Use of a forsythia muldschianae and preparation of a product for preventing and treating glycolipid metabolism disorder

Abstract

The present application belongs to the technical field of microbial inoculants, and particularly relates to a Phocaediola doleeri, and further discloses a use of the Phocaediola doleeri in preparing a product for preventing and treating glycolipid metabolic disorder. A YG0564 strain is screened from healthy adult feces, and is identified as the Phocaediola doleeri and classified and named as Phocaediola doleeri. The Phocaediola doleeri strain YG0564 contains cholesteryl sulfatase, and can significantly reduce blood fat and blood sugar in vivo and in vitro, and can obviously prevent and treat obesity and glycolipid metabolic disorder.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a strain of *Focusia dorsalis*, and further disclosing its use in preparing products for preventing and treating disorders of glucose and lipid metabolism. Background Technology

[0002] According to the "Report on Nutrition and Chronic Diseases of Chinese Residents (2020)," the overweight and obesity rate among Chinese residents continues to rise. The overweight and obesity rate among adults exceeds 50%, while the rates among children and adolescents aged 6-17 and under 6 years old reach 19% and 10.4%, respectively. Overweight and obesity have become a potential threat to residents' health.

[0003] Hyperlipidemia and glucose metabolism disorders are common complications associated with overweight and obesity. They typically refer to abnormal blood lipid and blood sugar levels caused by impaired glucose and lipid metabolism, often leading to common diseases such as atherosclerosis, heart failure, hypertension, or diabetes. They can also adversely affect other conditions, such as chronic inflammation, non-alcoholic liver disease, and Alzheimer's disease. Currently, clinical treatments for hyperlipidemia include statins, clofibrate, niacin, and bile acid chelating resins. However, these drugs can only control blood lipids within a certain range in the long term and cannot completely cure hyperlipidemia. They may also cause side effects such as nausea and vomiting, liver damage, ulcer induction, worsening of diabetes, and gout. Long-term use of hypoglycemic drugs, such as biguanides, sulfonylureas, and alpha-glucosidase inhibitors, can also lead to serious side effects such as gastrointestinal dysfunction and pancreatic failure. Therefore, there is an urgent need in this field to find natural and safe drugs or functional foods to prevent and treat glucose and lipid metabolism disorders.

[0004] Numerous studies have demonstrated the benefits of traditional probiotics in lowering blood lipids and blood sugar levels in hosts. Among these, Lactobacillus and Bifidobacterium are the most studied, exhibiting good lipid-lowering and blood sugar-lowering abilities in vitro, animal, and clinical trials. However, the abundance of traditional probiotics (Bifidobacterium and Lactobacillus) in the human body is relatively low, with significant individual variability, and their cholesterol-lowering mechanisms are singular, making them ineffective in preventing and treating high-fat and high-sugar diets.

[0005] In recent years, next-generation sequencing technology has greatly expanded the range of microorganisms that have potential benefits to the host; these microorganisms are known as next-generation probiotics (NGPs). NGPs are defined as "live symbiotic microorganisms identified through comparative microbiome analysis that, when given in adequate quantities, are beneficial to the health of the host," primarily from the genera *Ackermania*, *Bacteroides*, and *Femtobacter*. Recent studies have confirmed the lipid-lowering and blood sugar-lowering health effects of NGPs, particularly the latest paper reporting that the cholesterol sulfonase gene carried by the *Bacteroides polymorpha* strain *B. th* DSM2079 (…). Bt_0416It can directly act on cholesterol and convert and degrade it, providing strong evidence for the high lipid-lowering effect of NGPs. However, so far... Bt_0416 The presence and function of genes in other bacterial genera have not yet been verified. Therefore, the field looks forward to developing more probiotics and their preparations that have significant lipid-lowering and blood sugar-lowering effects in vivo and in vitro, and that can have significant preventive and therapeutic effects on obesity and glucose-lipid metabolism disorders. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a strain of *Focusia dorsalis* containing cholesterol sulfonase, which significantly reduces lipids and blood sugar in vivo and in vitro, and has significant preventive and therapeutic effects on obesity and disorders of glucose and lipid metabolism.

[0007] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned *Foscias dorsalis* in the preparation of products for the prevention and treatment of glucose and lipid metabolism disorders.

[0008] To address the aforementioned technical problems, this invention provides a regulator with targeted regulatory effects on the TGR5 / IL27 / FXR pathway and / or FGF15-CYP7A1 and SHP-HNF-4α pathways, used in the preparation of products that improve hyperglycemic and hyperlipidemia metabolic disorders or promote weight loss and fat reduction; wherein,

[0009] The regulator targets lipid metabolism based on the TGR5 / IL27 / FXR pathway and / or targets glucose metabolism based on the FGF15-CYP7A1 and SHP-HNF-4α pathways.

[0010] This invention also discloses the use of a formulation with targeted improvement of characteristic gut microbiota in disorders of glucose and lipid metabolism for the preparation of products with effects of improving hyperglycemic and hyperlipidemia metabolism disorders or weight loss and fat reduction; wherein,

[0011] The improvement of the characteristic gut microbiota of glucose and lipid metabolism disorders includes increasing the abundance of beneficial bacteria and / or decreasing the abundance of harmful bacteria;

[0012] Preferably, the characteristic gut microbiota for improving glucose and lipid metabolism disorders includes beneficial bacteria that target and drive the inhibition of obesity. Lactobacillus,Odoribacteraceae and Bacteroidales Abundance, reduction of harmful bacteria Vampirovibrio Abundance.

[0013] This invention discloses, in particular, a strain of *Focius dorsalis* YG0564, which is classified and named *Focius dorsalis*. Phocaeicola dorei It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27578 and deposit date of June 7, 2023.

[0014] The present invention also discloses the use of the aforementioned *Focusella dorsalis* YG0564 and its inactivated products and metabolites in the preparation of microbial preparations.

[0015] The present invention also discloses a microbial preparation, wherein the active ingredient of the microbial preparation comprises the cells of *Focusella dorsalis* YG0564 as described in claim 1, as well as its inactivated products and / or metabolites.

[0016] Specifically, in the microbial preparation, the effective bacterial count of *Focusia dorsalis* YG0564 is 5.0 × 10⁻⁶. 9 -1.0×10 11 CFU / day / person.

[0017] Specifically, the microbial preparation includes at least one of the following: powder, granules, pills, capsules, tablets, ointments, liquid preparations, gels, sprays, or solid beverages.

[0018] The present invention also discloses a method for preparing the microbial preparation, comprising the steps of culturing the *Foscias dorsalis* YG0564 as described in claim 1, and processing the selected dosage form according to conventional processes and by adding conventional excipients.

[0019] The present invention also discloses the use of the aforementioned *Focusia dorsalis* YG0564 and its inactivated products, metabolites, or the aforementioned microbial preparations in the preparation of functional products having at least one of the following (1)-(14):

[0020] (1) High expression of cholesterol sulfonase;

[0021] (2) Highly efficient cholesterol degradation;

[0022] (3) It efficiently degrades blood lipids and body fat;

[0023] (4) Prevention and treatment of metabolic disorders caused by high sugar and high fat;

[0024] (5) TGR5 / IL27 / FXR targets and regulates lipid metabolism;

[0025] (6) FGF15-CYP7A1 and SHP-HNF-4α target and regulate glucose metabolism;

[0026] (7) White fats undergo beige transformation;

[0027] (8) Activates thermogenesis of brown adipose tissue;

[0028] (9) High expression of BSH enzyme activity;

[0029] (10) Highly efficient degradation of conjugated bile acids;

[0030] (11) It produces a variety of short-chain fatty acids;

[0031] (12) Alleviates hepatocyte steatosis, inflammatory response and inhibits gluconeogenesis;

[0032] (13) Targeted inhibition of beneficial bacteria in obesity Lactobacillus,Odoribacteraceae and Bacteroidales Abundance, reduction of harmful bacteria Vampirovibrio Abundance;

[0033] (14) It inhibits the growth of body weight, liver weight, white fat weight, blood lipids and pro-inflammatory factors.

[0034] Specifically, the functional products include pharmaceuticals.

[0035] The present invention also discloses a microbial preparation for improving high sugar and high lipid metabolism disorders, wherein the active ingredient of the microbial preparation includes the *Focusia dorsalis* YG0564 and its inactivated products and / or metabolites;

[0036] Preferably, the effective bacterial count of *Focusia dorsalis* YG0564 is 2.0 × 10⁻⁶. 10 -8.0×10 10 CFU / day / person.

[0037] The present invention also discloses a microbial preparation with weight loss and fat reduction effects, wherein the active ingredients of the microbial preparation include the *Focusella dorsalis* YG0564 and its inactivated products and / or metabolites;

[0038] Preferably, the effective bacterial count of *Focusia dorsalis* YG0564 is 1.0 × 10⁻⁶. 10 -5.0×10 10 CFU / day / person.

[0039] This invention obtained a strain YG0564 from the feces of healthy adults, which was identified as *Focius dorsalis* and classified as […]. Phocaeicola dorei Experimental identification showed that the *Foscias dorsalis* strain YG0564 contains cholesterol sulfonase and exhibits significant lipid and blood sugar reduction effects both in vivo and in vitro, demonstrating a marked preventive and therapeutic effect on obesity and disorders of glucose and lipid metabolism.

[0040] The strain described in this invention is the first to discover a novel cholesterol sulfonase and its biosynthetic gene cluster in *Focusia dorsalis*. In vitro, *Focusia dorsalis* was found to have a high cholesterol sulfonase conversion effect, and mouse experiments confirmed that *Focusia dorsalis* can efficiently degrade blood lipids and body fat.

[0041] The strain described in this invention is the first to discover and verify that the *Foscias dorsalis* strain has a pathway that targets and regulates TGR5 / IL27 / FXR, thereby achieving beigeization of white adipose tissue and activating thermogenesis in brown adipose tissue, resulting in efficient weight loss.

[0042] The *Focusia dorsalis* strain YG0564 described in this invention has been experimentally proven to improve hyperlipidemia and hyperglycemia metabolic disorders. The *Focusia dorsalis* strain YG0564 of this invention improves hyperlipidemia and hyperglycemia disorders through a comprehensive mechanism, specifically manifested in:

[0043] a. The strain can highly express cholesterol sulfonase, which can directly participate in the degradation of cholesterol;

[0044] b. The strain has high BSH enzyme activity, which can efficiently degrade conjugated bile acids and regulate cholesterol metabolism through bile acids.

[0045] c. The strain can produce a variety of short-chain fatty acids, thereby regulating cholesterol metabolism;

[0046] d. The strain can alleviate hepatocellular steatosis, inflammatory response and inhibit gluconeogenesis;

[0047] e. The strains described can target and improve the characteristic gut microbiota of glucose and lipid metabolism disorders, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, and restore the health of the gut microbiota.

[0048] The *Focusia dorsalis* strain YG0564 described in this invention is a new generation of microbial preparations. These preparations can be made using *Focusia dorsalis* strain YG0564 as the active ingredient, following conventional methods. This not only addresses the problems of long-term drug use in treating hyperlipidemia and diabetes, such as damage to the gastrointestinal tract, liver, and pancreas, induction of ulcers, worsening of diabetes, and gout, but also effectively solves the shortcomings of existing traditional microbial products (including Bifidobacterium and Lactobacillus) in the human body, such as low abundance, significant individual variability, and a single cholesterol-lowering mechanism, lack of specificity, and inability to effectively prevent and treat hyperlipidemia and hyperglycemia and restore healthy intestinal flora, thus affecting the efficacy of the products.

[0049] The present invention relates to a microbial preparation for improving hyperglycemic and hyperlipidemic metabolic disorders and a microbial preparation for weight loss and fat reduction. The active ingredient of the microbial preparation includes *Focusia dorsalis* YG0564 and its inactivated products and / or metabolites. Other active ingredients may be added, or *Focusia dorsalis* YG0564 may be used as the only active ingredient. By utilizing the activity of *Focusia dorsalis* YG0564, the preparation can improve hyperglycemic and hyperlipidemic metabolic disorders and reduce weight and fat, and has the advantages of safe medication and significant improvement effect. Attached Figure Description

[0050] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0051] Figure 1 The results show the cholesterol degradation rates of strains LGG and YG0564 in Example 2;

[0052] Figure 2 The results of cholesterol sulfonation biosynthesis gene clusters in strains DSM2079 and YG0564 in Example 2;

[0053] Figure 3 The image shows the BT_0416 amino acid sequence alignment results of strains DSM2079 and YG0564 in Example 2, where the gray background represents conserved amino acid sequences.

[0054] Figure 4 For different strains in Example 2 BT_0416 Gene expression level detection results; the left image is a nucleic acid electrophoresis diagram, and the right image is a gene expression bar chart;

[0055] Figure 5 The results of the degradation ability of different strains of bile salts in Example 2 are shown.

[0056] Figure 6 This refers to the ability of different strains in Example 2 to produce short-chain fatty acids in the culture medium;

[0057] Figure 7 The results of mouse HFD modeling in Example 3; where A is the mouse weight and body size chart, BC is serum cholesterol and triglycerides, and CD is fasting blood glucose and insulin resistance index;

[0058] Figure 8 The image shows the mouse liver data results in Example 3; where A represents liver weight and volume, B represents liver triglyceride content, C represents liver pathology, and D represents gene expression levels.

[0059] Figure 9 The data results of white adipose tissue in mice in Example 3 are shown; where A is the weight and volume of white adipose tissue in the posterior abdominal wall, B is the microscopic examination results of white adipose tissue, and C is the gene expression map.

[0060] Figure 10 The data results of brown adipose tissue in mice in Example 3 are shown; where A is the microscopic examination result of brown adipose tissue, B is the expression map of lipid metabolism genes, and C is the expression map of inflammation regulation genes.

[0061] Figure 11 The results are shown in Example 3, which are the sequencing results of mouse gut microbial diversity. AB represents mouse gut microbial α-diversity; C represents the mouse gut microbial β-diversity PCoA plot; and D represents the species abundance results at the phylum level for each group.

[0062] Figure 12The results of the analysis of differences between groups of mouse gut microbiota genera and the correlation of physiological indicators in Example 3 are as follows: A is the species difference analysis at the genera level between the YG0564 and HFD groups; B is the correlation analysis between mouse body weight and organ weight and gut microbiota genera; C is the correlation analysis between mouse blood lipids and blood glucose and gut microbiota genera; D is the correlation analysis between mouse inflammatory markers and gut microbiota genera.

[0063] Figure 13 The results show the SCFA levels in the mouse cecal contents in Example 3. Detailed Implementation

[0064] In the following embodiments and accompanying drawings of the present invention, the strain YG0564 can be expressed as YGMCC 0564, YGMCC0564 or simply 0564, all of which represent the strain YG0564 screened in the present invention.

[0065] Example 1: Isolation and Identification of Strains

[0066] 1. Isolation of Fauciella dorsalis

[0067] Sample source

[0068] The strain used in this embodiment was isolated from the feces of healthy adults.

[0069] Isolation and screening of strains

[0070] Take 1g of fecal sample and place it in 9mL of PBS buffer (Landbridge), vortex to mix, and then perform a tenfold serial dilution with sterile physiological saline. Select 10... -6 10 -7 10 -8 Three dilution gradients were used, with 100 μL of each dilution evenly spread on Columbia blood agar medium. The culture was anaerobic at 37°C for 72 h until clear single colonies were formed. The colonies were then picked and purified on Columbia blood agar plates at least three times until the colony morphology on the plates was consistent.

[0071] 2. Identification of *Focusia dorsalis*

[0072] Colony characteristics

[0073] The selected strains, after being cultured on Columbia blood agar medium for 24-48 hours, had a diameter between 0.01-1.0 mm, smooth colony edges, a milky white color, and a moist surface. They were named YG0564.

[0074] 16S rRNA gene sequencing

[0075] The selected strains were sent to Shanghai Sangon Biotech for 16S rRNA gene sequencing. The sequencing results are shown in SEQ ID No. 1.

[0076]

[0077] The 16S rRNA sequence results were BLAST-aligned with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Based on the results and the strain's morphological characteristics, YG0564 was preliminarily identified as *Focius d'Orochaete*. Phocaeicola dorei ).

[0078] The strain selected in this embodiment is *Focius dorsalis* YG0564, which is classified and named *Focius dorsalis*. Phocaeicola dorei It was deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCCNo. 27578 and deposit date of June 7, 2023.

[0079] Example 2: In vitro screening for cholesterol reduction

[0080] 1. Cholesterol conversion efficiency test

[0081] Cholesterol culture medium preparation: Take 0.2g cholesterol, 2.0mL Tween-80, 0.2g sucrose ester, and 10.0mL glacial acetic acid, mix them, and then filter them sterilized by sonication (10min, 20% power) to obtain a cholesterol emulsion. Add the prepared cholesterol emulsion to fresh BHIS or MRS medium to a final concentration of 0.1 mg / mL.

[0082] LGG probiotics and the screened YG0564 strain were revived and activated three times. Fresh bacterial cultures were centrifuged, and the supernatant was discarded. The OD (600nm) value was adjusted to 1.5 using cholesterol medium, and the cultures were anaerobically incubated at 37°C for 4-48 hours. At different time points, bacterial cultures were centrifuged, and the supernatant was collected. Residual cholesterol in the supernatant was detected by high-performance liquid chromatography (HPLC). Cholesterol-free medium was used as a control. Each sample was tested in triplicate.

[0083] The liquid chromatography detection conditions are as follows: Sample preparation: Filter through a 0.22 μm organic filter membrane, and transfer the filtrate to a vial for use; Detection conditions: C18 reversed-phase column (4.6 mm × 150 mm, 5 μm); Mobile phase: 100% methanol (V / V, analytical grade); Detection wavelength: 205 nm; Flow rate: 2.0 mL / min; Column temperature: 40 ℃; Injection volume: 5 μL.

[0084] Cholesterol degradation rate = (control concentration - residual concentration) / control concentration × 100%, results are attached. Figure 1 As shown.

[0085] like Figure 1 The results show that both LGG probiotics and the YG0564 strain screened in this invention can significantly degrade cholesterol after co-incubation with it, and the cholesterol degradation capacity of the YG0564 strain is significantly higher than that of LGG at all time points. Specifically, the degradation capacity of LGG reaches its peak (19.4%) at 24 h and then tends to plateau; while the degradation capacity of YG0564 continues to increase at 24 h and 48 h, showing a maximum cholesterol degradation rate of 49.8%, indicating significant cholesterol conversion efficiency.

[0086] 2. Sulfonase gene prediction and testing

[0087] In this embodiment, to verify the potential cholesterol degradation mechanism of strain YG0564, the whole genome sequence of strain YG0564 was obtained.

[0088] After removing low-quality data from the sequencing platform, the genome of each strain was assembled using SOAPdenovo 2.0 and SPAdes 3.11, and Glimmer version 3.02 was used for coding sequence and GC content prediction. Functional annotation of the genome was performed using public databases such as KEGG, COG, and GO. Gene sequence and amino acid sequence alignment analysis was performed using CLUSTAL W2 and DNAMAN. Additionally, after obtaining homologous genes, targeted synthesis was carried out. BT_0416 Primers for the sulfonated cholesterol gene (see Table 1 below) were used, and the standard strain *Pseudomonas polymorpha* DSM2079 was used as a positive control. Total RNA was extracted from bacterial cultures co-incubated with cholesterol for 0 h and 2 h using the Quanshijin Total RNA Extraction Kit, and reverse transcribed into cDNA (Takara). The 16S gene was used as an internal control, and the 0 h sample was used as a control. After qPCR amplification and detection of the gene Ct value, the results were processed according to a 2... -△△Ct Gene expression folds were calculated, and the amplified PCR products were visualized using nucleic acid electrophoresis gels.

[0089] Table 1 qPCR primer design

[0090]

[0091] like Figure 2 The cholesterol sulfonation biosynthesis gene cluster shown was compared with that of DSM2079 using whole-genome bioinformatics. The results showed that YG0564 contains a cholesterol sulfonation biosynthesis gene cluster similar to that of DSM2079. The five key enzymes in the gene cluster and their amino acid homology are PAP dephosphorylase (65.8%), Na+, and Na+. +The presence of sulfate symporter (77.3%), adenylyl-sulfate kinase (82.1%), sulfate adenylyltransferase subunit 1 (83.3%), and cholesterol sulfotransferase BT_0416 (87.5%) suggests that YG0564 exhibits a similar yet different cholesterol sulfonation biosynthesis mechanism and efficiency compared to DSM2079.

[0092] like Figure 3 The amino acid sequence alignment results of BT_0416 show that the catalytic efficiency and stability of the enzyme vary significantly between species, depending on the amino acid sequence and composition of the enzyme protein. In strain YG0564, the amino acid sequence of the BT_0416 enzyme differs from that of DSM2079 by 46 amino acid residues, indicating species-specific structural differences in BT_0416 enzymes. The two BT_0416 enzymes may exhibit differences in catalytic efficiency and stability. Mutations in the enzyme protein amino acids towards valine (V) and glycine (G) are important factors in increasing the catalytic efficiency and thermostability of the enzyme protein. This example confirms three valine mutations (e.g., ...) in the BT_0416 enzyme of strain YG0564. Figure 3 The black dashed box in the middle indicates A44V, I270V, and I350V) and two glycine mutations (such as Figure 3 The residues marked with black solid boxes (K204G and N365G) are inferred to contribute to the higher catalytic efficiency and stability of BT_0416 in YG0564. To clarify this, future crystallographic studies and site-directed mutagenesis analysis of the BT_0416 enzyme in YG0564 will help to further understand its properties.

[0093] In this embodiment, after confirming the presence of cholesterol sulfonase, the cholesterol sulfonase gene was further detected before and after applying cholesterol stress to the strain. BT_0416 The expression of the gene in Bacteroides polymorpha DSM2079, F. dorsalis YG0564, and Lactobacillus rhamnosus LGG is shown in the attached figure. Figure 4 The image on the left is a nucleic acid electrophoresis diagram, and the image on the right is a gene expression bar chart.

[0094] The results showed that LGG cells did not contain the gene, while strain YG0564, like strain DSM2079, was able to successfully amplify it. Bt_416 Gene fragments. (See attached image) Figure 4 The results shown in the middle right figure demonstrate that the strain significantly upregulated this gene after 2 hours of cholesterol treatment, proving that... Bt_416 It participates in the cholesterol conversion process of the strain.

[0095] 3. BSH enzyme activity detection

[0096] The preserved YG0564 strain was removed from the cryogenic freezer, subcultured three times for activation and identification. The activated strain was inoculated into MRS or BHIS liquid medium at a 1% (v / v) inoculum and cultured overnight. OD600 values ​​were then measured, with each strain adjusted to OD600=1. The culture was centrifuged (5000 rpm, 5 min), the supernatant discarded, and the cells were washed once with 0.1 M HAc-NaAc buffer (pH 5.0), then resuspended in an equal volume. 400 μL of the bacterial suspension was mixed with 400 μL of a 20 mM bile salt mixture (TDCA+GDCA) and incubated at 37°C for 1 h. The control sample was prepared by mixing 400 μL of the 20 mM bile salt mixture with 400 μL of HAc-NaAc buffer. After the water bath, 800 μL of 15% TCA stop solution was added to terminate the reaction. After mixing, the mixture was centrifuged (5000 rpm, 5 min), and the supernatant was collected. The supernatant was filtered through a 0.22µm nylon membrane, and the filtrate was transferred to a vial for use in high performance liquid chromatography analysis.

[0097] Preparation of standards: Mix two 20mM bound bile salts (GDCA and TDCA) in equal proportions to obtain a 20mM bile salt mixture. The pH 5.0, 0.1M HAc-NaAc buffer is formulated as follows: 14.8mL of 0.2M HAc + 35.2mL of 0.2M NaAc, and dilute with water to 100mL.

[0098] Instrumentation conditions: Column: Agilent TC-18 C18 reversed-phase column (5µm, 4.6mm × 250mm); Detector: Diode array detector; Detection wavelength: 200nm; Injection volume: 20µL; Column temperature: 40℃; Column flow rate: 1mL / min; Mobile phase A: 7.5mmol / L tetrabutylammonium bisulfate in acetonitrile-water (60:40) solution (pH 2.5); Mobile phase B: 7.5mmol / L tetrabutylammonium bisulfate in acetonitrile-water (30:70) solution (pH 2.5); Gradient elution: Mobile phase A 10%, mobile phase B 90%, the proportion of mobile phase A increased from 10% to 70% within 30min.

[0099] Bile salt mixtures were prepared into concentration gradients of 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 mM using 0.1 M HAc-NaAc buffer (pH 5.0). A standard curve for bile salts was plotted with the peak area on the x-axis and the peak area on the y-axis for each bile salt concentration (mM), and the regression equation was calculated.

[0100] The bile salt degradation rate was detected and calculated as follows: (Bile salt content in the control group - Residual bile salt in the experimental group) / Bile salt content in the control group * %, with the results of the bile salt degradation ability of each strain shown in the appendix. Figure 5 As shown.

[0101] The results showed that the YG0564 strain screened in this invention significantly degraded both bound bile salt substrates, degrading 43.6% of GDCA and 30.5% of TDCA within 1 hour. Its BSH enzyme activity was significantly higher than that of the LGG strain (***). P <0.001).

[0102] 4. Test for ability to produce short-chain fatty acids

[0103] The preserved bacterial strain was activated by three generations. The activated bacterial solution was transferred to fresh culture medium and cultured anaerobicly overnight for 20 hours. The supernatant was then collected by centrifugation, and the levels of various short-chain fatty acids in the supernatant were detected by HPLC.

[0104] The yields of lactic acid, acetic acid, propionic acid, and butyric acid in the supernatant were determined using HPLC. The results are shown in the appendix. Figure 6 .

[0105] As can be seen, the levels of SCFAs in the LGG supernatant were significantly different from those of YG0564 and DSM 2079. Figure 6 As shown, LGG can only produce a large amount of lactic acid (124.31 mM in MRS medium), but cannot produce acetic acid, propionic acid, or butyric acid. HPLC analysis, however, confirmed that YG0564 can produce acetic acid, propionic acid, and butyric acid, exhibiting a variety of metabolic activities.

[0106] 5. Test for tolerance to artificial gastrointestinal fluid

[0107] Preparation of BHIS solid and liquid culture media: BHI liquid culture medium with or without 1.5% agar powder, add distilled water and mix well, autoclave, add heme chloride and vitamin K1, mix well and pour into plates.

[0108] Preparation of artificial gastric fluid at pH 3.0: Dissolve 3g of pepsin in 1000mL of sterile physiological saline (0.9% w / v) until the final enzyme concentration is 3g / L. Adjust the pH to 3 with hydrochloric acid. Filter the pepsin solution through a 0.22μm sterile filter membrane. Prepare and use immediately.

[0109] Preparation of artificial colonic fluid at pH 8.0: Dissolve 1g of trypsin in 1000mL of sterile physiological saline (0.9% w / v) until the final enzyme concentration is 1g / L. Adjust the pH to 8 with NaOH solution (add 1.1μL, 2M NaOH). Filter with a 0.22μm sterile filter membrane for sterilization. Prepare and use immediately.

[0110] The different bacterial strains were passaged three times for activation. 0.2 mL of the activated bacterial solution was used for viable cell counting (N0, 0 h). Another 1.2 mL of activated bacterial solution was centrifuged (5000 rpm, 5 min), the supernatant was discarded, and the solution was resuspended in 1.2 mL of simulated gastric fluid (pH 3.0) and anaerobically cultured at 37°C for 2 h. After 2 h of simulated gastric fluid treatment, 0.2 mL of bacterial solution was transferred to a blank EP tube, centrifuged (5000 rpm, 5 min), and the supernatant was discarded. One portion was resuspended in 0.2 mL PBS for viable cell counting (N1, gastric fluid treatment 2 h), and the other portion was resuspended in 1 mL of simulated colonic fluid (pH 8.0) and anaerobically cultured at 37°C for 4 h. After 4 h of simulated colonic fluid treatment, the solution was centrifuged (5000 rpm, 5 min), the supernatant was discarded, and the solution was resuspended in 1 mL PBS. The solution was then diluted and plated using a serial dilution plate counting method, with three replicates for each strain. Colony counting (N2) was performed after 48 h of incubation at 37°C.

[0111] Artificial gastric fluid survival rate (%) = [log CFU N1 / log CFU N0] * 100%;

[0112] Artificial intestinal fluid survival rate (%) = [log CFU N2 / log CFU N0] * 100%.

[0113] Good gastrointestinal tolerance is a fundamental condition for live bacteria to colonize the human gut and exert their effects. To evaluate the probiotic characteristics of these functional strains, such as gastrointestinal adaptation, this embodiment placed the activated strains in simulated gastric fluid for 2 hours and then in simulated intestinal fluid for another 4 hours, calculating the survival rate of the strains. The survival rate calculation results of different strains in simulated gastrointestinal fluid are shown in Table 2 below.

[0114] Table 2 Survival rates of different strains in artificial gastrointestinal fluid

[0115]

[0116] As shown in Table 2, the *Focusia dorsalis* YG0564 strain screened in this invention maintained a survival rate of 93.84% after exposure to gastric and intestinal fluid pressure, which is close to the tolerance of LGG. This demonstrates that the YG0564 strain screened in this invention not only has good cholesterol assimilation capacity but also exhibits good adaptability to the gastrointestinal environment.

[0117] Example 3: Effects of strain on improving glucose and lipid metabolism

[0118] 1. Animal Experiment Design

[0119] Strain preparation: First, the *Foscias dorsalis* YG0564 strain, preserved at -80℃, was taken out of its storage tube and activated at a 2% inoculum for third-generation culture expansion. Then, it was centrifuged at 7500 rpm / min for 10-15 min, followed by repeated pipetting with pre-chilled PBS. After washing once, the supernatant was discarded, leaving the bacterial cells at the bottom. Finally, it was resuspended in pre-chilled 20% glycerol solution to prepare a 1.6 × 10⁻⁶ bacterial culture. 10 CFU / mL cryopreservation solution was immediately placed in a -80℃ freezer for storage. Before gavage, the bacterial solution was diluted with medical saline to the required viable bacterial count (1×10⁻⁶) for each gavage group. 9 (CFU / mL), or mice can be gavaged with common food or drugs containing this bacterium.

[0120] 2. Animal experiments

[0121] The animals used in this embodiment of the animal experiment were specific pathogen-free (SPF) C57BL / 6J female mice (8 weeks old) purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. They were housed in a controlled environment at a temperature of 23±3℃ and a humidity of 50%±10% under a 12-hour light / 12-hour dark cycle. All mice were allowed free access to food and water, and chew sticks were provided in the housing environment.

[0122] The animal experiments in this study were approved by the Ethics Committee of the Beijing Genomics Institute, Chinese Academy of Sciences, with approval number PONY-2021-FL-17. Mice were randomly divided into 5 groups: normal control group (ND), model control group (HFD), positive drug control group (orlistat, a drug commonly used in clinical practice to improve glucose and lipid metabolism), positive live bacteria control group (LGG), and live bacteria drug group (YG0564), with 10 mice in each group.

[0123] After a one-week acclimatization period, except for the ND group, the other groups were fed a diet consisting of 48.5% HFD high-fat diet (Beijing Botai Hongda) with gradually increasing proportions until it was completely replaced, and this was continued for 8 weeks. Mice in the Orlistat group were administered 1.2 mg of the drug daily by gavage, and mice in the LGG group were administered 10 mg of the drug daily by gavage. 9 CFU LGG, YG0564 group, 10 gavages per day 9 CFU YG0564 was administered daily via gavage with PBS as a control in the ND and HFD groups. The mice were weighed weekly for 8 consecutive weeks. At week 8, fecal samples (4-5 pellets per mouse) were collected, flash-frozen in clean EP tubes with liquid nitrogen, and stored at -80°C. These samples were then sent to a testing company to analyze fecal microbial diversity.

[0124] After treatment, mice were fasted for 24 hours, and retroorbital hemorrhage was collected. Fasting blood glucose was measured as a endpoint, and serum was collected and stored at -80°C for subsequent serological testing. After euthanizing the mice, the skin was cut open to expose the scapula, and the butterfly-shaped brown adipose tissue was harvested. The abdominal and thoracic cavities were opened, and the contents of the liver, colon, cecum, posterior abdominal wall fat, and epididymal fat were collected. These were wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at -80°C. Small pieces of liver, posterior abdominal wall fat, and brown adipose tissue were fixed with 4% paraformaldehyde and stored at room temperature.

[0125] 3. Measurement Method

[0126] In this embodiment, the relevant parameters were measured according to the following indicators and methods.

[0127] a. Pathological examination

[0128] Fixed liver tissue was embedded in paraffin, sectioned into 5 mm sections, and stained with hematoxylin and eosin (H&E). The stained slides were observed under a light microscope. Hepatocellular steatosis and inflammatory infiltration were assessed using the following criteria.

[0129] Criteria for judging hepatocellular steatosis: Hepatic steatosis (0-3 points, based on parenchymal cells / total cells <5%, 5-33%, 33-66%, >66%), lobular inflammation (0-3 points, <2 lesions per 200X field, 2-4 lesions per 200X field, >4 lesions per 200X field), hepatocellular balloon-like changes (0-2 points, no changes, few balloon-like cells, many balloon-like cells / protrusions).

[0130] Fixed white fatty paraffin was embedded, sectioned into 5mm sections, and stained with hematoxylin and eosin (H&E). The stained slides were observed under an optical microscope, and the maximum diameter of cells was measured in 10 random fields of view for each slide using software.

[0131] b, oGTT and serum insulin assay

[0132] Blood samples were collected from mice by enucleation. A single drop of blood was placed on test strips, and the fasting blood glucose level was read using a glucometer. The remaining blood sample was left at room temperature for 30 minutes, then centrifuged at 4°C and 3000 rpm for 5 minutes. The supernatant was collected and centrifuged again at 4°C and 10000 rpm for 10 minutes. The separated serum was placed in a 1.5 mL EP tube. Insulin levels were measured according to the instructions of the serum insulin assay kit (Beyotime).

[0133] The HOMA-IR index is calculated as follows: fasting insulin (uU / mL) × fasting blood glucose (mmol / L) / 22.5.

[0134] c. Serum biochemical markers determination

[0135] Total cholesterol (TC) was measured in mouse serum samples using a fully automated biochemical analyzer (Mindray).

[0136] d. Gene expression level detection

[0137] Total RNA extraction from tissue samples: Add 50-100 mg of sample tissue to an RNase-free grinding tube, add 1 ml of Trans Zol Up, and use RNase-free tweezers to place an RNA-free steel ball to seal the tube tightly. Then grind the sample using a Bioprep-24R grinder. After grinding, remove the tube and let it stand at room temperature for 5 minutes. For adipose tissue, after standing, centrifuge at 12000 rpm for 10 minutes at 4°C, and transfer 500 μL of the middle layer to a new enzyme-free EP tube, avoiding aspirating the upper lipid layer and the lower solid material layer. Subsequent steps involve extracting total RNA from the tissue according to the kit (Quanshijin) instructions, and determining the RNA concentration and purity. Reverse transcription of the RNA samples was performed using the TaKaRa kit and the manufacturer's instructions. Finally, qPCR was performed using a Tiangen qPCR kit and a Roche quantitative PCR instrument. ® 480) The expression levels of each gene were detected.

[0138] e. Fecal 16s microbial diversity detection

[0139] Fresh mouse feces were collected and placed in sterile EP tubes, flash-frozen in liquid nitrogen, and stored at -80°C. Genomes were extracted, and 16S rRNA high-throughput sequencing was used to analyze the dominant species in the samples, obtaining the composition of the microbial community and the relative abundance differences among them.

[0140] f. Detection of SCFAs in cecal contents

[0141] Preparation of mixed standards: Take 9840 μL of n-butanol (HPLC grade), put it into a 15 mL centrifuge tube, add appropriate amounts of 8 short-chain fatty acid standards in sequence, vortex and mix well to obtain mixed standard stock solution A of 8 short-chain fatty acids.

[0142] Preparation of internal standard: Take 9990 μL of n-butanol (HPLC grade), put it into a 15 mL centrifuge tube, add 10 μL of internal standard 2-ethylbutyric acid, vortex to mix, and the internal standard stock solution B is obtained. Take the above mixed standard A and B solutions, dilute with n-butanol to prepare 7 working solutions of different concentrations, put them into injection vials, and perform GC-MS detection and analysis.

[0143] Sample preparation: Weigh 20 mg of cecal contents into a 2 ml grinding tube, add 800 μL of 0.5% phosphoric acid solution (containing 10 μg / mL internal standard 2-ethylbutyric acid). Freeze-grind the sample for 3 min (50 Hz), then sonicate for 10 min, and centrifuge at 13000 g for 15 min at 4 °C. Transfer 200 μL of the supernatant to a 1.5 mL centrifuge tube, then add 200 μL of n-butanol for extraction. Vortex for 10 s, sonicate at low temperature for 10 min, and centrifuge at 13000 g for 5 min at 4 °C. Transfer the supernatant to a vial for micro-extraction.

[0144] GC-MS detection: The analytical instrument used in this experiment was the Agilent Technologies Inc. (CA, UAS) 8890B-7000D GC / MSD system.

[0145] Chromatographic conditions: HP FFAP capillary column (30m×0.25mm×0.25μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas was high-purity helium (purity not less than 99.999%), flow rate was 1.0 mL / min, injection port temperature was 180℃; injection volume was 1 μL, split injection, split ratio 10:1, solvent delay for 2.5 min; temperature program: initial temperature of column oven was 80℃, temperature was programmed to 120℃ at 20℃ / min, temperature was programmed to 160℃ at 5℃ / min, and then run at 220℃ for 3 min.

[0146] Mass spectrometry conditions: Electron impact ion source (EI), ion source temperature 230℃, quadrupole temperature 150℃, transfer line temperature 230℃, electron energy 70 eV. Scan mode: Selected ion scan (SIM).

[0147] Data Analysis: Masshunter quantitative software (Agilent Technologies, v10.0.707.0) with default parameters was used to automatically identify and integrate the ionic fragments of the target short-chain fatty acids, supplemented by manual inspection. The detection concentration of each sample was calculated using a standard curve to determine the actual content of short-chain fatty acids in the sample.

[0148] 4. Test Results

[0149] like Figure 7 The data shown are the mouse HFD modeling data of this embodiment; where A is the mouse weight and body size, BC is the serum cholesterol and triglycerides, and CD is the fasting blood glucose and insulin resistance index.

[0150] From the appendix Figure 7The results show that after 8 weeks of high-fat diet induction, the HFD group experienced a significant increase in body weight and body size. The *Focusia dorsalis* YG0564 strain screened in this invention, like LGG and orlistat, can significantly reduce body weight and improve body shape. After continuous high-fat diet induction, serum TC levels in HFD mice were significantly increased compared to mice on a normal diet (***). P <0.001), and fasting blood glucose is elevated (*** P <0.001), increased insulin resistance (*) P <0.05), indicating disordered lipid and glucose metabolism. Results showed that orlistat and LGG intervention had no significant effect on serum cholesterol and insulin resistance index, only reducing triglycerides and fasting blood glucose to a certain extent. However, HFD mice treated with YG0564 live bacteria showed a significant reduction in circulatory cholesterol and triglyceride levels, while also reducing blood glucose and insulin resistance, significantly alleviating glucose and lipid metabolism disorders.

[0151] like Figure 8 The results of mouse liver data are shown in the figure; where A is liver weight and volume, B is liver triglyceride content, C is liver pathology, and D is gene expression level. In Figures C and D, the bars from left to right represent the results of the ND, HFD, Orlistat, LGG, and YG0564 groups, respectively.

[0152] From the appendix Figure 8 The results showed that after high-fat diet induction, the liver weight and volume of the HFD group increased significantly (Figure A), the TG content in the liver increased (Figure B), and HFD liver steatosis was obvious (***). P <0.001), increased intracellular lipid vacuoles, and inflammatory infiltration in multiple fields of view, manifesting as hepatic lobular inflammation (Figure C). *Focusia dorsalis* YG0564 significantly reduced liver weight and triglyceride accumulation. Simultaneously, interventions with orlistat, LGG, and YG0564 reduced hepatic steatosis to varying degrees, with YG0564 showing a significant alleviating effect on both hepatic steatosis and inflammatory infiltration (***). P <0.001).

[0153] like Figure 8As shown in Figure D, regarding glucose and lipid metabolism, on the one hand, after 8 weeks of high-fat diet induction, treatment with strain YG0564 further reduced hepatic fat synthesis (FAS), upregulated Srebp2 to inhibit hepatic cholesterol synthesis and accelerate bile acid metabolism, while reducing the inflammatory factor TNFα, alleviating hepatic lipid accumulation, inhibiting inflammation, and providing protection for the liver. On the other hand, we detected that FGF15 was upregulated in the colon, and the protein flowed back into the liver to inhibit CYP7A1 expression, and hepatic SHP was upregulated, thereby inhibiting HNF-4α, PEPCK, and G6PC1. At the same time, the glucocorticoid receptor gene NR3C1 was also significantly downregulated. These are the main molecules and enzymes in the hepatic gluconeogenesis pathway. This unified downregulation phenomenon was not observed in the orlistat and LGG treatment groups. Therefore, this example demonstrates that YG0564 can reduce hepatic gluconeogenesis in mice by acting on the FGF15-CYP7A1 and SHP-HNF-4α pathways, thereby reducing blood glucose production and alleviating glucose metabolism disorders.

[0154] like Figure 9 The results of mouse white adipose tissue data are shown; where A is the weight and volume of white adipose tissue in the posterior abdominal wall, B is the microscopic examination results of white adipose tissue, and C is the gene expression map. In Figure C, the bars from left to right represent the results of the ND, HFD, Orlistat, LGG, and YG0564 groups, respectively.

[0155] Depend on Figure 9 The results show that, for example Figure 9 According to the present invention, strain YG0564, like orlistat and LGG, can significantly reduce the mass and volume of white adipose tissue (*). P <0.05), significantly reduced lipid accumulation in adipocytes and decreased cell volume (e.g., ... Figure 9 B, *** P<0.001). Early studies have demonstrated that activation of bile acid G protein-coupled receptor 5 (TGR5) increases energy expenditure and reduces fat mass, thus contributing to weight loss. TGR5 is expressed in both white and brown adipose tissue, and bile acid activation of it increases energy expenditure and reduces diet-induced obesity. IL-27-IL-27Rα signaling plays a crucial role in improving thermogenesis, preventing diet-induced obesity, and improving insulin resistance. Mechanistic studies have shown that IL-27 directly targets adipocytes, activating p38 MAPK–PGC-1α signaling and stimulating UCP1 production. IL-27 plays an important role in coordinating metabolic programs and is a very promising target for anti-obesity immunotherapy. This invention found that the expression of both TGR5 and IL-27Rα mRNA was significantly upregulated in the adipose tissue of YG0564-treated mice. To confirm the thermogenesis mechanism related to energy expenditure associated with YG0564 treatment, this invention further investigated several markers associated with browning of white adipocytes in the tissue. The significant upregulation of Ucp1, Eiovl3, HSL, Pparα, and PGC-1α genes in the abdominal wall tissue treated with YG0564 indicates that the YG0564 strain can remodel white adipocytes into beige cells by regulating IL-27Rα and TGR5.

[0156] Farnesoid X receptor (FXR) is a nuclear receptor highly expressed in tissues such as the liver, intestine, and adipose tissue. Activation of FXR alters energy metabolism-related pathways. It not only acts as a bile acid receptor in the liver to regulate bile acid metabolism but also regulates the differentiation of preadipocytes into adipocytes, playing a regulatory role in energy metabolism in the liver, muscle, and adipose tissue. YG0564 significantly upregulated the expression of FXR, SHP, and Fgf15 genes in white adipose tissue, promoting energy metabolism in white adipose tissue by activating the FXR-FGF15 axis.

[0157] like Figure 10 Data on brown adipose tissue in mice; where A is the microscopic examination result of brown adipose tissue, B is the expression map of lipid metabolism genes, and C is the expression map of inflammation regulation genes; in Figures B and C, the bars from left to right represent the results of the ND, HFD, Orlistat, LGG, and YG0564 groups, respectively.

[0158] like Figure 10 As shown in Figure A (H&E staining microscopic results), treatment with strain YG0564 can reverse HFD-induced BAT albinism, reduce intracellular lipid accumulation, significantly decrease cell volume, and increase brown adipose tissue density (right figure***). P<0.001). Compared with the HFD group, IL-27Rα mRNA was highly expressed in the brown adipose tissue of mice treated with strain YG0564. To confirm the thermogenic mechanism related to energy consumption in strain YG0564 treatment, this example tested several biomarkers related to thermogenesis in brown adipose tissue. It was found that treatment with strain YG0564 significantly upregulated the expression of thermogenic genes Cidea and Elovl3 in brown adipose tissue, indicating that strain YG0564 can amplify the thermogenic metabolic effects of mouse brown adipocytes by regulating IL-27Rα. Simultaneously, leptin, a biomarker of white adipose tissue, was significantly downregulated after treatment with strain YG0564. HFD-fed mice also exhibited an inflammatory response in their brown adipose tissue, while the strain YG0564 screened in this invention significantly increased the levels of anti-inflammatory factors IL-4 and Foxp3, and decreased the production of pro-inflammatory factors TNF-α and IL-6, demonstrating a significant anti-inflammatory effect.

[0159] like Figure 11 16S sequencing results of the gut microbiota in mice showed that α-diversity of the gut microbiota decreased after induction of a high-fat diet under HFD. Figure 11 In this context, A represents the Shannon index; Figure 11 (B in the text refers to the Chao index). While orlistat treatment significantly reduces the obesity index, it further decreases gut microbiota diversity. In contrast, oral administration of Lactobacillus LGG and F. dorsalis YG0564 both increased gut microbiota α-diversity, and YG0564 also caused gut microbiota β-diversity (PCoA) to cluster towards the normal mouse ND group. Figure 11 Adding YG0564 (C) promotes a healthier gut microbiota. Further classification and abundance analysis showed that, compared to the ND group, the HFD group had higher relative abundances of Firmicutes and Actinobacteria, and lower relative abundances of Bacteroidetes. However, compared to the HFD group, the addition of YG0564 decreased the abundances of Verrucous and Proteobacteria, while increasing the abundances of Firmicutes and Actinobacteria. These changes indicate that YG0564 affects the relative abundance of different bacterial phyla in the gut microbiota of mice with disordered glucose and lipid metabolism.

[0160] like Figure 12The correlation analysis of intergroup differences and physiological indicators in the gut microbiota of mice at the genus level, as shown in 12A, further intergroup species difference analysis revealed that at the genus level, the YG0564 treatment group, compared with the HFD group, induced changes in the abundance of multiple genera, significantly reducing the abundance of harmful strains Vampirovibrio, Streptococcus, Erysipeiotrichaceae, Tidjianibacter, and Erysipelatoclostridium, and increasing the abundance of beneficial strains Lactobacillus, Bacteroidales, Odoribacteraceae, Adlercreutzia, and Prevotellamassilia. Further correlation analysis was conducted between physiological indicators such as mouse obesity, glucose and lipid metabolism, and inflammation and the abundance of gut microbiota genus (e.g., Figure 12 (BD), the results showed that the fungus genus was found. Lactobacillus, Odoribacteraceae, Bacteroidales,Limosalactobacillus,Duncaniella,Alistipes It showed a significant negative correlation with body weight, liver weight, white adipose tissue weight, blood lipids (TC, LDL, HLD), and pro-inflammatory factors (IL-1β, TNFα, IL-6, IL-8), and a positive correlation with brown adipose tissue weight and insulin, demonstrating significant lipid-lowering, weight-loss, and anti-inflammatory effects. Conversely, Desulfovibro, Faecalibaculum,Ileibacter Dubosiella,Vampirovibrio,Eubacterium,Allobaculum It exhibits a significant effect in promoting lipid accumulation, weight gain, and pro-inflammatory activity. Among these phenomena, the main beneficial bacteria are... Lactobacillus Odoribacteraceae,Bacteroidales and harmful bacteria Vampirovibrio All samples were identified as specific driver bacteria after treatment with YG0564, suggesting that the bacteria exert their therapeutic effect by regulating specific bacterial communities in cases of glucose and lipid metabolism disorders. This provides a promising strategy for further research and potential therapeutic interventions related to glucose and lipid metabolism disorders.

[0161] like Figure 13 Short-chain fatty acid (SCFA) levels in the cecal contents of mice from different groups were measured. Results showed that high-fat diet induction significantly reduced the levels of most SCFAs. After intervention with weight-loss drugs, LGG, or YG0564, SCFA levels significantly increased. In particular, YG0564 significantly increased the levels of acetic acid, propionic acid, butyric acid, valerate, isohexanoic acid, and hexanoic acid in the intestine.

[0162] In combination with the above Figure 6The in vitro molecular detection results show that YG0564 not only produces high levels of butyric acid and propionic acid, but also high levels of acetic acid. This promotes cross-feeding with other bacterial strains in the gut microbiota, further boosting the production of propionic and butyric acids. Evidence suggests that short-chain fatty acids (SCFAs) inhibit fat accumulation in white adipose tissue by synergistically inhibiting fat accumulation with GPR43. Propionic and butyric acids can stimulate the production of the satiety hormone leptin in adipocytes by activating cell surface FFAR3, and butyric acid intake alone can reduce visceral and hepatic fat accumulation. Therefore, YG0564 can exert a lipid-lowering effect in vivo by producing multiple SCFAs in synergy with gut microbiota.

[0163] In summary, the *Focusella dorsalis* strain YG0564 screened in this invention highly expresses cholesterol sulfonase, which can directly participate in cholesterol degradation; the strain has high BSH enzyme activity, which can efficiently degrade conjugated bile acids and regulate cholesterol metabolism through bile acids; the strain can produce a variety of short-chain fatty acids, thereby regulating cholesterol metabolism; the strain can alleviate hepatocyte steatosis and inflammatory response; the strain can reduce hepatic gluconeogenesis in mice by acting on the FGF15-CYP7A1 and SHP-HNF-4α pathways, thereby reducing blood glucose production and alleviating glucose metabolism disorders. The strain can target and improve the characteristic gut microbiota of glucose and lipid metabolism disorders, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, and restore the health of the gut microbiota.

[0164] Example 4

[0165] The *Foscias dorsalis* YG0564 described in this embodiment can be used to prepare various dosage forms of microbial preparations, such as powders, granules, solid beverages, and compressed candies.

[0166] The *Foscias dorsalis* YG0564 described in this embodiment can be supplemented with conventional excipients or other active ingredients, such as galactooligosaccharides, inulin, dietary fiber, and other beneficial ingredients. It can also be supplemented with flavoring functional sugar alcohols, fruit powders, or functional ingredients such as maltodextrin.

[0167] In the microbial preparation containing *Focusella dorsalis* YG0564 as the active ingredient described in this embodiment, the viable microbial count is controlled to be greater than 1 × 10⁻⁶. 10 CFU per person per day.

[0168] The method for preparing the microbial preparation containing *Focusella dorsalis* YG0564 as the active ingredient described in this embodiment can be achieved using conventional processes in the field.

[0169] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A strain of *Focusia dorsalis* YG0564, characterized in that, Its classification name is *Focius dorsalis*. Phocaeicola dorei It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27578 and deposit date of June 7, 2023.

2. The use of the *Focusella dorsalis* YG0564 according to claim 1 in the preparation of a medicament having at least one of the following effects (1)-(4): (1) Degrading blood lipids and body fat; (2) Prevention and treatment of metabolic disorders caused by high sugar and high fat; (3) Alleviates hepatocyte steatosis and inflammatory response; (4) Suppress body weight.

3. A drug for improving hyperglycemic and hyperlipidemic metabolic disorders, characterized in that, The active ingredient includes *Focusella dorsalis* YG0564 as described in claim 1.

4. A drug with weight loss and fat reduction effects, characterized in that, The active ingredient includes *Focusella dorsalis* YG0564 as described in claim 1.

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