Application of filter paper desolventizing clostridium ruminum in prevention and / or treatment of ulcerative colitis

Abstract

The invention provides application of filter paper rumen clostridium to prevention and / or treatment of ulcerative colitis, and belongs to the technical field of microorganisms. The rumen clostridium capable of decomposing the filter paper, disclosed by the invention, is a rumen clostridium capable of decomposing the filter paper, and the rumen clostridium capable of decomposing the filter paper is DSM 2782. Experiments prove that the bacterial strain improves colon shortening, colon bleeding, excrement character change and colon tissue damage caused by ulcerative colitis modeling, and has the effect of preventing and / or treating ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Clostridium rumenella in the prevention and / or treatment of ulcerative colitis. Background Technology

[0002] Ulcerative colitis is a chronic, idiopathic inflammatory disease of the colon, primarily affecting the rectal and colonic mucosa and submucosa. While mesalazine, corticosteroids, immunosuppressants, and biologics have shown some efficacy in treating ulcerative colitis, there is currently no cure. The condition is prone to recurrence, severely impacting patients' quality of life and significantly increasing the risk of colon cancer. Therefore, in-depth research and the search for targeted clinical interventions for ulcerative colitis are of significant medical and social importance.

[0003] Previous studies have found that the gut microbiota plays an important role in the development and progression of ulcerative colitis, and multiple experimental and clinical studies have also confirmed the high application potential of probiotic-based therapies. Currently, the most common probiotic preparations include Lactobacillus and Bifidobacterium, but these strains may not necessarily match the gut microbiota characteristics of ulcerative colitis patients, requiring further screening for suitable, individualized probiotics. Currently, there are no reports on the use of Clostridium rumen-derived *Ruminococcus* in the treatment of ulcerative colitis. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of Clostridium rumeniformis in the prevention and / or treatment of ulcerative colitis. Clostridium rumeniformis DSM 2782 improves colonic shortening, colonic bleeding, changes in stool characteristics, and colonic tissue damage caused by ulcerative colitis modeling, and has the effect of preventing and / or treating ulcerative colitis.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides the use of Clostridium rumenella in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0006] Preferably, the *Clostridium rumenella* is *Clostridium rumenella* (also known as *Clostridium rumenella*). Ruminiclostridium papyrosolvens DSM 2782, deposited at the German Center for Microbiology and Cell Culture.

[0007] The present invention provides a bacterial solution for the prevention and / or treatment of ulcerative colitis, the bacterial solution containing the Clostridium rumen-derived strain.

[0008] This invention provides a method for preparing a bacterial culture for the prevention and / or treatment of ulcerative colitis, comprising the following steps: inoculating the *Clostridium rumenella* into a liquid culture medium for culture, then transferring the culture mixture into a Columbia blood agar plate for culture, and adjusting the bacterial concentration.

[0009] Preferably, the liquid culture medium is DSMZ medium 289.

[0010] Preferred culture conditions in liquid culture medium include: anaerobic environment, culture temperature of 25-38℃, and culture time of 24-72h.

[0011] Preferred culture conditions in Columbia blood agar plates include: anaerobic environment, culture temperature of 25-38℃, and culture time of 24-72h.

[0012] Preferably, the solvent used to adjust the bacterial concentration is a phosphate buffer solution with a pH of 7.2-7.4.

[0013] Preferably, the adjusted bacterial concentration is 1×10⁻⁶. 8 -1×10 10 CFU / mL.

[0014] This invention provides the use of the bacterial solution or the bacterial solution obtained by the preparation method in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to propose the application of *Clostridium rumenella* in the prevention and / or treatment of ulcerative colitis. Animal experiments have verified that *Clostridium rumenella* DSM 2782 improved colonic shortening, colonic bleeding, changes in stool characteristics, and colonic tissue damage caused by ulcerative colitis modeling, demonstrating its role in the prevention and / or treatment of ulcerative colitis. Attached Figure Description

[0016] Figure 1 This image shows the colony morphology of Clostridium rumenella on a Columbia blood agar plate.

[0017] Figure 2 The results of using Clostridium rumeniol to improve DSS-induced colonic shortening are shown in the figure.

[0018] Figure 3 Figure showing the results of using Clostridium rumen-derived bacteria to improve colonic bleeding caused by DSS.

[0019] Figure 4 The results show that Clostridium rumenella strains improved DSS-induced colonic shortening with statistically significant differences.

[0020] Figure 5Image showing the results of using Clostridium rumeniol to improve DSS-induced colonic histological damage.

[0021] Figure 6 The results show that Clostridium rumenella strains improved DSS-induced colonic histological damage. Detailed Implementation

[0022] This invention provides the use of Clostridium rumenella in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0023] In this invention, the *Clostridium rumenella* is *Clostridium rumenella* (…). Ruminiclostridium papyrosolvens DSM 2782, deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (No. B89621, Chinese name: Clostridium difficile).

[0024] The present invention also provides a bacterial solution for the prevention and / or treatment of ulcerative colitis, the bacterial solution containing the Clostridium rumen-derived strain.

[0025] This invention also provides a method for preparing a bacterial culture for the prevention and / or treatment of ulcerative colitis, comprising the following steps: inoculating the *Clostridium rumenella* into a liquid culture medium and culturing it, then transferring the culture mixture into Columbia blood agar plates and culturing it, adjusting the bacterial concentration. Unless otherwise specified, the liquid culture medium and Columbia blood agar plates used in this invention are commercially available products well-known in the art. The liquid culture medium used in this invention is preferably DSMZ medium 289, prepared according to the DSMZ medium 289 disclosed by the German Center for Microbiology and Cell Culture.

[0026] In this invention, the culture conditions in the liquid culture medium include: an anaerobic environment, a culture temperature of 25-38℃, and a culture time of 24-72h; preferably, a culture temperature of 37℃ and a culture time of 48h; the culture conditions in the Columbia blood agar plate include: an anaerobic environment, a culture temperature of 25-38℃, and a culture time of 24-72h; preferably, a culture temperature of 37℃ and a culture time of 48h.

[0027] In this invention, the reagent used to adjust the bacterial suspension concentration is a phosphate buffer solution with a pH of 7.2-7.4. In this invention, round, milky-white colonies are picked or scraped from Columbia blood agar plates, placed in the phosphate buffer solution, and mixed thoroughly to prepare the desired bacterial suspension concentration. The bacterial suspension concentration adjusted in this invention is 1 × 10⁻⁶. 8 -1×10 10 CFU / mL, preferably 5×10⁻⁶8 -0.5×10 10 CFU / mL, further preferably 1×10⁻⁶ 9 CFU / mL.

[0028] This invention also provides the application of the bacterial culture or the bacterial culture obtained by the preparation method in the preparation of products for the prevention and / or treatment of ulcerative colitis. Animal experiments have verified that this strain improves colonic shortening, colonic bleeding, changes in stool characteristics, and colonic tissue damage caused by ulcerative colitis modeling, thus exhibiting a preventive and / or therapeutic effect on ulcerative colitis.

[0029] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0030] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] The strain used in the following examples is: Clostridium rumenella (…). Rumiclostridium papyrosolvens DSM2782, deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with the serial number B89621. The Chinese name of the purchased product is Clostridium difficile.

[0032] Example 1: Culture and identification of Clostridium rumenella from filter paper The liquid culture medium was prepared according to DSMZ Medium 289 from the German Center for Microbial and Cell Culture Collection (DSMZ). After mixing, it was autoclaved at 121°C for 30 min, cooled to below 50°C, and then placed in an anaerobic chamber composed of a mixed gas of 80% N2, 10% H2 and 10% CO2 overnight for deoxygenation.

[0033] In an anaerobic chamber, the purchased lyophilized Clostridium rumeniae powder was transferred to a deoxygenated liquid culture medium. After full activation and incubation at 37°C for 48 hours, powdery precipitate was visible in the liquid culture medium. After mixing by pipetting, the precipitate was streaked onto Columbia blood agar plates using an inoculation loop. The plates were then incubated at 37°C under anaerobic conditions for another 48 hours, resulting in round, milky-white colonies. Figure 1 .

[0034] Example 2: A mouse model of ulcerative colitis induced by dextran sulfate sodium (DSS) and alleviated by Clostridium rumenella var. rumenella. (1) Preparation of Clostridium rumen culture on filter paper The purchased lyophilized Clostridium rumeniol powder was transferred to the deoxygenated liquid culture medium prepared in Example 1. After being fully activated at 37°C and cultured for 48 hours, powdery precipitate was visible in the liquid culture medium. 200 μL of the mixture was taken and spread evenly on a Columbia blood agar plate with an L-shaped spreader. The plate was then cultured at 37°C under anaerobic conditions for 48 hours. Circular milky white colonies were then visible on the blood agar plate.

[0035] Bacterial counting was performed using a McFarland turbidimeter. 4 mL of sterile anaerobic phosphate-buffered saline (PBS) at pH 7.2 was added to a sterile tube. The McFarland turbidimeter was zeroed. Colonies from the culture were then scraped from a blood agar plate using a sterile swab and transferred to the sterile tube. The colonies were shaken off and the turbidity was measured again. The McFarland concentration (McF) was then determined. The formula is: 0.5 McFarland = 1.5 × 10⁻⁶. 8 The conversion formula for CFU / mL can be used to obtain the specific bacterial concentration. Collect colonies from a blood agar plate using a sterile cotton swab and adjust the bacterial concentration to 1×10⁻⁶. 9 CFU / mL was administered to mice via gavage.

[0036] (2) Clostridium rumenella for the treatment of mouse ulcerative colitis model Thirty 6-week-old male C57BL / 6J mice were pre-fed for one week and then divided into three groups: PBS group (control group), DSS group, and DSS+Clostridium rumenella group (DSS+Rp group). Mice in the PBS group were administered 200 μL / day of PBS by gavage for 17 days, with sterile water replaced by drinking water starting on day 10. Mice in the DSS+PBS group were administered 200 μL / day of PBS by gavage for 17 days, with DSS solution replaced by drinking water starting on day 10. Mice in the DSS+Rp group were administered 200 μL / day of Clostridium rumenella bacterial suspension by gavage for 17 days, with DSS solution replaced by drinking water starting on day 10.

[0037] After the experiment, anatomical sampling was performed to measure the length of the mouse colon in its natural state. The results are as follows: Figure 2 It was observed that supplementation with *Clostridium rumenella* improved the colonic shortening induced by the model. Longitudinal dissection of the mouse colon along the mesentery showed that supplementation with *Clostridium rumenella* improved colonic bleeding and changes in fecal characteristics induced by the model, as shown in the results. Figure 3 Statistical analysis of colon length among the groups showed that *Clostridium rumenii* significantly improved colon shortening. The results are as follows: Figure 4 .

[0038] The entire colon of mice was longitudinally dissected along the mesentery to create colon rolls, which were then fixed with 4% paraformaldehyde at room temperature for 48 hours. The colon rolls were then dehydrated, embedded in paraffin, sectioned, dewaxed, stained with hematoxylin and eosin (HE staining), dehydrated again, and mounted. Microscopic observation was then used to examine the pathological changes in the colon tissue of each group of mice and to evaluate the effect of *Clostridium rumeniformis* on intestinal histology. It was observed that supplementation with *Clostridium rumeniformis* reduced the histological damage caused by modeling, as shown in the results. Figure 5 Statistical analysis of pathological scores among the groups showed that *Clostridium rumenella* (filter paper) significantly improved colonic histological damage. The results are as follows: Figure 6 .

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of Clostridium rumenella in the preparation of products for the prevention and / or treatment of ulcerative colitis.

2. The application as described in claim 1, characterized in that, The *Clostridium rumenellae* mentioned is *Clostridium rumenellae* (…). Rumiclostridium papyrosolvens DSM 2782, deposited at the German Center for Microbiology and Cell Culture.

3. A bacterial solution for the prevention and / or treatment of ulcerative colitis, characterized in that, The bacterial solution contains Clostridium rumenaeum as described in claim 1 or 2.

4. A method for preparing a bacterial solution for the prevention and / or treatment of ulcerative colitis, characterized in that, The procedure includes the following steps: inoculating the *Clostridium rumenella* as described in any one of claims 1-3 into a liquid culture medium for culture, then transferring the culture mixture into a Columbia blood agar plate for culture, and adjusting the bacterial concentration.

5. The preparation method according to claim 4, characterized in that, The liquid culture medium is DSMZ medium 289.

6. The preparation method according to claim 4, characterized in that, The culture conditions in liquid culture medium include: anaerobic environment, culture temperature of 25-38℃, and culture time of 24-72h.

7. The preparation method according to claim 4, characterized in that, The culture conditions in Columbia blood agar plates include: anaerobic environment, culture temperature of 25-38℃, and culture time of 24-72h.

8. The preparation method according to claim 4, characterized in that, The solvent used to adjust the bacterial concentration is a phosphate buffer solution with a pH of 7.2-7.

4.

9. The preparation method according to claim 4, characterized in that, The bacterial culture concentration was adjusted to 1×10⁻⁶. 8 -1×10 10 CFU / mL.

10. The use of the bacterial solution as described in claim 3 or the bacterial solution obtained by the preparation method according to any one of claims 4-9 in the preparation of products for the prevention and / or treatment of ulcerative colitis.

Images