Intestinal Rosebaria sp. Capable of promoting intestinal bacteria to produce valeric acid and application of composition of intestinal Rosebaria sp. In relieving ulcerative colitis

Abstract

The invention discloses an application of intestinal Rosebaria capable of promoting intestinal bacteria to produce valeric acid and a composition of the intestinal Rosebaria in relieving ulcerative colitis, and belongs to the technical field of microorganisms. According to the intestinal Rosiberia NSP017 disclosed by the invention, the konjac glucomannan can be quickly utilized, and the composition of the intestinal Rosiberia and the konjac glucomannan can be used for improving the disease activity index and spleen enlargement of a mouse with ulcerative colitis, maintaining the integrity of an intestinal barrier, reducing the oxidative stress level, and improving the ulcerative colitis activity index and the spleen enlargement of the mouse with ulcerative colitis, so that the intestinal Rosiberia NSP017 can be used for treating ulcerative colitis. The level of a clinical diagnosis marker for colitis is remarkably reduced, the effect is superior to that of a clinical medicine mesalazine, and it is found that the intestinal Rosebawnella NSP017 can promote intestinal bacteria of mice with colitis to recover the capacity of producing valeric acid. The intestinal Rosibula sp. NSP017 and the composition of the intestinal Rosibula sp. NSP017 have very wide application prospects in the aspect of preparing foods, pharmaceutical compositions, health-care products or feed additives for relieving ulcerative colitis.

Description

Application of *Rosbyrus rosburyi* and its composition that promote valerate production by intestinal bacteria in the relief of ulcerative colitis Technical Field

[0001] This invention relates to the application of Rosbairi bacteria that can promote the production of valerate by intestinal bacteria and its composition in the relief of ulcerative colitis, belonging to the field of microbial technology. Background Technology

[0002] Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) with a complex pathogenesis involving multiple factors such as genetics, immunity, environment, and gut microbiota. Clinically, it mainly manifests as diarrhea, abdominal pain, mucus in stool, and bloody stool. Severe cases may present with systemic symptoms such as fever, anemia, weight loss, and malnutrition. Ulcerative colitis is characterized by its insidious nature, diverse and nonspecific symptoms, and continues to pose significant challenges to clinical treatment.

[0003] The gut microbiota, as the largest and most complex micro-ecosystem in the human body, plays an indispensable role in maintaining gut health. Extensive evidence suggests that gut microbiota dysbiosis is a core element in the occurrence and development of ulcerative colitis (UC). Compared to healthy individuals, the gut microbiota of UC patients undergoes significant alterations in both composition and function, exhibiting reduced diversity, a marked decrease in the richness and evenness of gut microbial species, and decreased ecosystem stability. The abundance of beneficial bacteria capable of producing short-chain fatty acids (especially butyrate), such as *Faecalibacterium prausnitzii* and *Roseburia intestinalis* from the Firmicutes phylum, is significantly reduced. The proportion of certain potentially pathogenic bacteria, such as those from the Proteobacteria phylum (e.g., *Escherichia coli*) and Fusobacteria phylum, is increased.

[0004] Supplementing with specific dietary fibers (such as fructooligosaccharides and inulin) or directly supplementing with beneficial live bacteria (such as certain strains of Lactobacillus and Bifidobacterium) can prevent and treat UC symptoms. Dietary fiber, as a substrate available to the gut microbiota, is degraded by beneficial bacteria, promoting the production of short-chain fatty acids, exerting high antioxidant activity, and inhibiting pathogen colonization in the gut. Probiotics can significantly alleviate inflammation by inhibiting the TLR4 / NF-κB pathway, and can also repair the intestinal barrier by increasing the expression levels of specific tight junction proteins and reducing goblet cell loss. In addition, microbiota-derived metabolites, fecal microbiota transplantation, and strategies based on microbiota interaction regulation have become promising treatment approaches for UC. However, in the process of using prebiotics, probiotic interventions, and fecal microbiota transplantation to treat UC, the introduction of dietary polysaccharides for adjuvant mediation and the precise targeting of probiotics with good therapeutic effects under polysaccharide mediation are currently hot research topics.

[0005] *Rhizobium* is a current hot topic in gut microbiota research and is considered an important candidate for next-generation probiotics. Variations in *Rhizobium* abundance are closely associated with the development of various diseases, including inflammatory bowel disease, irritable bowel syndrome, metabolic disorders, and neurological diseases. Furthermore, *Rhizobium* is a key butyrate producer and ecosystem engineer in the gut microbiota. Its abundance is broadly correlated with human health, playing a central role in maintaining the intestinal barrier, immune homeostasis, and metabolic health. Although technological challenges remain in its development into probiotics, it is undoubtedly one of the most promising genera in the field of microbial technology, with broad clinical application prospects.

[0006] Dietary fibers such as konjac glucomannan, pectin, inulin, and β-glucan can alleviate intestinal inflammation by relieving colonic atrophy, strengthening the intestinal barrier, reducing oxidative stress levels, and maintaining intestinal microecological balance. However, due to individual differences, only dietary fiber intervention may cause a few people to experience discomfort such as bloating and flatulence. Therefore, there is an urgent need to discover probiotic strains, such as *Lactobacillus*, that can both degrade cellulose to relieve bloating and flatulence symptoms and alleviate ulcerative colitis symptoms. Research on these strains and their combinations with dietary fiber can provide new ideas for the development of foods, pharmaceutical compositions, health products, or feed additives that alleviate ulcerative colitis. Summary of the Invention

[0007] This experiment, through extensive research, screened out a Rosbairi bacterium in the gut that can rapidly degrade konjac glucomannan and has 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.

[0008] This invention provides a strain of Roseburia intestinalis NSP017, which 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: 66955.

[0009] In one embodiment of the present invention, the *Roseburia intestinalis* NSP017 was isolated from a fecal sample of a healthy human from Nanchang, Jiangxi Province. Sequencing analysis of this strain revealed its 16S rDNA sequence as shown in SEQ ID No. 1. The sequenced data was then compared with the nucleic acid sequence of *Roseburia intestinalis* using NCBI. The results showed a 99.58% similarity to the nucleic acid sequence of *Roseburia intestinalis*. Therefore, this strain is identified as *Roseburia intestinalis* and named *Roseburia intestinalis* NSP017.

[0010] In one embodiment of the present invention, the intestinal Roseburia intestinalis NSP017 has the following properties: colony characteristics: Gram-negative rod-shaped bacteria, often slightly curved and spindle-shaped, with flagella, no spores, and a certain degree of motility.

[0011] Growth characteristics: This strain is a strict anaerobic bacterium that is sensitive to oxygen. It grows best at a temperature of 35-38℃ and at an optimal pH of 5.5-7.0. It grows well in glucose-containing media and enters the logarithmic phase in 12-24 hours and the stationary phase in 24-48 hours.

[0012] The present invention also provides a composition, characterized in that the composition contains the aforementioned Rosbyrate's bacterium NSP017 and prebiotics.

[0013] In one embodiment of the present invention, the composition contains at least 1 × 10⁻⁶ cells of *Rosbyrus spp.* NSP017.6 CFU / mL or 1×10 6 CFU / g.

[0014] 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.

[0015] In one embodiment of the present invention, the konjac glucomannan is obtained by water extraction from konjac powder.

[0016] 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.

[0017] The present invention also provides products containing the aforementioned Enterobacter rosbyresia NSP017 or the aforementioned composition.

[0018] In one embodiment of the present invention, the product is a microbial agent, food, pharmaceutical composition, health product, or feed additive.

[0019] In one embodiment of the present invention, the product contains at least 1 × 10⁻⁶ cells of Rosbyrateella NSP017. 6 CFU / mL or 1×10 6 CFU / g.

[0020] The present invention also provides the use of the above-mentioned Roseburia intestinalis NSP017 or the above-mentioned composition in the preparation of a medicament for the prevention and / or treatment of ulcerative colitis.

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

[0022] The present invention also provides the application of the aforementioned intestinal Rosbyrate's bacterium NSP017 in improving the effect of konjac glucomannan on colitis.

[0023] The present invention also provides the application of the aforementioned intestinal Rosbairi bacteria NSP017 in the preparation of valeric acid.

[0024] Beneficial effects: This invention screened out an intestinal Roseburia intestinalis strain NSP017 that can rapidly degrade konjac glucomannan, and this strain, or when combined with konjac glucomannan, can alleviate ulcerative colitis. Specifically, it demonstrates that: 1) the efficiency of degrading konjac glucomannan is higher than that of the standard strain Roseburia intestinalis DSM14610; 2) it can improve the disease activity index and splenomegaly in mice with ulcerative colitis, alleviating inflammation; 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 superoxide dismutase (SOD) in the colonic tissue of mice with ulcerative colitis, alleviating systemic and organ tissue oxidative damage; 5) it can promote the recovery of the ability of intestinal bacteria in mice with ulcerative colitis to produce valerate.

[0025] The intestinal Roseburia intestinalis NSP017 of the present invention can rapidly utilize konjac glucomannan. The combination of Roseburia intestinalis and konjac glucomannan can alleviate ulcerative colitis, with better effects than konjac glucomannan and the positive control drug mesalazine. It can restore the ability of intestinal bacteria in ulcerative colitis mice to produce valerate.

[0026] Therefore, the intestinal Roseburia intestinalis NSP017 of the present invention can be used to prepare pharmaceutical compositions, foods, health products or feed additives that can alleviate ulcerative colitis, and has a very broad application prospect.

[0027] Biological material deposit: A strain of *Roseburia intestinalis* NSP017, taxonomically named *Roseburia intestinalis*, was deposited on September 12, 2025, at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Institute of Microbiology, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 66955. Attached Figure Description

[0028] Figure 1: OD of Roseburia intestinalis NSP017 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).

[0029] Figure 2: Production of valeric acid during in vitro fermentation of feces from mice with ulcerative colitis using a combination of Roseburia intestinalis NSP017 and konjac glucomannan.

[0030] Figure 3: Changes in disease activity index (A) and spleen index (B) in mice with ulcerative colitis after intervention with Roseburia intestinalis NSP017 and its combination.

[0031] Figure 4: Changes in the tight junction proteins Occludin (A) and Claudin-1 (B) in mice with ulcerative colitis after intervention with Roseburia intestinalis NSP017 and its combination.

[0032] Figure 5: Changes in superoxide dismutase (SOD) content in mice with ulcerative colitis after intervention with Roseburia intestinalis NSP017 and its combination.

[0033] Figure 6: Valeric acid content in cecal contents of mice with ulcerative colitis after intervention with Roseburia intestinalis NSP017 and its composition.

[0034] Figure 7: Changes in disease activity index (A), spleen index (B), superoxide dismutase (SOD) content (C), and malondialdehyde (MDA) content (D) in mice with ulcerative colitis after sodium valerate intervention.

[0035] Among them, the model group was the DSS colitis mouse group, the mesalazine group was the DSS+ positive drug mesalazine group, the konjac glucomannan group was the DSS+ konjac glucomannan group, the NSP017 group was the DSS+ intestinal Rosbairi NSP017 group, and the NSP017+ konjac glucomannan group was the DSS+ intestinal Rosbairi NSP017+ konjac glucomannan group.

[0036] "*" 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

[0037] In the following examples, sodium dextran sulfate (DSS) was purchased from MPBio, mesalazine granules were purchased from Alpha Pharmaceuticals Ltd. (France), Rosbairi bacteria DSM14610 was purchased from the German Microbial Culture Collection (DSMZ), and sodium valerate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0038] The konjac glucomannan involved in the following examples is konjac glucomannan with a mannose content exceeding 50%, obtained through laboratory extraction. The extraction method is as follows: Konjac powder was soaked in petroleum ether overnight, and the petroleum ether was evaporated by centrifugation. Then, 45% ethanol solution was added at a certain ratio, and the mixture was stirred with a magnetic stirrer for 1.5 h. The supernatant was removed by centrifugation. After evaporating the ethanol from the lower precipitate, distilled water was added at a material-to-liquid ratio of 1:30 (w / v) to reconstitute the precipitate. The mixture was magnetically stirred at 70℃ for 2 h until fully dissolved. The supernatant was collected by centrifugation (4800 rpm, 10 min). This extraction was repeated three times, and the supernatants were combined. After concentrating the supernatant, anhydrous ethanol was slowly added until the final ethanol concentration reached 80% (v / v), and the mixture was placed at 4℃ for 24 h. After centrifugation (4800 rpm, 10 min), the precipitate was obtained, reconstituted with distilled water, and the ethanol was evaporated to remove it. 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%.

[0039] 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 liquid culture medium was obtained by sterilization at 121°C for 15 min.

[0040] 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.

[0041] The preparation methods of the solutions involved in the following examples are as follows: The preparation method of 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.

[0042] The detection methods involved in the following embodiments are as follows: The judgment criteria for the Disease Activity Index (DAI) are shown in Table 1: Table 1 Disease Activity Index (DAI) Scoring

[0043] Example 1: Isolation and screening of Roseburia intestinalis NSP017 1. Sample collection Fecal samples were collected from healthy human subjects in Nanchang, Jiangxi Province. The samples were placed in preservation tubes and stored in an insulated box with ice packs. They were then brought back to the laboratory and quickly placed in a -80℃ refrigerator for isolation and screening.

[0044] 2. Isolation and purification of Rosbairi bacteria (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. Repeat this process 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. Spread the medium and incubate it under anaerobic conditions at 37℃ for 48 h to obtain diluted spread plates.

[0045] (3) Purification culture: Select colonies of different morphologies from the diluted plating plate and streak them until pure single colonies with neat edges, slightly white, opaque, moist and smooth surfaces and consistent 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.

[0046] 3. Strain Preservation and Identification: 0.5 μL of the purified culture obtained above was used as a DNA identification template, and PCR was performed using bacterial 16S rDNA primers (see Table 2). Universal primers 27F (5'-AGAGTTTGATCCTGGCCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification of 16S rDNA.

[0047] Table 2 Primer Names

[0048] 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 NSP017 was Roseburia intestinalis, and it was named: Roseburia intestinalis NSP017.

[0049] Example 2: Degradation of konjac glucomannan by Roseburia intestinalis NSP017 1. Preparation of carbon-free YCFA medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) with konjac glucomannan as the sole carbon source. The medium was dissolved in distilled water, and 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 were added. After mixing evenly, the medium was sterilized at 121℃ for 15 min to obtain the medium with konjac glucomannan as the sole carbon source.

[0050] 2. Experimental Methods: *Roseburia intestinalis* NSP017, *Bacteroides thetaiotaomicron* BTH133, *Parabacteroides distasonis* PD197, *Bacteroides ovatus* BO373 from the laboratory bacterial bank, and the standard strain *Roseburia intestinalis* DSM14610 were streaked on YCFA solid medium. After single colonies grew, they were picked and inoculated into the medium prepared in step 1 using konjac glucomannan as the sole carbon source for adaptive growth. The culture was anaerobic at 37°C for 24 h to prepare a seed culture. The seed culture was then inoculated at a rate of 2% (v / v) into the medium prepared in step 1 using konjac glucomannan as the sole carbon source and anaerobic at 37°C for 24 h. The cultures were then compared at 0 h and 24 h. The growth rate in the culture medium was determined by measuring the absorbance of the culture medium at λ=600 nm at 24 h and the absorbance of the culture medium at λ=600 nm at 0 h. The initial OD of the culture medium was also determined. 600 The value is 0.2407. The result is shown in Figure 1.

[0051] Figure (A) shows the OD of Rosbyrates intestinal NSP017 after 24 h of anaerobic culture. 600 The growth rate reached 0.5049, higher than that of *Bacteroides multiforme* BTH133 (0.1441), *Pseudomonas diffusa* PD197 (0.1579), *Bacteroides ovalis* BO373 (0.2060), and *Rosbairi* DSM14610 (0.3814). Figure (B) shows that after 24 h of anaerobic culture, compared with the increase of 0.3814 in the standard strain *Rosbairi* DSM14610 (i.e., an increase of 1 fold), the OD of *Rosbairi* NSP017 was significantly higher. 600 It increased by 1.32 times, which is higher than the growth fold of Bacteroides multiforme BTH133 (0.38 times), Bacteroides digiri (PD197) (0.41 times), and Bacteroides ovalis (BO373) (0.54 times).

[0052] The above results indicate that the enteric Rosbyrates bacterium NSP017 strain of the present invention can utilize konjac glucomannan more quickly and efficiently than other strains, especially superior to the utilization effect of the standard strain enteric Rosbyrates bacterium DSM14610.

[0053] Example 3: The production of valeric acid during in vitro fermentation of feces from mice with ulcerative colitis using a combination of Roseburia intestinalis NSP017 and konjac glucomannan. 1. Preparation of fecal bacterial solution (1) Preparation of cryopreservative: Prepare a 50% glycerol solution with distilled water and add 0.1% cysteine ​​hydrochloride. After sterilization at 121°C for 15 min, the cryopreservative is obtained.

[0054] (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.

[0055] 2. Preparation of inoculum (1) Culture of bacterial strain: Streaking of *Rosbairi* NSP017 on YCFA solid medium, after single colonies grow, pick them and inoculate them into YCFA liquid medium, and culture them under anaerobic conditions at 37℃ for 24 h to prepare *Rosbairi* NSP017 bacterial suspension with a concentration of 1.0 × 10⁻⁶. 8 CFU / mL.

[0056] (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.

[0057] (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.

[0058] (4) By adding 20 μL of the above-obtained concentrated bacterial solution to each 1 mL of mouse fecal bacterial solution, the concentration of Rosbairi bacteria NSP017 in the intestine was prepared to be 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).

[0059] 3. Prepare carbon-free YCFA medium for in vitro fermentation. Inoculate the inoculum obtained in the above steps into the medium at a ratio of 2% and culture for 0, 12, 24, and 48 h. After each culture, take 1 mL of fermentation broth and store it at -80℃ for later use. Use carbon-free YCFA medium without inoculation of Rosbyrates intestinal NSP017 as a control (carbon-free medium group). Examine the changes in valeric acid content in the fermentation broth after inoculation with high and low doses of Rosbyrates intestinal NSP017. The culture medium with konjac glucomannan as the sole carbon source was prepared as in Example 2. The inoculum obtained in the above steps was inoculated into the culture medium at a ratio of 2%. 1 mL of fermentation broth at 0, 12, 24, and 48 h was stored at -80℃ for later use. The culture medium with konjac glucomannan as the sole carbon source but without inoculation of Rosbyrates intestinal NSP017 was used as a control (konjac glucomannan culture medium group). The changes in valeric acid content in the fermentation broth after inoculation with high and low doses of Rosbyrates intestinal NSP017 were investigated.

[0060] 4. Determination of Short-Chain Fatty Acids: Take the fermentation broth from step 3, centrifuge at 13000 rpm for 5 min, collect the supernatant and filter through a 0.22 μm aqueous filter membrane. Take 0.5-0.7 mL of the filtrate (using 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, collect the supernatant and filter through a 0.22 μm organic filter membrane, and determine the short-chain fatty acid content using a gas chromatograph. The results are shown in Figure 2.

[0061] Figure 2 shows that no valeric acid was produced in the carbon-free culture medium group and the konjac glucomannan culture medium group. However, after adding Rosbairi bacteria NSP017, valeric acid was produced in the fermentation broth of both the konjac glucomannan culture medium + low dose of Rosbairi bacteria NSP017 group and the konjac glucomannan culture medium + high dose of Rosbairi bacteria NSP017 group at 24 h and 48 h. The results indicate that the combination of Rosbairi bacteria NSP017 and konjac glucomannan can promote the recovery of the valeric acid production capacity of mouse fecal intestinal bacteria.

[0062] Example 4: Effects of Roseburia intestinalis NSP017 and its composition on disease activity index and spleen index in mice with ulcerative colitis 1. Preparation of intestinal Roseburia intestinalis NSP017 gavage culture: (1) Culture of strain: Roseburia intestinalis NSP017 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 intestinal Roseburia intestinalis NSP017 seed culture.

[0063] (2) After centrifuging the intestinal Rosbairi NSP017 seed culture prepared in step (1) at 5000 rpm for 10 min at 4℃, the precipitate was collected as intestinal Rosbairi NSP017 cells. The intestinal Rosbairi NSP017 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 yields the intestinal Rosbyrate's bacterium NSP017 gavage solution, which is administered immediately by gavage. Because Rosbyrate's bacterium NSP017 is strictly anaerobic, the bacterial solution needs to be prepared daily during the intervention period.

[0064] 2. Preparation of konjac glucomannan: Weigh konjac glucomannan according to the mouse body weight, dissolve it in water to obtain konjac glucomannan solution. The gavage dosage 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 mesalazine granules according to the mouse's body weight and dissolve them in a 5% (w / w) 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 cocktail: Ampicillin, metronidazole, vancomycin, and neomycin were weighed according to the mouse body weight and dissolved in water to obtain the 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 Experiment 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 + intestinal Rosbairi bacteria group (NSP017 group), and DSS + intestinal Rosbairi bacteria NSP017 + konjac glucomannan group (NSP017 + 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. During the experiment, the mice were weighed and scored daily. After the experiment, all mice were sacrificed, spleen tissue was collected, and the weight of the spleen tissue was measured. The spleen index was calculated based on the percentage of the mouse's body weight. The experimental results are shown in Figure 3.

[0071] Figure (A) shows that after the model group mice drank sterile distilled water containing 3% DSS for 7 consecutive days, the DAI value of the mice increased to 3.83 (p<0.0001). The DAI value of the normal group mice was 0.

[0072] After intervention with Rosbyrates bacterium NSP017, the DAI index of mice was significantly reduced to 0.71 (p<0.001); the DAI value of mice in the NSP017 + konjac glucomannan group decreased to 0.58 (p<0.0001), and the DAI value of mice in the konjac glucomannan group decreased to 2.29 (p<0.01), which was lower than the DAI value of 2.58 in the positive drug mesalazine group (p<0.05).

[0073] Figure (B) shows that the spleen index of the model group mice increased from 2.35 (normal group) to 4.80 (p<0.0001). After intervention with Rosbairi bacteria NSP017, the spleen index significantly decreased to 3.02 (p<0.0001). The spleen index of mice in the NSP017 + konjac glucomannan group decreased to 2.76 (p<0.0001), the spleen index of mice in the konjac glucomannan group decreased to 3.15 (p<0.0001), and the spleen index of mice in the positive drug mesalazine group was 3.50 (p<0.0001).

[0074] The above results indicate that the intestinal Rosbairi bacteria NSP017 and its composition with konjac glucomannan of the present invention can reduce the disease activity index and alleviate abnormal splenomegaly in mice with ulcerative colitis. Moreover, the composition of intestinal Rosbairi bacteria NSP017 and konjac glucomannan has the best effect, indicating that intestinal Rosbairi bacteria NSP017 and konjac glucomannan can synergistically alleviate the related symptoms in mice with ulcerative colitis.

[0075] Example 5: Effects of Roseburia intestinalis NSP017 and its composition on the intestinal barrier 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.

[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 shown in Figure 4.

[0077] 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, with the ocludin content in the colon of the model group mice as a reference.

[0078] After intervention with Rosbyrates rubella NSP017, the ocludin content in mice was 2.54 times that of the model group (p<0.05), and the ocludin content in mice in the konjac glucomannan group was 2.93 times that of the model group (p<0.01).

[0079] The ocludin content in mice in the NSP017+ konjac glucomannan group was 3.05 times that of the model group (p<0.001), which was 3.02 times higher than that in mice in the positive drug mesalazine group (p<0.001).

[0080] As shown in Figure (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.

[0081] After intervention with Rosbairi bacteria NSP017, the level of tight junction protein (Claudin-1) in mice was 5.57 times that of the model group (p<0.0001); the level of tight junction protein (Claudin-1) in mice in the NSP017 + konjac glucomannan group was 6.12 times that of the model group (p<0.0001), 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.

[0082] The above results indicate that the intestinal Rosbairi NSP017 and its composition with konjac glucomannan of the present invention can both upregulate the level of tight junction proteins and maintain the integrity of the intestinal barrier. Furthermore, the effect of intestinal Rosbairi NSP017 is superior to that of clinical drugs, and the effect of the composition of intestinal Rosbairi NSP017 and konjac glucomannan is superior to that of konjac glucomannan and clinical drugs.

[0083] Example 6: Effects of Roseburia intestinalis NSP017 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.

[0084] The content of superoxide dismutase (SOD) in colon tissue was determined using an ELISA kit (product of Shanghai Beyotime Biotechnology Co., Ltd.). The experimental results are shown in Figure 5.

[0085] As shown in Figure 5, the SOD content in the colonic tissue of the model group mice decreased from 431.14 U / mg protein (normal group) to 233.31 U / mg protein. After intervention with konjac glucomannan, the SOD content in the colonic tissue of mice was 365.47 U / mg. The SOD content in the colonic tissue of mice in the NSP017 group was 468.32 U / mg (p<0.01); the SOD content in the colonic tissue of mice in the NSP017+konjac glucomannan group was 524.28 U / mg (p<0.001), which was higher than the SOD content of 435.81 U / mg in the colonic tissue of mice in the positive drug mesalazine group.

[0086] The above results indicate that the intestinal Rosbairi bacteria NSP017 and its composition with konjac glucomannan of the present invention can alleviate the oxidative stress level in mice with ulcerative colitis, thereby alleviating colitis-related systemic and organ oxidative damage. Furthermore, the effect of intestinal Rosbairi bacteria NSP017 is superior to that of clinical drugs, and the effect of intestinal Rosbairi bacteria NSP017 and its composition with konjac glucomannan is superior to that of konjac glucomannan and clinical drugs.

[0087] Example 7: Restoration effect of Roseburia intestinalis NSP017 and its composition on valeric acid production in the intestinal flora of 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.

[0088] 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.

[0089] As shown in Figure 6, no valeric acid was produced in the cecal contents of the model group mice.

[0090] Valeric acid was produced in the cecal contents of mice in the NSP017 group and the NSP017+konjac glucomannan group, indicating that both intestinal Rosbairi bacteria NSP017 and its combination with konjac glucomannan can promote the recovery of valeric acid production capacity of intestinal flora in mice with ulcerative colitis. However, the in vitro fermentation results in Example 3 showed that only the combination of intestinal Rosbairi bacteria NSP017 and konjac glucomannan could promote valeric acid production in intestinal flora of colitis mice. The reason may be that there are differences between the in vivo environment and the in vitro environment. During in vitro fermentation, intestinal Rosbairi bacteria NSP017 in carbon-free culture medium has fewer available nutrients, and its acid production capacity may be worse than in the in vivo environment.

[0091] Example 8: Protective effect of valerate (calculated as sodium valerate) on mice with ulcerative colitis. 1. Preparation of sodium valerate solution: Weigh sodium valerate according to the weight of the mice and dissolve it in water. The gavage dose was 100 mg of sodium valerate per kilogram of mouse body weight, and the gavage volume was 0.2 mL.

[0092] 2. Animal Experiment Protocol: Twenty healthy male SPF-grade C57BL / 6J mice aged 6 weeks were randomly divided into two groups: a normal group and a sodium valerate group, with 10 mice in each group.

[0093] 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 7 days. The experimental procedure is shown in Table 4.

[0094] Table 4 Specific implementation plan for animal experiments

[0095] 3. During the DAI index and spleen index experiments, the mice were weighed and scored daily. After the experiment, all mice were sacrificed, spleen tissue was collected, and the weight of the spleen tissue was measured. The spleen index was calculated based on the percentage of the mouse's body weight. The experimental results are shown in Figure 7.

[0096] Figure (A) shows that after the model group mice drank sterile distilled water containing 3% DSS for 7 consecutive days, the DAI value of the mice increased to 3.61. After sodium valerate intervention, the DAI index of the mice decreased significantly to 1.00 (p<0.0001), which was significantly lower than that of the model group.

[0097] Figure (B) shows that the spleen index of the model group mice was 4.74. After sodium valerate intervention, the spleen index of the mice decreased to 3.83 (p<0.01), which was significantly lower than that of the model group.

[0098] The above results indicate that sodium valerate can reduce the disease activity index and alleviate abnormal splenomegaly in mice with ulcerative colitis.

[0099] 4. The specific implementation method for oxidative stress level is the same as in Example 4. After the experiment, the mice were sacrificed and serum and colon tissue samples were collected.

[0100] The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in colon tissue were determined using an ELISA kit (product of Shanghai Beyotime Biotechnology Co., Ltd.). The experimental results are shown in Figure 7.

[0101] As shown in Figure (C), the SOD content in the colon tissue of the model group mice was 10.21 U / mg protein. After intervention with sodium valerate, the SOD content in the colon tissue of the mice was 12.89 U / mg protein (p<0.05), which was significantly higher than that in the colon tissue of the model group mice.

[0102] As shown in Figure (D), the MDA content in the colon tissue of the model group mice was 0.0014 nmol / mg protein. After intervention with sodium valerate, the MDA content in the colon tissue of the mice was 0.0009 U / mg protein (p<0.01), which was significantly lower than the MDA content in the colon tissue of the model group mice.

[0103] The above results indicate that sodium valerate can alleviate oxidative stress levels in mice with ulcerative colitis, thereby alleviating colitis-related systemic and organ oxidative damage.

[0104] 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 Roseburia intestinalis NSP017 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: 66955.

2. A composition, characterized in that, The composition contains the intestinal Rosbyrates NSP017 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 number of Rosbyrate's bacterium NSP017 cells is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g; the mass percentage of the konjac glucomannan is not less than 0.2%.

4. The composition according to claim 3, characterized in that, The konjac glucomannan contains more than 50% mannose by mass.

5. A product containing the Enterobacter rosbyreii NSP017 of claim 1 or the composition of any one of claims 2 to 4, characterized in that, The products are microbial agents, food, pharmaceutical compositions, health products, or feed additives.

6. The product as described in claim 5, characterized in that, The product contains at least 1 × 10⁻⁶ cells of Rosbyrates intestinalis NSP017. 6 CFU / mL or 1×10 6 CFU / g.

7. The use of the Enterobacter rosbyresia NSP017 of claim 1 or the composition of any one of claims 2 to 4 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, or oral liquids.

9. The application of the intestinal Rosbyrate's bacterium NSP017 as described in claim 1 in improving the effect of konjac glucomannan on colitis.

10. The use of the intestinal Rosbairi bacteria NSP017 as described in claim 1 in the preparation of valeric acid.

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