Brucella EMF75-02B1B and its uses

Abstract

The present invention relates to the field of microbial medicine technology, and specifically discloses Blautia EMF75-02B1B and its uses. Blautia ([[]] Blautia sp.) EMF75-02B1B was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on September 2, 2024, with the deposit number GDMCC No: 65084. The present invention proves through a mouse model that Blautia EMF75-02B1B can significantly improve the disease activity index (DAI) of mice with dextran sulfate sodium-induced ulcerative colitis, relieve the weight loss and colon shortening of mice with ulcerative colitis, enhance the intestinal barrier function, and reduce the inflammatory response.

Description

Technical Field

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

[0002] Inflammatory Bowel Disease (IBD) is a group of chronic inflammatory diseases that affect the gastrointestinal tract, including Crohn's Disease (CD) and Ulcerative Colitis (UC). Globally, the incidence of IBD is on the rise, especially in industrialized countries. Although the exact cause of IBD has not been fully clarified, it is generally believed to be caused by the complex interaction between genetics, the immune system, environmental factors, and the gut microbiota.

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

[0004] Currently, the clinical treatment for Ulcerative Colitis (UC) mainly includes:

[0005] (1) Drug treatment

[0006] Salicylic acid drugs: such as Sulfasalazine (SASP) and Mesalazine, are used to treat mild to moderate UC patients, but may cause side effects such as gastrointestinal reactions, headache, allergic reactions, etc.

[0007] Glucocorticoids: such as Budesonide, are used for severe or fulminant UC patients, and may cause side effects such as metabolic disorders and water retention, and are usually only used as short - term emergency treatment.

[0008] Immunosuppressants: such as Cyclosporine, by inhibiting the production of IL - 2 by T cells, affect the progress of the immune response, and may cause nephrotoxicity and increase the risk of secondary infections, and are usually used as an adjuvant treatment.

[0009] Biological agents: such as drugs against tumor necrosis factor - α (TNF - α), Infliximab and Adalimumab, although they have significant efficacy, may cause side effects such as increased risk of infection and allergic reactions, and are costly.

[0010] (2) Surgical treatment

[0011] Surgical treatment may be required when drug treatment is ineffective or severe complications occur, such as colectomy.

[0012] However, these treatment methods are often accompanied by side effects or high costs, and not all patients can achieve satisfactory therapeutic effects. Summary of the Invention

[0013] The object of the present invention is to provide a Blautia EMF75 - 02B1B and its uses in view of the above - mentioned deficiencies of 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 intestinal barrier function and reduce inflammatory responses, and shows better therapeutic effects compared with existing therapeutic drugs, providing new insights and strategies for the treatment of IBD.

[0014] To achieve the above object, the present invention adopts the following technical solutions:

[0015] The first object of the present invention is to provide Blautia EMF75 - 02B1B, which was deposited on September 2, 2024 at the Guangdong Provincial Microbial Culture Collection Center, located at the 5th floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 65084.

[0016] The second object of the present invention is to provide a composition, which comprises Blautia with the deposit number GDMCC No: 65084.

[0017] The viable cell count of Blautia EMF75 - 02B1B contained in the above composition is (1 - 10000)×10 6 cfu / g.

[0018] The third object of the present invention is to provide the application of the above - mentioned Blautia EMF75 - 02B1B or composition in the preparation of drugs for preventing and alleviating inflammatory bowel disease.

[0019] Furthermore, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0020] Furthermore, the Blautia EMF75 - 02B1B can significantly improve the disease activity index of ulcerative colitis induced by dextran sulfate sodium.

[0021] Furthermore, the Blautia EMF75 - 02B1B can control colon shortening and reduce inflammatory responses.

[0022] Furthermore, the Blautia EMF75 - 02B1B can enhance the intestinal barrier function.

[0023] The fourth object of the present invention is to provide a drug, which comprises the cells or compositions of the above - mentioned Blautia EMF75 - 02B1B, and pharmaceutically acceptable excipients.

[0024] The Blautia EMF75 - 02B1B or its fermentation broth or composition can be used as the only active ingredient with the function of alleviating colitis, or can also contain other active ingredients. The drug provided by the present invention can be used alone, or can be compounded with other products having the function of alleviating colitis. The present invention has no special limitation on the viable cell concentration of Blautia in the product for alleviating colitis, and can be conventionally selected according to actual needs. The viable cell concentration of Blautia in the product is 1×10 8 ~1×10 10 CFU / mL. As an implementable mode, the colitis alleviation...

[0025] The dosage form of the drug can be solution, powder, granule, capsule or tablet.

[0026] The fifth object of the present invention is to provide a bacterial agent. The above - mentioned Blautia EMF75 - 02B1B strain is inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a cryoprotectant and then freeze - dried to obtain a Blautia EMF75 - 02B1B strain powder; the Blautia EMF75 - 02B1B strain powder is formulated according to the viable cell number ratio to obtain the bacterial agent.

[0027] Compared with the prior art, the beneficial effects brought by the technical solution provided by the present invention are:

[0028] The present invention isolated and screened a strain named Blautia EMF75 - 02B1B with the taxonomic name of Blautia sp001304935 from adult fecal microbial samples. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 2, 2024, with the deposit number GDMCC No: 65084. The strain Blautia EMF75 - 02B1B can effectively reduce the inflammatory response and show a remission effect in the treatment of intestinal inflammatory bowel disease. Specifically, Blautia EMF75 - 02B1B can significantly reduce the disease activity index (DAI) of a murine model of ulcerative colitis (UC) induced by DSS (dextran sulfate sodium). In addition, it can effectively control the weight loss and colon shortening in UC mice, while improving the intestinal barrier function and reducing the occurrence of inflammatory reactions. Description of the Drawings

[0029] Figure 1 Schematic diagram of the body weight monitoring results provided by the embodiments of the present invention;

[0030] Figure 2 Schematic diagram of the DAI score of the Blautia EMF75-02B1B group provided by the embodiments of the present invention;

[0031] Figure 3 Schematic diagram of the image of the colon tissue length provided by the embodiments of the present invention;

[0032] Figure 4 Schematic diagram of the statistical analysis of the colon length provided by the embodiments of the present invention;

[0033] Figure 5 Representative micrographs of H&E staining of colon tissues of four groups of mice provided by the embodiments of the present invention;

[0034] Figure 6 Schematic diagram of the histopathological score provided by the embodiments of the present invention;

[0035] Figure 7 Schematic diagram of the effect of measuring the expression of the anti-inflammatory factor TGF-β1 in colon tissues by ELISA;

[0036] Figure 8 Schematic diagram of the effect of measuring the expression of the inflammatory factor TNF-α in colon tissues by qRT-PCR technology;

[0037] Figure 9 Schematic diagram of the effect of measuring the expression of the anti-inflammatory factor IL-1β in colon tissues by qRT-PCR technology;

[0038] Figure 10 Schematic diagram of the effect of Blautia EMF75-02B1B on the intestinal tight junction protein ZO-1 provided by the embodiments of the present invention;

[0039] Figure 11 Schematic diagram of the effect of Blautia EMF75-02B1B on the intestinal tight junction protein Claudin-2 provided by the embodiments of the present invention. Detailed implementation manners

[0040] To make the objectives, technical solutions and advantages of the present invention clearer, the following further describes in detail the specific implementation manners of the present invention in conjunction with specific embodiments and the accompanying drawings. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchases.

[0041] A novel strain of Blautia EMF75 - 02B1B was isolated from the fecal samples of healthy adults. This strain can effectively reduce the inflammatory response and show a remission effect in the treatment of intestinal inflammatory bowel disease. Blautia EMF75 - 02B1B was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 2, 2024. The address of the depository is the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65084.

[0042] Example 1

[0043] Source, isolation and identification of Blautia EMF75 - 02B1B.

[0044] (1) Sample collection

[0045] Fecal samples from healthy adults were collected and transferred to sample tubes under aseptic conditions, and brought back to the laboratory for processing within 1 hour. All subjects were informed of the nature of sampling and the test and had given consent. The samples were strictly screened to exclude patient samples with the following conditions: those with a history of other types of cancer, those with a history of other serious systemic diseases or digestive system diseases, those with chronic viral infections, those with bacterial infections before surgery, and those who had received immunosuppressive treatments (such as chemotherapy, oral steroids, etc.) or other cancer - related treatments before surgery.

[0046] (2) Isolation and purification of Blautia

[0047] 0.2 g of fecal samples were mixed in 1 ml of sterile PBS, gradient - diluted and then spread on the culture medium, and cultured under anaerobic conditions at 37°C. Single colonies were selected for isolation.

[0048] (3) Strain preservation

[0049] The purified strain was cultured to a concentration of about 1×10 9 cfu / ml, and after adding 20% glycerol, it was stored long - term at - 80°C.

[0050] (4) Genomic DNA extraction and 16S rDNA identification

[0051] Genomic DNA was extracted using a commercial kit and PCR - amplified with 16S rDNA universal primers. After sequencing on the machine, it was compared with the EZbiocloud database. This strain is Blautia Blautia sp., and the sequencing sequence is shown in SEQ IDNO.1.

[0052] Example 2

[0053] Analysis of the genomic characteristics of Blautia EMF75 - 02B1B.

[0054] (1)Whole genome sequencing and assembly

[0055] The whole genome of Blautia sp. EMF75-02B1B was sequenced using high-throughput sequencing technology, resulting in 47 sequences with a total length of 3,252,807 bp.

[0056] (2)Genome annotation

[0057] The GTDB-Tk tool was used for genome classification, predicting 2,021 coding DNA sequences (CDSs), including 7 rRNA genes, 58 tRNA genes, and 2 tmRNA genes.

[0058] (3)Metabolic pathway analysis

[0059] Metabolic pathway annotation was performed through eggNOG-mapper and the KEGG database, revealing that this strain has strong metabolic functions and environmental adaptability.

[0060] Example 3

[0061] Safety assessment of Blautia sp. EMF75-02B1B.

[0062] (1)Detection of antibiotic resistance genes

[0063] The RGI tool and the CARD database were used to predict antibiotic resistance genes, and genes such as rpoB mutants, tet(W), dfrF, and ErmX were found. Genes related to antibiotic resistance are shown in Table 1.

[0064] Table 1.

[0065]

[0066] (2)Detection of virulence factor genes

[0067] The VFDB database was used to predict virulence factor genes, and genes related to bacterial adhesion, immune regulation, anti-phagocytosis, and nutrient / metabolic factors were found, as shown in Table 2.

[0068] Table 2. Results of virulence factor gene detection.

[0069]

[0070] Example 4

[0071] Therapeutic study of Blautia sp. EMF75-02B1B on a colitis mouse model.

[0072] (1)Establishment of animal model

[0073] In the animal experiment of this embodiment, 37 SPF healthy C57BL / 6 mice aged 6 - 8 weeks with an average body weight of 18 - 22 g were selected. The mice were adaptively fed in a laboratory environment at the SPF level for one week and randomly assigned to four different experimental groups. The grouping is as follows: normal control group, dextran sulfate sodium (DSS) treatment group, mesalazine (5-ASA) treatment group, and EMF-75 treatment group (Blautia EMF75-02B1B). The specific grouping details are shown in Table 3.

[0074] From day 1 to day 14, the mice in the EMF-75 treatment group were gavaged with 1×10 9 CFU of bacterial solution every day, while the mice in the mesalazine treatment group were gavaged with 200 mg / kg body weight of mesalazine (5-ASA) every day. Meanwhile, the mice in the DSS treatment group and the normal control group were gavaged with an equal amount of phosphate buffer solution (PBS). From day 8 to day 14, except for the normal control group, the mice in other groups freely drank a 2.5% dextran sulfate sodium (DSS) solution for 7 days. All the mice in each group showed (100%) diarrhea, decreased food intake, decreased body weight, decreased fur glossiness, and the inflammatory bowel disease model was successfully constructed.

[0075] Table 3. Mouse experiment design table.

[0076]

[0077] (2) Observation and detection indicators and treatments:

[0078] ① During the model establishment and gavage period, observe the changes in the body weight, food and water intake, fur glossiness, mental state, activity, and death of the mice, and determine the severity of enteritis in the mice according to the disease activity index (DAI). DAI includes the percentage of body weight loss, the degree of fecal diarrhea, and the presence of blood in the stool. The specific calculation method is: the sum of the scores of the percentage of body weight loss, the degree of fecal diarrhea, and the presence of blood in the stool; among them, the score for the percentage of body weight loss: 0 = no body weight loss; 1 = 1 - 5%; 2 = 5 - 10%; 3 = 10 - 20%; 4 = more than 20%; fecal score: 0 = solid fecal particles; 1 = solid fecal particles, easy to deform; 2 = unformed stool; 3 = loose stool; 4 = liquid stool; blood in the stool score: 0 = occult blood; negative detection; 1 = occult blood detection 1+, no visible blood in the stool; 2 = occult blood detection 2+; 3 = occult blood detection 3+, visible blood in the stool; 4 = severe blood in the stool. The specific DAI scoring criteria are shown in Table 4.

[0079] Table 4. DAI scoring criteria.

[0080]

[0081] Note: DAI = (weight loss score + stool consistency score + blood in stool score) / 3; The comprehensive DAI score ranges from 0 to 4 points, with 0 points representing normal and 4 points representing the highest degree of inflammatory activity.

[0082] ② On the 15th day of the experiment, the body weights of the mice were measured. After anesthesia, blood was collected by orbital puncture, and the mice were sacrificed by cervical dislocation. Subsequently, the collected blood samples were centrifuged to separate the serum, which was stored at -80 °C for subsequent biochemical index detection. The length of the colon of the mice was measured, and samples were taken for tissue sectioning. Some colon samples were used to make pathological sections for histological analysis under a microscope. The remaining colon tissues were frozen and stored at -80 °C for future experimental research.

[0083] ③ Distal colorectal tissues (about 2 cm from the anus) were taken to make Swiss rolls, fixed with 4% paraformaldehyde, routinely paraffin-embedded and sectioned to obtain colon tissue sections, which were stained with hematoxylin-eosin (H&E) to detect the pathological conditions and scored according to the following criteria: The pathological score includes 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 damage; 2 = 10 - 20% crypt damage; 3 = more than 20% crypt damage; Goblet cell reduction: 0 = none; 1 = mild; 2 = moderate; 3 = severe; Inflammatory cell infiltration: 0 = no infiltration; 1 = increased lamina propria inflammatory cells; 2 = inflammatory cells extending to the submucosa; 3 = transmural inflammatory cell infiltration. The specific pathological histological scoring criteria are shown in Table 5.

[0084] Table 5. Pathological histological scoring criteria.

[0085]

[0086] ④ Intestinal barrier function analysis

[0087] The mRNA expression levels of tight junction proteins (ZO-1 and Claudin-2) were evaluated by qRT-PCR, and the expressions of tight junction proteins (ZO-1 and Claudin-2) were analyzed by western blot to evaluate the recovery of intestinal barrier function.

[0088] ⑤ Determination of cytokine levels

[0089] The levels of anti-inflammatory factor (TGF-β1) in colon tissues were determined by ELISA; and the levels of pro-inflammatory factors (IL-1β and TNF-α) and anti-inflammatory factor (IL-10) in colon tissues were determined by qRT-PCR technology.

[0090] The results of body weight monitoring are as Figure 1 shown. As can be seen from the figure, the initial body weight (0 d) and the body weight after 14 d of intervention were monitored, and the body weight change rate was calculated. It can be seen that the body weight of mice with DSS-induced inflammatory bowel disease decreased. After continuous intragastric administration of EMF-75 bacterial solution for 14 days, compared with the model group, the EMF-75 group could significantly improve the body weight loss of DSS mice (P<0.05); compared with the 5-ASA group, there was no difference in the result of EMF-75 improving the body weight loss of mice compared with the 5-ASA group. The results show that the EMF-75 bacterial solution can effectively reduce the symptoms of body weight loss caused by inflammatory bowel disease.

[0091] The disease activity index (DAI) scores of four different treatment groups were detected and recorded. The DAI score results after the initial administration of 2.5% dextran sulfate sodium (7 d) and the final administration of 2.5% dextran sulfate sodium (14 d) were as Figure 2 shown. It can be seen that the disease activity index increased in the other 3 groups except the control group under the induction of dextran sulfate sodium; compared with the dextran sulfate sodium group, both 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, there was no difference in the result of the EMF-75 group reducing the disease activity index of mice compared with the mesalazine group. The results show that the EMF-75 group bacterial solution can effectively reduce the activity index caused by inflammatory bowel disease.

[0092] As Figure 3 shown, it is an image of the colon tissue length. As can be seen from the figure, the EMF-75 bacterial solution can significantly improve the colon shortening in mice treated with dextran sulfate sodium.

[0093] As Figure 4 shown, it is the statistical analysis result of the colon length. The colon length of the dextran sulfate sodium group (5.79±0.46 cm) in the figure 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 dextran sulfate sodium group (P<0.05), indicating that EMF-75 may reduce the colon shortening in mice with DSS-induced colitis.

[0094] As Figure 5As shown, these are representative micrographs of H&E staining of colon tissues from four groups of mice. The colon tissue structure of the control group mice is clear and intact; there is a certain degree of damage to the colon tissues of the mesalazine group and the EMF-75 group mice, but some structures are still retained; the colon tissue structure of the dextran sulfate sodium group mice is severely damaged and the inflammatory reaction is obvious.

[0095] As Figure 6 shown, these are the results of colon tissue pathological scoring. The pathological condition of the control group is good, while dextran sulfate sodium leads to an increase in 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. It is shown that EMF-75 may reduce the inflammatory level in mice with DSS-induced colitis.

[0096] As Figures 7 - 9 shown, these are the results of cell inflammatory factor expression. It can be seen that the levels of pro-inflammatory factors (IL-1β, TNF-α) in the EMF-75 group are significantly lower than those in the dextran sulfate sodium group (P<0.0001), while the anti-inflammatory factor (TGF-β1) in the EMF-75 group is significantly higher than that in the control group and the dextran sulfate sodium group. It is shown that EMF-75 can reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors to reduce the inflammatory factor level in murine colitis.

[0097] As Figures 10 - 11 shown, these are the result graphs of enhancing intestinal barrier function. It can be seen that the protein levels of occludin-2 and ZO-1 are significantly higher than those in the dextran sulfate sodium group (P<0.0005), indicating that EMF-75 can effectively maintain the integrity and function of the intestinal mucosal barrier.

[0098] Example 5

[0099] Preparation of a probiotic solution preparation containing Blautia EMF75-02B1B.

[0100] (1) Preparation of probiotic suspension

[0101] Inoculate a single colony of Blautia EMF75-02B1B into MRS anaerobic broth medium and culture anaerobically at 37 °C until the growth plateau phase, and collect the bacterial solution of Blautia EMF75-02B1B; centrifuge the bacterial solution at 3000×g for 10 minutes at 4 °C, discard the supernatant, collect the bacterial cells and resuspend them in physiological saline to a concentration of 1×10 9 CFU / mL to obtain the suspension of Blautia EMF75-02B1B.

[0102] (2) Probiotic preparation formula

[0103] Suspend the Blautia EMF75-02B1B in a suspension and mix it with a protective agent, nutritional supplements, etc. according to the viable count to prepare a probiotic preparation suitable for oral administration.

[0104] (3)Preparation stability test

[0105] Conduct a stability test on the prepared probiotic preparation to ensure the viability of probiotics during storage and use.

[0106] Example 6

[0107] Preparation of a freeze-dried probiotic powder preparation containing Blautia EMF75-02B1B.

[0108] (1)Probiotic cultivation

[0109] Inoculate a single colony of Blautia EMF75-02B1B into MRS anaerobic broth medium and culture it anaerobically at 37°C until the growth plateau phase. Collect the bacterial solution of Blautia EMF75-02B1B; centrifuge the bacterial solution at 3000×g for 10 minutes at 4°C, discard the supernatant, collect the bacterial cells, mix them with a protective agent, and then perform freeze-drying to obtain the Blautia EMF75-02B1B bacterial powder.

[0110] (2)Probiotic preparation formula

[0111] Mix the Blautia EMF75-02B1B strain bacterial powder with a protective agent, nutritional supplements, etc. according to the viable count to prepare a freeze-dried probiotic preparation suitable for oral administration.

[0112] (3)Preparation stability test

[0113] Conduct a stability test on the prepared probiotic preparation to ensure the viability of probiotics during storage and use.

[0114] Without conflict, the above-mentioned embodiments and the features in the embodiments in this article may be combined with each other.

[0115] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Blautia EMF75-02B1B, which was deposited in Guangdong Microbial Culture Collection Center on September 2, 2024. The address of the depository is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The taxonomic name is Blautia sp., 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 relieving ulcerative colitis.

4. 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.

5. 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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