Bacteroides xylanolyticum capable of promoting intestinal bacteria to produce propionic acid and application of composition of bacteroides xylanolyticum in relieving ulcerative colitis

Abstract

The invention discloses application of bacteroides xylanolyticum capable of promoting intestinal bacteria to produce propionic acid and a composition of the bacteroides xylanolyticum in relieving ulcerative colitis, and belongs to the technical field of microorganisms. The bacteroides xylanolyticus NSP016 disclosed by the invention can quickly utilize konjac glucomannan, and the composition of the bacteroides xylanolyticus and konjac glucomannan can improve the rise of histopathologic score of mice with ulcerative colitis, maintain the integrity of intestinal barriers, reduce the oxidative stress level and remarkably reduce the level of clinical diagnosis markers of colitis, so that the clinical diagnosis of colitis is facilitated. The effect is better than that of a clinical medicine mesalazine. The bacteroides xylanolyticum NSP016 and the composition thereof disclosed by the invention have very wide application prospects in the aspect of preparing foods, pharmaceutical compositions, health-care products or feed additives for relieving ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the application of Bacteroides xylana, which can promote propionic acid production by intestinal bacteria, and its composition in alleviating ulcerative colitis, belonging to the field of microbial technology. Background Technology

[0002] Inflammatory bowel disease (IBD) is a nonspecific, chronic inflammatory bowel disease primarily affecting the digestive system, mainly including two types: Crohn's disease and ulcerative colitis. The former affects any part of the gastrointestinal tract from the mouth to the anus, most commonly the terminal small intestine and colon, while the latter primarily affects the colon and rectum. The most prominent symptoms of ulcerative colitis are diarrhea and bloody, mucous stools. The etiology of this disease is insidious, and its clinical manifestations are diverse and lack specificity. Currently, the etiology is not fully understood, and the pathogenesis is complex and poorly elucidated, thus posing significant challenges to clinical treatment.

[0003] The gut microbiota is closely related to ulcerative colitis, with the core mechanism involving intestinal barrier disruption, abnormal immune activation, and exacerbated inflammatory responses caused by dysbiosis. Patients with ulcerative colitis exhibit significantly reduced gut microbiota diversity, with decreased beneficial bacteria (such as lactobacilli and bifidobacteria) and increased harmful bacteria (such as Escherichia coli and Clostridium perfringens). Excessive proliferation of some pathogenic bacteria can trigger inflammatory responses. Dysbiosis disrupts the integrity of the intestinal mucosal barrier, leading to increased permeability. Bacteria and their metabolites invade the intestinal wall, activating the immune system to release inflammatory factors (such as TNF-α and IL-6), forming a vicious cycle of "dysbiosis-barrier disruption-excessive immune response." Reduced production of gut microbiota metabolites, such as short-chain fatty acids, weakens their anti-inflammatory and mucosal repair effects; while the accumulation of harmful metabolites (such as pro-inflammatory substances) further exacerbates intestinal inflammation.

[0004] Currently, gut microbiota interventions, such as probiotics, prebiotics, and fecal microbiota transplantation, have become potential treatment strategies for ulcerative colitis (UC). Restoring gut microbiota balance can improve intestinal barrier function and immune status. Supplementing with specific dietary fibers (such as fructooligosaccharides and inulin) or directly supplementing with beneficial live bacteria (such as certain strains of lactobacillus and bifidobacteria) can prevent and treat UC symptoms. Dietary fiber, as a substrate that gut microbiota can utilize, is degraded by beneficial bacteria, promotes the production of short-chain fatty acids, exerts high antioxidant activity, and inhibits pathogen colonization in the gut.

[0005] Bacteroides are the most abundant bacteria in the human gut commensal flora, and they have close ties with the host's immune, metabolic, and nervous systems. Current research suggests that Bacteroides may become a "next-generation probiotic." For example, patent CN114933993A discloses a strain of Bacteroides xylanase (…). Bacteroides xylanisolvensNSP003 and its compound preparation with pectin can alleviate colitis in mice by reducing the disease activity index and maintaining the intestinal barrier. Besides pectin, other dietary fibers such as konjac glucomannan, inulin, and β-glucan can alleviate intestinal inflammation by reducing colonic atrophy, strengthening the intestinal barrier, reducing oxidative stress levels, and maintaining intestinal microecological balance. Whether probiotics and other dietary fibers can also synergistically intervene in ulcerative colitis is worth further exploration and could provide new ideas for the development of foods, drug compositions, health products, or feed additives to alleviate ulcerative colitis. Summary of the Invention

[0006] This experiment, through extensive research, screened out a strain of *Bacteroides xylana* capable of rapidly degrading konjac glucomannan and possessing an alleviating effect on ulcerative colitis. It was demonstrated that this strain, and its combination with konjac glucomannan, can improve symptoms such as increased disease activity index and splenomegaly in mice with ulcerative colitis in animal models. Simultaneously, it can maintain the integrity of the intestinal barrier in mice, reduce oxidative stress levels, and alleviate systemic and organ tissue oxidative damage. This indicates its significant importance and broad prospects in the intervention of ulcerative colitis.

[0007] This invention provides a strain of xylanbacterium ( Bacteroides xylanisolvens NSP016 was deposited on September 12, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66954.

[0008] In one embodiment of the present invention, the xylanobacterium ( Bacteroides xylanisolvens NSP016 was isolated from a fecal sample of healthy individuals from Nanchang, Jiangxi Province. Sequencing analysis revealed its 16S rDNA sequence to be shown in SEQ ID No. 1. The sequence was then compared with the nucleic acid sequence of *Bacteroides xylana* using NCBI. The results showed a 99.58% similarity to the nucleic acid sequence of *Bacteroides xylana*. Therefore, this strain is identified as *Bacteroides xylana*. Bacteroides xylanisolvens ), named: Bacteroides xylanopsinogen ( Bacteroides xylanisolvens ) NSP016.

[0009] In one embodiment of the present invention, the xylanobacterium ( Bacteroides xylanisolvens NSP016 has the following properties: Colony characteristics: The colony diameter is 0.5-2mm, round, with neat edges, opaque, grayish-white on the front, raised in the middle, smooth, moist in texture, and belongs to Gram-negative rod-shaped bacteria, without flagella and spores.

[0010] Growth characteristics: This strain is a strict anaerobe and is sensitive to oxygen. It grows best at a temperature of 35-38℃ and the optimal growth pH is 7.1-7.6. It grows well in glucose-containing medium and can enter the late logarithmic phase or stationary phase in 16-24 hours.

[0011] The present invention also provides a composition, characterized in that the composition contains the aforementioned Bacteroides xylanoplastinus NSP016 and prebiotics.

[0012] In one embodiment of the present invention, the composition contains at least 1 × 10⁻⁶ cells of Bacteroides xylana-NSP016. 6 CFU / mL or 1×10 6 CFU / g.

[0013] In one embodiment of the present invention, the prebiotic in the composition is konjac glucomannan, wherein the konjac glucomannan has a mannan content of more than 50%, and the konjac glucomannan is not less than 0.2% of the total mass of the composition.

[0014] In one embodiment of the present invention, the konjac glucomannan is obtained by water extraction from konjac powder.

[0015] In one embodiment of the present invention, the preparation method of the konjac glucomannan is as follows: Take dried konjac powder, soak it overnight in petroleum ether, and centrifuge to evaporate the petroleum ether. Then, add 45% ethanol solution in a certain proportion, stir with a magnetic stirrer for 1.5 h, and centrifuge to remove the supernatant. After evaporating the ethanol from the lower precipitate, add distilled water at a material-to-liquid ratio of 1:30 (w / v) to reconstitute, stir magnetically at 70℃ for 2 h until fully dissolved, centrifuge to collect the supernatant (4800 rpm, 10 min), repeat the extraction three times, and combine the supernatants. After concentrating the supernatant, slowly add anhydrous ethanol to a final ethanol concentration of 80% (v / v), and place at 4℃ for 24 h; centrifuge (4800 rpm, 10 min) to obtain the precipitate, reconstitute with distilled water, and evaporate to remove the ethanol. Add 0.3% (by volume) of thermostable α-amylase relative to the raw material volume, hydrolyze at 80℃ for 2 h to remove starch, add 0.2% papain and hydrolyze at 60℃ for 2 h to remove protein, inactivate in boiling water for 10 min after hydrolysis, then cool to room temperature, adjust pH to 4.5 with 0.1 M hydrochloric acid, place at 4℃ for 12 h, centrifuge to collect supernatant (4800 rpm, 10 min), dialyze with distilled water for 48 h, concentrate and freeze dry to obtain konjac glucomannan.

[0016] The present invention also provides products containing the aforementioned Bacteroides xylanase NSP016 or the aforementioned composition.

[0017] In one embodiment of the present invention, the product is a microbial agent, food, pharmaceutical composition, health product, or feed additive.

[0018] In one embodiment of the present invention, the product contains at least 1 × 10⁻⁶ cells of Bacteroides xylana-NSP016. 6 CFU / mL or 1×10 6 CFU / g.

[0019] The present invention also provides the above-mentioned xylan-degrading Bacteroides ( Bacteroides xylanisolvens ) NSP016. Use of the above composition in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0020] In one embodiment of the present invention, the product is a pharmaceutical product or a feed additive.

[0021] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, and oral liquids.

[0022] The present invention also provides the application of the aforementioned Bacteroides xylanaminase NSP016 in improving the effect of konjac glucomannan on colitis.

[0023] The present invention also provides the application of the aforementioned Bacteroides xylanoplastinus NSP016 in the preparation of propionic acid.

[0024] Beneficial effects: This invention screened out a strain of *Bacteroides xylana* capable of rapidly degrading konjac glucomannan. Bacteroides xylanisolvens NSP016, and this strain, when combined with konjac glucomannan, can alleviate ulcerative colitis, specifically in the following ways: 1) The efficiency of degrading konjac glucomannan is higher than that of the standard strain Bacteroides xylanolyticus DSM18836; 2) It can improve the pathological changes in the colonic tissue of mice with ulcerative colitis; 3) It can increase the level of tight junction protein in the colonic tissue of mice with ulcerative colitis, restoring the integrity of the intestinal barrier; 4) It can reduce the level of malondialdehyde (MDA), a lipid peroxide, in the colonic tissue of mice with ulcerative colitis, and alleviate systemic and organ tissue oxidative damage; 5) It can promote the production of propionic acid by intestinal bacteria in mice with ulcerative colitis.

[0025] The present invention is based on xylan-degrading Bacteroides ( Bacteroides xylanisolvensNSP016 can rapidly utilize konjac glucomannan. The combination of NSP016 and konjac glucomannan can alleviate ulcerative colitis, with better effects than konjac glucomannan and the positive control drug mesalazine. It can also promote the production of propionic acid by intestinal bacteria in mice with ulcerative colitis.

[0026] Therefore, the present invention contains xylanoidomyces ( Bacteroides xylanisolvens NSP016 can be used to prepare pharmaceutical compositions, food, health products or feed additives that can relieve ulcerative colitis, and has a very wide range of application prospects.

[0027] Preservation of biological materials A strain of xylanbacterium ( Bacteroides xylanisolvens NSP016, taxonomically named Bacteroides xylanopsinus Bacteroides xylanisolvens It was deposited on September 12, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with accession number GDMCC No: 66954. Attached image description: Figure 1 Bacteroides xylanophilus ( Bacteroides xylanisolvens OD of NSP016 in a medium with konjac glucomannan as the sole carbon source 600 Increase (A) and OD compared to standard strain 600 Increase by a factor of (B).

[0028] Figure 2 Bacteroides xylanophilus ( Bacteroides xylanisolvens The production of propionic acid in the in vitro fermentation of feces from mice with ulcerative colitis using NSP016 in carbon-free medium (A) and medium (B) with konjac glucomannan as the sole carbon source.

[0029] Figure 3 Bacteroides xylanophilus ( Bacteroides xylanisolvens (A) Morphology and pathological scores of colon tissue in mice with ulcerative colitis after intervention with NSP016 and its composition (B).

[0030] Figure 4 Bacteroides xylanophilus ( Bacteroides xylanisolvens Changes in the tight junction proteins Occludin (A) and Claudin-1 (B) in mice with ulcerative colitis after intervention with NSP016 and its composition.

[0031] Figure 5 Bacteroides xylanophilus ( Bacteroides xylanisolvens Changes in the content of malondialdehyde (MDA), a lipid peroxide, after intervention with NSP016 and its composition in mice with ulcerative colitis.

[0032] Figure 6 Bacteroides xylanophilus ( Bacteroides xylanisolvens The content of propionic acid in the cecal contents of mice with ulcerative colitis after NSP016 and its composition were treated.

[0033] Among them, the model group was the DSS colitis mouse group, the mesalazine group was the DSS+ positive drug mesalazine, the konjac glucomannan group was the DSS+ konjac glucomannan group, the NSP016 group was the DSS+ xylan-Bacteroides NSP016 group, and the NSP016+ konjac glucomannan group was the DSS+ xylan-Bacteroides NSP016+ konjac glucomannan group.

[0034] "*" indicates a significant difference from the model group (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001); "#" indicates a significant difference from the normal group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001, ####: p < 0.0001). Detailed Implementation

[0035] In the following examples, sodium dextran sulfate (DSS) was purchased from MPBio, mesalazine granules were purchased from Alpha Pharmaceuticals Ltd. in France, and Bacteroides xylanica DSM18836 was purchased from the German Microbial Culture Collection (DSMZ).

[0036] The konjac glucomannan used in the following examples is konjac glucomannan with a mannose content exceeding 50%, obtained through laboratory extraction. The extraction method is as follows: Take dried konjac powder, soak it in petroleum ether overnight, and centrifuge to evaporate the petroleum ether. Then, add 45% ethanol solution at a certain ratio, stir with a magnetic stirrer for 1.5 h, and centrifuge to remove the supernatant. After evaporating the ethanol from the lower precipitate, add distilled water at a material-to-liquid ratio of 1:30 (w / v) to reconstitute, stir magnetically at 70℃ for 2 h until fully dissolved, centrifuge to collect the supernatant (4800 rpm, 10 min), repeat the extraction three times, and combine the supernatants. After concentrating the supernatant, slowly add anhydrous ethanol to a final ethanol concentration of 80% (v / v), and place at 4℃ for 24 h. After centrifugation (4800 rpm, 10 min), the precipitate is obtained, reconstituted with distilled water, and evaporate to remove the ethanol. Thermoresistant α-amylase (0.3% by volume relative to the raw material) was added and hydrolyzed at 80℃ for 2 h to remove starch. Papain (0.2% by volume) was added and hydrolyzed at 60℃ for 2 h to remove protein. After hydrolysis, the enzyme was inactivated in boiling water for 10 min, then cooled to room temperature. The pH was adjusted to 4.5 with 0.1 M hydrochloric acid and placed at 4℃ for 12 h. The supernatant was collected by centrifugation (4800 rpm, 10 min), dialyzed against distilled water for 48 h, concentrated, and freeze-dried to obtain konjac glucomannan. The monosaccharide composition of konjac glucomannan was determined by high performance liquid chromatography-ion exchange chromatography. The content of mannose was 59.50%, glucose was 38.35%, and glucuronic acid was 2.16%.

[0037] The culture media involved in the following examples are as follows: Preparation of YCFA liquid culture medium: YCFA culture medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was prepared, dissolved in distilled water, and 1 g / L of anhydrous L-cysteine ​​hydrochloride, 10 μL / L of vitamin K1, and 5 mg / L of heme chloride were added. After mixing evenly, the mixture was sterilized at 121℃ for 15 min to obtain the liquid culture medium.

[0038] Preparation of YCFA solid culture medium: Add 1.5% agar to YCFA liquid culture medium, mix well, sterilize at 121℃ for 15 min to obtain the solid culture medium.

[0039] The solution preparation methods involved in the following examples are as follows: The method for preparing sterile distilled water with 3% DSS is as follows: Weigh 30 g of DSS, dissolve it in distilled water, and sterilize it in a high-pressure steam sterilizer at 121°C for 15 min to obtain the sterile distilled water with 3% DSS.

[0040] The detection methods involved in the following embodiments are as follows: The criteria for judging colonic histopathological scores are shown in Table 1: Table 1. Colonic histopathological score

[0041] Example 1: Bacteroides xylanolyticus ( Bacteroides xylanisolvens Separation and screening of NSP016 1. Sample collection Fecal samples were collected from healthy individuals in Nanchang, Jiangxi Province. The samples were placed in preservation tubes and stored in insulated boxes with ice packs. They were then brought back to the laboratory and quickly placed in a -80°C freezer for separation and screening.

[0042] 2. Isolation and purification of Bacteroides xylanoplastinus (1) Gradual dilution of fecal samples: In a sterile anaerobic environment, take about 1 g of the fecal sample collected in step 1 and add it to 9 mL of physiological saline to obtain the first gradient dilution. Take 1 mL of the first gradient dilution and add it to 9 mL of physiological saline to obtain the second gradient dilution. And so on, to prepare a total of 9 gradient dilutions; (2) Spreading culture: Take 100 μL of each of the above gradient dilutions and place them on YCFA solid medium. After spreading, incubate under anaerobic conditions at 37℃ for 48 h to obtain diluted spread plates.

[0043] (3) Purification culture: Select colonies of different morphologies from the diluted plating plate and streak them until pure single colonies with neat edges, grayish-white color, opaque appearance, moist and smooth surface and uniform morphology are obtained; select pure colonies and inoculate them into 5 mL of YCFA liquid medium and incubate them under anaerobic conditions at 37℃ for 24 h to obtain purified culture solution.

[0044] 3. Preservation and Identification of Microbial Strains Take 0.5 μL of the purified culture medium obtained above as a DNA identification template, and use the bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (see Table 2) to perform PCR amplification of 16S rDNA.

[0045] Table 2 Primer Names

[0046] After the PCR product was confirmed by nucleic acid electrophoresis analysis, the amplified product was sent to the company for sequencing. Its 16S rDNA sequence is shown in SEQ ID No. 1. The sequencing results were compared with the sequences in the NCBI database; the comparison results showed that the strain with the number NSP016 was *Bacteroides xylana*. Bacteroides xylanisolvens NSP016, named: Bacteroides xylanopsinogen ( Bacteroides xylanisolvens ) NSP016.

[0047] Example 2: Bacteroides xylanolyticus (Bacteroides xylanisolvens The degradation effect of NSP016 on konjac glucomannan 1. Preparation of culture medium using konjac glucomannan as the sole carbon source Prepare carbon-free YCFA medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), dissolve it in distilled water, and add 1 g / L of anhydrous L-cysteine ​​hydrochloride, 10 μL / L of vitamin K1, 5 mg / L of heme chloride, and 2 g / L of konjac glucomannan. Mix well and sterilize at 121℃ for 15 min to obtain the medium with konjac glucomannan as the sole carbon source.

[0048] 2. Experimental Methods Bacteroides xylanoplastinus ( Bacteroides xylanisolvens NSP016 and Bacteroides polymorpha in the laboratory bacterial bank Bacteroides thetaiotaomicron BTH133, Parabacterium difficile ( Parabacteroides distasonis PD197, Bacteroides ovalis ( Bacteroides ovatus BO373 and the standard strain Bacteroides xylanoplasmophilus DSM18836 were streaked on YCFA solid medium. After single colonies grew, they were picked and inoculated into the medium prepared in step 1 with konjac glucomannan as the sole carbon source for adaptive growth. The culture was carried out under anaerobic conditions at 37°C for 24 h to prepare seed liquid. The above seed culture was inoculated at a rate of 2% (v / v) into the culture medium prepared in step 1, which used konjac glucomannan as the sole carbon source. The medium was cultured anaerobically at 37°C for 24 h. Growth in the culture medium was measured at 0 h and 24 h (absorbance of the culture medium at λ=600 nm was measured at 24 h – absorbance at λ=600 nm was measured at 0 h). The initial OD of the culture medium was... 600 The value is 0.2407. The result is as follows: Figure 1 As shown.

[0049] Figure (A) shows the OD of Bacteroides xylanoplastinus NSP016 after 24 h of anaerobic culture. 600 The growth rate reached 0.2796, which was higher than that of *Bacteroides multiforme* BTH133 (0.1441), *Pseudomonas dignetii* PD197 (0.1579), *Bacteroides ovalis* BO373 (0.2060), and the standard strain *Bacteroides xylanoplasmosis* DSM18836 (0.2049). Figure (B) shows that after 24 h of anaerobic culture, compared with the standard strain *Bacteroides xylanoplasmosis* DSM18836 (i.e., a fold increase of 1), the OD of *Bacteroides xylanoplasmosis* NSP016 was significantly higher. 600It increased by 1.36 times, which is higher than the growth fold of Bacteroides multiforme BTH133 (0.71), Bacteroides digiri (PD197) (0.77), and Bacteroides ovalis (BO373) (1.01).

[0050] The above results indicate that the *Bacteroides xylanatum* NSP016 strain of the present invention can utilize konjac glucomannan more quickly and efficiently than other strains, and is particularly superior to the utilization effect of the standard strain *Bacteroides xylanatum* DSM18836.

[0051] Example 3: Bacteroides xylanolyticus ( Bacteroides xylanisolvens The production of propionic acid during in vitro fermentation of feces from mice with ulcerative colitis using a combination of NSP016 and konjac glucomannan. 1. Preparation of fecal microbial solution (1) Preparation of cryopreservation agent: Prepare a 50% glycerol solution with distilled water and add 0.1% cysteine ​​hydrochloride. After sterilization at 121°C for 15 min, the cryopreservation agent is obtained.

[0052] (2) Collect feces from ulcerative colitis mice that have been drinking sterile distilled water containing 3% DSS in the laboratory for 7 days. Homogenize the feces 1:9 (w / v) in sterile PBS solution with 0.1% L-cysteine ​​hydrochloride added. Remove impurities using a 100 μm cell filter. Mix the fecal bacteria solution with the cryopreservative prepared in step (1) at a ratio of 1:1 (v / v) to obtain the fecal bacteria solution.

[0053] 2. Preparation of inoculum (1) Strawberry culture: Bacteroides xylanaminase NSP016 was streaked on YCFA solid medium. After single colonies grew, they were picked and inoculated into YCFA liquid medium and cultured under anaerobic conditions at 37℃ for 24 h to prepare the intestinal Rosbairi bacteria suspension with a concentration of 1.0 × 10⁻⁶. 8 CFU / mL.

[0054] (2) Low-dose concentrated bacterial solution: Take 1 mL of the bacterial solution obtained in step (1), centrifuge at 5000 rpm for 10 min at 4℃, and resuspend the precipitate in 200 μL of sterile PBS solution with 0.1% L-cysteine ​​hydrochloride added to obtain a concentration of 5×10⁻⁶. 8 Prepare a low-dose concentrated bacterial solution at CFU / mL for later use.

[0055] (3) High-dose concentrated bacterial solution: Take 10 mL of the bacterial solution obtained in step (1) and perform subsequent processing according to step (2) to obtain a concentration of 5×10 9 High-dose concentrated bacterial solution (CFU / mL) is available for use.

[0056] (4) Add 20 μL of the above-obtained low or high dose concentrated bacterial solution to each 1 mL of mouse fecal bacterial solution to obtain a xylan-degradable Bacteroides NSP016 concentration of 1×10⁻⁶. 7 CFU / mL fecal microbiota solution (low-dose group) and concentration of 1×10 8 The inoculum solution obtained is a fecal microbial solution of CFU / mL (high-dose group).

[0057] 3. In vitro fermentation Prepare carbon-free YCFA medium. Inoculate the inoculum obtained in the above steps into the medium at a ratio of 2% and culture for 48 h. Then, take 1 mL of fermentation broth and store it at -80℃ for later use. Use carbon-free YCFA medium without inoculation of Bacteroides xylana-NSP016 as a control (carbon-free medium group). Examine the changes in propionic acid content in the fermentation broth after inoculation with high and low doses of Bacteroides xylana-NSP016. Figure 2 A). Prepare the culture medium with konjac glucomannan as the sole carbon source as in Example 2. Inoculate the culture medium with the inoculum obtained in the above steps at a ratio of 2% and culture for 48 h. Then, take 1 mL of fermentation broth and store it at -80℃ for later use. Use the culture medium with konjac glucomannan as the sole carbon source but without inoculation of Bacteroides xylolyticus NSP016 as the control (konjac glucomannan culture medium group). Examine the changes in propionic acid content in the fermentation broth after inoculation with high and low doses of Bacteroides xylolyticus NSP016. Figure 2 B).

[0058] 4. Determination of short-chain fatty acids Take the fermentation broth from step 3, centrifuge at 13000 rpm for 5 min, take the supernatant and filter it through a 0.22 μm aqueous filter membrane, take 0.5-0.7 mL of the filtrate (take the same amount), add 0.2 mL of 10% (v / v) sulfuric acid, vortex for 1 min to mix, add 0.4 mL of anhydrous diethyl ether, vortex to mix, let stand for 2 min, centrifuge at 13000 rpm for 2 min, take the supernatant and filter it through a 0.22 μm organic filter membrane, and use a gas chromatograph to determine the content of short-chain fatty acids.

[0059] Figure 2 (A) shows that the propionic acid production in the carbon-free culture medium group was 0.16 mg / mL, while the propionic acid content was increased to 0.20 mg / mL after adding a low dose of Bacteroides xylanaminase NSP016, and increased to 0.19 mg / mL after adding a high dose of Bacteroides xylanaminase NSP016, which was not significantly different from the carbon-free culture medium group.

[0060] Figure 2(B) shows that the propionic acid production in the konjac glucomannan medium group was 0.20 mg / mL, while the propionic acid content was increased to 0.31 mg / mL after the addition of a low dose of Bacteroides xylolyticus NSP016, and increased to 0.36 mg / mL after the addition of a high dose of Bacteroides xylolyticus NSP016 (p<0.05), which was significantly higher than that in the konjac glucomannan medium group (0.20 mg / mL).

[0061] The results showed that the combination of Bacteroides xylolyticus NSP016 and konjac glucomannan promoted the production of more propionic acid by the fecal intestinal flora of mice.

[0062] Example 4: Bacteroides xylanolyticus ( Bacteroides xylanisolvens Improvement of colonic tissue morphology in mice with ulcerative colitis by NSP016 and its composition 1. Preparation of Bacteroides xylanoplastin NSP016 gavage bacterial solution: (1) Culture of strain: Bacteroides xylana NSP016 was streaked on YCFA solid medium. After single bacteria grew, they were picked and inoculated into YCFA liquid medium and cultured under anaerobic conditions at 37℃ for 24 h to prepare Bacteroides xylana NSP016 seed culture.

[0063] (2) After centrifuging the *Bacteroides xylanoplastinus* NSP016 seed culture prepared in step (1) at 4℃ for 10 min at 5000 rpm, the precipitate was collected as *Bacteroides xylanoplastinus* NSP016 cells. The *Bacteroides xylanoplastinus* NSP016 cells were washed 1-2 times with sterile PBS solution (pH 7.2), and then resuspended in sterile PBS solution with 0.1% L-cysteine ​​hydrochloride added to make the bacterial concentration 1.0 × 10⁻⁶. 8 The CFU / mL concentration yielded the Bacteroides xylanoplastin NSP016 gavage solution, which was immediately administered via gavage. During the intervention period, the gavage solution was prepared daily to ensure the activity of Bacteroides xylanoplastin NSP016.

[0064] 2. Preparation of konjac glucomannan Weigh out konjac glucomannan according to the mouse's body weight, dissolve it in water to obtain konjac glucomannan solution. The gavage dose is 250 mg of konjac glucomannan per kilogram of mouse body weight, and the gavage volume is 0.2 mL.

[0065] 3. Preparation of mesalazine solution Weigh out the mesalazine granules according to the mouse's body weight, and dissolve them in a 5% sodium carboxymethyl cellulose aqueous solution to obtain the mesalazine solution. The gavage dose is 250 mg of mesalazine per kilogram of mouse body weight, and the gavage volume is 0.2 mL.

[0066] 4. Preparation of antibiotic cocktails Based on the mouse's body weight, ampicillin, metronidazole, vancomycin, and neomycin were weighed and dissolved in water to form an antibiotic cocktail solution. The concentrations of the four antibiotics were ampicillin 100 mg / kg; metronidazole 100 mg / kg; vancomycin 50 mg / kg; and neomycin 100 mg / kg. The gavage volume was 0.2 mL.

[0067] 5. Animal experimental protocol Seventy-two healthy male SPF-grade C57BL / 6J mice aged 6 weeks were randomly divided into 6 groups: normal group, ulcerative colitis group (model group), DSS+ positive drug mesalazine group (positive drug control, mesalazine group), DSS+ konjac glucomannan group (konjac glucomannan group), DSS+ xylan-Bacteroides group (NSP016 group), and DSS+ xylan-Bacteroides NSP016+ konjac glucomannan group (NSP016+ konjac glucomannan group), with 12 mice in each group.

[0068] Mice were housed in a standardized laboratory with a light / dark cycle of 12 h at 25±2℃ and 50±5% relative humidity. Growth maintenance feed was purchased from Beijing Keao Xieli Feed Co., Ltd. After 7 days of acclimatization feeding, the experiment lasted for 14 days. The experimental procedure is shown in Table 3.

[0069] Table 3 Specific implementation plan for animal experiments

[0070] 6. After the experiment, all mice were euthanized, and a 1 cm distal colon was taken and immersed in 4% paraformaldehyde solution for 24 hours to obtain fixed colon tissue samples. After dehydration, embedding, sectioning, dewaxing, and rehydration, the samples were stained with hematoxylin and eosin to obtain H&E sections. The sections were then observed and images were acquired using a pathological slide scanner, and scores were assigned according to Table 1. The experimental results are as follows: Figure 3 As shown.

[0071] Figure 3 A showed that the colon tissue structure of normal mice was clear, with intact mucosa, neatly arranged epithelial cells, intact crypts, and no obvious inflammatory cell infiltration. In contrast, the colon tissue structure of model mice was severely damaged, with almost complete mucosal destruction, disappearance of crypts, and obvious inflammatory cell infiltration.

[0072] Intervention with Bacteroides xylanaminase NSP016 effectively reduced inflammatory cell infiltration and maintained the integrity of intestinal mucosal epithelial cells. After combined intervention with NSP016 and konjac glucomannan, not only was inflammatory cell infiltration effectively reduced, but the colonic tissue cells were also more densely arranged, and the colonic tissue morphology was close to that of the normal group. The improvement effect was significantly better than that of the NSP016 group, the konjac glucomannan group, and the positive control drug mesalazine group.

[0073] Figure 3 B showed that the histopathological score of the model group mice increased from 1.17 (normal group) to 8.33 (p<0.0001). After intervention with Bacteroides xylana NSP016, the histopathological score significantly decreased to 3.50 (p<0.001). The histopathological score of the NSP016 + konjac glucomannan group decreased to 2.83 (p<0.0001), and the histopathological score of the konjac glucomannan group decreased to 4.67 (p<0.01). The effects were better than the histopathological score of 5.33 (p<0.05) in the positive drug mesalazine group.

[0074] The above results indicate that Bacteroides xylolyticus NSP016 and its composition with konjac glucomannan of the present invention can reduce the histopathological score of mice with ulcerative colitis and improve the morphology of colon tissue in mice with colitis. The composition of Bacteroides xylolyticus NSP016 and konjac glucomannan has the best effect.

[0075] Example 5: Bacteroides xylanoplasmosis ( Bacteroides xylanisolvens Effects of NSP016 and its composition on tight junction protein expression in colonic tissue of mice with ulcerative colitis The specific implementation method is the same as in Example 4. After the experiment, the mice were sacrificed and serum and colon tissue samples were collected.

[0076] The levels of tight junction proteins (Occludin and Claudin-1) in colon tissue were determined using an ELISA kit (product of Nanjing Fomax Biotechnology Co., Ltd.). The experimental results are as follows: Figure 4 As shown.

[0077] like Figure 4 As shown in Figure A, after the model group mice drank sterile distilled water containing 3% DSS for 7 consecutive days, the ocludin content in the colon of the model group mice was 2.43 times that of the model group mice, using the ocludin content in the colon of the model group mice as a reference.

[0078] After intervention with Bacteroides xylana NSP016, the ocludin content in mice was 2.92 times that of the model group (p<0.01), the ocludin content in mice in the NSP016+ konjac glucomannan group was 2.61 times that of the model group (p<0.01), and the DAI value in mice in the konjac glucomannan group decreased to 2.93 (p<0.01).

[0079] like Figure 4 As shown in B, after the model group mice drank sterile distilled water containing 3% DSS for 7 consecutive days, the tight junction protein (Claudin-1) content in the colon of the model group mice was compared with that in the normal group mice, which was 5.01 times that of the model group.

[0080] After intervention with Bacteroides xylanamin NSP016, the level of tight junction protein (Claudin-1) in mice was 4.59 times that of the model group (p<0.001); the level of tight junction protein (Claudin-1) in mice in the NSP016 + konjac glucomannan group was 4.88 times that of the model group (p<0.001), and the level of tight junction protein (Claudin-1) in mice in the konjac glucomannan group was 3.62 times that of the model group (p<0.05), which was higher than the 2.53-fold change in tight junction protein (Claudin-1) level in mice in the positive drug mesalazine group.

[0081] The above results indicate that Bacteroides xylanaminase NSP016 and its composition with konjac glucomannan can both upregulate the level of tight junction proteins and maintain the integrity of the intestinal barrier. Furthermore, the effects of Bacteroides xylanaminase NSP016 and its composition with konjac glucomannan are superior to those of konjac glucomannan and clinical drugs.

[0082] Example 6: Bacteroides xylanolyticus ( Bacteroides xylanisolvens Effects of NSP016 and its composition on oxidative stress levels in mice with ulcerative colitis The specific implementation method is the same as in Example 4. After the experiment, the mice were sacrificed and serum and colon tissue samples were collected.

[0083] The content of malondialdehyde (MDA), a lipid peroxide, in colon tissue was determined using an ELISA kit (product of Shanghai Beyotime Biotechnology Co., Ltd.). The experimental results are as follows: Figure 5 As shown.

[0084] like Figure 5 As shown, the MDA content in the colon tissue of the model group mice increased from 0.79 nmol / mg protein (normal group) to 1.51 nmol / mg protein.

[0085] After intervention with Bacteroides xylana NSP016, the MDA content in the colon tissue of mice decreased to 1.19 nmol / mg protein (p<0.01), while the MDA content in the colon tissue of mice in the konjac glucomannan group was 0.97 nmol / mg (p<0.0001).

[0086] The MDA content in the colon tissue of mice in the NSP016+ konjac glucomannan group was 0.88 nmol / mg (p<0.0001), which was lower than the MDA content in the colon tissue of mice in the positive drug mesalazine group (0.95 nmol / mg (p<0.0001)).

[0087] The above results indicate that Bacteroides xylolyticus NSP016 and its composition with konjac glucomannan can alleviate oxidative stress levels in mice with ulcerative colitis, thereby alleviating colitis-related systemic and organ oxidative damage. Furthermore, the composition of Bacteroides xylolyticus NSP016 and konjac glucomannan is more effective than konjac glucomannan and clinical drugs.

[0088] Example 7: Bacteroides xylanolyticus ( Bacteroides xylanisolvens The promoting effect of NSP016 and its composition on propionic acid production by intestinal flora in mice with ulcerative colitis The specific implementation method is the same as in Example 4. After the experiment, the mice were sacrificed and the contents of the cecum were collected.

[0089] Weigh the contents of the cecum, add sterile PBS at a ratio of 1:10, add 3 zirconium oxide grinding beads and grind, then prepare the sample according to the sample preparation method in step 4 of Example 3, and determine the content of short-chain fatty acids on the instrument.

[0090] like [[ID=5 As shown, the propionic acid content in the cecal contents of the model group mice decreased from 0.50 mg / mL (normal group) to 0.22 mg / mL (p<0.001).

[0091] After intervention with Bacteroides xylana-NSP016, the propionic acid content in the cecal contents of mice increased to 0.64 mg / mL (p<0.0001), the propionic acid content in the cecal contents of mice in the NSP016+konjac glucomannan group increased to 0.44 mg / mL (p<0.01), and the propionic acid content in the cecal contents of mice in the konjac glucomannan group increased to 0.36 mg / mL, all of which were higher than the 0.34 mg / mL in the positive drug mesalazine group.

[0092] The results show that Bacteroides xylolyticus NSP016 and its combination with konjac glucomannan can promote the production of more propionic acid by the intestinal flora of mice with ulcerative colitis, which is consistent with the in vitro fermentation results in Example 3.

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bacteroides xylanoplasmosis ( Bacteroides xylanisolvens NSP016 was deposited on September 12, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66954.

2. A composition, characterized in that, The composition contains Bacteroides NSP016 as described in claim 1 and prebiotics; the prebiotics include konjac glucomannan.

3. The composition according to claim 2, characterized in that, In the composition, the bacterial count of Bacteroides xylana-NSP016 is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g; the konjac glucomannan contains more than 50% mannose, and the konjac glucomannan accounts for no less than 0.2% of the total mass of the composition.

4. A product containing Bacteroides xylanase NSP016 as described in claim 1 or the composition as described in claim 2 or 3.

5. The product as described in claim 4, characterized in that, The products are microbial agents, food, pharmaceutical compositions, health products, or feed additives.

6. The product as described in claim 4 or 5, characterized in that, In the product, the bacterial count of Bacteroides xylanaminifera NSP016 is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

7. The use of Bacteroides xylanaminifera NSP016 as described in claim 1 or the composition as described in claim 2 or 3 in the preparation of a pharmaceutical or feed additive for the prevention and / or treatment of ulcerative colitis.

8. The application as described in claim 7, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, and oral liquids.

9. The application of Bacteroides xylanogene NSP016 as described in claim 1 in improving the effect of konjac glucomannan on colitis.

10. The use of Bacteroides xylanoplastinus NSP016 as described in claim 1 in the preparation of propionic acid.

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