Flavonifractor plautii xia-2 and use thereof

Abstract

Provided are a Flavonifractor plautii Xia-2 strain and the use thereof. The Flavonifractor plautii Xia-2 strain has a deposit number of GDMCC No: 62984. The Flavonifractor plautii Xia-2 strain and a derivative, culture medium or composition thereof have the effect of preventing, ameliorating or treating atherosclerosis. The Flavonifractor plautii Xia-2 strain can effectively reduce the blood lipid level, inhibit the formation of atherosclerotic plaque, reduce the local inflammation level in the plaque, has no significant impact on the body weight and glycometabolism of an animal, also causes no damage to the liver functions and renal functions of an animal, and has relatively good safety.

Description

A kind of Fusobacterium prausnitzii and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on July 24, 2023, with application number 202310906490.0 and invention name “A kind of Clostridium prausnitzii and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the technical field of microbial therapy, and in particular relates to a Fusobacterium prausnitzii and its application. Background Art

[0003] Among different types of cardiovascular disease, the burden of atherosclerotic cardiovascular disease (primarily including coronary heart disease and ischemic stroke) is rapidly increasing in the Chinese population, now accounting for over 60% of cardiovascular disease deaths. Therefore, controlling the growing burden of cardiovascular disease, especially atherosclerotic cardiovascular disease, is a daunting challenge.

[0004] Atherosclerosis (AS) is a disease characterized by the formation of patchy lipid deposits (atheromas or atherosclerotic plaques) on the walls of medium-sized or large arteries, leading to reduced or blocked blood flow. It is also a common pathological basis for coronary heart disease and ischemic stroke. Disturbances in glucose and lipid metabolism are key drivers of AS progression. Low-density lipoprotein cholesterol (LDL-C) in the blood is the primary component of atherosclerotic plaques. Elevated LDL-C levels are the most established risk factor for atherosclerosis and one of the most critical clinical therapeutic targets.

[0005] Currently, the prevention and treatment of AS mainly include drug intervention and lifestyle adjustment. Compliance with lifestyle interventions is often poor, such as long-term calorie restriction, regular exercise, or a low-salt and low-oil diet. Patients are usually unable to adapt to major changes in lifestyle, thus interrupting the treatment process. Long-term drug treatment may cause adverse reactions, such as statin intolerance causing abnormal liver function, increased blood sugar, or gastrointestinal discomfort. Some patients with contraindications need to change treatment methods. Even if they follow drug treatment, the blood lipid levels of some patients cannot be reduced to the expected target value, and there is still a large residual risk. Therefore, there is an urgent need to find early intervention measures with good compliance and obvious prevention and treatment effects.

[0006] Summary of the Invention

[0007] In view of this, the present application discloses a Fusobacterium prausnitzii and its application, which can effectively lower blood lipid levels, inhibit the formation of atherosclerotic plaques, and has good safety.

[0008] In a first aspect, the present application provides the Flavonifractor plautii Xia-2 strain, whose deposit number is GDMCC No: 62984.

[0009] It should be noted that the Fusobacterium plautii strain is deposited in Guangdong Provincial Microbiological Culture Collection Center and named Flavonifractor plautii Xia-2.

[0010] Specifically, the Fusobacterium prausnitzii strain can be cultured using GAM medium or common GAM modified medium.

[0011] The second aspect of the present application provides derivatives of the strain.

[0012] Preferably, the derivative is a living bacterium, a dead bacterium, a bacterial cell component, a culture, a lysate, an extract, an inactivated product or a combination thereof.

[0013] The third aspect of the present application provides a culture medium comprising the strain or the derivative.

[0014] Specifically, the culture includes the cell-free culture filtrate of Flavonifractor plautii Xia-2 and its progeny.

[0015] The fourth aspect of the present application provides a composition comprising the strain, the derivative or the culture medium.

[0016] Specifically, the composition is a composition or preparation for preventing, improving or treating atherosclerosis.

[0017] Preferably, the composition is a food composition, a dietary supplement composition, a nutritional composition, a health food composition, a pharmaceutical composition, a food composition for special medical purposes, a cosmetic composition or a feed composition.

[0018] Specifically, the food composition can be a solid beverage, candy or juice, or a dairy product (such as yogurt, flavored fermented milk, lactic acid bacteria beverage, or cheese).

[0019] Preferably, the composition is in the form of pills, tablets, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oily solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, or gel candies.

[0020] Preferably, the composition further comprises one or more of probiotics, postbiotics, prebiotics, antimicrobials, immunomodulators, minerals, vitamins, dietary fiber, proteins, carbohydrates, lipids, plant extracts, amino acids and milk substitutes.

[0021] Specifically, the composition is a solid, semisolid or liquid for oral administration or injection.

[0022] Preferably, the composition further comprises one or more of pharmaceutically acceptable excipients, food-acceptable excipients and metabolites.

[0023] More preferably, the pharmaceutically acceptable excipient is selected from one or more of an excipient, a disintegrant, a lubricant, a sweetener and a binder;

[0024] The metabolites are one or more of enzymes, cell structure components, exopolysaccharides and bacteriocins produced by the metabolism of the intestinal strain.

[0025] More preferably, the food-acceptable excipient is selected from one or more of minerals, vitamins, dietary fiber, prebiotics, proteins (such as enzymes), carbohydrates, lipids (such as fats), plant extracts (such as plant extracts), amino acids, immunomodulators and milk substitutes.

[0026] Specifically, the pharmaceutically acceptable excipients and the food-acceptable excipients are all existing conventional ingredients, and are not described in detail in this application.

[0027] As a preference, the Clostridium prausnitzii is 10 6 to 10 12 The presence of cfu / day.

[0028] Specifically, the Clostridium prausnitzii was 5×10 8 The presence of cfu / day.

[0029] The fifth aspect of the present application discloses the use of the strain, the derivative, the culture medium or the composition in preparing a preparation for preventing, improving or treating atherosclerosis.

[0030] Specifically, the preparation can be a pharmaceutical preparation, a probiotic preparation, or a food and / or feed additive preparation.

[0031] The sixth aspect of the present application discloses the use of the strain, the derivative, the culture medium or the composition in the preparation of a lipid-lowering preparation.

[0032] This application addresses the current lack of effective and compliant early intervention measures for atherosclerosis. The application provides a novel strain of Flavonifractor plautii Xia-2, deposited with GDMCC No. 62984. Experimental data demonstrate that this strain, its derivatives, culture medium, and composition, inhibit the progression of atherosclerosis, effectively lower blood lipid levels, and suppress the formation of atherosclerotic plaques, while also demonstrating a good safety profile. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a schematic diagram of an animal experiment provided in an embodiment of the present application;

[0034] FIG2 shows the determination of Flavonifractor plautii Xia-2 provided in the embodiment of the present application;

[0035] FIG3 shows the results of the embodiment of the present application showing that Flavonifractor plautii Xia-2 inhibits the formation of atherosclerotic plaques in mice;

[0036] FIG4 shows the results of the present invention's embodiment providing that Flavonifractor plautii Xia-2 reduces the level of inflammation in mouse plaques;

[0037] FIG5 shows the effects of Flavonifractor plautii Xia-2 intervention on body weight and glucose metabolism in mice provided in the examples of the present application;

[0038] FIG6 shows the effect of Flavonifractor plautii Xia-2 intervention on blood lipids in mice provided in the examples of the present application;

[0039] FIG7 shows the effects of Flavonifractor plautii Xia-2 intervention on the liver and kidneys of mice provided in the examples of the present application;

[0040] FIG8 shows the effects of inactivated bacteria of Flavonifractor plautii Xia-2 on the aorta and aortic sinus of mice after intervention with mice according to the embodiments of the present application;

[0041] FIG9 shows the effects of the key metabolite (allo-deoxycholic acid) of Flavonifractor plautii Xia-2 on the aorta and aortic sinus of mice after intervention with the mouse aorta, as provided in the examples of the present application. DETAILED DESCRIPTION

[0042] The present application provides a Clostridium prausnitzii and its application, which effectively solves the technical defect of the existing technology in that there is a lack of early intervention measures for atherosclerosis with good compliance and good prevention and treatment effects.

[0043] The following is a clear and complete description of the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0044] Among them, the animal model (AS model mouse) of the following examples:

[0045] Apoe - / - High-fat, high-cholesterol diet model: ApoE knockout mice, due to ApoE inactivation, are unable to recognize and bind to the relevant receptors for LDL, VLDL / IDL, resulting in delayed clearance of these lipoproteins and hyperlipidemia. High blood lipids induce oxidative modification of lipoproteins, promoting the development of AS lesions. Models can be established on both a standard diet and a high-fat, high-cholesterol diet, with a duration of approximately 8-12 weeks.

[0046] The Fusobacterium prautii (Flavonifractor plautii Xia-2, hereinafter referred to as FP) used in the following examples was isolated and identified by the applicant's team from healthy individuals. It was identified as belonging to the species Flavonifractor plautii. Its activity and gene sequence were verified by the Guangdong Provincial Center for Microbiological Collection and received a deposit number (GDMCC No. 62984).

[0047] Specifically, the deposit information of Flavonifractor plautii Xia-2 is as follows:

[0048] Collection center: Guangdong Provincial Microbiological Culture Collection Center (GDMCC);

[0049] Address of the Collection Center: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province;

[0050] Deposit number: GDMCC No:62984;

[0051] Depository: Sun Yat-sen University;

[0052] Address of the depository: Public Health Building, North Campus, Sun Yat-sen University, 74 Zhongshan 2nd Road, Yuexiu District, Guangzhou, Guangdong Province;

[0053] Taxonomic name: Flavonifractor plautii;

[0054] Deposit date: November 17, 2022;

[0055] The strain survival test time was November 17, 2022, and the test result was survival. HFHC in the following examples is a high-fat, high-cholesterol feed.

[0056] The FP live bacteria solution in the following examples was cultured anaerobically using Haibo Bio's GAM modified medium. The cultured bacteria were plated at a rate of 5 × 10 8 The total number of colonies per day was resuspended in sterile PBS (supplemented with cysteine ​​and vitamin B1) with a final concentration of 10% glycerol to maintain an anaerobic environment.

[0057] The FP inactivated bacterial solution of the following examples: the live bacteria obtained in the above manner were sterilized by autoclaving at 121°C for 15 minutes, and the inactivated strains were obtained by mixing 5×10 8 The total number of colonies per day was resuspended in sterile PBS (supplemented with cysteine ​​and vitamin B1) with a final concentration of 10% glycerol.

[0058] The key metabolite of FP in the following examples is isodeoxycholic acid. Through the combined analysis of metagenomics and targeted metabolomics, it was found that isodeoxycholic acid is the key metabolite of FP bacteria in exerting the AS protective effect.

[0059] The above-mentioned live FP bacteria, inactivated FP bacteria and active metabolites of FP were used to intervene in AS model mice, and the intervention period was 12 weeks.

[0060] Example

[0061] The present application provides animal experiments to verify the effects of Flavonifractor plautii Xia-2 in preventing, improving, or treating atherosclerosis, specifically including:

[0062] 1. Animal grouping and intervention design

[0063] The study plan is shown in Figure 1. 8-week-old male SPF-grade ApoE - / -Thirty-two mice were randomly divided into four groups, with eight mice in each group, namely the atherosclerosis model group (HFHC + sterile PBS, denoted as PBS); the FP live bacteria intervention group (HFHC + FP live bacteria solution, denoted as FP); the FP inactivated bacteria intervention group (HFHC + FP inactivated bacteria solution, denoted as inactivated FP) and the FP key metabolite group (HFHC + allodeoxycholic acid, denoted as FP metabolite). All four groups were fed with a high-fat, high-cholesterol diet HFHC for 12 weeks to establish an atherosclerosis model. The high-fat, high-cholesterol diet HFHC had a formula of 39.9% fat, 40% carbohydrates, 20% protein, and 1.25% cholesterol. The HFHC + PBS group was gavaged with sterile PBS solution every day, with a gavage volume of 200 μL; the FP live bacteria group was gavaged with 5×10 8 CFU of Fusobacterium prausnitzii FP was gavaged in a volume of 200 μL; mice in the FP inactivated intervention group were gavaged with 5×10 8 Mice in the FP active metabolite group received 200 μL of inactivated bacterial solution via oral gavage daily. Mice in the FP active metabolite group received 200 μL of allodeoxycholic acid daily via oral gavage. The intervention lasted 12 weeks. The animal room temperature was maintained at (22 ± 2)°C and the humidity was (45 ± 15)%. The mice were housed on a 12-hour day / night cycle (light on from 8:00 AM to 8:00 PM), had free access to food, and the bedding was kept dry and changed every 3-4 days.

[0064] 2. Bacterial colonization

[0065] Fecal DNA was extracted from the two groups of mice in the previous step, and the FP content was measured using fluorescent quantitative PCR. The results are shown in Figure 2. PBS in Figure 2 represents the PBS group, and FP represents the FP live bacteria intervention group. The FP live bacteria intervention group showed a significant increase in the FP bacteria content in the feces of mice, indicating that the FP live bacteria successfully colonized the mouse intestines and has the potential to become a new probiotic.

[0066] 3. FP live bacteria inhibit the formation of atherosclerotic plaques in mice

[0067] The aorta and aortic sinus of the two groups of mice were stained with Oil Red O. The results are shown in Figures 3A and 3B. PBS in Figure 3 refers to the PBS group, and FP refers to the FP live bacteria intervention group. In the FP live bacteria intervention group, the positive area of ​​the aorta gross specimens of mice decreased by 36.7% (Figure 3A), and the plaque area of ​​the aortic sinus decreased by 27.4% (Figure 3B). The differences were statistically significant, indicating that FP live bacteria intervention is beneficial to reducing the area of ​​atherosclerotic plaques in mice.

[0068] 4. FP live bacteria reduce inflammation levels in atherosclerotic plaques in mice

[0069] The expression of the proinflammatory cytokine MCP-1 and macrophage infiltration in AS plaques of the two groups of mice were measured. The results are shown in Figure 4. PBS in Figure 4 represents the PBS group, and FP represents the FP live bacteria intervention group. This indicates that FP live bacteria intervention significantly reduced the expression of the proinflammatory cytokine MCP-1 (Figure 4A) and macrophage infiltration (Figure 4B) in AS plaques of mice, thereby reducing the level of local inflammation in the mouse plaques. These results indicate that FP live bacteria intervention can effectively delay the progression of AS and reduce the severity of AS.

[0070] 5. After FP live bacteria intervention in mice, the effects of FP live bacteria on mouse body weight and glucose metabolism were determined

[0071] After the FP bacteria intervention, blood was taken from the tail vein of the mice to measure their blood sugar and evaluate their glucose metabolism. Before the mice were sacrificed, their body weight was weighed and their body composition was tested. After sacrifice, epididymal adipose tissue was collected to evaluate the obesity of the mice. As shown in Figure 5A-C, the PBS in Figure 5 is the PBS group, and FP is the FP live bacteria intervention group. The body weight, body fat percentage and epididymal fat content of the mice in the FP bacteria intervention group decreased. The glucose metabolism of the mice is shown in Figure 5D-E. The postprandial blood sugar and fasting blood sugar of the mice in the FP bacteria intervention group decreased slightly, and there was no significant improvement in the weight and glucose metabolism of the mice. The results show that the effect of FP bacteria on obesity and glucose metabolism in mice is not significant.

[0072] 6. After FP live bacteria intervention in mice, the effect of FP live bacteria on the blood lipids of mice was determined

[0073] Commercially available kits were used to measure serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) levels in mice in the PBS and FP groups to assess lipid metabolism. The results are shown in Figures 6A-D. PBS in Figure 6 represents the PBS group, and FP represents the FP live bacteria intervention group. The FP live bacteria intervention group showed a significant decrease in serum LDL-c levels, a slight decrease in serum TC and TG levels, and an increase in HDL-c levels. This indicates that FP live bacteria intervention significantly reduced blood lipid levels in mice and that FP live bacteria exerted its anti-atherosclerotic effect by regulating LDL-c levels.

[0074] 7. After FP live bacteria intervention in mice, the effects of FP live bacteria on the liver and kidneys of mice were determined

[0075] The safety of FP bacteria was measured by three aspects: changes in body weight and whether liver and kidney function was damaged. Kits were used to detect the levels of creatinine (Scr), urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in the PBS group and the FP group. The results are shown in Figure 7. The PBS in Figure 7 is the PBS group, and the FP is the FP live bacteria intervention group. There was no significant difference in the body weight of mice in the FP live bacteria intervention group and the PBS control group, as well as serum renal function-related indicators such as Scr and BUN, and liver damage-related indicators such as ALT and AST, and all were within the normal range. This shows that the intervention of FP live bacteria will not cause damage to the liver and kidney functions of mice. While effectively preventing atherosclerosis, it still has a certain degree of safety. The FP live bacteria intervention has good liver and kidney safety.

[0076] 8. FP-inactivated bacteria intervention reduced the area of ​​atherosclerotic plaques and lowered blood lipid levels in mice

[0077] The aorta and aortic sinus of the two groups of mice were stained with Oil Red O. The results are shown in Figures 8A and 8B. The PBS in Figure 8 represents the PBS group, and the inactivated FP represents the inactivated FP bacteria intervention group. The inactivated FP bacteria intervention group showed a 35% decrease in the positive area of ​​the aortic gross specimens (Figure 8A), and a 25% decrease in the area of ​​aortic sinus plaques (Figure 8B). These differences were statistically significant, indicating that inactivated FP bacteria intervention can also effectively reduce the area of ​​atherosclerotic plaques in mice. Furthermore, after inactivated FP bacteria intervention, the LDL-c levels of the mice were significantly reduced, while other lipid levels remained unchanged, indicating that inactivated FP bacteria has the effect of lowering low-density lipoprotein (LDL) (Figure 8C).

[0078] 9. Intervention of mice with FP's key metabolite (allo-deoxycholic acid) reduced atherosclerotic plaque area and lowered blood lipid levels

[0079] The aorta and aortic sinus of the two groups of mice were stained with Oil Red O. The results are shown in Figures 9A and 9B. The PBS in Figure 9 represents the PBS group, and the FP metabolite represents the FP key metabolite group. Treatment with the FP key metabolite (allo-deoxycholic acid) reduced the positive area of ​​the aorta gross specimens by 42% (Figure 9A) and the aortic sinus plaque area by 30% (Figure 9B). These differences were statistically significant, indicating that treatment with the FP key metabolite (allo-deoxycholic acid) can also effectively reduce the area of ​​atherosclerotic plaques in mice. Furthermore, treatment with the FP active metabolite (allo-deoxycholic acid) significantly reduced LDL-c levels in mice, while other lipid levels remained unchanged, indicating that the FP key metabolite (allo-deoxycholic acid) has the effect of lowering low-density lipoprotein (LDL) (Figure 9C).

[0080] In summary, the present application found that the live bacteria, inactivated bacteria and key metabolites of Flavonifractor plautii Xia-2 strain can effectively lower blood lipid levels, inhibit the formation of atherosclerotic plaques, reduce the level of local inflammation in plaques, and have no significant effect on the weight and glucose metabolism of animals. At the same time, they will not cause damage to the liver and kidney functions of animals and have good safety.

[0081] The above describes in detail the Fusobacterium prausnitzii provided by the present invention and its applications. The principles and implementation methods of the present invention are illustrated herein using specific examples. The description of the above examples is intended only to facilitate understanding of the methods and core concepts of the present invention. It should be noted that those skilled in the art will be able to make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A Flavonifractor plautii Xia-2 strain, whose deposit number is GDMCC No: 62984.

2. A derivative of the strain as claimed in claim 1.

3. The derivative according to claim 2, characterized in that The derivative is a living bacterium, a dead bacterium, a bacterial cell component, a culture, a lysate, an extract, an inactivated product or a combination thereof.

4. A culture medium, characterized in that It comprises the strain according to claim 1 or the derivative according to claim 2 or 3.

5. A composition, characterized in that It comprises the strain according to claim 1, the derivative according to claim 2 or 3, or the culture medium according to claim 4.

6. The composition according to claim 5, characterized in that The composition is a nutritional composition, a pharmaceutical composition, a food composition for special medical purposes, a cosmetic composition or a feed composition.

7. The composition according to claim 6, characterized in that The composition is in the form of pills, tablets, lyophilized powders, granules, capsules, aqueous solutions, alcohol solutions, oil solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, and gel candies.

8. The composition according to claim 6, characterized in that The composition also includes one or more of probiotics, postbiotics, prebiotics, antimicrobials, immunomodulators, minerals, vitamins, dietary fiber, proteins, carbohydrates, lipids, plant extracts, amino acids, and milk substitutes.

9. Use of the strain according to claim 1, the derivative according to claim 2 or 3, the culture medium according to claim 4 or the composition according to any one of claims 5 to 8 in preparing a preparation for preventing, improving or treating atherosclerosis.

10. Use of the strain according to claim 1, the derivative according to claim 2 or 3, the culture medium according to claim 4 or the composition according to any one of claims 5 to 8 in the preparation of a lipid-lowering preparation.

11. Use of the strain according to claim 1, the derivative according to claim 2 or 3, the culture medium according to claim 4 or the composition according to any one of claims 5 to 8 in preventing, improving or treating atherosclerosis.

12. Use of the strain according to claim 1, the derivative according to claim 2 or 3, the culture medium according to claim 4 or the composition according to any one of claims 5 to 8 in lowering blood lipids.

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