Weissella sinus ZMC030 and application of exopolysaccharide thereof in preventing and treating inflammatory bowel disease

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of Weissella cibaria ZMC030 and exopolysaccharides thereof in prevention and treatment of colitis. The preservation number of the Weissella sinus ZMC030 is CCTCC (China Center for Type Culture Collection) NO: M 20241261, and the Weissella sinus ZMC030 is preserved in the China Center for Type Culture Collection. The Weissella cibaria ZMC030 can secrete exopolysaccharide, and the secreted exopolysaccharide can relieve intestinal injury, reduce apoptosis of intestinal epithelial cells, effectively protect intestinal mucosa barriers and reduce the inflammation level; moreover, intestinal microorganisms and metabolites thereof can be regulated and controlled, the level of pro-inflammatory microorganisms is reduced, the level of anti-inflammatory microorganisms and short-chain fatty acid is increased, and ulcerative colitis symptoms of mice are finally relieved; in addition, the exopolysaccharide has no influence on the weight and the growth state of the mouse, has no damage to important visceral organs, and is high in safety. Therefore, the Weissella cibaria ZMC030 and the exopolysaccharide secreted by the Weissella cibaria ZMC030 can be used for preparing the medicine for relieving or / and treating the colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of Weissella sinensis ZMC030 and its extracellular polysaccharide in preventing and treating inflammatory bowel disease. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific, recurrent inflammatory bowel disease that mainly affects the digestive system. UC patients have a high risk of developing colorectal cancer. The World Health Organization has classified UC as one of the clinically intractable diseases. In recent years, with urbanization and the modernization of people's daily lives, the incidence of UC has increased year by year, and it has become a global public health challenge. UC is difficult to cure. Currently, commonly used clinical drugs mainly include salicylates, glucocorticoids, immunosuppressants, biological agents, and antibiotics. However, conventional drug treatments have disadvantages such as obvious side effects, poor patient tolerance, and easy recurrence. Therefore, seeking drugs with fewer adverse reactions and more effectiveness is of great significance to the clinical use and basic research of UC.

[0003] Microbial preparations have therapeutic potential for various diseases including UC, but live bacterial preparations have problems such as poor stability, low gastrointestinal survival rate, and possible safety hazards. Postbiotics are inanimate microorganisms, cell components, or metabolites secreted by microorganisms that are beneficial to host health, such as short-chain fatty acids, membrane proteins, and exopolysaccharides (EPS). They can play a regulatory role similar to probiotics, and postbiotics have advantages such as good stability and safety, easy storage and transportation, and may be a safer alternative to probiotics. Postbiotics can regulate intestinal flora, enhance intestinal epithelial barrier function, regulate local and systemic immunity, etc., and their application in various diseases including UC is increasingly valued.

[0004] Weissella cibaria is a type of lactic acid bacteria, which is widely found in saliva, breast milk, human gastrointestinal tract and traditional fermented foods. W. cibaria is a lactic acid bacteria that produces high EPS. It is currently mainly used as a thickener, stabilizer and gelling agent in food processing, cosmetics and pharmaceutical processing industries. In recent years, people have also begun to pay attention to the potential probiotic properties of W. cibaria EPS, including antibacterial, anti-tumor, antioxidant and obesity relief. However, the research on the probiotic function of EPS of this bacterium started late, and its application in diseases still needs to be further explored. Whether the EPS of W. cibaria can relieve UC and its related mechanisms have not been reported yet. Summary of the invention

[0005] The purpose of the present invention is to provide a use of Weissella sinensis ZMC030 and its exopolysaccharide in preventing and treating inflammatory bowel disease.

[0006] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0007] A first aspect of the present invention provides a Weissella sinensis, wherein the nucleic acid sequence of the Weissella sinensis contains a sequence shown in Sequence 1 (SEQ ID NO.1).

[0008] According to the above-mentioned Weissella cibaria, preferably, the Weissella cibaria is Weissella cibaria ZMC030, Weissella cibaria ZMC030 (Weissella cibaria ZMC030), the preservation number of the Weissella cibaria is CCTCCNO:M 20241261, the preservation institution is China Center for Type Culture Collection (CCTCC), and the address of the preservation institution is China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is June 17, 2024.

[0009] The screening and identification process of Weissella sinusoids ZMC030 is as follows: the feces of healthy infants and young children are resuspended in sterile physiological saline, shaken and mixed, and then allowed to stand to precipitate granular debris, the suspension is added to MRS liquid culture medium, and cultured at 37°C for 48 hours; the bacterial solution is gradient diluted and 10 -5 The dilution was spread on MRS solid medium and cultured at 37°C until a single colony was clearly grown. Primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify the 16S rDNA sequence of the single colony, and the amplified sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results of 16S rDNA were input into the BLAST database of NCBI to analyze the 16SrDNA sequence of the strain. Finally, the isolated strain was identified as Weissella cibaria based on homologous sequence analysis and named Weissella cibaria ZMC030. The strain was deposited in the China Center for Type Culture Collection, and the strain collection number was CCTCC M 20241261. The 16S rDNA sequence of Weissella cibaria ZMC030 is shown in Sequence 1 (SEQ ID NO.1).

[0010] The nucleotide sequence of sequence 1 is as follows:

[0011] gctttgggtgttacaaactctcatggtgtgacgggcggtgtgtacaagacccgggaacgtattcaccgcggcgtgctgatccgcgattactagcgattc

[0012] cgacttcatgtaggcgagttgcagcctacaatccgaactgagacgtactttaagagattagctcaccctcgcgggttggcaactcgttgtatacgccattgtagc

[0013] acgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttgtcaccggcagtctcactagagtgcccaactaaatgctgg

[0014] caactagtaataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtcaccttgtccccgaaggg

[0015] aacgctccatctctggagttgtcaagggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggtccccgtca

[0016] attcctttgagtttcaaccttgcggtcgtactccccaggcggagtgcttaatgcgttagctgcggcacttaagggcggaaaccctcaaacacctagcactcatc

[0017] gtttacggtgtggactaccagggtatctaatcctgtttgctacccacactttcgagcctcaacgtcagttacagtccagaaaagccgccttcgccactggtgttct

[0018] tccatatatctacgcatttcaccgctacacatggagttccactttcctctactgcactcaagtcatccagtttccaaagcaattcctcagtttgagctgagggctttc

[0019] acttcagacttaaataaccgtctgcgctcgctttacgcccaataaatccggataacgcttggaacatacgtattaccgcggctgctggcccgtattttagcccgtt

[0020] cctttctggtaagataccgtcacacattgaacagttactctcaatgtcattcttctcttacaacagtgttttacgagccgaaacccttcatcacacacgcggcgttgc

[0021] tccatcaggctttcgcccattgtggaagattccctactgctgcctcccgtaggagtatgggccgtgtctcagtcccattgtggccgatcagtctctcaactcggct

[0022] atgcatcatcgtcttggtgagccattacctcaccaactaactaatgcaccgcgggaccatctcttagtgatagcagaaccatcttttaagtagcaaccatgcggtt

[0023] gctattgttatacggtattagcatctgtttccaaatgttatcccctgctaagaggtaggtttcccacgtgttatcacccgttcgccactctttgcaatgtccatcgtc

[0024] atatctgagcaagctcttcaaatcagtgaaccacacaagcgtt.

[0025] The second aspect of the present invention provides an exopolysaccharide secreted by the Weissella sinensis described in the first aspect.

[0026] According to the above-mentioned extracellular polysaccharide, preferably, the extracellular polysaccharide is obtained by fermenting and culturing the Weissella sinensis ZMC030 to obtain a fermentation broth, and separating and purifying the fermentation broth.

[0027] According to the above-mentioned extracellular polysaccharide, preferably, the preparation method of the extracellular polysaccharide is: inoculate the Weissella vegetabilis ZMC030 described in the first aspect into a culture medium for fermentation culture to obtain a culture solution, remove the bacteria and remove the protein from the culture solution to obtain a supernatant; perform alcohol precipitation on the supernatant, collect the precipitate, and perform dialysis to remove impurities and freeze-dry the precipitate to obtain the extracellular polysaccharide. The extracellular polysaccharide is further purified by column chromatography through a DEAE-cellulose 52 chromatography column, and the fraction containing the polysaccharide is dialyzed to remove impurities and freeze-dried to obtain a purified extracellular polysaccharide.

[0028] According to the above-mentioned extracellular polysaccharide, preferably, the culture medium is MRS culture medium or mMRS culture medium (modified MRS culture medium).

[0029] According to the above-mentioned extracellular polysaccharide, preferably, the composition of the mMRS medium is: diammonium citrate 5g / L, sucrose 100g / L, peptone 10g / L, magnesium sulfate 0.2g / L, Tween 80 1ml / L, dipotassium hydrogen phosphate 2g / L, beef extract 10g / L, yeast extract 5g / L, anhydrous sodium acetate 5g / L, manganese sulfate 0.05g / L, and the balance is water; the fermentation culture temperature is 30°C to 37°C. More preferably, the culture temperature is 30°C.

[0030] According to the above-mentioned extracellular polysaccharide, preferably, the dialysis is performed in distilled water, and the dialysis molecular weight cut-off is 8000-14000Da.

[0031] According to the above-mentioned extracellular polysaccharide, preferably, the alcohol precipitation treatment is to add pre-cooled anhydrous ethanol to the supernatant, mix well and then stand at 4°C for 8h to 12h.

[0032] According to the above-mentioned extracellular polysaccharide, preferably, the specific operation of protein removal is: adding trichloroacetic acid to the culture solution after removing the bacteria, mixing evenly to obtain a mixed solution, placing the mixed solution at 4°C for 8h to 12h, centrifuging, and removing the precipitate. More preferably, the amount of trichloroacetic acid used is: the mass volume percentage concentration of trichloroacetic acid in the mixed solution is 4%.

[0033] According to the above-mentioned extracellular polysaccharide, preferably, the specific operation of removing the bacteria is: heating the fermentation liquid at 100°C for 15 minutes, cooling to room temperature, and centrifuging to remove the bacteria. More preferably, the centrifugation condition is: centrifugation at 4°C and 12000g for 30 minutes.

[0034] According to the above-mentioned extracellular polysaccharide, preferably, the crude extracellular polysaccharide freeze-dried powder can be further purified by column chromatography using a DEAE-cellulose 52 chromatography column, and deionized water or sodium chloride (0.1M, 0.3M and 0.5M) solution is used for gradient elution during column chromatography purification, and the eluate containing polysaccharide is collected; the eluate containing polysaccharide is dialyzed with deionized water and freeze-dried to obtain a purified extracellular polysaccharide. More preferably, during column chromatography purification, sodium chloride 0.1M is used for elution, the eluate containing polysaccharide is collected, and the collected

[0035] The third aspect of the present invention provides the use of the Weissella sinusoids or the culture of Weissella sinusoids described in the first aspect in any of the following:

[0036] (A1) Use in the preparation of products for preventing, alleviating or / and treating colitis;

[0037] (A2) Use in the preparation of a product for reducing the area of ​​intestinal epithelial ulcers;

[0038] (A3) Application in the preparation of a product for alleviating intestinal epithelial inflammatory cell infiltration;

[0039] (A4) Application in the preparation of a product for reducing apoptosis of intestinal epithelial cells;

[0040] (A5) Application in the preparation of products for protecting the intestinal mucosal barrier;

[0041] (A6) Application in products for regulating intestinal flora.

[0042] According to the above application, preferably, the inflammatory bowel disease is colitis; more preferably, the colitis is ulcerative colitis.

[0043] According to the above application, preferably, the product for preventing, alleviating or / and treating colitis is a medicine, a food additive, a functional food, a feed additive or a feed or a microbial agent.

[0044] The fourth aspect of the present invention provides the use of the extracellular polysaccharide described in the second aspect in any of the following:

[0045] (A1) Application in the preparation of products for preventing, alleviating or / and treating inflammatory bowel disease;

[0046] (A2) Use in the preparation of a product for reducing the area of ​​intestinal epithelial ulcers;

[0047] (A3) Application in the preparation of a product for alleviating intestinal epithelial inflammatory cell infiltration;

[0048] (A4) Application in the preparation of a product for reducing apoptosis of intestinal epithelial cells;

[0049] (A5) Application in the preparation of products for protecting the intestinal mucosal barrier;

[0050] (A6) Application in products for regulating intestinal flora.

[0051] According to the above application, preferably, the inflammatory bowel disease is colitis; more preferably, the colitis is ulcerative colitis.

[0052] According to the above application, preferably, the product for preventing, alleviating or / and treating colitis is a medicine, a food additive, a functional food, a feed additive or a feed.

[0053] The fifth aspect of the present invention provides a medicine comprising an active ingredient, wherein the active ingredient is the Weissella sinensis or a culture containing Weissella sinensis described in the first aspect above, or the exopolysaccharide or a fermentation broth containing exopolysaccharide described in the second aspect above; the medicine is used to prevent, alleviate and / or treat colitis or to reduce the area of ​​intestinal epithelial ulcers or to improve intestinal mucosal barrier disorders or to regulate intestinal flora.

[0054] According to the above-mentioned medicine, preferably, when the active ingredient is the Weissella sinensis or a culture containing Weissella sinensis as described in the first aspect above, the Weissella sinensis may be a living cell or a dead cell.

[0055] According to the above-mentioned drug, preferably, the drug is administered orally or by gavage.

[0056] According to the above-mentioned medicine, preferably, the medicine further comprises pharmaceutically acceptable excipients. More preferably, the excipients include excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants, lubricants or flavoring agents.

[0057] The sixth aspect of the present invention provides a functional food, which contains the Weissella sinensis or a culture containing Weissella sinensis described in the first aspect or the exopolysaccharide or a fermentation broth containing the exopolysaccharide described in the second aspect.

[0058] According to the above functional food, preferably, the functional food can reduce the area of ​​intestinal epithelial ulcers or alleviate intestinal epithelial inflammatory cell infiltration or reduce intestinal epithelial cell apoptosis or protect the intestinal mucosal barrier or regulate intestinal flora.

[0059] The seventh aspect of the present invention provides a food additive, which contains the Weissella sinensis or a culture containing Weissella sinensis described in the first aspect or the exopolysaccharide or a fermentation broth containing the exopolysaccharide described in the second aspect.

[0060] According to the above-mentioned food additives, preferably, the food additives can reduce the area of ​​intestinal epithelial ulcers or alleviate intestinal epithelial inflammatory cell infiltration or reduce intestinal epithelial cell apoptosis or protect the intestinal mucosal barrier or regulate the intestinal flora.

[0061] The eighth aspect of the present invention provides a feed additive, which contains the Weissella sinensis or a culture containing Weissella sinensis described in the first aspect or the exopolysaccharide or a fermentation broth containing the exopolysaccharide described in the second aspect.

[0062] According to the above-mentioned feed additive, preferably, the feed additive can reduce the area of ​​intestinal epithelial ulcers or alleviate intestinal epithelial inflammatory cell infiltration or reduce intestinal epithelial cell apoptosis or protect the intestinal mucosal barrier or regulate the intestinal flora.

[0063] According to the above feed additive, preferably, the feed additive is a feed additive for pets, poultry and livestock.

[0064] A ninth aspect of the present invention provides a feed, wherein the feed contains the Weissella sinensis or a culture containing Weissella sinensis described in the first aspect or the exopolysaccharide or a fermentation broth containing the exopolysaccharide described in the second aspect.

[0065] According to the above feed, preferably, the feed can reduce the area of ​​intestinal epithelial ulcers or alleviate intestinal epithelial inflammatory cell infiltration or reduce intestinal epithelial cell apoptosis or protect the intestinal mucosal barrier or regulate intestinal flora.

[0066] According to the above-mentioned feed, preferably, the feed is feed for feeding pets, poultry and livestock.

[0067] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0068] (1) The Weissella cibaria ZMC030 (W.cibariaZMC030) screened by the present invention and the exopolysaccharides secreted by it can reduce the area of ​​intestinal epithelial ulcers, reduce intestinal epithelial cell apoptosis, alleviate intestinal epithelial inflammatory cell infiltration, protect the intestinal mucosal barrier, and can effectively play a role in preventing, alleviating or / and treating colitis; and can also regulate intestinal microorganisms and their metabolites, increase the levels of intestinal anti-inflammatory microorganisms and short-chain fatty acids, and can play a role in alleviating the symptoms of colitis.

[0069] (2) The Weissella sinusoidalis ZMC030 of the present invention and the exopolysaccharide secreted by it have no effect on the weight and growth status of mice, no damage to the important organs of mice, and are highly safe.

[0070] (2) The Weissella sinusoids ZMC030 of the present invention is screened from the intestines of healthy infants, and its metabolite exopolysaccharide is also a natural component produced during the metabolism of lactic acid bacteria. From the perspective of clinical application, compared with other drugs for treating colitis, Weissella sinusoids ZMC030 and its exopolysaccharide are safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The results of the W.cibaria ZMC030 cultured on MRS and mMRS culture medium plates for the same time are compared; wherein A is an MRS plate and B is an mMRS plate;

[0072] Figure 2 This is a graph showing the detection result of the polysaccharide content in the eluate obtained by separation and purification of crude polysaccharide of W. cibaria ZMC030 by DEAE-cellulose 52 chromatography column;

[0073] Figure 3 The electron microscope scanning images of 030-EPS-2 polysaccharide; A is the surface microscopic morphology at 500X, and B is the surface microscopic morphology at 1000X;

[0074] Figure 4 This is the infrared spectrum scanning analysis result of 030-EPS-2 polysaccharide;

[0075] Figure 5 This is a graph showing the molecular weight test results of 030-EPS-2 polysaccharide;

[0076] Figure 6 This is the result of monosaccharide composition analysis of 030-EPS-2;

[0077] Figure 7 The results of the methylation analysis of 030-EPS-2 polysaccharide are shown in Figure 1, where A is the total ion current of methylation of 030-EPS-2 polysaccharide, and B is the percentage of sugar residues and the linkage mode of 030-EPS-2 polysaccharide;

[0078] Figure 8 The results of the nuclear magnetic resonance analysis of the polysaccharide structure of 030-EPS-2 polysaccharide; wherein A is the 1H NMR spectrum; B is the 13C NMR spectrum; C is the HSQC spectrum; D is the COSY spectrum; E is the HMBC spectrum; F is the NOESY spectrum; G is the structural formula of EPS-2;

[0079] Fig. 9 The effect of 030-EPS-2 polysaccharide on the body weight of mice with ulcerative colitis;

[0080] Fig.10 This is the test result of the effect of 030-EPS-2 polysaccharide on the colon length of mice;

[0081] Fig.11 The figure is the test result of the effect of 030-EPS-2 polysaccharide on mouse spleen, wherein the left figure is a food photo of mouse spleen, and the right figure is the test result of mouse spleen weight;

[0082] Fig.12 The results of the detection of the effect of 030-EPS-2 polysaccharide on colon injury are shown in Figure 1, wherein A is the HE staining result and B is the TUNEL staining result;

[0083] Fig.13 The effect of 030-EPS-2 polysaccharide on intestinal mucosal barrier; A is the result of PAS staining, B is the result of Alcian blue staining, C is the result of immunofluorescence staining for Claudin-1, and D is the result of immunofluorescence staining for Occludin;

[0084] Fig.14 The effect of 030-EPS-2 polysaccharide on inflammatory factors in colon tissue of colitis mice;

[0085] Fig.15 This is the test result of 030-EPS-2 polysaccharide regulating intestinal flora and short-chain fatty acids; among them, A is Bacteroidota, B is Proteobacteria, C is Muribaculaceae, D is Escherichia-Shigella, and E is propionic acid;

[0086] Fig.16 This is the test result of the effect of 030-EPS-2 on the body weight of mice;

[0087] Fig.17 The test results of AST, ALT, CREA-S and UREA are shown in the figure;

[0088] Fig.18 This is the HE staining result of mouse heart, liver, spleen, lung and kidney;

[0089] Fig.19 The results are a comparison of the effects of extracellular polysaccharides of Weissella sinusoids D-2 and Weissella sinusoids 030 in alleviating ulcerative colitis in mice; wherein, A is the statistical result of mouse weight, and B is the measurement result of mouse colon length. DETAILED DESCRIPTION

[0090] 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 in conjunction with specific embodiments.

[0091] The following detailed descriptions are all exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit 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, it indicates the presence of features, steps, operations, parts and / or combinations thereof.

[0092] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0093] Example 1: Acquisition and identification of W. cibaria ZMC030

[0094] Healthy infant feces were resuspended in sterile saline, shaken and mixed, and then allowed to settle granular debris, added to MRS liquid culture medium, and cultured at 37°C for 48h; the bacterial solution was diluted in gradients and 10 -5 The diluted solution was spread onto MRS solid medium and cultured at 37°C until obvious single colonies grew.

[0095] Primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify the 16S rDNA sequence of a single colony, and the amplified sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of W. cibaria ZMC030 obtained by sequencing is shown in Sequence 1.

[0096] The sequencing results of 16S rDNA were input into the BLAST database of NCBI to analyze the 16S rDNA sequence of the strain. Finally, the isolated strain was identified as Weissella cibaria based on homologous sequence analysis and named Weissella cibaria ZMC030. The strain was deposited in the China Center for Type Culture Collection with the strain collection number of CCTCC NO:M 20241261 and the preservation date of June 17, 2024.

[0097] Example 2: Acquisition of extracellular polysaccharides from W. cibaria ZMC030

[0098] 1. Optimization of fermentation medium for W. cibaria ZMC030

[0099] W.cibaria ZMC030 was streaked onto MRS solid culture plates and mMRS solid culture plates by streaking, and then the solid culture plates were cultured at 30°C for 24h-48h to observe the secretion of extracellular polysaccharides by W.cibaria ZMC030 on the MRS solid culture plates and mMRS solid culture plates. Figure 1 As shown in A and B.

[0100] Depend on Figure 1 As shown in A and B, compared with the single colony on MRS medium, W.cibaria ZMC030 showed obvious "slime" on the mMRS solid medium containing sucrose, indicating that W.cibaria ZMC030 can use sucrose to produce EPS, and the amount of polysaccharide secreted is significantly higher. Therefore, mMRS is preferred for fermentation of W.cibaria ZMC030.

[0101] 2. Extracellular polysaccharide acquisition

[0102] Place the W.cibaria ZMC030 cryopreservation tube in a 37°C water bath to thaw, dip 1-2 drops of bacterial solution with a sterile inoculation loop, inoculate by three-zone streak method, and let stand in a 37°C constant temperature incubator for 48 hours, and a single colony can be observed. Pick the obtained single colony with a micropipette tip and inoculate it into 5ml liquid MRS culture medium for culture. The culture condition is to stand at 37°C for 24 hours, and observe that the bacterial solution becomes turbid. Inoculate the bacterial solution into mMRS liquid culture medium at a 1% (v / v) inoculation amount, and let stand at 30°C for 48 hours to obtain a fermentation liquid. Boil the above fermentation liquid in a 100°C water bath for 15 minutes to achieve the purpose of inactivating bacteria and enzymes, and cool to room temperature. Centrifuge the boiled bacterial solution at 12000g and 4°C in a high-speed refrigerated centrifuge for 30 minutes, discard the bacterial body and other precipitates, and obtain the supernatant. Add 80% (m / v) trichloroacetic acid (TCA) to the supernatant to a final concentration of 4% (m / v) to remove protein. Place in a 4°C refrigerator for 12 hours and centrifuge at 4°C, 12000g for 30 minutes in a high-speed refrigerated centrifuge to collect the supernatant. Repeat this step 2 to 3 times to remove protein impurities as much as possible. Precool anhydrous ethanol at 4°C in advance, add three times the volume (v / v) of cold anhydrous ethanol to the supernatant after removing protein and other impurities, place in a 4°C refrigerator for 12 hours (the purpose is to precipitate extracellular polysaccharides with alcohol), and centrifuge at 4°C, 12000g for 30 minutes to obtain the precipitate, which is the crude extracellular polysaccharide. Dissolve the crude extracellular polysaccharide precipitate with pure water, and repeat the alcohol precipitation step 2 to 3 times. After the alcohol precipitation, the extracellular polysaccharide obtained by alcohol precipitation was redissolved, and then dialyzed in distilled water (dialysis molecular weight cutoff is 8000-14000Da) for 72 hours at 4°C to remove impurities, and the water was changed every 8 hours; the polysaccharide solution after dialysis was vacuum freeze-dried to obtain crude extracellular polysaccharide freeze-dried powder, and weighed. It was found by weighing that after W.cibaria ZMC030 was statically cultured at 30°C for 48 hours, the yield of crude extracellular polysaccharide in the fermentation broth was 24.2g / L, indicating that the extracellular polysaccharide yield of W.cibaria ZMC030 strain was high.

[0103] The crude extracellular polysaccharide lyophilized powder was prepared into a 10 mg / mL crude polysaccharide aqueous solution, then loaded onto a DEAE-cellulose 52 chromatography column (2.6 x 30 cm), eluted with deionized water and sodium chloride solution (0.1 M, 0.3 M and 0.5 M), and the corresponding eluate was collected (10 ml / tube). The sugar content in the eluate was detected by the phenol-sulfuric acid method. The experimental results are shown in Figure 2. Figure 2 shown.

[0104] Depend on Figure 2It can be seen that after the crude polysaccharide of W.cibaria ZMC030 was separated and purified by DEAE-cellulose 52 chromatography column, elution peaks appeared in deionized water and 0.1M sodium chloride eluent, respectively, and were named 030-EPS-1 and 030-EPS-2. 030-EPS-1 and 030-EPS-2 were dialyzed with deionized water (dialysis cutoff was 8000-14000Da), freeze-dried, and the yields of 030-EPS-1 and 030-EPS-2 were calculated. It was found by calculation that the yields of polysaccharides in 030-EPS-1 and 030-EPS-2 were 8.6% and 53.2%, respectively. In view of the fact that the yield of polysaccharides in the eluate of 030-EPS-1 was too low to meet the subsequent animal experiments, the polysaccharides in the eluate of 030-EPS-2 were selected for subsequent characterization analysis and functional identification.

[0105] Example 3: Identification and Characterization of Exopolysaccharide 030-EPS-2

[0106] 1. Scanning electron microscope observation of morphology

[0107] Take an appropriate amount of the 030-EPS-2 polysaccharide obtained in Example 2, dissolve it in ethanol, and fix it under an electron microscope for observation after ultrasonic cleaning, sampling, infrared drying, gold spraying (10nm), and gluing. The surface micromorphology of the lyophilized powder of extracellular polysaccharide was observed at 500 and 1000 times magnification at an accelerating voltage of 5.0 kV. The scanning electron microscopy results are shown in Figure 2. Figure 3 shown.

[0108] Depend on Figure 3 It can be seen that the surface of 030-EPS-2 polysaccharide is relatively smooth and has a loose porous network structure.

[0109] 2. Fourier transform infrared scanning

[0110] Accurately weigh an appropriate amount of the 030-EPS-2 polysaccharide obtained in Example 2 and mix it with potassium bromide in a certain proportion. Compress the mixed particles using a conventional potassium bromide (KBr) tableting method and then use a Fourier transform infrared spectrometer to measure the sample at 4000 cm -1 -500cm -1 The results are as follows Figure 4 shown.

[0111] Depend on Figure 4 It can be seen that the FT-IR spectrum analysis results of 030-EPS-2 show that 030-EPS-2 exhibits unique absorption peak characteristics of sugars. -1 The absorption bands / peaks at 2927.33 and 1353.38 cm-1 are attributed to the -OH stretching vibration peaks of polysaccharide molecules. -1The sharp peak at 1639.32cm is the CH stretching vibration of methyl or methylene in the sugar molecule. -1 The stretching vibration of C=O double bond is 1155.16cm -1 The peak at 950-1200 cm is the stretching vibration peak of COC in the sugar ring. -1 The peaks within the range are the fingerprints of polysaccharides, which can reflect the structural differences of polysaccharides. -1 The peak at 914.41 cm is the stretching vibration of the COH bond in the pyran ring. -1 、844.45cm -1 and 765.62cm -1 The peak at is caused by the α-glycosidic bond. The above results indicate that 030-EPS-2 may be an α-configuration pyranose.

[0112] 3. Determination of polysaccharide molecular weight by gel permeation chromatography (HPLC / GPC)

[0113] After 030-EPS-2 was fully dissolved in deionized water, it was filtered through a 0.22 μm filter membrane and subjected to ultrasonic treatment before sample analysis. The GPC system was an Agilent 1260 Infinity II GPC / SEC system, with PL aquagel-OH (7.5 × 50 mm, 8 μm) and PL aquagel-OH (7.5 × 300 mm, 8 μm) columns connected in series, equipped with a differential refractive index detector (1260 Infinity II). The test temperature was 45 °C, and the mobile phase was 0.1 M NaNO 3 , the flow rate is 1mL / min. The polysaccharide molecular weight results are as follows Figure 5 shown.

[0114] Depend on Figure 5 It can be seen that the HPLC / GPC analysis results show that the molecular weight of 030-EPS-2 polysaccharide is 8.45×10 5 Da.

[0115] 4. Monosaccharide composition analysis

[0116] Separate different monosaccharide components by chromatographic column, analyze monosaccharide composition, and quantitatively analyze monosaccharide content by external standard method. Take appropriate amount of mannose, arabinose, ribose, rhamnose, galacturonic acid, glucuronic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, xylose, fucose, galactose and other reference substances, dissolve and dilute with water, and mix the reference solution with a concentration of 50μg / mL. Pipette 250μl mixed reference solution into 5mL EP tube, add 250μL 0.6mol / LNaOH, 500μL of 0.4mol / l PMP-methanol, 70℃, 1h. Ice water bath for 10 minutes; 500μl (0.3mol / L) HCl neutralization, 1mL chloroform, vortex for 1min, centrifuge at 3000r / min for 10min, take supernatant, and extract 3 times. The supernatant is used for HPLC.

[0117] Accurately weigh about 5 mg of 030-EPS-2 polysaccharide sample into a 10 mL ampule, add 5 mL of TFA (2 mol / l), seal the tube, and acid hydrolyze at 120°C for 4 h. Take out and add methanol nitrogen to evaporate TFA, and re-dissolve with 5 mL of water. Take 250 μL of sample solution into a 5 mL EP tube. Follow the same steps as above.

[0118] The chromatographic conditions for HPLC analysis were as follows: Dionex TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. Mobile phase A (H2O), mobile phase B (0.1MNaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5ml / min; column temperature 30℃; elution gradient: 0minA phase / B phase / C phase (95:5:0, V / V), 26minA phase / B phase / C phase (85:5:10, V / V), 42minA phase / B phase / C phase (85:5:10, V / V), 42.1minA phase / B phase / C phase (60:0:40, V / V), 52minA phase / B phase / C phase (60:40:0, V / V), 52.1minA phase / B phase / C phase (95:5:0, V / V), 60minA phase / B phase / C phase (95:5:0, V / V). The results are as follows: Figure 6 shown.

[0119] Depend on Figure 6 It can be seen from the qualitative and quantitative analysis by HPLC that the monosaccharide composition of 030-EPS-2 polysaccharide is glucan composed of glucose, which is a neutral polysaccharide.

[0120] 5. Methylation analysis

[0121] Weigh 030-EPS-2 polysaccharide sample (5 mg), dissolve it in 1 mL of pure water, add 1 ml of 100 mg / mL 1-cyclohexyl-2-morpholineethylcarbodiimide methyl p-toluenesulfonate, and react for 2 hours. After adding 1 mL of 2M imidazole, divide the sample into two equal parts, add 1 mL of 30 mg / mL NaBH4 and 1 mL of 30 mg / mL NaBD4, respectively, and react for 3 hours. Add 100 μL of glacial acetic acid to terminate the reaction. After dialysis for 48 hours, freeze-dry the sample for subsequent treatment. Add 500 μL of DMSO to the sample to dissolve, add 1 mg of NaOH, and incubate for 30 minutes. Add 50 μL of iodomethane solution and react for 1 hour. Add 1 ml of water and 2 ml of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Aspirate the lower dichloromethane phase and blow dry with nitrogen. Add 100μL 2M TFA, react at 121℃ for 90min, and evaporate to dryness at 30℃. Add 50mL 2M ammonia, 50μL 1M NaBD4, mix well, and react at room temperature for 2.5h. Add 20μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250μL methanol, and blow dry with nitrogen. Add 250μL acetic anhydride, vortex mix, react at 100℃ for 2.5h, add 1mL water and let stand for 10min. Add 500μL dichloromethane, vortex mix, centrifuge, discard the aqueous phase, and repeat washing with water 3 times. Take the lower dichloromethane phase and detect it on GC-MS. The GC-MS chromatographic system uses an Agilent gas chromatograph system (Agilent7890A; Agilent Technologies), and the chromatographic column is BPX70 (30m×0.25mm×0.25μm, SGE, Australia). The injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high-purity helium; the initial temperature of the column oven was 140°C for 2.0 min, and then the temperature was raised to 230°C at 3°C / min and maintained for 3 min. Figure 7 shown.

[0122] Depend on Figure 7 It can be seen that the monosaccharide residues of 030-EPS-2 polysaccharide are composed of linear residues of →6)-glcp-(1→(91.06%), terminal residues of T-Glcp-(1→(5.58%), and branched residues of →3,6)-glcp-(1→(3.36%), among which →6)-glcp-(1→ is the main component. It is speculated that 030-EPS-2 is an α-(1→6)-glucan with a branch located at the O-3 position of glucose.

[0123] 6. Nuclear Magnetic Resonance Analysis

[0124] After fully dissolving an appropriate amount of 030-EPS-2 polysaccharide with D2O, a polysaccharide solution with a concentration of 150 mg / mL was prepared. The solution was transferred to an NMR tube, and the amount of liquid added was 0.5 mL. The NMR tube was placed in an NMR spectrometer to scan the one-dimensional 1H spectrum, 13C spectrum, and two-dimensional COSY, HSQC, HMBC and NOESY spectra. The target was quantitatively analyzed using a Bruker (Germany) 500MHz NMR spectrometer with a scanning temperature of 25°C. Liquid probe QXI 1H / 31P / 13C / 15N 5mm quad-resonance reverse detection probe (Z-gradient, ATMAcc), technical parameters: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating) BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM) technical parameters: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1. 030-EPS-2 NMR analysis results are as follows Figure 8 shown.

[0125] Depend on Figure 8 From A and C, we can see that the chemical shifts of the corresponding anomeric hydrogen are δ4.91, 4.92, and 5.26 ppm, respectively, corresponding to the H-1 of the three sugar residues; Figure 8 From B and C, we can see that multiple signal peaks were identified in the anomeric carbon region of the sample, and it was determined that there were three anomeric signals in the sample: δ4.91 / 97.68, 5.26 / 99.29, 4.92 / 97.83 ppm, which were confirmed to be the H1 / C1 signals of three glycosidic bonds; Figure 8 From D, we can see the chemical shifts of C and H in the three sugar residues and the substitution positions of each residue on the sugar ring. Figure 8 E and F show the structure and connection mode of sugar residues in the polysaccharide. Finally, based on the analysis of 1D and 2D NMR information and methylation results, it was inferred that the polysaccharide was mainly composed of →6)-α-D-Glcp-(1→ and a small amount of →3,6)-α-D-Glcp-(1→ connected to each other to form the main chain, and the main branch chain was composed of α-D-Glcp-(1→ connected to the O-3 position of the sugar residue →3,6)-α-D-Glcp-(1→.

[0126] Example 4: Animal experiments verifying the efficacy of exopolysaccharide 030-EPS-2

[0127] 1. Experimental animal grouping and establishment of mouse ulcerative colitis model

[0128] Thirty SPF female C57BL / 6J mice aged 6-8 weeks (purchased from Beijing Sibeifu Experimental Animal Co., Ltd.) were randomly divided into three groups, namely NC group, DSS group, and EPS / DSS group, with 10 mice in each group.

[0129] Method for establishing mouse ulcerative colitis model:

[0130] Mice in the NC group and the DSS group were gavaged with normal saline (gavage volume was mouse body weight * 10 μL), the infusion volume of normal saline was 10 μL / kg body weight, and it was infused once a day; mice in the EPS / DSS group were gavaged with 030-EPS-2 polysaccharide solution (gavage volume was mouse body weight * 10 μL), and it was infused once a day, wherein the 030-EPS-2 polysaccharide solution was prepared with normal saline as solvent, and the concentration of the 030-EPS-2 polysaccharide solution was 10 mg / ml; after one week of continuous gavage intervention, the NC group drank normal water, and the mice in the DSS group and the EPS / DSS group drank 3% DSS water freely, and drank it for 6 days to construct the ulcerative colitis model, and then changed to drinking normal water for two days. The first day of giving mice DSS water was recorded as day 0, and the mice were killed until day 8, and the experiment was terminated.

[0131] 2. Effect of 030-EPS-2 on the body weight of mice

[0132] The weight of mice in each group was recorded from day 0 until mice were killed on day 8. The mice provided statistical results such as Fig. 9 shown.

[0133] Depend on Fig. 9 It can be seen that compared with the NC group, the body weight of mice in the DSS group decreased significantly, and 030-EPS-2 polysaccharide intervention can significantly alleviate the weight loss of DSS-treated mice.

[0134] 3. Effect of 030-EPS-2 polysaccharide on colon length in mice

[0135] After the mice were killed on the eighth day, the length of the complete colon was measured. Fig.10 shown.

[0136] Depend on Fig.10 It can be seen that the length of the colon of mice in the DSS group was significantly shortened, while 030-EPS-2 polysaccharide significantly increased the length of the colon of DSS-treated mice.

[0137] 4. Effects of 030-EPS-2 on the spleen of mice

[0138] After the mice were killed, the spleens were taken and weighed. Fig.11 shown.

[0139] Depend on Fig.11It can be seen that DSS will cause an increase in the spleen weight ratio of mice, while 030-EPS-2 polysaccharide can effectively alleviate this phenomenon.

[0140] 5. Effects of 030-EPS-2 polysaccharide on colonic injury

[0141] After the mice were killed, the intestinal segments of about 0.5-1 cm in the distal colon of the mice were taken and fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin for the preparation of wax blocks. The paraffin sections of the colon were stained with Hematoxylin & Eosin (HE) and TUNEL. The results are as follows Fig.12 shown.

[0142] Depend on Fig.12 As shown in Figure A, the intestinal epithelial structure of mice in the DSS group was disordered, the villi and crypt structures were significantly damaged, goblet cells were lost, large-area ulcers were generated, and there was a large amount of inflammatory cell infiltration, which had typical pathological characteristics of colitis. After intervention with 030-EPS-2 polysaccharide, the intestinal epithelial structure of DSS-induced mice was significantly improved, the ulcer surface muscles were reduced, and the inflammatory cell infiltration was also improved. Fig.12 As shown in Figure B, the apoptosis of colon epithelial cells in mice in the DSS group increased significantly, while the number of apoptotic cells was significantly reduced after treatment with 030-EPS-2 polysaccharide.

[0143] 6. Effects of 030-EPS-2 polysaccharide on intestinal mucosal barrier

[0144] Alcian blue staining, PAS staining, and immunofluorescence staining were performed on paraffin sections of the colon to analyze the expression and distribution of tight junction proteins (Claudin-1 and Occludin). Fig.13 shown.

[0145] Depend on Fig.13 It can be seen that the PAS staining results showed that DSS can destroy goblet cells, and the number of goblet cells was significantly reduced. Correspondingly, the Alcian blue staining results showed that the secretion of mucin was also significantly reduced, and the mucus layer was severely damaged. 030-EPS-2 treatment increased the number of goblet cells and the content of mucin. DSS disrupted the distribution of tight junction proteins (Claudin-1 and Occludin) in intestinal epithelial cells and reduced the expression level, indicating that the intestinal epithelial barrier structure was destroyed. 030-EPS-2 polysaccharide intervention alleviated this phenomenon. The above results show that 030-EPS-2 can effectively improve the intestinal barrier disorder caused by DSS and protect the integrity of the intestinal barrier.

[0146] 7. Inflammatory factor detection

[0147] After the mice were killed, the colon tissues of the mice were obtained, cut into pieces, added to pre-cooled PBS containing PMSF, processed by a freezing grinder, centrifuged at 12000×g for 15 min, and the supernatant was obtained to obtain colon tissue lysate. The levels of inflammatory factors (IL-1β, IFN-γ, IL-6, TNF-α, MPO and IL-10) in the colon tissue lysate were detected. Fig.14 shown.

[0148] Depend on Fig.14 It can be seen that compared with the control group, the levels of pro-inflammatory factors in the colon of mice in the DSS group, including IL-6, IL-1β, TNF-α, IFN-γ and MPO, increased, and the level of anti-inflammatory factor IL-10 decreased. After intervention with 030-EPS-2 polysaccharide, the levels of pro-inflammatory factors IL-6, IL-1β, TNF-α, IFN-γ and MPO decreased, while the level of anti-inflammatory factor IL-10 increased. These results indicate that 030-EPS-2 polysaccharide can alleviate DSS-induced inflammatory response in colon tissue.

[0149] 8. Targeted metabolome sequencing of intestinal flora and short-chain fatty acids

[0150] After the mice were killed, the colon contents of the mice were collected. The collected colon contents of the mice were placed in cryopreservation tubes and sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for intestinal flora and short-chain fatty acid targeted metabolome sequencing, and the results were analyzed. Fig.15 shown.

[0151] Depend on Fig.15It can be seen that at the phylum level, compared with normal mice, the abundance of Bacteroidota in colitis mice was significantly reduced, and the abundance of Proteobacteria increased. After treatment with 030-EPS-2 polysaccharide, the abundance of Bacteroidota increased, and the abundance of Proteobacteria decreased significantly. At the genus level, the abundance of Muribaculaceae in the DSS group mice decreased significantly, and Escherichia-Shigella was significantly enriched. 030-EPS-2 polysaccharide intervention can increase the abundance of Muribaculaceae and reduce the content of Escherichia-Shigella. Escherichia-Shigella of the Proteobacteria phylum is the main source of endotoxins and can promote the body's inflammatory response. Muribaculaceae of the Bacteroidota phylum can ferment complex polysaccharides to produce short-chain fatty acids, which are positively correlated with the level of propionic acid in the host body that has anti-inflammatory effects. The results of short-chain fatty acid targeted metabolomics showed that DSS significantly reduced the content of propionic acid in the feces of mice with enteritis, and 030-EPS-2 polysaccharide treatment significantly increased the level of propionic acid in the feces of mice with enteritis. This indicates that 030-EPS-2 polysaccharide can reduce the level of pro-inflammatory bacteria in mice with enteritis, increase the level of anti-inflammatory bacteria and propionic acid, and thus alleviate the symptoms of ulcerative colitis in mice.

[0152] Example 5: Safety Analysis of 030-EPS-2 Polysaccharide

[0153] Twenty female C57BL / 6J mice aged 6-8 weeks were randomly divided into two groups, namely NC group and EPS group, with 10 mice in each group. Among them, the mice in the NC group were gavaged with normal saline (the gavage volume was the mouse body weight * 10μL), and the mice in the EPS group were gavaged with 030-EPS-2 polysaccharide solution (the gavage volume was the mouse body weight * 10μL), once a day, for one month, and the mice were weighed every day during the gavage. Among them, the 030-EPS-2 polysaccharide solution was prepared using normal saline as the solvent, and the concentration of the PS-2 polysaccharide solution was 10mg / ml. One month after gavage, the blood of the mice was collected to detect AST, ALT, CREA-S, and UREA, and the effects of 030-EPS-2 on liver and kidney function were analyzed. At the same time, the heart, liver, spleen, lung, kidney and colon tissues were collected, prepared into wax blocks, and HE staining was performed. The mouse weight results are shown in the figure. Fig.16 Shown

[0154] Depend on Fig.16 It can be seen that continuous intragastric administration of 030-EPS-2 polysaccharide solution for one month has no effect on the body weight of mice.

[0155] The test results of AST, ALT, CREA-S and UREA are as follows Fig.17 shown.

[0156] Depend on Fig.17 It can be seen that continuous intragastric administration of 030-EPS-2 polysaccharide solution for one month has no effect on the liver and kidney functions of mice.

[0157] HE staining results Fig.18 shown.

[0158] Depend on Fig.18 It can be seen that continuous intragastric administration of 030-EPS-2 polysaccharide solution for one month has no significant effect on the main organs of mice such as the heart, liver, spleen, lungs, and kidneys.

[0159] Example 6: Comparison of the efficacy of exopolysaccharides of Weissella sinensis ZMC030 and other exopolysaccharides of Weissella sinensis

[0160] The functions of exopolysaccharides from different lactic acid bacteria sources are often different. In order to further prove the functional differences of different polysaccharides, the 030-EPS-2 polysaccharide secreted by the edible sinus Weissella ZMC030 of the present invention was compared with the exopolysaccharide secreted by the edible sinus Weissella D-2 (for the preparation of the exopolysaccharide of the edible sinus Weissella D-2, please refer to the invention patent with patent number ZL 202310908858.7 or article DOI: 10.1038 / s41598-023-47943-7). The specific experiments are as follows:

[0161] Experimental animal grouping and establishment of mouse ulcerative colitis model:

[0162] Forty SPF female C57BL / 6J mice aged 6-8 weeks (purchased from Beijing Sibeifu Experimental Animal Co., Ltd.) were randomly divided into 4 groups, namely NC group, DSS group, D-2-EPS / DSS group, and 030-EPS-2 / DSS group, with 10 mice in each group.

[0163] Method for establishing mouse ulcerative colitis model:

[0164] The mice in the NC group and the DSS group were gavaged with normal saline (the gavage volume was the mouse body weight*10μL) once a day; the mice in the D-2-EPS / DSS group were gavaged with Weissella sinus D-2 exopolysaccharide solution (the gavage volume was the mouse body weight*10μL) once a day, wherein the Weissella sinus D-2 exopolysaccharide solution was prepared by dissolving Weissella sinus D-2 exopolysaccharide in normal saline, and the concentration of Weissella sinus D-2 exopolysaccharide was 100 mg / ml; the mice in the 030-EPS-2 / DSS group were gavaged with Weissella sinus D-2 exopolysaccharide solution (the gavage volume was the mouse body weight*10μL) once a day. The polysaccharide solution of 030-EPS-2 polysaccharide secreted by ZMC030 was gavaged (gavage volume was mouse body weight*10μL) once a day, wherein the 030-EPS-2 polysaccharide solution was prepared with normal saline as solvent, and the concentration of the 030-EPS-2 polysaccharide solution was 100mg / ml; after continuous gavage intervention for one week, the NC group drank normal water, and the mice in the DSS group, D-2-EPS / DSS group and 030-EPS / DSS group drank 3% DSS water freely for 6 days to construct an ulcerative colitis model, and then changed to drinking normal water for two days. The first day of giving mice DSS water was recorded as day0, and the mice were killed until day8, ending the experiment.

[0165] The weight of mice in each group was recorded from day 0 until the mice were killed on day 8. After the mice were killed, the complete colon of the mice was taken to measure the length of the mice. The statistical results of the weight of mice and the measurement results of the colon length are shown in the figure. Fig.19 shown.

[0166] Depend on Fig.19 It can be seen that although they are both exopolysaccharides of Weissella sinusitis, the exopolysaccharide secreted by Weissella sinusitis D-2 cannot alleviate the weight loss and colon shortening of mice with enteritis, while the exopolysaccharide secreted by Weissella sinusitis 030 effectively alleviates the symptoms. This shows that different strains of the same bacteria produce different functions of exopolysaccharides, and not all exopolysaccharides of Weissella sinusitis have the effect of relieving ulcerative colitis.

[0167] In summary, the extracellular polysaccharide of ZMC030 of Eriobotrya japonica can reduce the area of ​​intestinal epithelial ulcers, reduce intestinal epithelial cell apoptosis, alleviate intestinal epithelial inflammatory cell infiltration, protect the intestinal mucosal barrier, and can also regulate intestinal microorganisms and their metabolites, increase the levels of intestinal anti-inflammatory microorganisms and short-chain fatty acids. Therefore, the Eriobotrya japonica ZMC030 of the present invention and the extracellular polysaccharides secreted by it can be used to prevent, alleviate or / and treat inflammatory bowel disease, especially colitis; at the same time, the extracellular polysaccharide of ZMC030 of Eriobotrya japonica has no effect on the weight and growth status of mice, does not damage the important organs of mice, and has high safety.

[0168] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A Weissella sinusoidalis, characterized in that The nucleic acid sequence of Weissella sinensis contains the sequence shown in Sequence 1.

2. The Weissella sinusitis according to claim 1, characterized in that The described Weissella sinusitis is Weissella sinusitis ZMC030, and the preservation number of Weissella sinusitis ZMC030 is CCTCC NO: M 20241261.

3. An extracellular polysaccharide, characterized in that The extracellular polysaccharide is obtained by fermenting and culturing the Weissella sinusitis described in claim 1 or 2 to obtain a fermentation broth, and then separating and purifying the fermentation broth.

4. The extracellular polysaccharide according to claim 3, characterized in that The preparation method of the extracellular polysaccharide comprises the following steps: inoculating the Weissella sinusoidalis into a culture medium for fermentation culture to obtain a fermentation liquid, removing the bacteria and removing the protein from the fermentation liquid to obtain a supernatant; subjecting the supernatant to an alcohol precipitation treatment, collecting the precipitate, and subjecting the precipitate to a dialysis treatment for impurity removal and freeze-drying to obtain the extracellular polysaccharide.

5. The extracellular polysaccharide according to claim 4, characterized in that The culture medium is MRS culture medium or mMRS culture medium; the fermentation culture temperature is 30° C. to 37° C.; and the dialysis cut-off molecular weight is 8000 to 14000 Da.

6. Use of the Weissella sinusitis or the culture of Weissella sinusitis according to claim 1 or 2 or the exopolysaccharide according to any one of claims 3 to 5 in any of the following: (A1) Use in the preparation of products for preventing, alleviating or / and treating inflammatory bowel disease; (A2) Use in the preparation of products for reducing the area of ​​intestinal epithelial ulcers; (A3) Application in the preparation of products for alleviating intestinal epithelial inflammatory cell infiltration; (A4) Use in the preparation of products for reducing apoptosis of intestinal epithelial cells; (A5) Application in the preparation of products for protecting the intestinal mucosal barrier; (A6) Application in products that regulate intestinal flora.

7. The application according to claim 6, characterized in that: The product is a medicine, a food additive, a functional food, a feed additive or a feed.

8. A drug, characterized in that The drug comprises an effective ingredient, wherein the effective ingredient is the Weissella sinusitis described in claim 1 or 2 or a culture containing Weissella sinusitis or an extracellular polysaccharide described in any one of claims 3 to 5; the drug is used to prevent, alleviate or / and treat colitis or to reduce the area of ​​intestinal epithelial ulcers or to improve intestinal mucosal barrier disorders or to regulate intestinal flora.

9. A functional food, characterized in that: The food contains the Weissella vesica according to claim 1 or 2 or a culture containing the Weissella vesica or the exopolysaccharide according to any one of claims 3 to 5.

10. A feed, characterized in that: The feed contains the Weissella sinensis according to claim 1 or 2 or a culture containing Weissella sinensis or the exopolysaccharide according to any one of claims 3 to 5.

11. A food additive, characterized in that: The food additive contains the Weissella vegetativea described in claim 1 or 2 or a culture containing Weissella vegetativea or the exopolysaccharide described in any one of claims 3 to 5.

12. A feed additive, characterized in that: The feed additive contains the Weissella sinensis according to claim 1 or 2 or a culture containing Weissella sinensis or the exopolysaccharide according to any one of claims 3 to 5.

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