; where, compared with Control: * P < 0.05, ** P < 0.001; compared with Model: * P < 0.05;

[0041] Figure 9 Results of HE staining and alveolitis score of lung tissues of mice in each experimental group at the 16th week. Among them, the left figure is a representative picture of HE staining of lung tissues of experimental mice in each group (scale bar = 20μm); the right figure is the alveolitis score of lung tissues of experimental mice in each group Results; Control represents the Control group, Model represents the Model group, X - 5 extracellular polysaccharide represents the extracellular polysaccharide treatment group, and aminophylline represents the aminophylline treatment group; n = 8, compared with Control: * P < 0.05, ** P < 0.001;

[0042] Figure 10 Effects of Weissella cibaria X - 5 extracellular polysaccharide at different concentrations on the activity of MHS cells ( ); compared with Control, * P < 0.05;

[0043] Figure 11 Effects of Weissella cibaria X - 5 extracellular polysaccharide at different concentrations on the inflammation of MHS cells ( ); compared with Control, * P < 0.05. Detailed implementation manners

[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with specific embodiments.

[0045] The following detailed descriptions are all exemplary and are intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, components, and / or combinations thereof.

[0046] For the experimental methods without specific conditions indicated in the following examples, conventional techniques in this technical field are adopted, or the conditions recommended by the manufacturer are followed; for the reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained commercially.

[0047] Example 1: Obtaining and identification of Weissella cibaria W.cibaria X-5

[0048] The fermented pickled Chinese cabbage was shredded, ground, added with sterile PBS, and shaken and resuspended. The supernatant was taken and serially diluted to 10 -5 , and this dilution was added to the MRS liquid medium for lactic acid bacteria growth, and cultured statically at 37°C for 48 h. The bacterial solution was taken and serially diluted to 10 -5 , then the dilution was taken and spread on the MRS solid medium, and cultured statically at 37°C until obvious single colonies grew. The single colonies were picked into the MRS liquid medium, and after culturing statically at 37°C for 24 h, 0.5 ml of the bacterial solution was taken, centrifuged at 5000 g for 5 min at 4°C, and after removing the supernatant, the bacterial pellet was resuspended with sterile PBS.

[0049] The 16S rDNA sequence of the bacteria was amplified using primers (27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R: 5’-TACGGCTACCTTGTTACGACTT-3’), and the amplified sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of Weissella cibaria W.cibaria X-5 obtained by sequencing is shown in Sequence 1.

[0050] The sequencing results of 16S rDNA were input into the BLAST database of NCBI for identification. Finally, it was confirmed by homologous sequence analysis that the isolated strain was Weissella cibaria, named Weissella cibaria X-5 (W.cibaria X-5), and this strain was deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number of the strain was CCTCC NO: M 20232334.

[0051] Example 2: Obtaining of extracellular polysaccharide of W.cibaria X-5

[0052] 1. Optimization of the Fermentation Medium for W. cibaria X-5

[0053] W. cibaria X-5 was streaked onto MRS solid culture plates and mMRS solid culture plates by the method of streak plating, and then the solid culture plates were placed at 37 °C for 24 h - 48 h to observe the extracellular polysaccharide secretion of W. cibaria X-5 on the MRS solid culture plates and mMRS solid culture plates. The experimental results are shown in Figure 1 A and B below.

[0054] As can be seen from Figure 1 A and B below, compared with the extracellular polysaccharide of Weissella cibaria X-5 grown on MRS medium, the extracellular polysaccharide of Weissella cibaria X-5 grown in mMRS medium is more viscous, indicating an increase in the extracellular polysaccharide production of Weissella cibaria X-5. Therefore, mMRS is preferably used for the fermentation treatment of W. cibaria X-5.

[0055] 2. Obtaining Extracellular Polysaccharide

[0056] The cryopreservation tube of Weissella cibaria X-5 was thawed in a 37°C water bath. A sterile inoculation loop was used to pick up the bacterial solution and inoculated by the three-zone streaking method. It was statically cultured in a 37°C incubator for 48 h, and single colonies could be observed. A micropipette tip was used to pick up the obtained single colonies and inoculated into 5 ml of liquid MRS medium for culture. The culture conditions were static culture at 37°C for 24 h, and the bacterial solution became turbid. The bacterial solution was inoculated into a conical flask of modified MRS liquid medium (mMRS medium) at an inoculation amount of 2% (v / v) and statically cultured at 30°C for 48 h until the medium became viscous to obtain the bacterial strain fermentation broth. The above bacterial solution was boiled in a 100°C water bath for 15 min to inactivate bacteria and enzymes, and then cooled to room temperature. If the bacterial solution was viscous, sterilized deionized water could be taken to dilute the bacterial solution. The boiled bacterial solution was centrifuged at 12,000 g and 4°C for 30 min in a high-speed refrigerated centrifuge, and the precipitate such as bacterial cells was discarded to obtain the supernatant. Trichloroacetic acid (TCA) at 80% (m / v) was added to the supernatant until the final concentration of trichloroacetic acid was 4% (m / v) to remove proteins. After standing in a 4°C refrigerator for 12 h, it was centrifuged at 4°C and 12,000 g for 30 min in a high-speed refrigerated centrifuge, and the supernatant was collected. This step was repeated 2 - 3 times to remove protein impurities as much as possible. Absolute ethanol was pre-cooled at 4°C in advance. Three volumes (v / v) of cold absolute ethanol were added to the supernatant after removing protein and other impurities, and it was left standing in a 4°C refrigerator for 12 h. The purpose was to precipitate extracellular polysaccharides by alcohol. It was centrifuged at 4°C and 12,000 g for 30 min to obtain the precipitate. The extracellular polysaccharide precipitate was dissolved in pure water, and the alcohol precipitation step was repeated 2 - 3 times. After completing the alcohol precipitation, the extracellular polysaccharides obtained by alcohol precipitation were redissolved, and then dialyzed in distilled water at 4°C (the dialysis cut-off molecular weight was 8000 - 14000 Da) for 48 - 72 h to remove impurities, and the water was changed every 8 h. The dialyzed polysaccharide solution was freeze-dried under vacuum to obtain the extracellular polysaccharide freeze-dried powder (as Figure 1 shown in C of Figure 1 . As can be seen from C of

[0057] Example 3: Identification and Characterization of the Extracellular Polysaccharide of Weissella cibaria X-5

[0058] 1. Observation of morphology by scanning electron microscopy

[0059] An appropriate amount of the extracellular polysaccharide freeze-dried powder of Weissella cibaria obtained in Example 2 was dissolved in an ethanol solution, and after steps such as ultrasonic cleaning, sampling, infrared drying, sputtering with gold (10 nm), and coating, it was fixed and observed under an electron microscope. Under an accelerating voltage of 3.0 kV, the surface microtopography of the extracellular polysaccharide freeze-dried powder was observed at magnification multiples of 1000, 2000, 5000, and 10000 times. The scanning electron microscopy results are as Figure 2 shown.

[0060] As can be seen from Figure 2It can be seen that the extracellular polysaccharide of Weissella cibaria X-5 has a regular porous structure.

[0061] 2. Ultraviolet spectrum scanning

[0062] Accurately weigh an appropriate amount of the freeze-dried powder of the extracellular polysaccharide of Weissella cibaria obtained in Example 2 and dissolve it in ultrapure water to prepare a sample with a concentration of 500 μg / ml. Then, use a spectrophotometer to scan the characteristic absorption peaks in the wavelength range of 200-800 nm to observe the ultraviolet absorption of the extracellular polysaccharide from Weissella cibaria at different wavelengths. The results are as Figure 3 shown.

[0063] From Figure 3 it can be seen that the ultraviolet spectrum scanning results show that the ultraviolet absorption spectral curve of the extracellular polysaccharide of Weissella cibaria X-5 is smooth and flat after 280 nm, and no obvious absorption peaks are found, indicating that there are no proteins and nucleic acids in the extracellular polysaccharide sample of Weissella cibaria X-5.

[0064] 3. Infrared spectrum scanning

[0065] Accurately weigh an appropriate amount of the freeze-dried powder of the extracellular polysaccharide of Weissella cibaria obtained in Example 2, mix it with potassium bromide in a certain proportion, compress the mixed particles by the conventional potassium bromide (KBr) tablet pressing method, and use a Fourier transform infrared spectrometer to measure the absorption spectrum of the sample in the range of 500 cm -1 -4500 cm -1 The results are as Figure 4 shown.

[0066] From Figure 4 it can be seen that the spectrum in the wavelength range of 500 cm -1 -4500 cm -1 reveals the vibrations of functional groups in the extracellular polysaccharide of Weissella cibaria X-5. The broad and strong peak at 3421 cm -1 is the characteristic peak of the stretching vibration of the hydroxyl O-H in the carbohydrate component, indicating that the extracellular polysaccharide of Weissella cibaria X-5 is a carbohydrate substance.

[0067] The sharp peak at the absorption peak at 2928 cm -1 is the stretching vibration of the C-H single bond in the sugar ring, and the sharp peak at the absorption peak at 2077 cm -1 is the stretching vibration of the C=O double bond. The sharp peak at the absorption peak at 1640 cm -1 is the stretching vibration of the carboxyl group (COO-). The absorption peak between 1419 cm -1 and 1350 cm -1 is attributed to the bending vibration of the CH bond. The fingerprint region of polysaccharides is generally considered to be between 1000 cm -1 and 1200 cm -1The absorption peaks between indicate that the polysaccharide has broad absorption peaks at 1155 and 1205 cm -1 which are caused by the stretching vibrations of the C-OH linkage and the C-O-C glycosidic bond of the sugar ring, indicating that the polysaccharide contains pyranose ring sugar residues. The peak at 916 cm -1 is caused by the asymmetric ring stretching vibration of the pyranose ring, also proving the existence of the pyranose ring sugar in the polysaccharide. The absence of a peak at 890 cm -1 confirms the absence of the β-configuration of the glycosidic bond, while the peak at 846 cm -1 is caused by the stretching vibration of the α-anomeric carbon, indicating the α-configuration of the glycosidic bond in the polysaccharide. Therefore, the FT-IR spectrum shows that the extracellular polysaccharide of Weissella cibaria X-5 has pyranose ring sugar residues and α-type glycosidic bonds.

[0068] 4. HPLC Analysis of Monosaccharides and Uronic Acids in the Extracellular Polysaccharide of Weissella cibaria X-5

[0069] Different monosaccharide components were separated by a chromatographic column, and the monosaccharide content in the monosaccharide components of different samples was quantitatively analyzed by the external standard method. Weigh appropriate amounts of reference substances such as mannose, glucuronic acid, glucose, xylose, arabinose, and galactose, and dissolve and dilute each with water to a concentration of 50 μg / ml to form a mixed reference solution. Add 250 μl of the mixed reference solution, 250 μl of 0.6 mol / l NaOH solution, and 500 μl of 0.4 mol / l PMP-methanol solution to a 5 ml EP tube, mix well to form a mixed solution, place the mixed solution in an environment at 70 °C for 1 h, then in an ice-water bath for 10 minutes, add 500 μl (0.3 mol / l) HCl to the above mixed solution for neutralization, then add 1 ml of chloroform, vortex for 1 minute, centrifuge at 3000 r for 10 minutes, take the supernatant, and extract 3 times. Analyze the supernatant by HPLC.

[0070] Accurately weigh about 5 mg of the freeze-dried powder of the extracellular polysaccharide of Weissella cibaria obtained in Example 2 into a 10 ml ampoule, add 5.0 mL of TFA (2 mol / l), seal the tube, and hydrolyze with acid at 120 °C for 4 h. Take out and add methanol to blow-dry the TFA by nitrogen, and re-dissolve with 5.0 ml of water. Take 250 μl of the sample solution into a 5 mL EP tube. The following steps are the same as above.

[0071] The chromatographic conditions for HPLC analysis are as follows: The instrument is Shimadzu LC-20AD, the chromatographic column is Xtimate C18 4.6*200 mm 5 μm, the column temperature is 30 °C, the flow rate is 1.0 ml / min, the detection wavelength is 250 nm, the injection volume is 20 μl, and the mobile phase is 0.05 M potassium dihydrogen phosphate solution (adjust the PH to 6.70 with sodium hydroxide solution)-acetonitrile.

[0072] The detection results are as Figure 5 shown.

[0073] It can be seen from Figure 5 that the high performance liquid chromatography analysis results show that the extracellular polysaccharide of Weissella cibaria X-5 is dextran.

[0074] Example 4: Animal experiment to verify the efficacy of extracellular polysaccharide of W. cibaria X-5 in improving chronic obstructive pulmonary disease

[0075] 1. Experimental animals and breeding environment:

[0076] Thirty-two SPF-grade C57BL / 6J mice (body weight: 20 ± 2 g; animal quality certificate number: 110324221101674161) were purchased from Beijing Speifo Experimental Animal Co., Ltd. (SCK[Beijing]2019-0010). The mice were allowed to adapt to the environment for 7 days after purchase.

[0077] Breeding environment: The mice were bred in an IVC-II type animal breeding cage. The laboratory room temperature was maintained at about 25 degrees, the humidity was maintained at about 50%, the ventilation frequency was maintained at 12 times / hour, the noise was lower than 60 db, and a regular day-night cycle was maintained. The mice were allowed to freely eat sterilized water and feed; the purification operation system was regularly checked to keep the environment quiet.

[0078] 2. Construction of the mouse chronic obstructive pulmonary disease model:

[0079] The method for constructing the mouse chronic obstructive pulmonary disease model was as follows: A mouse model of chronic obstructive pulmonary disease was prepared by continuous cigarette smoke exposure combined with repeated bacterial infection. Specifically, 20 μl of Klebsiella pneumoniae liquid (bacterial liquid concentration: 5×10 6 CFU / ml) was instilled into the nasal cavity of the mice once every 7 days; at the same time, cigarette smoke inhalation was combined. Each time, the smoke concentration in the smoking box was reached (3000 ± 500) ppm, 40 min / time, twice a day (once in the morning and once in the evening), for a total of 8 weeks.

[0080] 3. Experimental grouping and treatment:

[0081] Thirty-two SPF-grade C57BL / 6J mice were randomly divided into a blank control group (Control group), a model group (Model group), an extracellular polysaccharide treatment group, and an aminophylline treatment group (i.e., the positive control group), with 8 mice in each group. The blank control group was not treated. Mice in the model group (Model group), the extracellular polysaccharide treatment group, and the aminophylline treatment group were modeled according to the above method for constructing a mouse model of chronic obstructive pulmonary disease, and were normally raised after modeling. Mice in the blank control group and the model group were orally administered 0.9% sodium chloride solution (dosage: 0.2 ml / day / animal) from the 9th week, for 8 consecutive weeks. The extracellular polysaccharide treatment group and the aminophylline treatment group were given drug interventions from the 9th week. Among them, the extracellular polysaccharide treatment group was given an extracellular polysaccharide solution (100 mg / kg / d, 0.5 mL / 100 g, qd) by gavage, and the aminophylline treatment group was given an aminophylline solution (50 mg / kg / d, 0.5 mL / 100 g, qd) by gavage. The drug intervention lasted for 8 weeks, and the experiment ended. Samples were taken after the gavage ended. The drug dose was calculated using the equivalent dose coefficient conversion formula. The formula is: D 小鼠 = D 人 ×(HI 小鼠 / HI 人 )×(W 小鼠 / W 人 ) 2 / 3 , where D: dose, W: body weight, HI: body shape coefficient.

[0082] 4. Effects of extracellular polysaccharide of W. cibaria X-5 on the growth of mice:

[0083] During the modeling period of the mouse model of chronic obstructive pulmonary disease and the drug intervention period, the feeding, mental state, and activity responses of mice in the Control group, Model group, extracellular polysaccharide treatment group, and aminophylline treatment group were closely observed until the end of the experiment.

[0084] The results showed that during the modeling period from the 1st week to the 8th week, mice in the Control group were in good health, with bright eyes, normal mental state, good fur gloss, normal diet, activities, urination, and defecation. During the experiment, the body weight of the mice remained at a normal level or increased slightly, and no death occurred. Starting from the 3rd week, mice in the model group showed listlessness, curled up and less active, and yellowish fur; after the 6th week, the fur became dull and dry, and some showed accelerated breathing and nasal secretions; at the 8th week, the modeled mice had poor spirits, dull eyes, more hair loss, audible gurgling sounds in the throat, and purple and dark nose and lips.

[0085] After the successful preparation of the model, drug intervention was carried out. The results showed that during the drug intervention period from the 9th week to the 16th week, there were no obvious changes in mice in the Control group, and the symptoms of mice in the Model group were not relieved, and some mice were emaciated. Starting from the 11th week, the mental states of mice in the extracellular polysaccharide treatment group and the aminophylline treatment group were both improved, and the wheezing was reduced.

[0086] 5. Effects of extracellular polysaccharide of Weissella cibaria X-5 on the body weight of mice

[0087] Record the initial body weights of the Control group and the Model group, as well as the body weight changes at the 4th and 8th weeks after the start of modeling. The results are as Figure 6 shown

[0088] Starting from the 9th week, the extracellular polysaccharide treatment group and the aminophylline group were given the corresponding drugs for intervention for 8 weeks. Record the body weight changes of the mice in the Control group, Model group, extracellular polysaccharide group, and aminophylline group at the 9th, 12th, and 16th weeks. The results are as Figure 7 shown

[0089] As can be seen from Figure 6 and 7 , the body weights of the mice in each group increased with time. Starting from the fourth week, the body weights of the mice in the Model group were significantly lower than those in the Control group (P<0.05). After four weeks and eight weeks of drug treatment, compared with the mice in the Model group, the body weights of the mice in the extracellular polysaccharide group and the aminophylline group increased (P<0.05), and the increase in body weight was significantly restored. This shows that the extracellular polysaccharide of Weissella cibaria X-5 has a significant improvement effect on the body weight of mice with chronic obstructive pulmonary disease.

[0090] 6. Improvement of pulmonary function in mice with chronic obstructive pulmonary disease by extracellular polysaccharide of Weissella cibaria X-5

[0091] After successful modeling, the extracellular polysaccharide treatment group and the aminophylline group were given the corresponding drugs for intervention for 8 weeks. After the drug treatment, measure the changes in pulmonary function indexes EF50 (Explratory Flow, 50% tidal volume expiratory flow), TV (Tldal Volume, tidal volume), and MV (Mlnute Ventllatlon Volume, maximum minute ventilation volume). The results are as Figure 8 shown

[0092] As can be seen from Figure 8 , compared with the Control group, EF50, TV, and MV in the Model group were still significantly decreased (P<0.01); compared with the Model group, EF50 and MV in the extracellular polysaccharide group increased significantly (P<0.05), and TV increased significantly (P<0.05); compared with the Model group, EF50, MV, and TV in the aminophylline group all increased but not significantly. This shows that the extracellular polysaccharide of Weissella cibaria X-5 has a significant improvement effect on various indexes of mouse pulmonary function.

[0093] 7. Study on the Effect of Extracellular Polysaccharide of Weissella cibaria X-5 on Lung Tissue Injury in Mice with Chronic Obstructive Pulmonary Disease:

[0094] At the end of the experiment (after successful modeling, 8 weeks of drug intervention, and the end of the experiment), the mice were sacrificed and their lung tissues were collected. HE staining was performed respectively, and the staining results are as Figure 9 shown.

[0095] It can be Figure 9 seen that compared with the Control group, the alveolar walls of the lung tissues of the Model group mice were significantly thickened, and there were severe fractures and fusions. Obvious inflammatory exudation was visible in the alveolar cavity and the alveolitis score was significantly increased (P < 0.01). The extracellular polysaccharide group and the aminophylline group treatments could effectively improve the above symptoms (P < 0.01).

[0096] The above results indicate that the extracellular polysaccharide of Weissella cibaria X-5 can significantly improve various problems such as thickening, fracture, fusion, and inflammatory exudation of the alveolar walls in the lung tissues of mice with chronic obstructive pulmonary disease, and at the same time reduce the alveolitis score. This shows that the extracellular polysaccharide of Weissella cibaria X-5 can be used to relieve and treat chronic obstructive pulmonary disease.

[0097] Example 5: Verification of the Anti-inflammatory Effect of Extracellular Polysaccharide of Weissella cibaria X-5 in the Inflammatory Response of Chronic Obstructive Pulmonary Disease

[0098] 1. Experimental Cells

[0099] Cell line: Mouse alveolar macrophage (MHS) cell strain, purchased from Wuhan Punosai Life Science Co., Ltd. The essence of COPD is chronic inflammatory response in the lungs, and the main pathological manifestations are chronic bronchitis and emphysema. Macrophages are differentiated from monocytes and play phagocytic, immune, and secretory roles in the lungs. Under normal circumstances, MHS has an anti-inflammatory effect, and when tissue is damaged, MHS can play a pro-inflammatory role to maintain tissue homeostasis.

[0100] 2. Effect of Extracellular Polysaccharide of Weissella cibaria X-5 on the Cell Viability of MHS

[0101] The experimental groups included: experimental group (EPS X-5 group) and control group (Control group). In the experimental group (EPS X-5 group), extracellular polysaccharide of Weissella cibaria X-5 was added to the cells. In the EPS X-5 group, different concentrations of extracellular polysaccharide of Weissella cibaria were added according to different functional tests. No extracellular polysaccharide was added to the Control group.

[0102] The MHS cells in good growth state were inoculated into 96-well plates at a density of (100 μL / well), incubated in an incubator at 37 °C and 5% CO2 for about 24 h. When the cells grew to cover about 80% of the bottom of the culture dish, different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) of extracellular polysaccharide solution of Weissella cibaria X-5 were added to the experimental groups at 200 μL each, and 200 μL of PBS was added to the Control group, and induced for 48 h; CCK8 reagent (10 μL / well) was added and allowed to act for 2 h, and the absorbance at a wavelength of 450 nm was detected with an enzyme-linked immunosorbent assay reader. The experimental results are as Figure 10 shown.

[0103] It can be Figure 10 seen that compared with the Control group, the extracellular polysaccharide of Weissella cibaria X-5 at a concentration of 1.0 mg / ml could significantly reduce the viability of MHS cells (P < 0.05); the extracellular polysaccharide of Weissella cibaria X-5 at concentrations of 0.2, 0.4, 0.6, and 0.8 mg / ml had no obvious effect on the viability of MHS cells.

[0104] 3. Identification of the degree of chronic inflammatory response of mouse alveolar macrophage (MHS) cell line and the improvement effect of extracellular polysaccharide on inflammatory response

[0105] The mouse alveolar macrophages (MHS) were grouped, including: LPS group, high-concentration extracellular polysaccharide group, low-concentration extracellular polysaccharide group, COPD group, and healthy control group.

[0106] Among them, the LPS group was added with a medium containing LPS at a concentration of 100 ng / ml and intervened for 24 h; the high-concentration extracellular polysaccharide group was added with a medium containing LPS at a concentration of 100 ng / ml and extracellular polysaccharide at a concentration of 800 μg / ml; the low-concentration extracellular polysaccharide group was added with a medium containing LPS at a concentration of 100 ng / ml and extracellular polysaccharide at a concentration of 200 μg / ml, and intervened for 24 h. The COPD group and the healthy control group: no drugs were used, and they were cultured in the original environment for 24 h. After the intervention of each group, the cells and supernatants were collected for PCR detection of the indexes of inflammatory factors (IL-6, TNF-α, IL-1β). The detection results are as Figure 11 shown.

[0107] It can be Figure 11It can be seen that compared with the healthy control group, the contents of inflammatory factors (IL-6, TNF-α, IL-1β) in the COPD group were significantly increased (P < 0.05); the contents of inflammatory factors in the LPS group were higher than those in the COPD group (P < 0.05); compared with the LPS group, the contents of inflammatory factors in the exopolysaccharide group were significantly down-regulated (P < 0.05), and the down-regulation amplitude in the high-concentration exopolysaccharide group was greater (P < 0.05). This shows that the exopolysaccharide of Weissella cibaria X-5 can significantly improve the inflammatory response of MHS cells.

[0108] In summary, the exopolysaccharide of Weissella cibaria X-5 can significantly improve various problems such as thickening, rupture, fusion, and inflammatory exudation of the alveolar wall in the lung tissue of mice with chronic obstructive pulmonary disease, and reduce the alveolitis score; when the exopolysaccharide of Weissella cibaria X-5 is at a concentration of 0.8 mg / ml, it has no damage to cells and can significantly improve the inflammatory level of macrophages in the inflammatory response of chronic obstructive pulmonary disease. Therefore, the exopolysaccharide of Weissella cibaria X-5 can be used for the prevention, alleviation, and treatment of chronic obstructive pulmonary disease.

[0109] The functions of the above embodiments are to illustrate the substantial content of the present invention, but do not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. Weissella esculenta ( Weissella cibaria ), characterized in that The nucleic acid sequence of Weissella cibaria contains the sequence shown in Sequence 1; the Weissella cibaria is Weissella cibaria X-5, and the deposit number of Weissella cibaria X-5 is CCTCC NO: M 20232333.

2. An extracellular polysaccharide secreted by the Weissella cibaria according to claim 1; the preparation method of the extracellular polysaccharide is: inoculating the Weissella cibaria into a medium for fermentation culture to obtain a culture solution, removing the bacteria from the culture solution, and removing proteins from the culture solution to obtain a supernatant; performing alcohol precipitation on the supernatant, collecting the precipitate, and subjecting the precipitate to dialysis for impurity removal and freeze-drying to obtain the extracellular polysaccharide; the medium is MRS medium or mMRS medium; the fermentation culture temperature is 30°C to 37°C; the dialysis cut-off molecular weight is 8000 to 14000 Da.

3. The exopolysaccharide according to claim 2, characterized in that, The alcohol precipitation treatment is to add absolute ethanol to the supernatant, mix evenly, and stand at 4°C for 12 h to 18 h; the specific operation of protein removal is: adding trichloroacetic acid to the culture solution after removing the bacteria, mixing evenly, standing at 4°C for 12 h to 18 h, and centrifuging to remove the precipitate.

4. Use of the Weissella cibaria according to claim 1 or the extracellular polysaccharide according to claim 2 or 3 in the preparation of a drug for preventing, alleviating or / and treating obstructive pulmonary disease.

5. A drug for preventing, alleviating or / and treating obstructive pulmonary disease, characterized in that, Containing an active ingredient, the active ingredient is the Weissella cibaria according to claim 1 or a culture containing Weissella cibaria or the extracellular polysaccharide according to claim 2 or 3.

6. The drug according to claim 5, characterized in that, The drug is administered by oral administration, gavage administration or parenteral administration.

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