<i>Bacteroides fragilis </i>for relieving endotoxin infection and application thereof

Abstract

The disclosure discloses Bacteroides fragilis for relieving endotoxin infection and application thereof, and belongs to the technical field of microorganisms. The Bacteroides fragilis CCFM1020 of the disclosure has low immunogenicity, and can reduce the content of pro-inflammatory factors and increase the concentration of anti-inflammatory factors in the blood of hosts infected with endotoxin, up-regulate the number of Foxp3.sup.+ regulatory T cells and stabilize the composition of the intestinal microbiota. A pharmaceutical composition of the Bacteroides fragilis CCFM1020 for relieving endotoxin infection has broad application prospects.

Claims

1. A composition comprising: a bacterium of Bacteroides fragilis with a preservation number of GDMCC No: 60342, preserved at the Guangdong Microbial Culture Collection Center (GDMCC) on Mar. 29, 2018, a cryoprotective agent, wherein the cytoprotective agent comprises a phosphate buffer solution, and a powder cryopreserve agent, wherein active Bacteroides fragilis is present in the composition at 10.sup.6 CFU/g or higher.

2. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

3. The composition of claim 2, wherein the pharmaceutically acceptable carrier comprises one or more fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

4. The composition of claim 3, wherein the fillers comprise excipient diluents or excipient absorbents.

5. The composition of claim 3, wherein the binders are viscous substances that are added to a drug when the drug has no viscosity or insufficient viscosity, so as to facilitate granulation.

6. The composition of claim 3, wherein the wetting agents are liquid that are added to a drug when the drug has no viscosity.

7. The composition of claim 3, wherein the disintegrants are excipients that are added to tablets to promote rapid disintegration into fine particles in gastrointestinal fluid.

8. The composition of claim 3, wherein the lubricants are chemical substances that improve the mobility of tablets during processing and demoulding; and the flavoring agents are excipients used in drugs to improve or shield the undesirable smell and taste.

9. The composition of claim 3, wherein the pharmaceutical composition is in a form of granules, capsules, tablets, pills or oral liquid.

10. A method of regulating intestinal microbiota in a subject in need thereof, which comprises administering to the subject a pharmaceutically effective amount of the composition of claim 1, wherein the subject is suffering from an endotoxin infection.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows the concentration of immune factors in the blood and colon tissues of mice after intragastric administration of Bacteroides fragilis CCFM1020.

(2) FIG. 2 shows the regulating effects of Bacteroides fragilis CCFM1020 on the level of immune factors in the blood of mice infected with endotoxin; * indicates that there is a significant difference between the Bacteroides fragilis CCFM1020 group and the endotoxin model group, * indicates p<0.05, and ** indicates p<0.01.

(3) FIG. 3 shows the regulating effects of Bacteroides fragilis CCFM1020 on the level of regulatory T cells in the spleen of mice infected with endotoxin; * indicates that there is a significant difference between the Bacteroides fragilis CCFM1020 group and the endotoxin model group, and ** indicates p<0.01; # indicates that there is a significant difference between the endotoxin infection group and the control group, and ## indicates p<0.01.

(4) FIG. 4 shows the intestinal microbiota of mice in the endotoxin infection group; D1LPSCTRL: the intestinal microbiota of mice in the endotoxin infection group on the 1st day of the experiment; D3LPSCTRL: the intestinal microbiota of mice in the endotoxin infection group on the 3rd day of the experiment; and D5LPSCTRL: the intestinal microbiota of the mice in the endotoxin infection group on the 5th day of the experiment.

(5) FIG. 5 shows the regulating effects of Bacteroides fragilis CCFM1020 on the composition of the intestinal microbiota of mice infected with endotoxin; D1LPSBF: the intestinal microbiota of mice in the endotoxin infection group with the intervention of Bacteroides fragilis CCFM1020 on the 1st day of the experiment; D3LPSBF: the intestinal microbiota of mice in the endotoxin infection group with the intervention of Bacteroides fragilis CCFM1020 on the 3rd day of the experiment; and D5LPSBF: the intestinal microbiota of mice in the endotoxin infection group with the intervention of Bacteroides fragilis CCFM1020 on the 5th day of the experiment.

(6) FIG. 6 shows the relative abundance of Allobaculum in the intestinal microbiota of mice infected with endotoxin and in the intestinal microbiota of mice in the endotoxin infection group with the intervention of Bacteroides fragilis CCFM1020.

(7) FIG. 7 shows the relative abundance of Clostridiales in the intestinal microbiota of mice infected with endotoxin and in the intestinal microbiota of mice in the endotoxin infection group with the intervention of Bacteroides fragilis CCFM1020.

(8) FIG. 8 shows the relative abundance of Lactobacillus in the intestinal microbiota of mice infected with endotoxin and in the intestinal microbiota of mice in the endotoxin infection group with the intervention of Bacteroides fragilis CCFM1020.

PRESERVATION PROOF OF THE BIOLOGICAL MATERIALS

(9) A novel Bacteroides strain, with a taxonomic name of Bacteroides fragilis, has been preserved at the Guangdong Microbial Culture Collection Center on Mar. 29, 2018, with the preservation address of 5.sup.th Floor, Building 59, Courtyard 100, Xianlie Middle Road, Guangzhou, and the preservation number of GDMCC No: 60342.

DETAILED DESCRIPTION

Example 1: Culture and Preservation of Bacteroides fragilis CCFM1020

(10) 1. Preparation of a culture medium: brain heart infusion BHI culture medium (such as a product of Qingdao Haibo Biotechnology Co., Ltd.) was prepared in distilled water. Cysteine hydrochloride (1 g/L), hemin (0.01 g/L) and vitamin K1 (0.002 g/L) were added and mixed uniformly. The pH was adjusted to 7.0, and sterilization was carried out at 115-121° C. for 15-20 min to obtain the culture medium.

(11) 2. Culture method: Bacteroides fragilis CCFM1020 was inoculated according to an inoculation amount of 2-4% based on the weight of the above culture medium, and anaerobically cultured at 37° C. for 12-18 h to reach a stationary phase.

(12) 3. Preparation of protective agent: cysteine hydrochloride (1 g/L) and glycerol (200 g/L) were weighed and uniformly dissolved in distilled water, and the mixed solution was sterilized at 115-121° C. for 15-20 min to obtain the protective agent.

(13) 4. Preservation method: liquid culture of Bacteroides fragilis CCFM1020 in the stationary phase was washed with a sterile phosphate buffer solution (pH 7.2) for 1-2 times, and then resuspend with the protective agent to reach the concentration of 10.sup.10 CFU/mL, so as to obtain the Bacteroides fragilis CCFM1020 cryopreserved agent, and the Bacteroides fragilis CCFM1020 cryopreserved agent was preserved at −80° C. for later use.

Example 2: Tolerance Dose Experiment in Mice with Intragastric Administration of Bacteroides fragilis CCFM1020

(14) 10 of 6-8 week old healthy female C57BL6 mice intragastrically administered with 0.1 mL of the cryopreserved agent of Bacteroides fragilis CCFM1020 (10.sup.10 CFU/mL) prepared as Example 1 once every 24 hours. The mice were observed for 5 days and the body weight and death of the mice were recorded.

(15) Results are shown in Table 1, which indicated that feeding the Bacteroides fragilis CCFM1020 at the concentration of 10.sup.9 CFU did not cause any negative effects on the mice. The mice grew normally, no death occurred, and the mice had no pathological symptoms.

(16) TABLE-US-00001 TABLE 1 Influence of 10.sup.9 CFU of Bacteroides fragilis CCFM1020 on the body weight of mice Time (d) 1 2 3 4 5 Body 20.49 ± 20.92 ± 21.20 ± 21.49 ± 21.77 ± 0.5 0.7 0.5 0.6 0.3 weight (g) 3 7 3 8 6 Death None None None None None

Example 3: Immune Tolerance Experiment in Mice with Intragastric Administration of Bacteroides fragilis CCFM1020

(17) 20 of 6-8 week old healthy female C57BL6 mice were randomly divided into two groups: a negative control group and a Bacteroides fragilis CCFM1020 intervention group, each group contained 10 mice. Mice in the Bacteroides fragilis CCFM1020 intervention group were intragastrically administered with 0.1 mL of the above Bacteroides fragilis CCFM1020 cryopreserved agent (10.sup.10 CFU/mL) once every 24 hours, while mice in the control group were intragastrically administered with 0.1 mL of the protective agent of the above cryopreserved agent once every 24 hours. All mice were sacrificed after 5 days of continuous intragastric administration. After blood and colon tissue were taken, lysed and homogenized with RIPA lysate (a product of Beyotime Biotechnology Co., Ltd.), the concentration of immune factors was determined with an ELISA kit (a product of Nanjing SenBeiJia Biological Technology Co., Ltd.).

(18) Experimental results are shown in FIG. 1, which indicated that the contents of the immune factors of TNF-α, IL-6, IL-10 and CXCL-2 in the blood and the immune factors of sIgA and IgE in the colon tissues of mice in the Bacteroides fragilis CCFM1020 intervention group were not significantly different from those of mice in the control group. Therefore, the results of Example 3 show that Bacteroides fragilis CCFM1020 of the disclosure does not stimulate the immune system of mice and has low immunogenicity.

Example 4: Regulating Effects of Bacteroides fragilis CCFM1020 on the Level of Immune Factors in the Blood of Mice Infected with Endotoxin

(19) 30 of 6-8 week old healthy female C57BL6 mice were randomly divided into three groups: a negative control group, an endotoxin infection model group and a Bacteroides fragilis CCFM1020 treatment group, each group contained 10 mice. Mice in the endotoxin infection group and the Bacteroides fragilis CCFM1020 treatment group were intraperitoneally injected with 0.1 mL of an endotoxin solution (0.1 mg/kg) diluted with normal saline once every 24 hours. Mice in the control group and the endotoxin infection group were intragastrically administered with 0.1 mL of the protective agent of the cryopreserved agent prepared in Example 1 once every 24 hours. The mice in the Bacteroides fragilis CCFM1020 treatment group were intragastrically administered with 0.1 mL of the Bacteroides fragilis CCFM1020 cryopreserved agent (10.sup.10 CFU/mL) prepared in Example 1 once every 24 hours. All mice were sacrificed after 5 consecutive days. Blood was collected and the concentration of immune factors was determined by using an ELISA kit (a product of Nanjing SenBeiJia Biological Technology Co., Ltd.).

(20) Experimental results are shown in FIG. 2, which indicated that endotoxin infection increased the content of the pro-inflammatory factor TNF-α in the blood of mice from 522.93 ng/L to 566 ng/L, while decreased the anti-inflammatory factor IL-10 from 686.83 ng/L to 567.68 ng/L. After treatment with Bacteroides fragilis CCFM1020, the content of TNF-α in the blood of mice was significantly reduced to 450 ng/L (p<0.05), and the content of IL-10 was increased to 692 ng/L (p<0.01), making the levels of the above immune factors approaching those of normal mice. The above results indicate that Bacteroides fragilis CCFM1020 of the disclosure can relieve the inflammatory response caused by endotoxin infection.

Example 5: Regulating Effects of Bacteroides fragilis CCFM1020 on the Content of Regulatory T Cells in the Spleen of Mice Infected with Endotoxin

(21) 30 of 6-8 week old healthy female C57BL6 mice were randomly divided into three groups: a negative control group, an endotoxin infection model group and a Bacteroides fragilis CCFM1020 treatment group, each group contained 10 mice. Mice in the endotoxin infection group and the Bacteroides fragilis CCFM1020 treatment group were intraperitoneally injected with 0.1 mL of an endotoxin solution (0.1 mg/kg) diluted with normal saline once every 24 hours. Mice in the control group and the endotoxin infection group were intragastrically administered with 0.1 mL of the protective agent of the cryopreserved agent once every 24 hours. The mice in the Bacteroides fragilis CCFM1020 treatment group were intragastrically administered with 0.1 mL of the Bacteroides fragilis CCFM1020 cryopreserved agent (10.sup.10 CFU/mL) once every 24 hours. All mice were sacrificed after 5 consecutive days. After the spleen tissue was taken, lysed and homogenized with red blood cell lysate, CD4, CD25 and surface molecule Foxp3 were labeled by using a mouse regulatory T cell staining kit (a product of eBioscience company), and the levels of the regulatory T cells was detected by flow cytometry (FACSCalibur, BD company).

(22) Experimental results are shown in FIG. 3, which indicated that endotoxin infection significantly reduced the Foxp3.sup.+ regulatory T cells in the spleen of mice from 7.69% to 5.69% (p<0.01). After treatment with Bacteroides fragilis CCFM1020, the content of the regulatory T cells in the spleen of mice increased significantly to 7.73% (p<0.01), which was close to the level of mice in the control group. The results of Example 5 indicate that the Bacteroides fragilis CCFM1020 of the disclosure can relieve the inflammatory response caused by endotoxin infection.

Example 6: Regulating Effects of Bacteroides fragilis CCFM1020 on the Composition of the Intestinal Microbiota of Mice Infected with Endotoxin

(23) 30 of 6-8 week old healthy female C57BL6 mice were randomly divided into three groups: a negative control group, an endotoxin infection model group and a Bacteroides fragilis CCFM1020 treatment group, each group contained 10 mice. Mice in the endotoxin infection group and the Bacteroides fragilis CCFM1020 treatment group were intraperitoneally injected with 0.1 mL of an endotoxin solution (0.1 mg/kg) diluted with normal saline once every 24 hours. Mice in the control group and the endotoxin infection group were intragastrically administered with 0.1 mL of the protective agent of the cryopreserved agent once every 24 hours. The mice in the Bacteroides fragilis CCFM1020 treatment group were intragastrically administered with 0.1 mL of the Bacteroides fragilis CCFM1020 cryopreserved agent (10.sup.10 CFU/mL) once every 24 hours. All mice were sacrificed after 5 consecutive days. During the experiment, the mouse feces was taken before intragastric administration on the first day and after intragastric administration on the third day and the fifth day. After extracting the genomic DNA of fecal bacteria with a Fast DNA Spin Kit for Feces (a product of MP Biomedicals Company), the 16s V3-V4 region sequence was amplified by PCR, and the differences in the composition of the intestinal microbiota in the feces samples were determined by next-generation sequencing.

(24) Experimental results are shown in FIGS. 4 to 8. The abundance of Bacteroidetes and Firmicutes in the intestines of mice infected with endotoxin were 50% and 40% respectively, wherein Bacteroides S24-7 accounted for 42.7%, Lactobacillus accounted for 12%, Clostridiales accounted for 8.2%, and the content of Allobaculum related to colitis increased significantly to 10%, with a decrease in the diversity of intestinal microbiota. After treatment with Bacteroides fragilis CCFM1020, it was found that the abundance of Bacteroidetes and Firmicutes in the fecal microbiota of mice were adjusted to 60% and 30% respectively, wherein the variations of Bacteroides, Clostridiales and Allobaculum were greatly reduced, the abundance of which was 38.3%, 8.7% and 0.5% respectively, and the content of Lactobacillus increased to about 15%. Therefore, the results of Example 6 showed that the Bacteroides fragilis CCFM1020 of the disclosure can improve the disorders of the intestinal microbiota caused by LPS, increase the diversity of the intestinal microbiota and stabilize the balance of the intestinal microbiota, thereby prevent a series of diseases that may be caused by the imbalance of the intestinal microbiota caused by endotoxin.

(25) The above examples showed that the Bacteroides fragilis CCFM1020 of the disclosure can help relieve the imbalance of the composition of the intestinal microbiota and the immune response caused by endotoxin infection in mice.

Example 7: Preparation of Capsule Products Containing Bacteroides fragilis CCFM1020 of the Disclosure

(26) The Bacteroides fragilis CCFM1020 of the disclosure was cultured anaerobically in BHI culture medium at 37° C. for 24 hours. The liquid culture was centrifuged at 5000 rpm for 15 min at 4° C., and washed 1-2 times with a sterile phosphate buffer solution (pH 7.2). The bacteria were resuspended using the above protective agent to make the final concentration of the bacteria reach 10.sup.10 CFU/mL. The bacterial suspension was added to sodium alginate solution, and mixed thoroughly to make cells evenly dispersed. Then the mixed solution was squeezed into a calcium chloride solution to form colloidal particles. After static solidification for 30 min, the colloidal particles were filtered and collected. The collected colloidal particles were freeze-dried for 48 hours to obtain powder containing the Bacteroides fragilis CCFM1020 of the disclosure. The powder was encapsulated with commercially available medicinal capsules to obtain the capsule product.

Example 8: Preparation of Tablets Using Bacteroides fragilis CCFM1020 of the Disclosure

(27) 25.7 parts by weight of the Bacteroides fragilis CCFM1020 bacterial powder preparation of the disclosure prepared by a freeze-drying method, 55.0 parts by weight of starch, 4.5 parts by weight of a cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 part by weight of talc, 1.0 part by weight of sucrose and 1.0 part by weight of water were individually weighed and mixed. The mixed material was made into wet granules by a conventional method, and then the wet granules were compressed with a tablet press produced by Zhongnan Pharmaceutical Machinery. The tablets were dried by a small medicine dryer produced by Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd., and then the tablets were packaged to obtain the tablets of the disclosure.

(28) Although the disclosure has been disclosed as above in preferred examples, it is not intended to limit the disclosure. Without departing from the spirit and scope of the disclosure, anyone familiar with this technology can make various changes and modifications. Therefore, the protection scope of the disclosure should be defined by the claims.