Bile acid metabolizing bacteria for preventing and treating inflammatory bowel disease and application thereof

Abstract

The present invention provides a bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease and its application. The present invention provides a bacterium, which includes any one, two or three of the following strains: Clostridium sp. with a deposit number of GDMCC No: 62707; Eubacterium limosum with a deposit number of GDMCC No: 62708; Bacteroides ovatus with a deposit number of GDMCC No: 62709. The bacterium of the present invention can convert primary bile acid into secondary bile acid, and improve the inflammatory symptoms of DSS-induced ulcerative colitis mice.

Description

Technical Field

[0001] The present invention relates to a bacterium for preventing and treating inflammatory bowel disease such as ulcerative colitis and its application. Specifically, the present invention relates to a bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease, a composition containing the bacterium and related applications. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, recurrent inflammatory disease of the intestine. Patients often have persistent ulcers in the mucosa of the small intestine and large intestine. It is mainly divided into two types, ulcerative colitis and Crohn's disease (CD). Possible causes of IBD include: genetic susceptibility, environmental factors such as intestinal flora, social behaviors such as smoking and diet, and increased risk of IBD is associated with early childhood exposure to antibiotics. The relative prevalence of IBD varies greatly by geographic region, and its pathological features are similar but also significantly different. IBD has a high recurrence rate, a long course of disease, is difficult to cure, and has a poor prognosis. Currently, there is no cure for it.

[0003] The pathophysiological mechanism of IBD is not very clear. It usually occurs after the immune system produces an excessive immune response to the normal intestinal flora, triggering a series of inflammatory events that damage and destroy the intestinal wall. The intestinal mucosal barrier separates the commensal flora from the intestinal epithelium. The immune cells in the mucosa inhibit the accumulation and translocation of the commensal flora. Some IBD-related gene mutations can lead to impaired mucosal barriers or immune responses, causing disorders of intestinal flora and immune balance. To date, a large number of literature has reported the association between IBD and dysbiosis. The mechanisms of the influence of flora changes on intestinal inflammation include: regulating Treg cells and other immune cells, regulating pro-inflammatory and anti-inflammatory factors, and directly invading intestinal epithelial cells. As the largest immune organ, the current research on IBD focuses on short-chain fatty acids, while bile acids are mostly believed to be related to metabolic diseases such as diabetes and fatty liver. Few studies have explored the relationship between IBD and bile acids. Therefore, studying the relationship between inflammation and bile acids and flora during IBD will provide new strategies for the prevention and treatment of IBD.

[0004] Similar schemes currently available include: Honda et al. screened 17 bacteria that can regulate immune responses through specific culture media for the treatment of IBD; Daniel van et al. analyzed the commercial strain library through bioinformatics and designed two synthetic bacterial colonies, GUT-103 and GUT-108, for the treatment of IBD.

[0005] The synthetic bacterial strains involved in previous studies were chloroform-resistant strains obtained by adding chloroform to the culture medium, which lacked rational design. The strains screened based on bioinformatics analysis also lacked in vitro metabolic detection and verification of the intestinal flora metabolites of interest. Summary of the invention

[0006] One object of the present invention is to provide bacteria that can prevent and treat inflammatory bowel disease.

[0007] Another object of the present invention is to provide applications of the screened bacteria.

[0008] In the study, the inventors of this case, based on the human microbiome array data, targeted the phenomenon of lack of secondary bile acids in the feces of IBD patients, and screened bacteria, especially synthetic bacteria, that can metabolize conjugated primary bile acids into secondary bile acids by combining bioinformatics analysis and in vitro metabolic detection. They further administered the designed bacteria to the DSS-induced mouse model by oral administration, and verified the improvement of the enteritis indicators of mice by the bacteria, proving that the bacteria of the present invention are bile acid metabolizing bacteria, and the bacterial community of the present invention is a synthetic bacterial community that can improve the symptoms of IBD.

[0009] Specifically, the present invention provides three strains capable of metabolizing conjugated primary bile acids into secondary bile acids, which are respectively named Clostridium sp.DA266, Eubacterium limosum DA510, and Bacteroidesovatus DA668. These three strains have been deposited, among which:

[0010] Strain Clostridium sp.DA266, deposit date: August 18, 2022; deposit unit: Guangdong Microbiological Culture Collection Center (GDMCC); deposit unit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070; deposit number: GDMCCNo:62707; classification name: Clostridium sp. The strain Clostridium sp.DA266 of the present invention is also called Clostridium sp.:GDMCC-62707.

[0011] Strain Eubacterium limosum DA510, deposit date: August 18, 2022; deposit unit: Guangdong Microbiological Culture Collection Center (GDMCC); deposit unit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070; deposit number: GDMCC No: 62708; classification name: Eubacterium limosum. The strain Eubacterium limosum DA510 of the present invention is also called Eubacteriumlimosum: GDMCC-62708.

[0012] Strain Bacteroides ovatus DA668, deposit date: August 18, 2022; deposit unit: Guangdong Microbiological Culture Collection Center (GDMCC); deposit unit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070; deposit number: GDMCCNo:62709; classification name: Bacteroides ovatus. The strain Bacteroides ovatus DA668 of the present invention is also called Bacteroides ovatus:GDMCC-62709.

[0013] The research of the present invention shows that any one or more combinations of the strains of the present invention, Clostridium sp.DA266, Eubacterium limosumDA510, and Bacteroides ovatus DA668, all have the ability to metabolize and convert conjugated primary bile acids into secondary bile acids, and can improve IBD symptoms. All three bacteria of the present invention can be called bile acid metabolizing bacteria.

[0014] Thus, in one aspect, the present invention provides a bacterium comprising any one, two or three of the following strains:

[0015] Clostridium sp. with a deposit number of GDMCC No: 62707;

[0016] Eubacterium limosum with a deposit number of GDMCC No: 62708;

[0017] Bacteroides ovatus with the deposit number of GDMCC No:62709.

[0018] In other words, the bacteria of the present invention may be a pure culture of a single species of bacteria, or may be a bacterial group including two or more species of bacteria.

[0019] According to a specific embodiment of the present invention, the bacteria of the present invention include Clostridium sp. with a deposit number of GDMCC No: 62707 and Eubacterium limosum with a deposit number of GDMCC No: 62708.

[0020] According to a specific embodiment of the present invention, the bacteria of the present invention include Eubacterium limosum with a deposit number of GDMCC No: 62708 and Bacteroides ovatus with a deposit number of GDMCC No: 62709.

[0021] According to a specific embodiment of the present invention, the bacteria of the present invention include Clostridium sp. with a deposit number of GDMCC No: 62707 and Bacteroides ovatus with a deposit number of GDMCC No: 62709.

[0022] According to a specific embodiment of the present invention, the bacteria of the present invention include Clostridium sp. with a deposit number of GDMCC No: 62707, Eubacterium limosum with a deposit number of GDMCC No: 62708, and Bacteroides ovatus with a deposit number of GDMCC No: 62709.

[0023] According to a specific embodiment of the present invention, in the bacteria of the present invention, the Clostridium sp with a deposit number of GDMCC No: 62707 has a complete bai gene cluster.

[0024] According to a specific embodiment of the present invention, in the bacteria of the present invention, the Eubacterium limosum: GDMCC-62708 strain with a deposit number of GDMCC No: 62708 has 7-α / β HSDH enzyme.

[0025] According to a specific embodiment of the present invention, in the bacteria of the present invention, the Bacteroides ovatus:GDMCC-62709 strain has BSH enzyme.

[0026] According to a specific embodiment of the present invention, when the bacteria of the present invention include two or three strains, the quantitative ratio between the strains can be 0.01-100:0.01-100, preferably 0.1-10:0.1-10, and more preferably 0.1-10:1.

[0027] According to a specific embodiment of the present invention, the bacteria of the present invention can be a solid or liquid bacterial preparation. The bacteria can be prepared into a bacterial preparation by any feasible method in the relevant field. Preferably, the bacteria in the bacterial preparation are live bacteria.

[0028] On the other hand, the present invention also provides a composition, which includes the bacteria of the present invention (i.e., one, two or three of Clostridium sp with a deposit number of GDMCC No: 62707, Eubacterium limosum with a deposit number of GDMCC No: 62708, and Bacteroides ovatus with a deposit number of GDMCC No: 62709). The composition of the present invention has the ability to metabolize and convert conjugated primary bile acids into secondary bile acids due to the inclusion of the bacteria, and can improve IBD symptoms. The composition of the present invention can be called a bile acid metabolism composition. Preferably, the composition may also include other active substances and / or excipients that have the same function as the bacteria of the present invention or are beneficial to the function of the bacteria of the present invention (e.g., bile acid metabolism). According to some specific embodiments of the present invention, the composition may be a pharmaceutical composition. The pharmaceutical composition may also include conventional excipients in the pharmaceutical field.

[0029] On the other hand, the present invention also provides use of the bacteria or the composition of the present invention in preparing a composition for preventing and / or treating inflammatory bowel disease.

[0030] According to a specific embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis.

[0031] On the other hand, the present invention also provides the use of the bacteria or the composition of the present invention in the preparation of a composition for alleviating weight loss and / or colon shortening in individuals with inflammatory bowel disease.

[0032] On the other hand, the present invention also provides use of the bacteria or the composition of the present invention in the preparation of a composition for improving bile acid metabolism in individuals with inflammatory bowel disease.

[0033] On the other hand, the present invention also provides the use of the bacteria or the composition of the present invention in the preparation of a composition for improving the intestinal flora of an individual with inflammatory bowel disease.

[0034] According to a specific embodiment of the present invention, the composition for preventing and / or treating inflammatory bowel disease of the present invention may be a live bacteria preparation or an inactivated bacteria preparation. Preferably, the composition for preventing and / or treating inflammatory bowel disease is an oral preparation. The composition may be a pharmaceutical composition. The pharmaceutical composition may also include conventional excipients in the pharmaceutical field.

[0035] According to a specific embodiment of the present invention, when the composition includes two or three bacteria of the present invention, the quantity ratio between the bacteria can be 0.01-100:0.01-100, preferably 0.1-10:0.1-10, more preferably 0.1-10:1, for example, the quantity of each bacteria is substantially the same.

[0036] According to a specific embodiment of the present invention, the bacteria of the present invention are grown at a rate of 1×10 5 CFU~1×10 13 The amount of CFU is used to prepare the composition. Preferably, the bacteria of the present invention are used at a rate of 1×10 7 CFU~1×10 10 The amount of CFU is used to prepare the composition.

[0037] Specific experiments of the present invention show that one or more of the three bacteria of the present invention can convert primary bile acid into secondary bile acid, which can improve the inflammatory symptoms of DSS-induced ulcerative colitis mice. It can be reasonably expected that the secondary metabolites of the combination of the three bacteria of the present invention, short-chain fatty acids, their derivatives and / or culture fluids, also have certain applications in preventing and / or alleviating inflammatory bowel disease; the abundance of the three bacterial combinations of the present invention and / or their metabolites can be used as markers to diagnose inflammatory bowel disease. Further, the technology of the present invention can use intestinal flora as a target for disease and drug administration, and use bile acid as a material basis to explore the molecular mechanism of improving IBD by strains that metabolize and produce secondary bile acids. Based on multi-omics analysis, the changes in the flora structure, differential gene expression, and metabolic pathway regulation of the animal model before and after the administration of strains that can metabolize and produce secondary bile acids are systematically explained, and the molecular mechanism of bile acid and strains that can metabolize bile acid for the treatment of ulcerative colitis is systematically explained. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Bile acids and short-chain fatty acids were shown to protect against intestinal inflammation in a mouse model.

[0039] Figure 2a to Figure 2e Displays the test results of bacterial flora screening based on bioinformatics analysis and the verification of in vitro metabolic detection of three bacteria.

[0040] Figure 3a to Figure 3g Test results showing the effects of three bacteria on body weight changes and colon length in enteritis mice.

[0041] Figure 4a and Figure 4b The results of the fecal bile acid content measurement after the three bacteria acted on mice with enteritis.

[0042] Figure 5a and Figure 5b Showing the results of the measurement of short-chain fatty acid content in the feces of mice with enteritis after the three bacteria were treated.

[0043] Figure 6 Metagenomic sequencing results showing the effect of the three-bacteria combination on the intestinal flora of mice with enteritis.

[0044] Deposit of microorganisms for patent procedure:

[0045] (1) Strain Clostridium sp.DA266

[0046] Deposit date: August 18, 2022;

[0047] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0048] Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, 510070;

[0049] Deposit number: GDMCC No:62707;

[0050] Classification and nomenclature: Clostridium sp.

[0051] (2) Strain Eubacterium limosum DA510

[0052] Deposit date: August 18, 2022;

[0053] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0054] Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, 510070;

[0055] Deposit number: GDMCC No:62708;

[0056] Taxonomic name: Eubacterium limosum.

[0057] (III) Bacteroides ovatus DA668

[0058] Deposit date: August 18, 2022;

[0059] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0060] Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, 510070;

[0061] Deposit number: GDMCC No:62709;

[0062] Taxonomic nomenclature: Bacteroides ovatus. DETAILED DESCRIPTION

[0063] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the relevant art. The method operations not specifically noted in the embodiments are performed according to the conventional operations of the prior art in the relevant field or the operations recommended by the manufacturer's instructions.

[0064] Example 1: Protection of the compound against mouse enteritis model

[0065] This example is mainly based on the protection of the compound on the mouse enteritis model. After adaptive feeding, the mice were divided into two groups, namely the bile acid and SCFA group and the DSS group. The model was established 10 days after the preventive administration, that is, the drinking water was replaced with 2% DSS water, and the weight of the mice was recorded every day. The mice were sacrificed on the seventh day of modeling. The results are shown in Figure 1 Compared with the control group, the weight loss of mice in the bile acid and SCFA group on the seventh day of modeling was significantly reduced, indicating that the combined use of bile acid and SCFA has a good protective effect on enteritis in mice.

[0066] Example 2, strain screening

[0067] This example is mainly based on the combination of bioinformatics analysis and in vitro metabolic detection to screen for synthetic bacteria that can metabolize conjugated primary bile acids into secondary bile acids.

[0068] The strain library screened in the present invention is 48 strains of bacteria isolated and cultured from healthy human intestinal feces samples in the laboratory in the early stage. All strains were purified by monoclonal cloning and their species were determined by 16S RNA sequencing ( Figure 2a );

[0069] Bioinformatics analysis: Screening of strains with 7-α dehydroxylase, BSH enzyme, and 7-α / β HSDH enzyme. The screening database comes from the strains isolated and purified from healthy human intestinal feces samples in the laboratory in the early stage, and the whole genome sequencing was performed. The analysis results are as follows: there are two strains with a complete bai gene cluster, and the in vitro metabolic capacity is tested. One of them is named Clostridium sp. DA266 in the present invention; there is only one strain with 7α / β, which is named Eubacteriumlimosum DA510 in the present invention; and BSH exists in many strains. Based on the literature report that Bacteroides ovatus has a good effect in treating enteritis, the present invention finally selected a strain named Bacteroides ovatus DA668. These three strains have been preserved, including: strain Clostridium sp.DA266, preservation date: August 18, 2022; preservation unit: Guangdong Microbiological Culture Collection Center (GDMCC); preservation unit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070; preservation number: GDMCC No: 62707; classification name: Clostridium sp. The strain Clostridium sp.DA266 of the present invention is also called Clostridium sp: GDMCC-62707. Strain Eubacterium limosum DA510, deposit date: August 18, 2022; deposit unit: Guangdong Microbiological Culture Collection Center (GDMCC); deposit unit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070; deposit number: GDMCC No: 62708; classification name: Eubacterium limosum. The strain Eubacterium limosum DA510 of the present invention is also called Eubacterium limosum: GDMCC-62708. Strain Bacteroides ovatus DA668, deposit date: August 18, 2022; deposit unit: Guangdong Microbiological Culture Collection Center (GDMCC); deposit unit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070; deposit number: GDMCC No: 62709; classification name: Bacteroides ovatus. The strain Bacteroides ovatus DA668 of the present invention is also called Bacteroides ovatus: GDMCC-62709.The present invention selects three strains of Clostridium sp: GDMCC-62707, Eubacterium limosum: GDMCC-62708, and Bacteroides ovatus: GDMCC-62709 to form a synthetic bacterial community for subsequent in vitro metabolic experiments.

[0070] In vitro metabolic testing verifies whether the screened strains have the mentioned enzymes: ① Use brain heart infusion BHI medium to revive the screened strains, then transfer the revived bacterial solution to a new medium containing 50μmol primary conjugated bile acid TCDCA / GCDCA, culture for 48 hours, and collect the culture medium; ② Take 200μL of the collected culture medium, add an equal volume of ethyl acetate, vigorously vortex for 15 minutes, centrifuge at 10000rpm for 5 minutes, aspirate the supernatant ethyl acetate layer into a new 1.5 ml centrifuge tube, dry it, and then dissolve it with 200μL 50% methanol, centrifuge it at 10000rpm for 10 minutes, filter it through a 0.22um filter membrane, collect the filtrate in a liquid sample bottle, and store it in a -80℃ refrigerator. ③ The results of liquid chromatography-mass spectrometry analysis are as follows Figure 2b , Figure 2c , Figure 2d , Figure 2e As shown, the substrate TCDCA / GCDCA was metabolically converted by the three bacteria co-culture to produce secondary bile acids UDCA and LCA.

[0071] Example 3: Protection of the combined bacteria SBE against DSS-induced enteritis model in mice

[0072] After adaptive feeding, 48 9-week-old C57 mice were randomly divided into 6 groups, 8 mice in each group, including single bacteria group (Clostridium fulgidus group, Eubacterium group, Bacteroides ovatus group), combination bacteria group (two bacteria combination, three bacteria combination), and control group. The total amount of bacteria in all the groups was the same, that is, the single bacteria group was gavaged with 1×10 9 CFU, Clostridium sp:GDMCC-62707 and Eubacterium limosum:GDMCC-62708 were injected 5×10 8 CFU, three bacteria combination group Clostridium sp: GDMCC-62707, Eubacterium limosum: GDMCC-62708, Bacteroides ovatus: GDMCC-62709 were injected 3.3×10 8CFU, the control group was gavaged with the same volume of blank culture medium for 10 consecutive days. On the 10th day, the feces of mice in each group were collected and immediately placed in a liquid nitrogen tank and brought back to the laboratory for storage at -80°C. Then the drinking water of the mice was replaced with water containing 2% DSS, and a DSS-induced enteritis model was established in mice. The mice were allowed to drink water freely, and gavage was continued. The weight of the mice was recorded every day. The model was established for 7 consecutive days, and the feces of mice in each group were collected. The mice were sacrificed, serum and colon were collected, and the length of the mouse colon was recorded. The feces, colon and blood were stored in a -80°C refrigerator. The changes in mouse weight and colon length were as follows Figure 3a to Figure 3g The three-bacteria combination was better than the single-bacteria group and the two-bacteria combination in alleviating weight loss and colon shortening in mice.

[0073] Example 4: Effect of the three-bacteria combination on fecal bile acid and short-chain fatty acids in mice with enteritis

[0074] Results of determination of bile acid content in feces of mice (feeding scheme is the same as in Example 3) Figure 4a and Figure 4b ) It can be seen that compared with the control group, the content of secondary bile acids DCA, UDCA and LCA in the 3strains group after intragastric administration increased. This result is consistent with the conclusion of the present invention that 3strains can metabolize and convert conjugated primary bile acids into secondary bile acids in in vitro detection. In addition, the amount of short-chain fatty acids propionic acid and butyric acid in the 3strains group after intragastric administration also increased ( Figure 5a and Figure 5b ).

[0075] Example 5: Effect of the three-bacteria combination on the intestinal flora of mice with enteritis

[0076] Results of fecal metagenomic sequencing from mice (feeding regimen was the same as in Example 3) Figure 6 ) It can be seen that the abundance of some bacteria in the intestinal flora changed compared with the DSS-control group after oral administration of the three bacteria.

[0077] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A bacterial preparation comprising the following three strains: The deposit number is GDMCC No: 62707 Clostridium sp.; The deposit number is GDMCC No: 62708 Eubacterium limosum ; The deposit number is GDMCC No: 62709 Bacteroides ovatus .

2. The bacterial preparation according to claim 1, wherein The deposit number is GDMCC No: 62707 Clostridium sp. has a complete bai gene cluster; The deposit number is GDMCC No: 62708 Eubacterium limosum The strain has a 7-α / β HSDH enzyme; and The deposit number is GDMCC No: 62709 Bacteroides ovatus The strain has BSH enzyme.

3. The bacterial preparation according to claim 1 or 2, which is a solid or liquid bacterial preparation.

4. A composition comprising the bacterial preparation according to any one of claims 1 to 3.

5. Use of the bacterial preparation according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a composition for preventing and / or treating inflammatory bowel disease, wherein the bacteria in the bacterial preparation or composition are live bacteria.

6. The use according to claim 5, wherein: The inflammatory bowel disease is ulcerative colitis.

7. The use according to claim 5, wherein: The composition for preventing and / or treating inflammatory bowel disease is an oral preparation.

8. The use according to any one of claims 5 to 7, wherein: The bacterial preparation according to any one of claims 1 to 3 is administered at a rate of 1×10 5 CFU~1×10 13 The amount of CFU is used to prepare the composition.

9. The use according to any one of claims 5 to 7, wherein: The bacterial preparation according to any one of claims 1 to 3 is administered at a rate of 1×10 7 CFU~1×10 10 The amount of CFU is used to prepare the composition.

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