WO2024066079A1 - Bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease and use thereof - Google Patents

Bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease and use thereof Download PDF

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WO2024066079A1
WO2024066079A1 PCT/CN2022/139762 CN2022139762W WO2024066079A1 WO 2024066079 A1 WO2024066079 A1 WO 2024066079A1 CN 2022139762 W CN2022139762 W CN 2022139762W WO 2024066079 A1 WO2024066079 A1 WO 2024066079A1
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bacteria
inflammatory bowel
bowel disease
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戴磊
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中国科学院深圳先进技术研究院
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    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
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Definitions

  • 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.
  • IBD Inflammatory bowel disease
  • CD ulcerative colitis and Crohn’s disease
  • Possible causes of IBD include: genetic susceptibility, environmental factors such as intestinal flora, social behaviors such as smoking and diet, and an 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 the disease or treatment.
  • IBD 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.
  • 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.
  • 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.
  • 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.
  • One object of the present invention is to provide bacteria that can prevent and treat inflammatory bowel disease.
  • Another object of the present invention is to provide applications of the screened bacteria.
  • 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.
  • the present invention provides three strains that can metabolize conjugated primary bile acids into secondary bile acids, which are named Clostridium sp. DA266, Eubacterium limosum DA510, and Bacteroides ovatus DA668 in the present invention. These three strains have been preserved, among which:
  • 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: 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 research of the present invention shows that any one or more combinations of the strains of the present invention, Clostridium sp. DA266, Eubacterium limosum DA510, and Bacteroides ovatus DA668, 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.
  • the present invention provides a bacterium comprising any one, two or three of the following strains:
  • 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.
  • the bacteria of the present invention include Clostridium sp. with a preservation number of GDMCC No: 62707 and Eubacterium limosum with a preservation number of GDMCC No: 62708.
  • the bacteria of the present invention include Eubacterium limosum with a preservation number of GDMCC No: 62708 and Bacteroides ovatus with a preservation number of GDMCC No: 62709.
  • the bacteria of the present invention include Clostridium sp. with a preservation number of GDMCC No: 62707 and Bacteroides ovatus with a preservation number of GDMCC No: 62709.
  • the bacteria of the present invention include Clostridium sp. with a preservation number of GDMCC No: 62707, Eubacterium limosum with a preservation number of GDMCC No: 62708, and Bacteroides ovatus with a preservation number of GDMCC No: 62709.
  • the Clostridium sp with the preservation number GDMCC No: 62707 has a complete bai gene cluster.
  • the Eubacterium limosum:GDMCC-62708 strain with a preservation number of GDMCC No: 62708 has 7- ⁇ / ⁇ HSDH enzyme.
  • the Bacteroides ovatus:GDMCC-62709 strain has BSH enzyme.
  • the quantitative ratio between the strains may be 0.01-100:0.01-100, preferably 0.1-10:0.1-10, and more preferably 0.1-10:1.
  • 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.
  • the bacteria in the bacterial preparation are live bacteria.
  • 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.
  • 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).
  • the composition may be a pharmaceutical composition or a food composition, and the food composition may be, for example, a health food, a food for special medical purposes, or a therapeutic diet.
  • the pharmaceutical composition or food composition may also include conventional excipients in the field of medicine or food.
  • 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.
  • the inflammatory bowel disease is ulcerative colitis.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition for preventing and/or treating inflammatory bowel disease is an oral preparation.
  • the composition may be a pharmaceutical composition or a food composition, and the food composition may be, for example, a health food, a food for special medical purposes, or a therapeutic diet.
  • the pharmaceutical composition or food composition may also include conventional excipients in the field of medicine or food.
  • the number 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 number of each bacteria is substantially the same.
  • the bacteria of the present invention are used to prepare the composition in an amount of 1 ⁇ 10 5 CFU to 1 ⁇ 10 13 CFU per day.
  • the bacteria of the present invention are used to prepare the composition in an amount of 1 ⁇ 10 7 CFU to 1 ⁇ 10 10 CFU per day.
  • the present invention also provides a method for preventing and/or treating inflammatory bowel disease, which comprises administering an effective amount of the bacteria of the present invention or the composition of the present invention to a subject.
  • the prevention and/or treatment of inflammatory bowel disease comprises:
  • the bacteria of the present invention are administered to a subject in an amount of 1 ⁇ 10 5 CFU to 1 ⁇ 10 13 CFU, preferably 1 ⁇ 10 7 CFU to 1 ⁇ 10 10 CFU per day.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 shows the protective effects of bile acids and short-chain fatty acids on a mouse model of enteritis.
  • Figures 2a to 2e show the test results of the bacterial flora screening based on bioinformatics analysis and the verification of the in vitro metabolic detection of three bacteria.
  • Figures 3a to 3g show the test results of the effects of the three bacteria on body weight changes and colon length in enteritis mice.
  • Figures 4a and 4b show the results of the determination of fecal bile acid content in mice with enteritis after the three bacteria acted on them.
  • Figures 5a and 5b show the results of the determination of short-chain fatty acid content in feces of mice with enteritis after the three bacteria acted on them.
  • FIG6 shows the metagenomic sequencing results of the effect of the three-bacteria combination on the intestinal flora of enteritis mice.
  • GDMCC Guangdong Microbiological Culture Collection Center
  • GDMCC Guangdong Microbiological Culture Collection Center
  • Taxonomic nomenclature Eubacterium limosum.
  • GDMCC Guangdong Microbiological Culture Collection Center
  • Classification name Bacteroides ovatus.
  • This example is mainly based on the protection of the compound on the mouse enteritis model.
  • 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. This shows that the combination of bile acid and SCFA has a better protective effect on enteritis in mice.
  • 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.
  • the strain library screened in the present invention is 48 strains of bacteria obtained by isolation and culture in 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);
  • Bioinformatics analysis Screening of strains with 7- ⁇ dehydroxylase, BSH enzyme, and 7- ⁇ / ⁇ HSDH enzyme.
  • the screening database was derived from strains isolated and purified from healthy human intestinal feces samples in our laboratory in the early stage, and 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 was tested. One of them was named Clostridium sp. DA266 in the present invention; there was only one strain with 7 ⁇ / ⁇ , which was named Eubacterium limosum DA510 in the present invention; and BSH existed in many strains.
  • Bacteroides ovatus has a good effect in treating enteritis
  • the present invention finally selected a strain named Bacteroides ovatus DA668.
  • the 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 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.
  • In vitro metabolic detection verifies whether the selected strains have the mentioned enzymes: 1 Use brain heart infusion BHI medium to revive the selected strains, then transfer the revived bacterial solution to a new medium containing 50 ⁇ mol primary conjugated bile acid TCDCA/GCDCA, culture for 48 hours, and collect the culture solution; 2 Take 200 ⁇ L of the collected culture solution, add an equal volume of ethyl acetate, vigorously mediate for 15 minutes, centrifuge at 10000rpm for 5 minutes, draw the supernatant ethyl acetate layer into a new 1.5 ml centrifuge tube, dry it, and then dissolve it with 200 ⁇ L 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°C refrigerator.
  • mice 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.
  • single bacteria group Clotridium fulgidus group, Eubacterium group, Bacteroides ovatus group
  • combination bacteria group two-bacteria combination, three-bacteria combination
  • control group 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 bacterial load of all bacterial groups was the same, i.e., the single bacterial group was gavaged with 1 ⁇ 10 9 CFU per day, the two bacterial combination groups were gavaged with 5 ⁇ 10 8 CFU of Clostridium sp:GDMCC-62707 and Eubacterium limosum:GDMCC-62708 respectively, the three bacterial combination groups were gavaged with 3.3 ⁇ 10 8 CFU of Clostridium sp:GDMCC-62707, Eubacterium limosum:GDMCC-62708, and Bacteroides ovatus:GDMCC-62709 respectively, and the control group was gavaged with the same volume of blank culture medium for 10 consecutive days.
  • 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 the DSS-induced mouse enteritis model was established. The mice were allowed to drink water freely, and the gavage was continued. The weight of the mice was recorded every day. The model was established for 7 consecutive days. The feces of the mice in each group were collected, the mice were sacrificed, the serum and colon were collected, the length of the mouse colon was recorded, and the feces, colon and blood were stored in a -80°C refrigerator. The weight change and colon length of the mice are shown in Figures 3a to 3g. The effect of the three-bacteria combination on alleviating the weight loss and colon shortening of the mice is better than that of the single bacteria group and the two-bacteria combination.
  • Example 4 Effect of the three-bacteria combination on fecal bile acid and short-chain fatty acids in mice with enteritis
  • Example 5 Effect of the three-bacteria combination on the intestinal flora of mice with enteritis

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Abstract

The present invention provides a bile acid metabolizing bacterium for preventing and treating an inflammatory bowel disease and use thereof. The present invention provides a bacterium which comprises any one, two or three of the following strains: Clostridium sp. with the collection number of GDMCC No: 62707, Eubacterium limosum with the collection number of GDMCC No: 62708, and Bacteroides ovatus with the collection number of GDMCC No: 62709. The bacterium of the present invention can convert primary bile acids into secondary bile acids, ameliorating the inflammation symptoms of DSS-induced ulcerative colitis in mice.

Description

防治炎症性肠病的胆汁酸代谢菌及其应用Bile acid metabolizing bacteria for preventing and treating inflammatory bowel disease and application thereof 技术领域Technical Field
本发明是关于一种防治炎症性肠病例如溃疡性结肠炎的菌及其应用，具体而言，本发明是关于一种防治炎症性肠病的胆汁酸代谢菌、包含所述菌的组合物及相关应用。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.
背景技术Background technique
炎症性肠病(inflammatory bowel disease,IBD)是慢性的反复发作的肠道炎症性疾病，患者常常在小肠和大肠黏膜处有持续性的溃疡，主要分为两种类型，溃疡性结肠炎和克罗恩病(Crohn’s disease,CD)。IBD的可能病因包括：遗传易感性、肠道菌群等环境因素、吸烟和饮食等社会行为，此外，IBD风险增加与儿童早期接触抗生素有关。IBD的相对流行率因地理区域的不同而有很大差异，其病理特征类似但也有明显区别。IBD复发高，病程长，难治愈，预后差，目前尚无可根治的药物或治疗方法。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 an 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 the disease or treatment.
IBD的病理生理机制尚不十分清楚，通常发生于免疫系统对正常存在于肠道的菌群产生过激的免疫响应后，激起一连串的破坏和摧毁肠壁的炎症事件。肠道粘膜屏障将共生菌群与肠道上皮分隔，粘膜处的免疫细胞抑制共生菌群的积累及易位，一些IBD相关基因突变可导致粘膜屏障或免疫应答受损，引起肠道菌群及免疫平衡的失调。迄今，已有大量文献报道了IBD与菌群失调的关联，菌群变化对肠道炎症的影响机制包括：调节Treg细胞其它免疫细胞、调节促炎及抑炎因子、直接侵入肠道上皮细胞等。肠道作为最大的免疫器官，目前关于IBD的研究多聚焦短链脂肪酸，而胆汁酸则多认为与糖尿病和脂肪肝等代谢性疾病相关，鲜少有研究去探讨IBD与胆汁酸的关系，因此，研究IBD过程中的炎症与胆汁酸及菌群的关联，将对IBD的预防和治疗提供新的策略。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.
目前已有的相似方案包括：Honda等通过特定培养基筛选到17种能调节免疫反应的细菌用于治疗IBD；Daniel van等通过生信分析商用菌株库，设计了GUT-103和GUT-108两个合成菌群用于治疗IBD。Similar schemes currently available include: Honda et al. screened 17 types of 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.
以往研究中所涉及到的合成菌群的菌株是通过在培养基中加氯仿获得的能耐氯 仿的菌株，缺乏理性设计。而基于生信分析所筛选的菌株，对所关注的肠道菌群代谢物也缺乏体外代谢检测验证。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.
发明内容Summary of the invention
本发明的一个目的在于提供可以防治炎症性肠病的菌。One object of the present invention is to provide bacteria that can prevent and treat inflammatory bowel disease.
本发明的另一目的在于提供所筛选得到的菌的应用。Another object of the present invention is to provide applications of the screened bacteria.
本案发明人在研究中，基于人类微生物数组数据，针对IBD患者粪便缺乏次级胆汁酸的现象，通过基于生信分析和体外代谢检测相结合筛选到了能把结合型初级胆汁酸代谢成次级胆汁酸的菌特别是合成菌群，并进一步把所设计的菌灌胃用于DSS-诱导小鼠模型，验证了该菌对小鼠肠炎指标的改善情况，证明了本发明的菌为胆汁酸代谢菌，本发明的菌群为能改善IBD症状的合成菌群。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.
具体而言，本发明提供了三株能把结合型初级胆汁酸代谢成次级胆汁酸的菌株，本发明中分别命名为Clostridium sp.DA266、Eubacterium limosum DA510、Bacteroides ovatus DA668。这三株菌株均已进行了保藏，其中：Specifically, the present invention provides three strains that can metabolize conjugated primary bile acids into secondary bile acids, which are named Clostridium sp. DA266, Eubacterium limosum DA510, and Bacteroides ovatus DA668 in the present invention. These three strains have been preserved, among which:
菌株Clostridium sp.DA266，保藏日期：2022年08月18日；保藏单位：广东省微生物菌种保藏中心(GDMCC)；保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；保藏编号：GDMCC No:62707；分类命名：Clostridium sp.。本发明的菌株Clostridium sp.DA266亦称为Clostridium sp.:GDMCC-62707。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: GDMCC No: 62707; classification name: Clostridium sp.. The strain Clostridium sp.DA266 of the present invention is also called Clostridium sp.: GDMCC-62707.
菌株Eubacterium limosum DA510，保藏日期：2022年08月18日；保藏单位：广东省微生物菌种保藏中心(GDMCC)；保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；保藏编号：GDMCC No:62708；分类命名：Eubacterium limosum。本发明的菌株Eubacterium limosum DA510亦称为Eubacterium limosum:GDMCC-62708。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.
菌株Bacteroides ovatus DA668，保藏日期：2022年08月18日；保藏单位：广东省微生物菌种保藏中心(GDMCC)；保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；保藏编号：GDMCC No:62709；分类命名：Bacteroides ovatus。本发明的菌株Bacteroides ovatus DA668亦称为Bacteroides ovatus:GDMCC-62709。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.
本发明的研究表明，本发明的菌株Clostridium sp.DA266、Eubacterium limosum DA510、Bacteroides ovatus DA668中的任意一种或多种的组合，均具有将结合型初级胆汁酸代谢转化成次级胆汁酸的能力，能改善IBD症状。本发明的这三种菌均可称为胆汁酸代谢菌。The research of the present invention shows that any one or more combinations of the strains of the present invention, Clostridium sp. DA266, Eubacterium limosum DA510, and Bacteroides ovatus DA668, 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.
从而，一方面，本发明提供了一种菌，其包括以下菌株中的任意一种、两种或三种：Thus, in one aspect, the present invention provides a bacterium comprising any one, two or three of the following strains:
保藏编号为GDMCC No：62707的Clostridium sp.；Clostridium sp. with a deposit number of GDMCC No: 62707;
保藏编号为GDMCC No：62708的Eubacterium limosum；Eubacterium limosum with a deposit number of GDMCC No: 62708;
保藏编号为GDMCC No：62709的Bacteroides ovatus。Bacteroides ovatus with the deposit number GDMCC No: 62709.
换而言之，本发明的菌，可以是单种菌的纯培养物，也可以是包括两种或三种以上菌的菌群。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.
根据本发明的具体实施方案，本发明的菌包括保藏编号为GDMCC No：62707的Clostridium sp.以及保藏编号为GDMCC No：62708的Eubacterium limosum。According to a specific embodiment of the present invention, the bacteria of the present invention include Clostridium sp. with a preservation number of GDMCC No: 62707 and Eubacterium limosum with a preservation number of GDMCC No: 62708.
根据本发明的具体实施方案，本发明的菌包括保藏编号为保藏编号为GDMCC No：62708的Eubacterium limosum以及保藏编号为GDMCC No：62709的Bacteroides ovatus。According to a specific embodiment of the present invention, the bacteria of the present invention include Eubacterium limosum with a preservation number of GDMCC No: 62708 and Bacteroides ovatus with a preservation number of GDMCC No: 62709.
根据本发明的具体实施方案，本发明的菌包括保藏编号为GDMCC No：62707的Clostridium sp.以及保藏编号为GDMCC No：62709的Bacteroides ovatus。According to a specific embodiment of the present invention, the bacteria of the present invention include Clostridium sp. with a preservation number of GDMCC No: 62707 and Bacteroides ovatus with a preservation number of GDMCC No: 62709.
根据本发明的具体实施方案，本发明的菌包括保藏编号为GDMCC No：62707的Clostridium sp.以及保藏编号为GDMCC No：62708的Eubacterium limosum以及保藏编号为GDMCC No：62709的Bacteroides ovatus。According to a specific embodiment of the present invention, the bacteria of the present invention include Clostridium sp. with a preservation number of GDMCC No: 62707, Eubacterium limosum with a preservation number of GDMCC No: 62708, and Bacteroides ovatus with a preservation number of GDMCC No: 62709.
根据本发明的具体实施方案，本发明的菌中，所述保藏编号为GDMCC No：62707的Clostridium sp具有完整bai基因簇。According to a specific embodiment of the present invention, in the bacteria of the present invention, the Clostridium sp with the preservation number GDMCC No: 62707 has a complete bai gene cluster.
根据本发明的具体实施方案，本发明的菌中，所述保藏编号为GDMCC No：62708的Eubacterium limosum:GDMCC-62708菌株具有7-α/βHSDH酶。According to a specific embodiment of the present invention, among the bacteria of the present invention, the Eubacterium limosum:GDMCC-62708 strain with a preservation number of GDMCC No: 62708 has 7-α/βHSDH enzyme.
根据本发明的具体实施方案，本发明的菌中，所述Bacteroides ovatus:GDMCC-62709菌株具有BSH酶。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.
根据本发明的具体实施方案，本发明的菌中包括两种或三种菌株时，各菌株之间的数量比可以为0.01-100：0.01-100，优选为0.1-10：0.1-10，更优选为0.1-10： 1。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 may be 0.01-100:0.01-100, preferably 0.1-10:0.1-10, and more preferably 0.1-10:1.
根据本发明的具体实施方案，本发明的菌，其可以为固态或液态菌制剂。可以采用所属领域中任何可行的方式将所述菌制备成菌制剂。优选地，所述菌制剂中的菌为活菌。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.
另一方面，本发明还提供了一种组合物，其包括本发明所述的菌(即保藏编号为GDMCC No：62707的Clostridium sp、保藏编号为GDMCC No：62708的Eubacterium limosum、保藏编号为GDMCC No：62709的Bacteroides ovatus中的一种、两种或三种)。本发明的组合物因包括所述菌而具有将结合型初级胆汁酸代谢转化成次级胆汁酸的能力，能改善IBD症状。本发明的组合物可以称为胆汁酸代谢组合物。优选地，所述组合物还可包括与本发明的菌具有相同或有利于本发明的菌的功能(例如胆汁酸代谢)的其他活性物质，和/或辅料。根据本发明的一些具体实施方案，所述组合物可以为药物组合物或是食品组合物，所述的食品组合物例如可以是保健食品、特殊医学用途的食品或是治疗膳食。所述的药物组合物或是食品组合物中还可包括药物或食品领域中常规的辅料等。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 or a food composition, and the food composition may be, for example, a health food, a food for special medical purposes, or a therapeutic diet. The pharmaceutical composition or food composition may also include conventional excipients in the field of medicine or food.
另一方面，本发明还提供了本发明所述的菌或所述的组合物在制备用于预防和/或治疗炎症性肠病的组合物中的应用。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.
根据本发明的具体实施方案，所述炎症性肠病为溃疡性结肠炎。According to a specific embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis.
另一方面，本发明还提供了本发明所述的菌或所述的组合物在制备用于缓解炎症性肠病个体体重减轻和/或结肠缩短的组合物中的应用。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.
另一方面，本发明还提供了本发明所述的菌或所述的组合物在制备用于改善炎症性肠病个体的胆汁酸代谢的组合物中的应用。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.
另一方面，本发明还提供了本发明所述的菌或所述的组合物在制备用于改善炎症性肠病个体的肠道菌群的组合物中的应用。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.
根据本发明的具体实施方案，本发明的所述用于预防和/或治疗炎症性肠病的组合物可以为活菌制剂或灭活菌制剂。优选地，所述用于预防和/或治疗炎症性肠病的组合物为口服制剂。所述的组合物可以为药物组合物或是食品组合物，所述的食品组合物例如可以是保健食品、特殊医学用途的食品或是治疗膳食。所述的药物组合物或是食品组合物中还可包括药物或食品领域中常规的辅料等。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 or a food composition, and the food composition may be, for example, a health food, a food for special medical purposes, or a therapeutic diet. The pharmaceutical composition or food composition may also include conventional excipients in the field of medicine or food.
根据本发明的具体实施方案，当所述组合物中包括两种或三种本发明的菌时， 各菌之间的数量比可以为0.01-100：0.01-100，优选为0.1-10：0.1-10，更优选为0.1-10：1，例如各菌数量基本相同。According to a specific embodiment of the present invention, when the composition includes two or three bacteria of the present invention, the number 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 number of each bacteria is substantially the same.
根据本发明的具体实施方案，本发明所述的菌按照每天1×10 5CFU～1×10 13CFU的量用于制备所述组合物。优选地，本发明所述的菌按照每天1×10 7CFU～1×10 10CFU的量用于制备所述组合物。 According to a specific embodiment of the present invention, the bacteria of the present invention are used to prepare the composition in an amount of 1×10 5 CFU to 1×10 13 CFU per day. Preferably, the bacteria of the present invention are used to prepare the composition in an amount of 1×10 7 CFU to 1×10 10 CFU per day.
另一方面，本发明还提供了一种预防和/或治疗炎症性肠病的方法，该方法包括给予受试者有效量的本发明所述的菌或本发明所述的组合物。On the other hand, the present invention also provides a method for preventing and/or treating inflammatory bowel disease, which comprises administering an effective amount of the bacteria of the present invention or the composition of the present invention to a subject.
根据本发明的具体实施方案，所述预防和/或治疗炎症性肠病包括：According to a specific embodiment of the present invention, the prevention and/or treatment of inflammatory bowel disease comprises:
缓解炎症性肠病个体体重减轻和/或结肠缩短；Relieve weight loss and/or colon shortening in individuals with inflammatory bowel disease;
改善炎症性肠病个体的胆汁酸代谢；和/或Improving bile acid metabolism in individuals with inflammatory bowel disease; and/or
改善炎症性肠病个体的肠道菌群。Improving the intestinal flora in individuals with inflammatory bowel disease.
根据本发明的具体实施方案，本发明所述的菌按照每天1×10 5CFU～1×10 13CFU优选1×10 7CFU～1×10 10CFU的量给予受试者。 According to a specific embodiment of the present invention, the bacteria of the present invention are administered to a subject in an amount of 1×10 5 CFU to 1×10 13 CFU, preferably 1×10 7 CFU to 1×10 10 CFU per day.
本发明的具体实验表明，本发明的三种菌中的一种或多种均能将初级胆汁酸转化成次级胆汁酸，可改善DSS-诱导的溃疡性结肠炎小鼠的炎症症状。可以合理预期，本发明的这三种菌的组合的次级代谢产物短链脂肪酸、其衍生物和/或培养液也具有一定的预防和/或缓解炎症性肠病的应用；本发明的这三种菌组合的丰度和/或其代谢物可作为标志物诊断炎症性肠病。进一步，本发明的技术可以肠道菌群作为疾病和给药的靶点，以胆汁酸为物质基础去探讨代谢产生次级胆汁酸的菌株改善IBD的分子机制，基于多组学分析给与能代谢产生次级胆汁酸菌株前后动物模型菌群结构变化、差异基因表达、代谢通路调控，系统阐述胆汁酸及能代谢胆汁酸菌株治疗溃疡性结肠炎的分子机制。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.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示胆汁酸和短链脂肪酸对肠炎小鼠模型的保护。Figure 1 shows the protective effects of bile acids and short-chain fatty acids on a mouse model of enteritis.
图2a至图2e显示基于生信分析筛选菌群的测试结果和显示三菌体外代谢检测验证。Figures 2a to 2e show the test results of the bacterial flora screening based on bioinformatics analysis and the verification of the in vitro metabolic detection of three bacteria.
图3a至图3g显示三菌对肠炎小鼠体重变化和结肠长度影响的测试结果。Figures 3a to 3g show the test results of the effects of the three bacteria on body weight changes and colon length in enteritis mice.
图4a和图4b显示三菌对肠炎小鼠作用后的粪便胆汁酸含量测定结果。Figures 4a and 4b show the results of the determination of fecal bile acid content in mice with enteritis after the three bacteria acted on them.
图5a和图5b显示三菌对肠炎小鼠作用后的粪便短链脂肪酸含量测定结果。Figures 5a and 5b show the results of the determination of short-chain fatty acid content in feces of mice with enteritis after the three bacteria acted on them.
图6显示三菌组合对肠炎小鼠肠道菌群的影响的宏基因组测序结果。FIG6 shows the metagenomic sequencing results of the effect of the three-bacteria combination on the intestinal flora of enteritis mice.
用于专利程序的微生物保藏：Deposit of microorganisms for patent procedure:
(一)菌株Clostridium sp.DA266(I) Clostridium sp. DA266
保藏日期：2022年08月18日；Deposit date: August 18, 2022;
保藏单位：广东省微生物菌种保藏中心(GDMCC)；Depository: Guangdong Microbiological Culture Collection Center (GDMCC);
保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, 510070;
保藏编号：GDMCC No:62707；Deposit number: GDMCC No:62707;
分类命名：Clostridium sp.。Classification and nomenclature: Clostridium sp.
(二)菌株Eubacterium limosum DA510(ii) strain Eubacterium limosum DA510
保藏日期：2022年08月18日；Deposit date: August 18, 2022;
保藏单位：广东省微生物菌种保藏中心(GDMCC)；Depository: Guangdong Microbiological Culture Collection Center (GDMCC);
保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, 510070;
保藏编号：GDMCC No:62708；Deposit number: GDMCC No:62708;
分类命名：Eubacterium limosum。Taxonomic nomenclature: Eubacterium limosum.
(三)菌株Bacteroides ovatus DA668(III) Bacteroides ovatus DA668
保藏日期：2022年08月18日；Deposit date: August 18, 2022;
保藏单位：广东省微生物菌种保藏中心(GDMCC)；Depository: Guangdong Microbiological Culture Collection Center (GDMCC);
保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, 510070;
保藏编号：GDMCC No:62709；Deposit number: GDMCC No:62709;
分类命名：Bacteroides ovatus。Classification name: Bacteroides ovatus.
具体实施方式Detailed ways
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及附图对本发明的技术方案进行以下详细说明，应理解这些实施例仅用于说明 本发明而不用于限制本发明的范围。除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。实施例中未特别注明的方法操作，按照所属领域现有技术的常规操作或商厂说明书建议的操作进行。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 drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended 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 generally 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.
实施例1、化合物对小鼠肠炎模型的保护Example 1: Protection of the compound against mouse enteritis model
本实施例主要基于化合物对小鼠肠炎模型的保护。经过适应性喂养后，把小鼠分成2个组，分别是bile acid and SCFA组，以及DSS组,提前预防给药10天后开始造模，即把饮用水换成2％DSS水，每天记录小鼠体重造模的第七天牺牲小鼠。结果见图1，与control组比，造模第七天bile acid and SCFA组小鼠体重下降显著降低。说明bile acid与SCFA联合用药对小鼠的肠炎保护性较好。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. This shows that the combination of bile acid and SCFA has a better protective effect on enteritis in mice.
实施例2、菌株筛选Example 2, strain screening
本实施例主要基于生信分析和体外代谢检测相结合筛选能把结合型初级胆汁酸代谢成次级胆汁酸的合成菌群。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.
本发明所筛选的菌株库是本实验室前期在健康人肠道粪便样本中通过分离培养得到的48株菌，所有的菌株都经过单克隆纯化，16S rna测序鉴定确定其物种(图2a)；The strain library screened in the present invention is 48 strains of bacteria obtained by isolation and culture in 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);
生信分析：筛选具有7-α脱羟基酶、BSH酶、7-α/βHSDH酶菌株的筛选，筛选的数据库来源于本实验室前期从健康人体肠道粪便样本中分离纯化得到的菌株，并进行了全基因组测序，分析结果如下，具有完整bai基因簇的有两株菌，给予体外代谢能力的检测，其中一株本发明命名为Clostridium sp.DA266；有7α/β的菌株只有一株，本发明命名为Eubacterium limosum DA510；而BSH则存在于很多菌株中，基于文献报道卵形拟杆菌有较好的治疗肠炎的作用，最终本发明选择了一株命名为Bacteroides ovatus DA668。这三株菌株均已进行了保藏，其中：菌株Clostridium sp.DA266，保藏日期：2022年08月18日；保藏单位：广东省微生物菌种保藏中心(GDMCC)；保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；保藏编号：GDMCC No:62707；分类命名：Clostridium sp.。本发明的菌株Clostridium sp.DA266亦称为Clostridium sp:GDMCC-62707。菌株Eubacterium limosum DA510，保藏日期：2022年08月18日；保藏单位：广东省微生物菌种保藏中心(GDMCC)；保藏单位地址：中国广州 市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；保藏编号：GDMCC No:62708；分类命名：Eubacterium limosum。本发明的菌株Eubacterium limosum DA510亦称为Eubacterium limosum:GDMCC-62708。菌株Bacteroides ovatus DA668，保藏日期：2022年08月18日；保藏单位：广东省微生物菌种保藏中心(GDMCC)；保藏单位地址：中国广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，邮编：510070；保藏编号：GDMCC No:62709；分类命名：Bacteroides ovatus。本发明的菌株Bacteroides ovatus DA668亦称为Bacteroides ovatus:GDMCC-62709。本发明选择了Clostridium sp:GDMCC-62707，Eubacterium limosum:GDMCC-62708，Bacteroides ovatus:GDMCC-62709这三株菌组成合成菌群去进行后续体外代谢实验。Bioinformatics analysis: Screening of strains with 7-α dehydroxylase, BSH enzyme, and 7-α/β HSDH enzyme. The screening database was derived from strains isolated and purified from healthy human intestinal feces samples in our laboratory in the early stage, and 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 was tested. One of them was named Clostridium sp. DA266 in the present invention; there was only one strain with 7α/β, which was named Eubacterium limosum DA510 in the present invention; and BSH existed in many strains. Based on literature reports that Bacteroides ovatus has a good effect in treating enteritis, the present invention finally selected a strain named Bacteroides ovatus DA668. The 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 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.
体外代谢检测验证所筛选设计的菌株是否具有所提及的酶：①用脑心浸出液BHI培养基将所筛选的菌株复苏，然后把复苏好的菌液转接到新的含有50μmol初级结合型胆汁酸TCDCA/GCDCA培养基中，培养48小时，收集培养液；②取200μL所收集的培养液，加入等体积乙酸乙酯，剧烈斡旋15分钟，10000rpm离心5分钟，吸取上清乙酸乙酯层至新的1.5毫升的离心管中，晾干，再用200μL 50％甲醇溶解，10000rpm离心10分钟，过0.22um的滤膜，滤液收集于液相样品瓶中，存放-80℃冰箱。③液相色谱质谱分析结果如图2b、图2c、图2d、图2e所示，底物TCDCA/GCDCA被3菌共培养代谢转化产生次级胆汁酸UDCA和LCA。In vitro metabolic detection verifies whether the selected strains have the mentioned enzymes: ① Use brain heart infusion BHI medium to revive the selected 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 solution; ② Take 200μL of the collected culture solution, add an equal volume of ethyl acetate, vigorously mediate for 15 minutes, centrifuge at 10000rpm for 5 minutes, draw 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 shown in Figures 2b, 2c, 2d, and 2e. The substrate TCDCA/GCDCA is metabolized and converted by the co-culture of the three bacteria to produce secondary bile acids UDCA and LCA.
实施例3、组合菌SBE对DSS诱导小鼠肠炎模型的保护Example 3: Protection of the combined bacteria SBE against DSS-induced enteritis model in mice
经过适应性喂养后，把48只9周龄的C57小鼠随机分成6组，每组8只，分别是单菌组(闪烁梭菌组，真杆菌组，卵形拟杆菌组)，组合菌组(两菌组合，三菌组合)，以及对照组。所有给菌组总的菌量相同，即单菌组每天灌胃1×10 9CFU，两菌组合组Clostridium sp:GDMCC-62707，Eubacterium limosum:GDMCC-62708分别灌5×10 8CFU，三菌组合组Clostridium sp:GDMCC-62707，Eubacterium limosum:GDMCC-62708，Bacteroides ovatus:GDMCC-62709每天分别灌3.3×10 8CFU，对照组灌胃相同体积的空白培养基，连续灌胃10天，在第10天收集各组小鼠的粪便，立即放入液氮罐带会实验室-80℃冰箱保存。然后把小鼠的饮用水换成含2％DSS的水，DSS-诱导造小鼠肠炎模型，让小鼠自由饮水，继续灌胃，并每天记录小鼠的体重，连续造模7天，收集各组小鼠粪便，牺牲小鼠，收集血清，结 肠，记录小鼠结肠长度，粪便，结肠和血液存放-80℃冰箱保存。小鼠体重变化和结肠长度如图3a至图3g，三菌组合缓解小鼠体重减轻和结肠缩短的效果比单菌组和二菌组合好。 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 bacterial load of all bacterial groups was the same, i.e., the single bacterial group was gavaged with 1×10 9 CFU per day, the two bacterial combination groups were gavaged with 5×10 8 CFU of Clostridium sp:GDMCC-62707 and Eubacterium limosum:GDMCC-62708 respectively, the three bacterial combination groups were gavaged with 3.3×10 8 CFU of Clostridium sp:GDMCC-62707, Eubacterium limosum:GDMCC-62708, and Bacteroides ovatus:GDMCC-62709 respectively, and the control group was gavaged with the same volume of blank culture medium for 10 consecutive days. On the 10th day, the feces of the 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 the DSS-induced mouse enteritis model was established. The mice were allowed to drink water freely, and the gavage was continued. The weight of the mice was recorded every day. The model was established for 7 consecutive days. The feces of the mice in each group were collected, the mice were sacrificed, the serum and colon were collected, the length of the mouse colon was recorded, and the feces, colon and blood were stored in a -80℃ refrigerator. The weight change and colon length of the mice are shown in Figures 3a to 3g. The effect of the three-bacteria combination on alleviating the weight loss and colon shortening of the mice is better than that of the single bacteria group and the two-bacteria combination.
实施例4、三菌组合对肠炎小鼠粪便胆汁酸和短链脂肪酸的影响Example 4: Effect of the three-bacteria combination on fecal bile acid and short-chain fatty acids in mice with enteritis
从小鼠(喂养方案同实施例3)粪便中胆汁酸含量测定结果(图4a和图4b)可以看出，相较于对照组，灌胃三菌组合3strains组次级胆汁酸DCA，UDCA和LCA含量都升高了，该结果于本发明在体外检测中发现3strains能把结合型初级胆汁酸代谢转化成次级胆汁酸的结论是一致的。此外，3菌组合灌胃组短链脂肪酸丙酸和丁酸的量也升高了(图5a和图5b)。From the results of the bile acid content measurement in the feces of mice (the feeding scheme is the same as in Example 3) (Figures 4a and 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 oral administration of the three-bacteria combination increased, which is consistent with the conclusion of the present invention that 3strains can metabolize 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 3-bacteria combination oral administration group also increased (Figures 5a and 5b).
实施例5、三菌组合对肠炎小鼠肠道菌群的影响Example 5: Effect of the three-bacteria combination on the intestinal flora of mice with enteritis
从小鼠(喂养方案同实施例3)粪便宏基因组测序的结果(图6)可以看出，给予三菌灌胃相比于DSS-control组，肠道菌群中一些菌的丰度发生了变化。From the results of fecal metagenomic sequencing of mice (feeding regimen is the same as in Example 3) ( FIG. 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.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。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 (13)

  1. 一种菌，其包括以下菌株中的任意一种、两种或三种：A bacterium comprising any one, two or three of the following strains:
    保藏编号为GDMCC No：62707的Clostridium sp.；Clostridium sp. with a deposit number of GDMCC No: 62707;
    保藏编号为GDMCC No：62708的Eubacterium limosum；Eubacterium limosum with a deposit number of GDMCC No: 62708;
    保藏编号为GDMCC No：62709的Bacteroides ovatus。Bacteroides ovatus with the deposit number GDMCC No: 62709.
  2. 根据权利要求1所述的菌，其中，所述保藏编号为GDMCC No：62707的Clostridium sp.具有完整bai基因簇；The bacteria according to claim 1, wherein the Clostridium sp. with the deposit number of GDMCC No: 62707 has a complete bai gene cluster;
    所述保藏编号为GDMCC No：62708的Eubacterium limosum:GDMCC-62708菌株具有7-α/βHSDH酶；和/或The Eubacterium limosum:GDMCC-62708 strain with a deposit number of GDMCC No: 62708 has a 7-α/β HSDH enzyme; and/or
    所述Bacteroides ovatus:GDMCC-62709菌株具有BSH酶。The Bacteroides ovatus:GDMCC-62709 strain has BSH enzyme.
  3. 根据权利要求1或2所述的菌，其为固态或液态菌制剂。The bacteria according to claim 1 or 2, which is a solid or liquid bacterial preparation.
  4. 一种组合物，其包括权利要求1-3任一项所述的菌；优选地，所述组合物为药物组合物或食品组合物。A composition comprising the bacteria according to any one of claims 1 to 3; preferably, the composition is a pharmaceutical composition or a food composition.
  5. 权利要求1-3任一项所述的菌或权利要求4所述的组合物在制备用于预防和/或治疗炎症性肠病的组合物中的应用；Use of the bacteria 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;
    优选地，所述炎症性肠病为溃疡性结肠炎。Preferably, the inflammatory bowel disease is ulcerative colitis.
  6. 权利要求1-3任一项所述的菌或权利要求4所述的组合物在制备用于缓解炎症性肠病个体体重减轻和/或结肠缩短的组合物中的应用。Use of the bacteria according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a composition for alleviating weight loss and/or colon shortening in individuals with inflammatory bowel disease.
  7. 权利要求1-3任一项所述的菌或权利要求4所述的组合物在制备用于改善炎症性肠病个体的胆汁酸代谢的组合物中的应用。Use of the bacteria according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a composition for improving bile acid metabolism in individuals with inflammatory bowel disease.
  8. 权利要求1-3任一项所述的菌或权利要求4所述的组合物在制备用于改善炎症性肠病个体的肠道菌群的组合物中的应用。Use of the bacteria according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a composition for improving the intestinal flora of an individual with inflammatory bowel disease.
  9. 根据权利要求5-8任一项所述的应用，其中，所述用于预防和/或治疗炎症性肠病的组合物为活菌制剂或灭活菌制剂；优选地，所述用于预防和/或治疗炎症性肠病的组合物为口服制剂。The use according to any one of claims 5 to 8, wherein the composition for preventing and/or treating inflammatory bowel disease is a live bacteria preparation or a killed bacteria preparation; preferably, the composition for preventing and/or treating inflammatory bowel disease is an oral preparation.
  10. 根据权利要求5-9任一项所述的应用，其中，权利要求1-3任一项所述的菌按照每天1×10 5CFU～1×10 13CFU优选1×10 7CFU～1×10 10CFU的量用于制备所述组合物。 The use according to any one of claims 5 to 9, wherein the bacteria according to any one of claims 1 to 3 is used to prepare the composition in an amount of 1×10 5 CFU to 1×10 13 CFU, preferably 1×10 7 CFU to 1×10 10 CFU per day.
  11. 一种预防和/或治疗炎症性肠病的方法，该方法包括给予受试者有效量的权利要求1-3任一项所述的菌或权利要求4所述的组合物。A method for preventing and/or treating inflammatory bowel disease, comprising administering an effective amount of the bacteria according to any one of claims 1 to 3 or the composition according to claim 4 to a subject.
  12. 根据权利要求11所述的方法，其中，所述预防和/或治疗炎症性肠病包括：The method according to claim 11, wherein the prevention and/or treatment of inflammatory bowel disease comprises:
    缓解炎症性肠病个体体重减轻和/或结肠缩短；Relieve weight loss and/or colon shortening in individuals with inflammatory bowel disease;
    改善炎症性肠病个体的胆汁酸代谢；和/或Improving bile acid metabolism in individuals with inflammatory bowel disease; and/or
    改善炎症性肠病个体的肠道菌群。Improving the intestinal flora in individuals with inflammatory bowel disease.
  13. 根据权利要求11或12所述的方法，其中，权利要求1-3任一项所述的菌按照每天1×10 5CFU～1×10 13CFU优选1×10 7CFU～1×10 10CFU的量给予受试者。 The method according to claim 11 or 12, wherein the bacteria according to any one of claims 1 to 3 are administered to the subject in an amount of 1×10 5 CFU to 1×10 13 CFU, preferably 1×10 7 CFU to 1×10 10 CFU per day.
PCT/CN2022/139762 2022-09-28 2022-12-16 Bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease and use thereof WO2024066079A1 (en)

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