Brawler's bacteria EMF75-02B1B and application thereof

Abstract

The invention relates to the technical field of microbial medicines, and particularly discloses Brawler's bacteria EMF75-02B1B and an application of the Brawler's bacteria EMF75-02B1B. The invention discloses Blauria sp. EMF75-02B1B, which is preserved in the Guangdong Microbial Culture Collection Center on September 2, 2024, and the preservation number of the Blauria sp. EMF75-02B1B is GDMCC No: 65084. A mouse model proves that the Brawler bacteria EMF75-02B1B can obviously improve the disease activity index (DAI) of a dextran sodium sulfate induced ulcerative colitis mouse, relieve the weight loss and colon shortening degree of the ulcerative colitis mouse, enhance the intestinal barrier function and relieve inflammatory response.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial medicine, and specifically relates to Blautia EMF75-02B1B and uses thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a class of chronic inflammatory diseases that affect the gastrointestinal tract, including Crohn's disease (CD) and ulcerative colitis (UC). The incidence of IBD is increasing worldwide, especially in industrialized countries. Although the exact cause of IBD is not fully understood, it is generally believed to be caused by a complex interaction between genetics, the immune system, environmental factors, and the intestinal microbiota.

[0003] With the development of metagenomics and microbiome, the relationship between intestinal microecology and host health has gradually been revealed. The balance of intestinal flora has a potential impact on the prevention and treatment of inflammatory diseases. Specific probiotic strains may have a positive effect on intestinal barrier function by regulating the intestinal environment and the host's metabolic pathways.

[0004] At present, clinical treatments for ulcerative colitis (UC) mainly include: (1) Drug treatment Salicylic acid drugs: such as sulfasalazine (SASP) and mesalazine, are used to treat patients with mild to moderate UC, but may cause side effects such as gastrointestinal reactions, headaches, and allergic reactions.

[0005] Glucocorticoids: such as betamethasone (Budesonide), used in patients with severe or fulminant UC, may cause side effects such as metabolic disorders and water retention, and are usually only used as a short-term emergency treatment.

[0006] Immunosuppressants: such as cyclosporine, which affect the progression of the immune response by inhibiting the production of IL-2 by T cells, may cause nephrotoxicity and increase the risk of secondary infection, and are usually used as an adjuvant treatment.

[0007] Biological agents: such as the anti-tumor necrosis factor-α (TNF-α) drugs infliximab and adalimumab, although they are effective, they may lead to side effects such as increased risk of infection and allergic reactions, and are costly.

[0008] (2) Surgical treatment When medical treatment is ineffective or serious complications occur, surgical treatment, such as colectomy, may be necessary.

[0009] However, these treatments are often accompanied by side effects or high costs, and not all patients can achieve satisfactory results. Summary of the invention

[0010] The purpose of the present invention is to provide a Blautia EMF75-02B1B and its use in view of the above-mentioned deficiencies in the prior art. Blautia EMF75-02B1B can significantly improve the disease activity index (DAI) of UC mice induced by DSS (dextran sulfate sodium), control the weight loss and colon shortening of UC mice, improve the intestinal barrier function and reduce the inflammatory response, and compared with the existing therapeutic drugs, it shows a better therapeutic effect, providing new insights and strategies for the treatment of IBD.

[0011] To achieve the above object, the present invention adopts the following technical solution: The first object of the present invention is to provide Blautia EMF75-02B1B, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65084.

[0012] The second object of the present invention is to provide a composition, which comprises Blautia with a deposit number of GDMCC No:65084.

[0013] The amount of live bacteria of Blautia EMF75-02B1B contained in the above composition is (1~10000)×10 6 cfu / g.

[0014] The third object of the present invention is to provide the use of the above-mentioned Blautia EMF75-02B1B or the composition in the preparation of drugs for preventing and alleviating inflammatory bowel disease.

[0015] Furthermore, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0016] Furthermore, the Blautia EMF75-02B1B can significantly improve the disease activity index of ulcerative colitis induced by sodium dextran sulfate.

[0017] Furthermore, the Blautia EMF75-02B1B can control colon shortening and reduce inflammatory response.

[0018] Furthermore, the Blautia EMF75-02B1B can enhance the intestinal barrier function.

[0019] The fourth object of the present invention is to provide a health food, which comprises the bacteria or composition of the above-mentioned Blautia EMF75-02B1B, and a food-acceptable auxiliary material.

[0020] The fifth object of the present invention is to provide a medicine, which comprises the above-mentioned Blautia EMF75-02B1B bacteria or composition, and pharmaceutically acceptable excipients.

[0021] The Blautia EMF75-02B1B or fermentation liquid or composition can be used as the only active ingredient with the function of relieving colitis, or it can contain other active ingredients. The drug provided by the present invention can be used alone or in combination with other products with the function of relieving colitis. The present invention does not specifically limit the concentration of live Blautia bacteria in the colitis relieving product, and it can be conventionally selected according to actual needs. The live Blautia bacteria concentration in the product is 1×10 8 ~1×10 10 CFU / mL. As an embodiment, the colitis is relieved.

[0022] The dosage form of the drug can be solution, powder, granules, capsule or tablet.

[0023] The sixth object of the present invention is to provide a bacterial agent, wherein the above-mentioned Blautia EMF75-02B1B strain is inoculated into a culture medium for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged separately, mixed with a protective agent and freeze-dried to obtain Blautia EMF75-02B1B strain powder; the Blautia EMF75-02B1B strain powder is prepared according to the ratio of live bacteria count to obtain the bacterial agent.

[0024] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: The present invention isolates and screens a strain of microorganisms from adult feces samples, and the taxonomic name is Blautia The strain of sp001304935, Blautia EMF75-02B1B, was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 2, 2024, with a deposit number of GDMCC No: 65084. The strain Blautia EMF75-02B1B can effectively reduce the inflammatory response and show a relieving effect in the treatment of intestinal inflammatory bowel disease. Specifically, Blautia EMF75-02B1B can significantly reduce the disease activity index (DAI) of the ulcerative colitis (UC) mouse model induced by DSS (dextran sulfate sodium). In addition, it can effectively control the weight loss and colon shortening of UC mice, while improving the intestinal barrier function and reducing the occurrence of inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of weight monitoring results provided by an embodiment of the present invention; Figure 2 A schematic diagram of DAI scoring of the Blautia EMF75-02B1B group provided in an embodiment of the present invention; Figure 3 A schematic diagram of the length of colon tissue provided by an embodiment of the present invention; Figure 4 A schematic diagram of the statistical analysis of colon length provided by an embodiment of the present invention; Figure 5 Representative micrographs of H&E staining of colon tissues of four groups of mice provided in the embodiments of the present invention; Figure 6 A schematic diagram of histopathological scoring provided by an embodiment of the present invention; Figure 7 Schematic diagram of the effect of ELISA on the expression of anti-inflammatory factor IL-10 in colon tissue; Figure 8 Schematic diagram of the effect of ELISA on the expression of anti-inflammatory factor TGF-β1 in colon tissue; Fig. 9 Schematic diagram of the effect of qRT-PCR technology on the expression of inflammatory factor TNF-α in colon tissue; Fig.10 Schematic diagram of the effect of qRT-PCR technology on the expression of anti-inflammatory factor IL-1β in colon tissue; Fig.11 A schematic diagram of the effect of Blautia EMF75-02B1B on intestinal tight junction protein ZO-1 provided in an embodiment of the present invention; Fig.12 A schematic diagram of the effect of Blautia EMF75-02B1B on the intestinal tight junction protein Claudin-2 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0027] The present invention isolates a new strain of Blautia EMF75-02B1B from fecal samples of healthy adults. The strain can effectively reduce inflammatory response and show a relieving effect in the treatment of intestinal inflammatory bowel disease. Blautia EMF75-02B1B was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65084.

[0028] Example 1 Origin, isolation and identification of Blautia sp. EMF75-02B1B.

[0029] (1) Sample collection Fecal samples from healthy adults were collected and transferred to sample tubes under sterile conditions and brought back to the laboratory for processing within 1 hour. All subjects were informed of the nature of the sampling and the experiment and their consent was obtained. The samples were strictly screened, and samples from patients with the following conditions were excluded: patients with a history of other types of cancer, other serious systemic diseases or digestive system diseases, chronic viral infections, bacterial infections before surgery, and those who had received immunosuppressive therapy (such as chemotherapy, oral steroids, etc.) or other cancer-related treatments before surgery.

[0030] (2) Isolation and purification of Blautia 0.2 g of fecal sample was mixed in 1 ml of sterile PBS, spread on the culture medium after gradient dilution, cultured under anaerobic conditions at 37°C, and single colonies were selected for isolation.

[0031] (3) Preservation of bacterial strains The purified strain was cultured to a concentration of about 1×10 9 cfu / ml, and after adding 20% ​​glycerol, they were stored at -80°C for long term storage.

[0032] (4) Genomic DNA extraction and 16S rDNA identification Genomic DNA was extracted using a commercial kit and amplified by PCR using universal 16S rDNA primers. The strain was sequenced and compared with the EZbiocloud database. Blautia sp., the sequencing sequence is shown in SEQ ID NO.1.

[0033] Example 2 Analysis of the genomic characteristics of Blautia EMF75-02B1B.

[0034] (1) Whole genome sequencing and assembly The whole genome of Blautia EMF75-02B1B was sequenced using high-throughput sequencing technology, and 47 sequences were obtained with a total length of 3,252,807 bp.

[0035] (2) Genome annotation The GTDB-Tk tool was used for genome classification, and 2021 coding DNA sequences (CDS) were predicted, including 7 rRNA genes, 58 tRNA genes, and 2 tmRNA genes.

[0036] (3) Metabolic pathway analysis Metabolic pathway annotation was performed using eggNOG-mapper and the KEGG database, and it was found that the strain had strong metabolic functions and environmental adaptability.

[0037] Example 3 Safety evaluation of Blautia sp. EMF75-02B1B.

[0038] (1) Antibiotic resistance gene detection The antibiotic resistance genes were predicted using the RGI tool and the CARD database, and genes such as rpoB mutant, tet(W), dfrF, and ErmX were found. The genes associated with antibiotic resistance are shown in Table 1.

[0039] Table 1.

[0040] (2) Virulence factor gene detection The VFDB database was used to predict virulence factor genes, and genes related to bacterial adhesion, immune regulation, antiphagocytosis, and nutritional / metabolic factors were found, as shown in Table 2.

[0041] Table 2. Virulence factor gene detection results.

[0042]

[0043] Example 4 Study on the therapeutic effect of Blautiae EMF75-02B1B on colitis mouse model.

[0044] (1) Establishment of animal model The animal experiment in this embodiment selected 37 6-8 week SPF healthy C57BL / 6 mice with an average weight of 18-22g. The mice were adaptively raised in an SPF-grade laboratory environment for one week and randomly assigned to four different experimental groups. The grouping is as follows: normal control group, dextran sodium sulfate (DSS) treatment group, mesalazine (5-ASA) treatment group and EMF-75 treatment group (Blauterella EMF75-02B1B), and the specific grouping is shown in Table 3.

[0045] From day 1 to day 14, mice in the EMF-75-treated group received 1×10 -9 The mice in the DSS-treated group and the normal control group were gavaged with an equal amount of phosphate buffered saline (PBS). From the 8th to the 14th day, except for the normal control group, the mice in other groups drank 2.5% dextran sulfate sodium (DSS) solution freely for 7 days. All the mice in each group had diarrhea (100%), decreased food intake, decreased weight, decreased fur gloss, and the inflammatory bowel disease model was successfully established.

[0046] Table 3. Mouse experiment design table.

[0047]

[0048] (2) Observation and detection indicators and treatment: ① During the modeling and oral gavage, the weight changes, food and water intake, hair gloss, mental state, activity and death of mice were observed, and the severity of enteritis in mice was determined according to the disease activity index (DAI). DAI includes the percentage of weight loss, the degree of fecal diarrhea and blood in the stool. The specific calculation method is: the sum of the scores of the percentage of weight loss, the degree of fecal diarrhea and blood in the stool; among them, the percentage of weight loss score: 0 = no weight loss; 1 = 1-5%; 2 = 5-10%; 3 = 10-20%; 4 = more than 20%; feces score: 0 = solid feces pellets; 1 = solid feces pellets, easy to deform; 2 = unformed stool; 3 = loose stool; 4 = liquid stool; blood in the stool score: 0 = occult blood; negative test; 1 = occult blood test 1+, no visible blood in the stool; 2 = occult blood test 2+; 3 = occult blood test 3+, visible blood in the stool; 4 = severe blood in the stool. The specific DAI scoring criteria are shown in Table 4.

[0049] Table 4. DAI scoring criteria.

[0050]

[0051] Note: DAI = (weight loss score + stool characteristics score + blood in stool score) / 3; the comprehensive score of DAI is between 0 and 4, with 0 representing normal and 4 representing the highest inflammatory activity.

[0052] ②On the 15th day of the experiment, the mice were weighed, blood was collected from their eyes after anesthesia, and the mice were killed by cervical dislocation. Subsequently, the collected blood samples were centrifuged to separate the serum and stored at -80°C for subsequent biochemical index testing. The length of the mouse colon was measured, and samples were taken for tissue sections. Some colon samples were used to make pathological sections for histological analysis under a microscope. The remaining colon tissue was frozen and stored at -80°C for future experimental studies.

[0053] ③ The distal colorectal tissue (about 2 cm away from the anus) was made into a Swiss roll and fixed with 4% paraformaldehyde before routine paraffin embedding and sectioning to obtain colon tissue sections. Hematoxylin-eosin (H&E) staining was performed to detect the pathological condition and score the score. The criteria were as follows: Pathological score included epithelial loss, crypt damage, goblet cell reduction and inflammatory cell infiltration; epithelial loss: 0 = no pathological change; 1 = 0-5% epithelial loss; 2 = 5-10% epithelial loss; 3 = more than 10% epithelial loss; crypt damage: 0 = crypt intact; 1 = 0-10% crypt damaged; 2 = 10-20% crypt damaged; 3 = more than 20% crypt damaged; goblet cell reduction: 0 = none; 1 = mild; 2 = moderate; 3 = severe; inflammatory cell infiltration; 0 = no infiltration; 1 = increased inflammatory cells in the lamina propria; 2 = inflammatory cells extending to the submucosal layer; 3 = transmural inflammatory cell infiltration. The specific histopathological scoring criteria are shown in Table 5.

[0054] Table 5. Pathological histological scoring criteria.

[0055]

[0056] ④Intestinal barrier function analysis The mRNA expression levels of tight junction proteins (ZO-1 and Claudin-2) were evaluated by qRT-PCR, and the expression of tight junction proteins (ZO-1 and Claudin-2) was analyzed by western blot to evaluate the recovery of intestinal barrier function.

[0057] ⑤ Cytokine level determination ELISA was used to determine the levels of anti-inflammatory factors (TGF-β1) in colon tissues; and qRT-PCR was used to determine the levels of pro-inflammatory factors (IL-1β and TNF-α) and pro-inflammatory factors (IL-10) in colon tissues.

[0058] The results of weight monitoring are as follows Figure 1 As shown in the figure, the initial body weight (0d) and the body weight after 14d of intervention were monitored, and the body weight change rate was calculated. It can be seen that DSS-induced inflammatory bowel disease mice will lose weight. After continuous intragastric administration of EMF-75 bacterial solution for 14 days, compared with the model group, the EMF-75 group can significantly improve the weight loss of DSS mice (P<0.05); compared with the 5-ASA group, the results of EMF-75 improving the weight loss of mice were no different from those of the 5-ASA group. The results show that EMF-75 bacterial solution can effectively reduce the weight loss symptoms caused by inflammatory bowel disease.

[0059] The disease activity index (DAI) scores of the four different treatment groups were detected and recorded. The DAI scores after the initial administration of 2.5% dextran sulfate sodium (7 days) and the final administration of 2.5% dextran sulfate sodium (14 days) were monitored. Figure 2 As shown. It can be seen that the disease activity index of the other three groups except the control group increased under the induction of dextran sulfate sodium; compared with the dextran sulfate sodium group, the EMF-75 group and the mesalazine group could significantly reduce the disease activity index of DSS mice (P<0.0001); compared with the mesalazine group, the results of reducing the disease activity index of mice in the EMF-75 group were no different from those in the mesalazine group. The results show that the bacterial solution of the EMF-75 group can effectively reduce the activity index caused by inflammatory bowel disease.

[0060] like Figure 3 The image shown is the length of colon tissue. It can be seen from the figure that EMF-75 bacterial solution can significantly improve the shortening of the colon in mice treated with sodium dextran sulfate.

[0061] like Figure 4 Figure 2 shows the results of statistical analysis of colon length. The colon length of the sodium dextran sulfate group (5.79±0.46 cm) was significantly shorter than that of the control group (8.35±0.52) cm (P<0.0001). The colon length of the EMF-75 group (7.15±0.86 cm) was significantly longer than that of the sodium dextran sulfate group (P<0.05), indicating that EMF-75 may reduce colon shortening in DSS-induced colitis mice.

[0062] like Figure 5 The following are representative micrographs of H&E staining of colon tissues of mice in the four groups. The colon tissue structure of mice in the control group was clear and intact; the colon tissue of mice in the mesalazine group and EMF-75 group was damaged to a certain extent, but some structures were still retained; the colon tissue structure of mice in the sodium dextran sulfate group was severely damaged, and the inflammatory response was obvious.

[0063] like Figure 6The figure shows the results of colon tissue pathology scoring. The pathological condition of the control group was good, while sodium dextran sulfate caused an increase in the pathological score. Mesalazine and EMF-75 may have a certain effect on improving the pathological condition, and the effect of EMF-75 may be better than that of mesalazine. This shows that EMF-75 may reduce the inflammatory level of DSS-induced colitis mice.

[0064] like Figure 7-10 The figure shows the expression results of cellular inflammatory factors. It can be seen that the levels of pro-inflammatory factors (IL-10, IL-1β, TNF-α) in the EMF-75 group were significantly lower than those in the sodium dextran sulfate group (P<0.0001), while the anti-inflammatory factor (TGF-β1) in the EMF-75 group was significantly higher than that in the control group and the sodium dextran sulfate group. This shows that EMF-75 can reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors to reduce the level of inflammatory factors in colitis in mice.

[0065] like Figure 11-12 As shown in the figure, it is the result of enhancing the intestinal barrier function. It can be seen that the levels of occludin-2 and ZO-1 proteins were significantly higher than those in the sodium dextran sulfate group (P<0.0005), indicating that EMF-75 can effectively maintain the integrity and function of the intestinal mucosal barrier.

[0066] Example 5 Preparation of microecological solution preparation containing Blautia EMF75-02B1B.

[0067] (1) Preparation of probiotic suspension A single colony of Blautia EMF75-02B1B was inoculated into MRS anaerobic broth medium and cultured anaerobically at 37°C until the growth plateau phase, and the bacterial suspension of Blautia EMF75-02B1B was collected; the bacterial suspension was centrifuged at 3000×g and 4°C for 10 minutes, the supernatant was removed, the bacterial cells were collected and resuspended with physiological saline to a concentration of 1×10 9 CFU / mL, and a suspension of Blautia EMF75-02B1B was obtained.

[0068] (2) Microecological preparation formula The suspension of Blautia EMF75-02B1B is mixed with a protective agent, a nutritional supplement, etc. according to the number of live bacteria to prepare a microecological preparation suitable for oral administration.

[0069] (3) Preparation stability test The prepared microecological preparations are subjected to stability tests to ensure the activity of probiotics during storage and use.

[0070] Example 6 Preparation of microecological freeze-dried bacterial powder preparation containing Blautia EMF75-02B1B.

[0071] (1) Probiotic culture A single colony of Blautia EMF75-02B1B was inoculated into an MRS anaerobic broth medium, cultured anaerobically at 37°C until the growth plateau phase, and the bacterial liquid of Blautia EMF75-02B1B was collected; the bacterial liquid was centrifuged at 3000×g and 4°C for 10 minutes, the supernatant was removed, the bacterial bodies were collected and mixed with a protective agent, and then freeze-dried to obtain Blautia EMF75-02B1B bacterial powder.

[0072] (2) Microecological preparation formula The bacterial powder of the Blautia EMF75-02B1B strain is mixed with a protective agent, a nutritional supplement, etc. according to the number of live bacteria to prepare a microecological freeze-dried powder preparation suitable for oral administration.

[0073] (3) Preparation stability test The prepared microecological preparations are subjected to stability tests to ensure the activity of probiotics during storage and use.

[0074] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Blautia EMF75-02B1B, which was deposited in Guangdong Microbiological Culture Collection Center on September 2, 2024. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 65084.

2. A composition, characterized in that The composition comprises Blautia sp. EMF75-02B1B with a deposit number of GDMCC No:65084.

3. Use of the Blautia EMF75-02B1B according to claim 1 or the composition according to claim 2 in the preparation of a medicament for preventing and alleviating inflammatory bowel disease.

4. The use according to claim 3, characterized in that The inflammatory bowel disease is ulcerative colitis or Crohn's disease.

5. The use according to claim 3, characterized in that The Blautiae EMF75-02B1B can significantly improve the disease activity index of ulcerative colitis induced by sodium dextran sulfate.

6. The use according to claim 3, characterized in that The Blautiae EMF75-02B1B is able to control colon shortening and reduce inflammatory responses.

7. The use according to claim 3, characterized in that The Blautia EMF75-02B1B can enhance the intestinal barrier function.

8. A health food, characterized in that: The health food comprises the bacterial body of Blautia EMF75-02B1B according to claim 1 or the composition according to claim 2, and a food-acceptable auxiliary material.

9. A drug, characterized in that The drug comprises the bacteria of Blautia EMF75-02B1B according to claim 1 or the composition according to claim 2, and pharmaceutically acceptable excipients.

10. A bacterial agent, characterized in that: The Blautia EMF75-02B1B strain described in claim 1 is inoculated into a culture medium for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged separately, mixed with a protective agent, and then freeze-dried to obtain Blautia EMF75-02B1B strain powder; the Blautia EMF75-02B1B strain powder is prepared according to the ratio of live bacteria count to obtain the bacterial agent.

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