Use of a filter paper-dissolving clostridium ramosum in preventing and / or treating ulcerative colitis

Abstract

The application provides an application of filter paper-lysing Clostridium ramosum in preventing and / or treating ulcerative colitis, and belongs to the technical field of microorganisms. Ruminiclostridium papyrosolvens The filter paper-lysing Clostridium ramosum is Clostridium ramosum (Clostridium ramosum) DSM 2782. It is proved by experiments that the strain improves the colon shortening, colon hemorrhage, stool 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] The application belongs to the technical field of microorganisms, and particularly relates to an application of filter paper-tolerant Clostridium leptum in preventing and / or treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis is a chronic idiopathic inflammatory disease mainly involving the mucosa and submucosa of the rectum and colon. Although mesalazine, corticosteroids, immunosuppressants and biological agents have certain effects on ulcerative colitis, there is currently no cure, and the disease is prone to recurrence, which seriously affects the quality of life of patients and significantly increases the risk of colon cancer. Therefore, it is of great medical and social significance to further explore and find targeted clinical interventions for ulcerative colitis.

[0003] Previous studies have found that intestinal flora plays an important role in the occurrence and development of ulcerative colitis, and many experiments and clinical studies have confirmed that probiotic-based therapy has high application prospects. The most common probiotic preparations are lactobacilli and bifidobacteria, etc. The above strains may not meet the intestinal flora characteristics of patients with ulcerative colitis, and further screening of suitable individualized probiotics is still needed. There is no relevant report on the use of filter paper-tolerant Clostridium leptum in treating ulcerative colitis. SUMMARY

[0004] Therefore, the application provides an application of filter paper-tolerant Clostridium leptum in preventing and / or treating ulcerative colitis. Filter paper-tolerant Clostridium leptum DSM 2782 improves colon shortening, colon bleeding, fecal character change, and colon tissue damage caused by ulcerative colitis modeling, and has the effect of preventing and / or treating ulcerative colitis.

[0005] To solve the above technical problems, the application provides the following technical solutions.

[0006] The application provides an application of filter paper-tolerant Clostridium leptum in preparing a product for preventing and / or treating ulcerative colitis.

[0007] Preferably, the filter paper-tolerant Clostridium leptum is filter paper-tolerant Clostridium leptum (Clostridium leptum) DSM 2782, which is preserved in the German Collection of Microorganisms and Cell Cultures. Ruminiclostridium papyrosolvens The application provides a filter paper-tolerant Clostridium leptum DSM 2782, which is preserved in the German Collection of Microorganisms and Cell Cultures.

[0008] The application provides a bacterial liquid for preventing and / or treating ulcerative colitis, which contains the filter paper-tolerant Clostridium leptum.

[0009] The application provides a preparation method of a bacteria solution for preventing and / or treating ulcerative colitis, which comprises the following steps: inoculating the Ruminococcus albus into a liquid culture medium for culture, and then transferring the culture mixture into a Columbia blood plate for culture, and adjusting the concentration of the bacteria solution.

[0010] Preferably, the liquid culture medium is DSMZ medium 289.

[0011] Preferably, the culture condition in the liquid culture medium comprises: an anaerobic environment, a culture temperature of 25-38 DEG C, and a culture time of 24-72 h.

[0012] Preferably, the culture condition in the Columbia blood plate comprises: an anaerobic environment, a culture temperature of 25-38 DEG C, and a culture time of 24-72 h.

[0013] Preferably, the solvent for adjusting the concentration of the bacteria solution is a phosphate buffer solution with a pH of 7.2-7.4.

[0014] Preferably, the concentration of the bacteria solution is 1x10 8 1x10 10 CFU / mL.

[0015] The application provides application of the bacteria solution or the bacteria solution obtained by the preparation method in preparing a product for preventing and / or treating ulcerative colitis.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The application first proposes application of Ruminococcus albus in preventing and / or treating ulcerative colitis. Through animal experiment verification, the Ruminococcus albus DSM 2782 improves colon shortening, colon hemorrhage, change of stool character, colon tissue damage caused by DSS, and has the effect of preventing and / or treating ulcerative colitis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a colony shape diagram of Ruminococcus albus on a Columbia blood plate.

[0019] Figure 2 It is a result diagram of Ruminococcus albus improving colon shortening caused by DSS.

[0020] Figure 3 It is a result diagram of Ruminococcus albus improving colon hemorrhage caused by DSS.

[0021] Figure 4 It is a result diagram of Ruminococcus albus improving colon shortening caused by DSS with statistical difference.

[0022] Figure 5Figure for resolving filter paper Clostridium ramosum to improve DSS-induced colonic histological injury results.

[0023] Figure 6 Figure for resolving filter paper Clostridium ramosum to improve DSS-induced colonic histological injury with statistical difference results. DETAILED DESCRIPTION

[0024] The application provides a use of resolving filter paper Clostridium ramosum in preparation of a product for preventing and / or treating ulcerative colitis.

[0025] In the application, the resolving filter paper Clostridium ramosum is Clostridium ramosum (Clostridium ramosum) DSM 2782, which is preserved in the German Collection of Microorganisms and Cell Cultures (DSMZ) and purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (No. B89621, Chinese name: Clostridium ramosum). Ruminiclostridium papyrosolvens ) DSM 2782, which is preserved in the German Collection of Microorganisms and Cell Cultures (DSMZ) and purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (No. B89621, Chinese name: Clostridium ramosum).

[0026] The application also provides a bacteria liquid for preventing and / or treating ulcerative colitis, which contains the resolving filter paper Clostridium ramosum.

[0027] The application also provides a preparation method of the bacteria liquid for preventing and / or treating ulcerative colitis, which comprises the following steps: inoculating the resolving filter paper Clostridium ramosum into a liquid culture medium for culture, then transferring the culture mixture into a Columbia blood plate for culture, and adjusting the concentration of the bacteria liquid. The liquid culture medium and the Columbia blood plate in the application are both from commercially available products well known in the art, unless otherwise specified. The liquid culture medium in the application is preferably DSMZ medium 289, which is prepared according to the DSMZ medium 289 disclosed by the German Collection of Microorganisms and Cell Cultures.

[0028] In the application, the culture conditions in the liquid culture medium include: an anaerobic environment, a culture temperature of 25-38°C, and a culture time of 24-72h; preferably, the culture temperature is 37°C, and the culture time is 48h; the culture conditions in the Columbia blood plate include: an anaerobic environment, a culture temperature of 25-38°C, and a culture time of 24-72h; preferably, the culture temperature is 37°C, and the culture time is 48h.

[0029] In the application, the reagent used for adjusting the concentration of the bacteria liquid is a phosphate buffer solution with a pH of 7.2-7.4. In the application, the round and milky white colonies on the Columbia blood plate are picked or scraped, and then mixed uniformly in the phosphate buffer solution to prepare the bacteria liquid with a required concentration. The concentration of the bacteria liquid adjusted in the application is 1×10 8 -1×10 10 CFU / mL, preferably 5×108 -0.5 x 10 10 CFU / mL, further preferably 1 x 10 9 CFU / mL.

[0030] The present application also provides the use of the bacterial liquid or the bacterial liquid obtained by the preparation method in the preparation of a product for preventing and / or treating ulcerative colitis. The animal experiment verifies that the strain improves the colon shortening, colon bleeding, stool character change, and colon tissue damage caused by the modeling of ulcerative colitis, and has the effect of preventing and / or treating ulcerative colitis.

[0031] In the present application, all components or reagents or culture media are commercially available products well known to those skilled in the art, unless otherwise specified.

[0032] The technical solutions in the present application will be described clearly and completely in combination with the examples in the present application. Obviously, the described examples are only some of the examples of the present application, but not all. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0033] The strain used in the following examples is: Fibrobacter succinogenes (Fibrobacter succinogenes) DSM 2782, deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ), purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with the number B89621, and the Chinese name is Fibrobacter succinogenes. Ruminiclostridium papyrosolvens

[0034] Example 1 Culture and identification of Fibrobacter succinogenes

[0035] The liquid medium is prepared according to the DSZM Medium 289 of the German Collection of Microorganisms and Cell Cultures (DSMZ), mixed and sterilized at 121°C for 30 min, cooled to below 50°C, and then placed in an anaerobic box composed of 80% N2, 10% H2 and 10% CO2 mixed gas to deoxygenate overnight.

[0036] The purchased Fibrobacter succinogenes freeze-dried powder is transferred to the deoxygenated liquid medium in the anaerobic box, and after being fully activated and cultured at 37°C for 48 h, powdery precipitate is visible in the liquid medium. After mixing by blowing, the inoculation ring is streaked onto the Columbia blood plate, and then cultured at 37°C in an anaerobic environment for 48 h. Round, milky white colonies are visible. Figure 1 .

[0037] ​Example 2 Ruminococcus flavefaciens strain 091 alleviates dextran sodium sulfate (DSS)-induced ulcerative colitis model in mice

[0038] (1) Preparation of Ruminococcus flavefaciens strain 091 solution

[0039] The purchased Ruminococcus flavefaciens strain 091 freeze-dried powder was transferred to the oxygen-free liquid medium prepared in Example 1, and after 48 h of activation and culture at 37°C, a powdery precipitate was observed in the liquid medium. 200 μL of the mixture was taken to a Columbia blood plate and evenly smeared with an L-shaped applicator stick. After 48 h of culture at 37°C in an anaerobic environment, round milky-white colonies appeared on the blood plate.

[0040] The bacterial count was performed using a McFarland turbidimeter. 4 mL of sterile anaerobic phosphate buffer solution (PBS) with pH 7.2 was added to a sterile tube, and the McFarland turbidimeter was adjusted to zero. Then, the colonies cultured in the blood plate were scraped with a sterile cotton swab and shaken up and down in the above-mentioned sterile tube to shake off the colonies. The turbidity was measured again to obtain the specific McFarland concentration unit (McF). According to the conversion formula of 0.5 McF = 1.5 x 10 8 CFU / mL, the specific bacterial solution concentration can be obtained. The colonies in the blood plate were scraped with a sterile cotton swab, and the bacterial solution concentration was adjusted to 1 x 10 9 CFU / mL for mouse gavage.

[0041] (2) Ruminococcus flavefaciens strain 091 treatment of ulcerative colitis model in mice

[0042] After pre-feeding for one week, 30 six-week-old C57BL / 6J male mice were grouped into three groups: PBS group (control group), DSS group, and DSS + Ruminococcus flavefaciens strain 091 group (DSS + R.p group). The PBS group mice were given 200 μL / d of PBS gavage treatment for 17 days, and from the 10th day, the drinking water was changed to sterile water. The DSS + PBS group mice were given 200 μL / d of PBS gavage treatment for 17 days, and from the 10th day, the drinking water was changed to DSS solution. The DSS + R.p group was given 200 μL / d of Ruminococcus flavefaciens strain 091 solution gavage treatment for 17 days, and from the 10th day, the drinking water was changed to DSS solution.

[0043] After the experiment, the mice were dissected and sampled, and the colon length of the mice in natural state was measured, and the results are shown in Figure 2 , which shows that the addition of Ruminococcus flavefaciens strain 091 improves the colon shortening caused by modeling. The colon of the mice was longitudinally dissected along the mesentery, and it was found that the addition of Ruminococcus flavefaciens strain 091 improved the colon bleeding and fecal character change caused by modeling, and the results are shown in Figure 3 . Statistical analysis of the colon length among the groups showed that Ruminococcus flavefaciens strain 091 had a statistically significant improvement in colon shortening, and the results are shown inFigure 4 .

[0044] After the mouse whole colon was dissected longitudinally along the mesentery, the colon roll was prepared, then fixed with 4% paraformaldehyde at room temperature for 48 h, and then the colon roll was dehydrated, paraffin-embedded, sectioned, dewaxed, hematoxylin-eosin stained (HE staining), dehydrated, and mounted. Then the pathological changes of the colon tissues of the mice in each group were detected by microscope observation, and the influence of Clostridium xylanolyticum on intestinal histology was evaluated. It can be seen that the histological damage caused by modeling is reduced after supplementing Clostridium xylanolyticum, and the results are as follows Figure 5 . Statistical analysis of the pathological scores between groups showed that the improvement of Clostridium xylanolyticum on colon histological damage had statistical difference, and the results were as follows Figure 6 .

[0045] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. The use of Clostridium rumen-derived bacteria in the preparation of products for the prevention and / or treatment of ulcerative colitis, characterized in that, The *Ruminiclostridium papyrosolvens* strain mentioned is *Ruminiclostridium papyrosolvens* DSM2782, deposited at the German Center for Microbiology and Cell Culture.

2. The use of a bacterial culture containing Clostridium rumenii DSM 2782 as described in claim 1 in the preparation of products for the prevention and / or treatment of ulcerative colitis.

3. The application as described in claim 2, characterized in that, The method for preparing the bacterial culture includes the following steps: inoculating the *Clostridium rumenella* of claim 1 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.

4. The application as described in claim 3, characterized in that, The liquid culture medium is DSMZ medium 289.

5. The application as described in claim 3, characterized in that, The culture conditions in liquid culture medium include: anaerobic environment, culture temperature of 25-38℃, and culture time of 24-72h.

6. The application as described in claim 3, 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.

7. The application as described in claim 3, characterized in that, The solvent used to adjust the bacterial concentration is a phosphate buffer solution with a pH of 7.2-7.

4.

8. The application as described in claim 3, characterized in that, Adjust the bacterial concentration to 1×10 8 -1×10 10 CFU / mL.

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