A malic acid-enriched strictly anaerobic Faecalibacterium rodentium RFM0224 for preventing obesity and its application

Abstract

The present invention discloses a strain of malic acid-enriched strictly anaerobic Faecalibacterium rodentium RFM0224 for preventing obesity and its application, belonging to the technical field of intestinal microorganism-host interaction and its application. The present invention isolates a strain of Faecalibacterium rodentium RFM0224 for the first time, which is a Gram-positive bacterium and an endogenous strain in the intestine of mammals. It can reduce the accumulation of white adipose tissue in the body under a high-fat diet, reduce the levels of total cholesterol (TC) and triglycerides (TG) in serum, and can significantly downregulate the expression of colon inflammatory cytokines IL-6, IL-1β and TNF-αmRNA. This lays a foundation for the use of this strain and the bacterial agent developed based on this strain in the preparation of products related to the prevention and treatment of intestinal inflammation, obesity and hyperlipidemia.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a malic acid-enriched strictly anaerobic Faecalibacterium rodentium RFM0224 for preventing obesity and an application thereof. Background Art

[0002] The intestinal flora regulates the host's metabolism and health by converting complex carbohydrates and proteins that enter the intestine into microbial metabolites and derivatives such as short-chain fatty acids and secondary bile acids, affecting the host's energy homeostasis, obesity, appetite, blood sugar regulation, insulin sensitivity, inflammation, and endocrine regulation.

[0003] Ultra-processed foods are food ingredients and additives obtained through chemical processing that are combined into convenient and delicious sweet drinks, dairy snacks (ice cream, milkshakes, etc.), French fries, desserts, biscuits and other high-sugar or high-fat foods; these foods can lead to an increase in the prevalence of obesity-related chronic diseases such as metabolic diseases, cardiovascular diseases and inflammation. Malic acid can significantly reduce fat accumulation and intestinal inflammation, and differences in intestinal flora can interfere with the effects of diet on host metabolic activity and health status. Therefore, there is an urgent need to identify strains in the intestinal flora that can mediate the lipid-lowering and anti-inflammatory benefits of malic acid to help provide precise treatment for patients with obesity and related chronic diseases.

[0004] Faecalibacterium rodentium is an anaerobic bacterium. CN 117281840 A discloses the use of Faecalibacterium rodentium in the preparation of a preparation for protecting hematopoietic stem cells and a preparation for inhibiting radiation-mediated apoptosis of hematopoietic stem cells. The strain can be used to prepare drugs for treating radiation diseases, intestinal homeostasis and organ transplant immunity. In addition, existing studies have shown that Faecalibacterium rodentium also has the effect of maintaining the homeostasis of duodenal epithelial cells, inhibiting the growth of intestinal tumors, and affecting the aging of the body. However, there are currently no reports on the effects of Faecalibacterium rodentium on obesity and intestinal inflammation in mice induced by a high-fat diet. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a malic acid-enriched strictly anaerobic Faecalibacterium rodentium RFM0224 for preventing obesity and its application.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a strain of Faecalibaculum rodentium RFM0224 is provided. The strain was deposited on February 2, 2024 in the General Microbiological Center of China Microorganism Culture Collection Committee (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and its deposit number is CGMCC No. 29854. The systematic classification name of the strain is: Faecalibaculum rodentium.

[0008] In a second aspect, the present invention provides a bacterial agent containing the above-mentioned Faecalibacterium rodentium RFM0224.

[0009] The third aspect of the present invention provides the use of the above-mentioned Faecalibacterium rodentium RFM0224 or the above-mentioned bacterial agent containing Faecalibacterium rodentium RFM0224 in the preparation of products or medicines for preventing and treating hyperlipidemia.

[0010] The hyperlipidemia is induced by a high-fat diet.

[0011] The application is achieved by reducing the triglyceride and total cholesterol content in serum.

[0012] In a fourth aspect, the present invention provides the use of the above-mentioned Faecalibacterium rodentium RFM0224 or the above-mentioned bacterial agent containing Faecalibacterium rodentium RFM0224 in the preparation of products or medicines for preventing and treating intestinal inflammation.

[0013] The intestinal inflammation is induced by a high-fat diet.

[0014] The application is achieved by down-regulating the mRNA expression level of inflammatory markers.

[0015] The inflammatory markers are interleukin-6, interleukin-1β and tumor necrosis factor-α.

[0016] In a fifth aspect, the present invention provides the use of the above-mentioned Faecalibacterium rodentium RFM0224 or the above-mentioned bacterial agent containing Faecalibacterium rodentium RFM0224 in the preparation of a product for treating obesity induced by a high-fat diet.

[0017] Beneficial effects of the present invention:

[0018] The present invention obtains a strain of Faecalibacterium rodentium RFM0224 for the first time through malic acid enrichment and separation. The strain is a Gram-positive bacterium and an endogenous strain in the intestine of mammals. The present invention has found that the strain can reduce the increase of white adipose tissue in the body induced by a high-fat diet, reduce the levels of total cholesterol (TC) and triglycerides (TG) in serum, and can significantly downregulate the expression of colon inflammatory cytokines IL-6, IL-1β and TNF-α mRNA, which lays a foundation for using the strain and the bacterial agent developed based on the strain in the preparation of products for preventing and treating intestinal inflammation, obesity and hyperlipidemia induced by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Changes in relative abundance of Faecalibaculum for malic acid treatment

[0020] Figure 2 This is a colony map of Faecalibacterium rodentium RFM0224 provided by the present invention.

[0021] Figure 3 This is a Gram staining image of Faecalibacterium rodentium RFM0224 provided by the present invention.

[0022] Figure 4 Effects of Faecalibacterium rodentium RFM0224 on the mass of epididymal white adipose tissue (eWAT); ** indicates a very significant difference (P<0.01)

[0023] Figure 5 The figure shows the effect of Faecalibacterium rodentium RFM0224 on serum triglyceride (TG); where * indicates significant difference (P<0.05), and black dots represent outliers.

[0024] Figure 6 The figure shows the effect of Faecalibacterium rodentium RFM0224 on serum total cholesterol (TC); where * indicates significant difference (P<0.05), and black dots represent outliers.

[0025] Figure 7 The results show the effect of Faecalibacterium rodentium RFM0224 on the mRNA expression of intestinal inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α); * indicates significant difference (P<0.05), and black dots represent outliers. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0027] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0028] The DSM 104 medium used in the embodiment is prepared as follows: 5 g of trypticase peptone, 5 g of peptone, 10 g of yeast extract, 5 g of beef extract, 5 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 1 ml of Tween-80, 40.00 ml of saline solution (0.25 g of CaCl2·2H2O, 0.50 g of MgSO4·7H2O, 1.00 g of K2HPO4, 1.00 g of KH2PO4, 10.00 g of NaHCO3, 2.00 g of NaCl dissolved in 1000.00 ml of distilled water) are added to 950 ml of ultrapure water, and the mixture is sterilized at 121° C. for 15 min under high pressure, and then 0.50 g of sterile Cysteine-HCl·H2O and 0.2 ml of vitamin K1 solution (0.1 ml of vitamin K1 dissolved in 20 ml of 95% ethanol, filter sterilized), 10.00 ml of hemin solution (50 mg of hemin was dissolved in 1 ml of 1N NaOH, distilled water was added to 100 ml, and filter sterilized), and reduced under strict anaerobic conditions for 12 hours.

[0029] Example 1: Isolation and identification of Faecalibacterium rodentium RFM0224

[0030] Seven-week-old male Wistar rats were randomly divided into two groups (Control group and Malate group), with 6 rats in each group, and the same feeding conditions were maintained. The Control group was gavaged with 3 ml of saline every day, and the Malate group was gavaged with 3 ml of saline containing 34 mg of malic acid every day for 30 days. The rats were anesthetized with ether, and the cecal contents were taken for 16S rRNA gene sequencing analysis. It was found that compared with the Control group, the relative abundance of Faecalibaculum in the Malate group increased ( Figure 1 )

[0031] Based on this, under strict anaerobic conditions (85% N2, 10% H2, 5% CO2), the frozen stock solution of the cecal contents of Wistar rats in the Malate group was evenly suspended in 50 ml of pre-reduced DSM 104 medium and serially diluted to 10 -5100 μL was inoculated on a DSM 104 agar plate (15 g agar was added to the DSM 104 medium before sterilization). The plate was grown at 37°C for 4 days, and a single colony was picked and cultured on a new DSM 104 agar plate for 4 days. This was repeated 3 times to obtain a pure bacterial isolate. The colony of the pure bacterial isolate was milky white, indicating a smooth round ( Figure 2 ). Gram staining results showed that it was a Gram-positive bacterium, and its morphology was rod-shaped under an optical microscope ( Figure 3 ).

[0032] The genomic DNA of the pure bacterial isolate was extracted, and its 16S rRNA gene was amplified and sequenced using the primers shown in SEQ ID NO:1-SEQ ID NO:2. The sequencing result is shown in SEQ ID NO:3.

[0033] SEQ ID NO: 1: AGAGTTTGATCMTGGCTCAG;

[0034] SEQ ID NO:2: GGTTACCTTGTTACGACTT.

[0035] SEQ ID NO:3:

[0036] TTCGACGGCTCACTCCTCTAATGAGGTCATGCCACCGGCTTCGGGCATTACCGACTC

[0037] CCATGGTTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGC

[0038] TGATCCGCGATTACTAGCGATTCCAACTTCATGGAGGCGGGTTGCAGCCTCCAATCC

[0039] GAACTGGGACGGCTTTTATGAGATTCGCTCGACATCCCTGTCTCCGCTGCTCTTTGTA

[0040] GCCGCCATTGTAGTACGTGTGTAGCCCAGGCCATAAGGGGCATGATGATTTGACGT

[0041] CATCCCCGCCTTCCTCCGGTTTGTCACCGGCAGTCTGGCATGAGTCCTCAGCTTTTCC

[0042] TGTTAGCAACATGCCACAAGGGTTGCGCTCGTTGCGGGACTTGACCCAACATCTCA

[0043] CGACACGAGCTGACGACAACCATGCACCACCTGTGTGGATGTTGACCTCCGATGCA

[0044] TCTCTGCATCTTCGCATCCCATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGA

[0045] ATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAC

[0046] ACTTGCGTGCATACTCCCCAGGCGGAGGACTCACTGCGTTAACTTCAGCACTGAGG

[0047] TTTCACCCCCAACACTTGGTCCTCATCGTTTACGGCGTGGACTACTAGGGTATCTAA

[0048] TCCTATTTGCTCCCCACGCTTTCGTGCCTCAGCGTCAGCAACAGGCCAGGCGGCCGC

[0049] CTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTTACCGCTACACATGGAGTTCC

[0050] ACCGCCCTCTCCTGCTCTCCAGTCCTCCAGTTTCCAAAGCCATGCATGAGTTGAGCT

[0051] CATGCGTTTCACTCCAGACTTGCAGGACCGCCTGCGCACCCTTTACGCCCAATCATT

[0052] CCGGATAACGCTCGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGT

[0053] GACTTTCTGGCGGGGCACCATCAGTCAGCACCCATTTCCTGATGCTGCCTTTTTTCC

[0054] CCCGCAACAGAGCTTTACGACCCGAAGGCCTTCTTCACTCACGCGGCATCGCTCGTT

[0055] CAGGCTTGCGCCCATTGACGAAAATTCCCTACTGCTGCCTCCCGCAGGAGTCTGGGC

[0056] CGTGTCCAGTCCCAATGTGGCCGTCCGCCCTCTCAGGCCGGCTATGCATCATCGTC

[0057] TTGGTGGGCCTTTACCCCGCCCAACCAACTAATGCACCGCAGGTCCATCCGCGCCCCA

[0058] TCCCCTAAAGGATGTTTCACAGAAAGAAGATGCCTCCTTCCTGTACATCGGGATTTG

[0059] TTCTCCGTTTCCAGAGCGTATTCCCGGTGCGCGGGCAGGTTCCCTACGTGTTACTCA

[0060] CCCGTTCGCCACTAGAGACCCGAAGGTCTCTCGTTCGACTTGC

[0061] The obtained sequence was compared with the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the identified pure bacterial isolate was determined to be Faecalibacterium rodentium. The isolated pure bacterial isolate has been deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), with the deposit name RFM0224, the deposit date is February 2, 2024, and the deposit number is CGMCC No. 29854.

[0062] Example 2: Functional study of Faecalibacterium rodentium RFM022

[0063] 1. Experimental Methods

[0064] Seven-week-old male C57BL / 6J mice were randomly divided into two groups (HFP+PBS group, n=6; HFP+FR group, n=7), with free access to high-fat diet (HFD, 45% of energy from fat) and sterile water. The HFP+PBS group was gavaged with 200 μL of phosphate buffered saline (PBS) every two days, and the HFP+FR group was gavaged with 200 μL of PBS containing Faecalibacterium rodentium RFM0224 every two days (the Faecalibacterium rodentium RFM0224 cultured in pre-reduced DSM 104 medium for 18 hours was centrifuged at 12000 rpm for 2 minutes and diluted with PBS to OD 600 nm=0.8) for 60 days, the mice were anesthetized with ether, their colon and white adipose tissue were taken, and their blood was extracted.

[0065] The extracted white adipose tissue of mice was weighed, and the changes in eWAT mass of the two groups of mice were observed.

[0066] The extracted mouse blood was centrifuged at 4°C, 3000r for 15 minutes to obtain serum, and the contents of TG and TC in the extracted mouse serum were detected using a serum biochemical analyzer (Hitachi, model 7020).

[0067] The extracted mouse colon tissue was fully ground in liquid nitrogen, and its RNA was extracted using an RNA extraction kit (Tiangen, China), and its cDNA was obtained using a reverse transcription kit (Aikerui, China). β-actin (primers such as SEQ ID NO: 4-5) was used as a housekeeping gene, and the relative expression level was calculated using the 2-ΔΔCt method to detect the mRNA expression levels of inflammatory markers IL-6 (primers such as SEQ ID NO: 6-7), IL-1β (primers such as SEQ ID NO: 8-9), and TNF-α (primers such as SEQ ID NO: 10-11) in mouse colon tissue.

[0068] SEQ ID NO:4: CTATTGGCAACGAGCGGTTCC;

[0069] SEQ ID NO:5: GCACTGTGTTGGCATAGAGGTC.

[0070] SEQ ID NO:6: GCGACAGATTGGCGAGTTCTAC;

[0071] SEQ ID NO:7: GAGAGGAGACTTCACAGAGGATACC.

[0072] SEQ ID NO:8:TCATTTCCACGATTTCCCAGAGAAC;

[0073] SEQ ID NO:9:TCCACGGGAAAGACACAGGTAG.

[0074] SEQ ID NO: 10: CACGCTTCTCTGTCTACTGAACTTC;

[0075] SEQ ID NO: 11: TGGGCTACAGGCTTGTCACTC.

[0076] 2. Experimental results

[0077] The extracted white adipose tissue of mice was weighed, and the results showed that Faecalibacterium rodentium RFM022 significantly reduced the increase in the mass of eWAT in mice ( Figure 4 ).

[0078] The results of mouse serum tests showed that Faecalibacterium rodentium RFM022 significantly prevented the increase of TG and TC levels in mouse serum ( Figure 5 , Figure 6 ).

[0079] The results of the mRNA expression level detection of inflammatory markers in mouse colon tissue showed that Faecalibacterium rodentium RFM022 significantly downregulated the expression of colon inflammatory cytokines IL-6, IL-1β and TNF-α mRNA ( Figure 7 ).

[0080] In summary, Faecalibacterium rodentium RFM022 significantly reduced the serum TG and TC content that promote atherosclerosis and epididymal white adipose tissue, and downregulated the expression of intestinal inflammatory cytokines IL-6, IL-1β and TNF-α mRNA. Therefore, the Faecalibacterium rodentium RFM022 isolated and obtained by the present invention, and the bacterial agent prepared using the strain have important application prospects in the preparation of products for preventing and treating obesity, intestinal inflammation, hyperlipidemia and other related chronic diseases.

[0081] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Faecalibaculum rodentium RFM0224 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 2, 2024, and its deposit number is CGMCC No.29854.

2. A bacterial agent containing the Faecalibacterium rodentium RFM0224 according to claim 1.

3. Use of the Faecalibacterium rodentium RFM0224 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for treating hyperlipidemia.

4. The use according to claim 3, characterized in that: The hyperlipidemia is induced by a high-fat diet.

5. The use according to claim 3, characterized in that: The application is achieved by reducing the triglyceride and total cholesterol content in serum.

6. Use of the Faecalibacterium rodentium RFM0224 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for treating intestinal inflammation induced by a high-fat diet.

7. Use of the Faecalibacterium rodentium RFM0224 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for treating obesity induced by a high-fat diet.

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