Cheese-clostridium casei lc001 for reducing cholesterol by regulating intestinal fxr-fgf15 axis and application thereof

Abstract

The application discloses a strain of Brevibacterium casei LC001 for reducing cholesterol by regulating an intestinal FXR-FGF15 axis and an application thereof, and relates to the field of Brevibacterium casei. The application constructs a functional strain with the abilities of bile acid metabolism reconstruction, signal pathway intervention and environmental response regulation by performing genetic engineering modification and metabolic regulation treatment on the Brevibacterium casei. The strain can improve the activity of bile salt hydrolase, promote the generation of secondary bile acid, thereby reducing the activation level of the FXR receptor. Meanwhile, the strain can realize multi-level regulation on the FXR-FGF15 signal axis by secreting FGF15 regulating factors or constructing a bile acid response type expression system, and further combined with a complex microbial population system, can form a synergistic enhancement effect, and significantly improve the cholesterol reduction efficiency.

Description

Technical Field

[0001] This invention relates to the field of Lactobacillus casei, and more particularly to a strain of Lactobacillus casei LC001 that reduces cholesterol by regulating the intestinal FXR-FGF15 axis and its applications. Background Technology

[0002] Disorders of cholesterol metabolism are a significant factor in the development of atherosclerosis and related cardiovascular diseases. Existing research indicates that gut microbiota can participate in cholesterol homeostasis regulation by modulating bile acid metabolism. Among these, the farnesoid X receptor (FXR) and its downstream fibroblast growth factor 15 (FGF15) signaling axis play a crucial role in bile acid feedback regulation and cholesterol metabolism. However, most probiotics in current technologies only possess limited bile salt hydrolysis capabilities and have weak ability to regulate bile acid composition, making it difficult to effectively influence the FXR-FGF15 signaling pathway. Furthermore, existing strains lack targeted regulatory mechanisms for this signaling axis, failing to achieve a synergistic effect from bile acid metabolism remodeling to signal transduction intervention, resulting in unstable or low-efficiency cholesterol-lowering effects. Summary of the Invention

[0003] Therefore, in order to address the above-mentioned shortcomings, this invention provides a strain of Lactobacillus casei LC001 that reduces cholesterol by regulating the intestinal FXR-FGF15 axis and its applications.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a strain of *Lactobacillus casei* LC001 that lowers cholesterol by regulating the intestinal FXR-FGF15 axis, wherein *Lactobacillus casei* LC001 is deposited at the China General Microbiological Culture Collection Center, classified and named as *Lactobacillus casei*, with the accession number CGMCC. NO.20587, Deposit Date: September 1, 2020, Deposit Address: China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Deposit Result: Viable, and the strain, after genetic engineering modification and / or metabolic regulation treatment, meets at least one or more of the following characteristics: bile salt hydrolase activity increased by ≥20% compared to the starting strain; under bile salt culture conditions, the proportion of secondary bile acids in total bile acids increased by ≥10%; under 0.1–0.5 mM bile acid induction conditions, the expression level of metabolites regulating the FXR signaling pathway increased by ≥1.5 times; secretes active factors that can reduce FGF15 expression or inhibit FGF15 receptor binding; has bile acid adsorption capacity, with an adsorption rate of ≥15%, and possesses sustained-release properties; thereby reducing FXR expression level by ≥20% and / or FGF15 expression level by ≥20%, and achieving a serum cholesterol reduction effect.

[0005] Preferably, the strain achieves a bile acid debinding rate of ≥40% by introducing or enhancing the bile salt hydrolase encoding genes bsh1 and / or bsh2.

[0006] Preferably, the strain has the ability to convert primary bile acids into deoxycholic acid and / or lithocholic acid, so that the proportion of secondary bile acids reaches ≥35%.

[0007] Preferably, the strain is equipped with a bile acid-inducible expression system, including a bile acid-responsive promoter and a metabolic regulatory gene linkage structure, which induces the expression of short-chain fatty acids or FXR-inhibiting metabolites when bile acid concentration increases.

[0008] Preferably, the active factor is a polypeptide with a length of 5–50 amino acids, which can competitively bind to the FGF15 receptor FGFR4, thereby reducing the receptor binding rate by ≥30%.

[0009] Preferably, the strain is modified with cell surface polysaccharides or proteins to achieve an adsorption rate of 15%–40% for bound bile acids and to achieve delayed release in the intestinal environment.

[0010] A microbial composition comprising the above-mentioned strains, and at least one secondary bile acid producing bacterium and / or short-chain fatty acid producing bacterium; wherein each strain is compounded in a mass or quantity ratio of (0.5–2):(0.5–2):1, thereby increasing the proportion of secondary bile acids in vivo by ≥15% and decreasing FXR expression by ≥30%.

[0011] Preferably, the short-chain fatty acid producing bacteria are butyric acid bacteria, and the secondary bile acid producing bacteria are intestinal bacteria with 7α-dehydroxylation ability.

[0012] The strains or compositions of the present invention can be used to prepare products for regulating the FXR-FGF15 signaling axis, preferably for reducing serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) by more than 15%; the products can be food, health food or pharmaceutical preparations.

[0013] The beneficial effects of this invention are as follows: By genetically engineering and metabolically regulating Lactobacillus casei, this invention constructs a functional strain that combines bile acid metabolism remodeling, signal pathway intervention, and environmental response regulation. This strain can increase the activity of bile salt hydrolase and promote the production of secondary bile acids, thereby reducing the activation level of FXR receptors. At the same time, by secreting FGF15 regulatory factors or constructing a bile acid-responsive expression system, it can achieve multi-level regulation of the FXR-FGF15 signaling axis. Furthermore, combined with a complex microbial community system, it can form a synergistic enhancement effect and significantly improve the cholesterol-lowering efficiency. Detailed Implementation

[0014] To further explain the technical solution of the present invention, a detailed description is provided below through specific embodiments.

[0015] This invention provides a strain of *Lactobacillus casei* LC001 that lowers cholesterol by regulating the intestinal FXR-FGF15 axis and its applications. Based on the already deposited *Lactobacillus casei* LC001, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) under the classification and name *Lactobacillus casei*, accession number CGMCC. NO.20587, deposit date: September 1, 2020, deposit address: China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposit result: viable. Through functional enhancement modification, it was able to acquire the ability to regulate the intestinal FXR-FGF15 signaling axis. The functional enhancement was mainly achieved through the following technical pathways: enhanced bile acid metabolism, regulation of bile acid responsive expression, construction of FGF15 signaling intervention ability, regulation of bile acid adsorption and release, and construction of a multi-bacterial synergistic regulation system. Through the above technical means, the strain can reduce the activation level of FXR receptors by regulating bile acid composition and further inhibit FGF15 expression, thereby achieving the technical effect of lowering cholesterol.

[0016] Example 1, BSH Enhanced: Construction of engineered bacteria, vector: pMG36e, inserted genes: bsh1, bsh2, electroporation parameters: 2.0kV / 200Ω / 25μF, screening for positive bacteria: erythromycin resistance + PCR verification; functional detection: bile acid transformation, conditions: 0.3% bile salts, culture for 24h, results: debinding rate: 42%, secondary bile acids: 38%; FXR-FGF15 detection, FXR decreased by 28%, FGF15 decreased by 23%; animal experiments, TC decreased by 18%, LDL decreased by 15%.

[0017] Example 2, Enhanced bile acid conversion: Based on Example 1, a secondary bile acid conversion-related enzyme system (7α-dehydroxylation-related gene fragment) was introduced; Functional verification: Bile acid analysis, debinding rate: 46%, secondary bile acid ratio: 42%; FXR-FGF15, FXR decreased by 32%, FGF15 decreased by 27%; Animal experiments, TC decreased by 20%, LDL decreased by 17%.

[0018] Example 3, Bile Acid-Responsive Engineered Bacteria: System construction, bile acid-inducible promoter, downstream of which short-chain fatty acid metabolism-related genes are linked; Induction experiments, concentrations set: 0 mM, 0.2 mM, 0.5 mM; Results, under 0.5 mM conditions: functional metabolites: ↑2.3 times, FXR decreased: 35%, FGF15 decreased: 30%; Animal experiments, TC decreased: 22%, LDL decreased: 18%.

[0019] Example 4, FGF15 antagonistic peptide secretion: The gene encoding the FGF15 receptor competitive binding peptide was inserted, and the secretion signal peptide was used to export it to the extracellular space; expression verification: the concentration of secreted peptide was detected by ELISA, and expression was verified by Western blot; functional verification: receptor binding experiment: FGF15 receptor binding rate decreased by about 40%; signal detection: FXR decreased by 40%, FGF15 decreased by 42%; animal experiment: TC decreased by 24%, LDL decreased by 20%.

[0020] Example 5, Synergistic System of Compound Microbial Community: Microbial Community Composition: LC001-BSH, secondary bile acid bacteria, butyric acid bacteria, ratio: 1:1:1 (10 9 CFU / mL); Animal experiment design: High-fat model mice, administered by gavage for 4 weeks;

[0021] Results: FXR decreased by 45%, FGF15 decreased by 38%, secondary bile acids decreased by 48%, TC decreased by 31%, and LDL decreased by 26%.

[0022] Comparative analysis: For bacterial culture, preserved *Lactobacillus casei* LC001 was anaerobically cultured in MRS medium at 37°C for 24 h. For bile acid metabolism experiments, 0.3% bile salts (GCA:TCA = 1:1) were added to the culture medium, and the culture was continued for 24 h. Bile acid composition was determined by HPLC. Results: Debinding rate: approximately 12%; Secondary bile acid proportion: approximately 12%. Cell experiments: IEC-6 cells treated with bacterial supernatant for 48 h: FXR expression: 1.00 (baseline); FGF15 expression: 1.00. Animal experiments: High-fat mice administered via gavage for 4 weeks: TC decreased by approximately 3%; LDL decreased by approximately 2%.

[0023] A comprehensive comparison of FXR-FGF15 axis and cholesterol regulation is shown in the table below:

[0024] Comparative Example Original LC001 1 1 12% 3% 2% Example 1 BSH Enhancement (2 Weights) 0.72（↓28%） 0.77（↓23%） 38% 18% 15% Example 2 Enhanced bile acid conversion (weight 3) 0.68（↓32%） 0.73（↓27%） 42% 20% 17% Example 3 Responsive expression system (weight 4) 0.65（↓35%） 0.70（↓30%） 41% 22% 18% Example 4 FGF15 antagonistic peptide (weight 5) 0.60（↓40%） 0.58（↓42%） 40% 24% 20% Example 5 Complex microbial community (right 7) 0.55（↓45%） 0.62（↓38%） 48% 31% 26%

[0025] The following table compares bile acid metabolism capabilities:

[0026] Comparative Example 12% 78% 12% none Example 1 42% 52% 38% obvious Example 2 46% 48% 42% Strong Example 3 45% 49% 41% powerful Example 4 43% 50% 40% Strong (non-bile acid dominant) Example 5 50% 42% 48% Significant

[0027] The blood lipid indicators from animal experiments are shown in the table below:

[0028] Comparative Example 6.8 4.2 1.2 — Example 1 5.6 3.6 1.3 17.60% Example 2 5.4 3.5 1.32 20.60% Example 3 5.2 3.4 1.35 23.50% Example 4 5 3.3 1.36 26.50% Example 5 4.7 3.1 1.4 30.90%

[0029] The comparison results of the five embodiments and comparative examples above show that each functional enabling module of the present invention can exert different degrees of regulatory effect on the FXR-FGF15 signaling axis, and ultimately affect cholesterol metabolism levels. Firstly, the comparative results show that the unmodified LC001 strain has no significant effect on FXR and FGF15 expression, with cholesterol decreasing by less than 5%, indicating that the original preserved strain does not possess the ability to regulate the FXR-FGF15 axis. Secondly, Examples 1 and 2 significantly altered the composition of bile acids by enhancing bile acid metabolism, with the proportion of secondary bile acids increasing from 12% to 38%–42%, while FXR expression decreased by 28%–32%, indicating that bile acid metabolic remodeling is the fundamental pathway for regulating FXR. Furthermore… In Example 3, by constructing a bile acid-responsive expression system, the regulatory effect was enhanced in a high bile acid environment, resulting in a decrease in FXR expression to 0.65, indicating that this regulatory method has environmental adaptability and dynamic regulatory ability. In Example 4, by secreting FGF15 antagonistic peptides, the downstream signaling pathway of FXR was directly intervened, resulting in a 42% decrease in FGF15 expression, which was significantly higher than in other examples, indicating that this pathway can enhance the regulation of the FXR-FGF15 axis at the signal transduction level. Finally, in Example 5, by constructing a complex microbial community system, a multi-mechanism synergistic effect was achieved, resulting in a decrease in FXR expression to 0.55 and a 30.9% decrease in total cholesterol, which was significantly better than single-function modification, indicating that there is a significant synergistic enhancement effect between different functional modules.

[0030] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus casei* LC001 that lowers cholesterol by regulating the intestinal FXR-FGF15 axis, characterized by: The *Lactobacillus casei* LC001 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.20587. After genetic engineering modification and / or metabolic regulation, the strain meets at least one or more of the following characteristics: bile salt hydrolase activity is increased by ≥20% compared to the starting strain; under bile salt culture conditions, the proportion of secondary bile acids in total bile acids is increased by ≥10%; under 0.1–0.5 mM bile acid induction conditions, the expression level of metabolites regulating the FXR signaling pathway is increased by ≥1.5 times; it secretes active factors that can reduce FGF15 expression or inhibit FGF15 receptor binding; it has bile acid adsorption capacity with an adsorption rate of ≥15% and exhibits sustained-release properties; thereby reducing FXR expression level by ≥20% and / or FGF15 expression level by ≥20%, and achieving a serum cholesterol reduction effect.

2. The *Lactobacillus casei* LC001 strain that lowers cholesterol by regulating the intestinal FXR-FGF15 axis according to claim 1, characterized in that: The strain achieves a bile acid debinding rate of ≥40% by introducing or enhancing the bile salt hydrolase encoding genes bsh1 and / or bsh2.

3. The *Lactobacillus casei* LC001 strain according to claim 1, which lowers cholesterol by regulating the intestinal FXR-FGF15 axis, is characterized in that: The strain has the ability to convert primary bile acids into deoxycholic acid and / or lithocholic acid, so that the proportion of secondary bile acids reaches ≥35%.

4. The *Lactobacillus casei* LC001 strain that lowers cholesterol by regulating the intestinal FXR-FGF15 axis according to claim 1, characterized in that: The strain has constructed a bile acid-inducible expression system, including a bile acid-responsive promoter and a metabolic regulatory gene linkage structure, which induces the expression of short-chain fatty acids or FXR-inhibiting metabolites when bile acid concentration increases.

5. The *Lactobacillus casei* LC001 strain that lowers cholesterol by regulating the intestinal FXR-FGF15 axis according to claim 1, characterized in that: The active factor is a polypeptide with a length of 5–50 amino acids, which can competitively bind to the FGF15 receptor FGFR4, reducing the receptor binding rate by ≥30%.

6. The *Lactobacillus casei* LC001 strain according to claim 1, which lowers cholesterol by regulating the intestinal FXR-FGF15 axis, is characterized in that: The strain is modified with cell surface polysaccharides or proteins to achieve an adsorption rate of 15%–40% for bound bile acids and to achieve delayed release in the intestinal environment.

7. A microbial composition, characterized in that: Includes the strains described in any one of claims 1-6, and at least one secondary bile acid producing bacterium and / or short-chain fatty acid producing bacterium; The strains were combined in a mass or quantity ratio of (0.5–2):(0.5–2):1, which increased the proportion of secondary bile acids in vivo by ≥15% and reduced FXR expression by ≥30%.

8. The composition according to claim 7, characterized in that: The short-chain fatty acid producing bacteria are butyric acid bacteria, and the secondary bile acid producing bacteria are intestinal bacteria with 7α-dehydroxylation ability.

9. The use of the strain according to any one of claims 1-6 or the composition according to any one of claims 7-8 in the preparation of a product for regulating the intestinal FXR-FGF15 signaling axis.

10. The application according to claim 9, characterized in that: The product is used to lower serum total cholesterol and / or low-density lipoprotein cholesterol by ≥15%.