New applications of metabolites from a strain of Bacillus sarcodactylis

Abstract

This invention discloses a novel application of the metabolites of *Bacillus salsaformis*. *Bacillus salsaformis* ZHT-9 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20241011, located at the China Center for Type Culture Collection (Wuhan University). This invention utilizes the metabolites of *Bacillus salsaformis* to feed SPF-grade mice, revealing that the metabolites have a significant lipid-lowering function in the animal's blood, and also exhibit a unique "eat more but don't gain weight" phenomenon in a mouse model.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a new application of the metabolites of a strain of Bacillus sabolicii. Background Technology

[0002] In recent years, research on the application of probiotics in areas such as lowering blood lipids has become increasingly in-depth. Studies have shown that specific strains (such as lactic acid bacteria and bifidobacteria) can reduce serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) through mechanisms such as regulating the balance of intestinal flora, producing short-chain fatty acids (SCFAs), inhibiting cholesterol synthase activity, and promoting bile acid excretion.

[0003] For example, animal studies have shown that feeding probiotic-containing diets can reduce total cholesterol (TC) levels in high-fat model rats by 15%-30%. However, due to a lack of specificity in strain screening, the effects are unstable and species-specific. Furthermore, probiotics are susceptible to the effects of processing, storage, and the gastrointestinal environment (such as gastric acid and bile salts), leading to low colonization efficiency and limited practical efficacy. Live bacteria may also trigger abnormal immune responses or carry the risk of transferring drug-resistant genes.

[0004] Compared to live bacteria preparations, probiotic metabolites have a greater safety advantage:

[0005] Risk controllability: Metabolites do not rely on live bacteria colonization, thus avoiding the potential infection risks and horizontal gene transfer risks associated with live bacteria;

[0006] High dosage controllability: The product can be added directly in quantitative quantities, avoiding the activity fluctuations of live bacteria affected by environmental factors (such as pH and antibiotics);

[0007] Low toxicological burden: Purified metabolites (such as specific fatty acids) have been toxicologically validated and have no significant risk of liver or kidney damage or inflammation at effective doses and are easily eliminated through metabolism.

[0008] In summary, probiotic metabolites have a better safety profile in lipid-lowering applications. Therefore, developing more probiotic metabolite products has significant clinical application value. Summary of the Invention

[0009] The purpose of this invention is to provide a new application of the metabolites of a strain of Bacillus sabolicii.

[0010] To address the problems existing in the prior art, the technical solution adopted in this invention is:

[0011] This invention provides the use of the metabolites of Bacillus salsa ZHT-9 in the preparation of products for the treatment and / or prevention of lipid-lowering and weight regulation.

[0012] Furthermore, the preservation number of the *Bacillus sarcodactylis* is CCTCC NO: M20241011.

[0013] Furthermore, the method for preparing the metabolites of *Bacillus salsa* ZHT-9 includes the following steps:

[0014] ① Inoculate Bacillus sabolicus ZHT-9 onto TSA medium and culture at 37±1℃ for 24±1h to activate the strain. Pick a single colony and inoculate it into TSB medium. Culture at 37±1℃ and 180±5 rpm with shaking for 16±1h to prepare seed culture. Inoculate the fresh seed culture into fermentation medium at an inoculation rate of (1±0.1)% (v / v) and ferment at 37±1℃ and 150±5 rpm for 16±1h to obtain Bacillus sabolicus ZHT-9 fermentation broth.

[0015] ② Centrifuge the fermentation broth at 8000±100 rpm for 10±1 min at 4±0.5℃ to remove the cells, collect the supernatant, and repeat the above steps 1-3 times to obtain a crude extract of sterile metabolites.

[0016] Furthermore, the fermentation medium is TSB medium or NB medium.

[0017] Furthermore, the viable bacteria concentration in the fermentation broth is (7.53±4.38) x 10⁻⁶. 7 cfu / ml.

[0018] Furthermore, the product includes pharmaceuticals.

[0019] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0020] Furthermore, the dosage forms of the drug include tablets, capsules, granules, oral solutions, water injections, powder injections, lyophilized powder injections, sprays, suppositories, or pills.

[0021] Furthermore, the routes of administration of the drug include oral, injection, inhalation spray, or rectal administration.

[0022] The advantages and beneficial effects of this invention are:

[0023] Bacillus sarfusae ( Bacillus safensis ZHT-9 metabolites have significant lipid-lowering effects. Mouse experiments showed that the metabolites of this strain can effectively reduce blood triglyceride (TG) and total cholesterol (TC) levels, thereby improving lipid metabolism. This discovery fills a gap in the research field of bacterial metabolites' lipid-lowering function.

[0024] Metabolites of *Bacillus salsaformis* significantly regulate the relationship between food intake and weight gain in mice. Experimental results showed that mice ingesting *Bacillus salsaformis* metabolites experienced increased food intake, but their weight gain was significantly lower than the control group, demonstrating a "eat more, don't gain weight" effect. This unique metabolic regulation provides a novel approach and method for the prevention and treatment of obesity and related metabolic diseases.

[0025] Compared to whole bacteria, Bacillus safoetida metabolites offer several advantages in practical applications, including well-defined composition, high safety, direct mechanism of action, strong stability, precise dosage control, and ease of storage and transportation. These characteristics make them more suitable for development and application as drug components, possessing greater clinical translational value and market potential.

[0026] Preservation instructions:

[0027] Strain name: Bacillus safranin Bacillus safensis ZHT-9;

[0028] Accession number: CCTCC NO: M20241011;

[0029] Category Naming: Bacillus safensis ;

[0030] Deposit date: May 20, 2024;

[0031] Depository: China Center for Type Culture Collection;

[0032] Address of the depositary institution: Wuhan University, Wuhan, China. Attached Figure Description

[0033] Figure 1 The colony morphology of Bacillus sabovella ZHT-9;

[0034] In the figure: a shows the morphology of ZHT-9 colony plates; b shows the morphology of ZHT-9 colonies under Gram staining microscopy (oil immersion 100×, eyepiece 10×).

[0035] Figure 2 The growth curve of Bacillus sabinatus ZHT-9;

[0036] Figure 3 To construct a phylogenetic tree of this bacterium using Neighbou-joining clustering based on pangeny;

[0037] Figure 4 The figure shows the classification of OTU numbers for the control group, ZQ group, and ZDX group:

[0038] (A) Cluster pie chart of OTUs in the control group, (B) Cluster pie chart of OTUs in the ZQ group, (C) Cluster pie chart of OTUs in the ZDX group;

[0039] Figure 5 The figure shows a bar chart of species distribution for the control group, ZQ group, and ZDX group.

[0040] (A) Relative abundance of cecal microbes at the phylum level in the control group, ZQ group, and ZDX group; (B) Relative abundance of cecal microbes at the genus level in the control group, ZQ group, and ZDX group; (C) Relative abundance of cecal microbes at the species level in the control group, ZQ group, and ZDX group. Each color represents a species, the length of the color block (bar chart) represents the relative abundance ratio of the species, Others represents the sum of the remaining species; Unclassified represents species without taxonomic annotations. Detailed Implementation

[0041] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] The Bacillus sarcodactylis ZHT-9 of this invention is abbreviated as ZHT-9.

[0044] Strain source: The Bacillus sarcodactylus ZHT-9 used for isolation and screening in this invention was derived from Chenghua Black Pigs from a pig farm in Qionglai City, Sichuan Province.

[0045] TSB and TSA media were used for the culture of bacteria ZHT-9.

[0046] SPF-grade mice were 7 weeks old and purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0047] Taq DNA polymerase, dNTPs, and bacterial genomic DNA extraction kits were all purchased from Takara Bio Ltd.

[0048] The blood analyzer is a Seamaty-120VP, used to detect triglyceride and total cholesterol levels in the blood.

[0049] Example 1

[0050] 1. Isolation, purification and identification of Bacillus salsa ZHT-9

[0051] 1.1 Isolation, purification, staining, and microscopic examination of the strain

[0052] Intestinal contents of Chenghua black pigs were spread on TSA plates, and single colonies were picked and purified using the streak plate method. Single colonies with good growth were picked from the purified bacterial culture medium to obtain strain ZHT-9, which was then Gram-stained. The morphological characteristics of the bacteria were observed under a light microscope.

[0053] like Figure 1 As shown in Figure a, after the strain grew on TSA medium for 24 hours, the colony diameter was 2-5 mm, the colony was milky white, the edge was smooth, and the center was wrinkled.

[0054] 1.2 Physiological and biochemical identification:

[0055] This bacterium is Gram-positive (G+), with rod-shaped cells, 1-1.5 µm long and 0.3-0.5 µm wide. Figure 1 b).

[0056] 1.3 Determination of growth curve

[0057] Fresh single colonies were picked from TSA medium (OXOID, UK) and inoculated into bacterial bottles containing TSB medium (OXOID, UK). The bottles were then incubated on a shaker at 37 ℃ and 180 rpm / min. The colonies were removed every 2 hours and analyzed based on bacterial OD. 630 Plot the bacterial growth curve. After culturing in TSB for 12 hours, bacterial growth enters the plateau phase (…). Figure 2 ).

[0058] 1.4 Molecular identification and preservation of bacteria

[0059] Genomic DNA was extracted from the isolated and purified strain ZHT-9 using a bacterial genomic DNA extraction kit (Takara). The extracted DNA was then amplified by 16S rRNA gene PCR and sequenced by a sequencing company. Bacterial 16S rDNA was amplified using primers 27F / 1492R, with upstream primer 27F (SEQ ID NO. 1: 5'-AGAGTTTGAT CCTGG CTCAG-3') and downstream primer 1492R (SEQ ID NO. 2: 5'-GGTTA CCTTG TTACG ACTT-3'). The PCR reaction system (50 µl) consisted of: 10 µl of 5×PS Buffer, 5 µl of 10 µmol / L 27F, 5 µl of 10 µmol / L 1492R, 4 µl of 2.5 µmol / L dNTPs, 4.5 µl of bacterial template, 1.5 µl of Primer STAR, and 20 µl of H2O. PCR reaction conditions: 98℃ for 5 min; 98℃ for 10 sec, 55℃ for 5 sec, 72℃ for 60 sec, this step was repeated 30 times; 72℃ for 10 min. The strain was sent to Sangon Biotech Co., Ltd. for whole genome sequencing.

[0060] A phylogenetic tree for this bacterium was constructed using Neighbou-joining clustering based on pangeny, such as... Figure 3 As shown.

[0061] 1.5 Identification Results:

[0062] The amplified 16S rRNA gene fragment of this bacterium was 1456 bp in size. Sequencing yielded the 16S rRNA sequence shown in SEQ ID NO. 3. Comparison with bacteria in the NCBI database revealed that the bacterium belongs to the Bacillus subtilis family. Through whole-genome sequencing, strain ZHT-9 was identified as *Bacillus safranin*. The genome accession information for this bacterium is JAZDWY010000000, and the species name is... Bacillus safensis .

[0063] Bacillus sarcodactylis ZHT-9 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20241011, located at Wuhan University, Wuhan, China, on May 20, 2024.

[0064] SEQ ID NO. 3:

[0065]

[0066] 2. Bacterial scale-up culture and preparation of metabolites

[0067] ① *Bacillus sabolicus* ZHT-9 was inoculated onto TSA solid medium and cultured at 37℃ for 24 h to activate the strain. A single colony was picked and inoculated into TSB medium, and cultured with shaking at 37℃ and 180 rpm for 16 h to prepare a seed culture. The fresh seed culture was then inoculated into TSB fermentation medium at a rate of 1% v / v and fermented at 37℃ and 150 rpm for 16 h to obtain the *Bacillus sabolicus* ZHT-9 fermentation broth. The viable cell concentration in the fermentation broth was (7.53±4.38) x 10⁻⁶. 7 cfu / ml.

[0068] ② Centrifuge the fermentation broth at 8000 rpm for 10 min at 4℃ to remove the bacterial cells, collect the supernatant, and repeat the above steps twice to obtain a crude extract of sterile Bacillus salsa ZHT-9 metabolites.

[0069] 3. In vitro antibody screening of Bacillus salsa ZHT-9

[0070] 3.1 Acid resistance test

[0071] The pH of TSB medium (OXOID, UK) was adjusted to 2.0 and 4.0, respectively, and sterilized at 121 °C for 15 min. 100 ml of the fermentation broth in the logarithmic growth phase was added to 900 ml of TSB medium at the corresponding pH and cultured for 3 h. Then, it was serially diluted and plated onto TSA medium and cultured at 37 °C for 24 h. The number of viable strains was determined by plate counting the following day.

[0072] 3.2 Bile salt tolerance test

[0073] Prepare 0.2% and 0.5% porcine bile salt TSB medium, filter through a 0.22 μm filter into sterile wide-mouth bottles, and use porcine bile salt-free TSB medium as a negative control. Incubate 100 ml of the fermentation broth in the logarithmic growth phase in 900 ml of porcine bile salt-concentrated TSB medium for 2 h, then serially dilute and plate onto TSA medium and incubate at 37 ℃ for 24 h. The number of viable strains is determined by plate counting the following day.

[0074] Table 1. Effect of low pH on the survival rate of strain ZHT-9

[0075]

[0076] As shown in Table 1, the survival rate of the strain of this application reached 52.94% after culturing in a medium with pH 2.0 for 3 hours; and the survival rate of the strain of this application reached 88.24% after culturing in a medium with pH 4.0 for 3 hours, indicating that the strain of this application has a certain acid resistance.

[0077] Table 2. Effects of 0.2% and 0.5% concentrations of porcine bile salts on the survival rate of strain ZHT-9.

[0078]

[0079] As shown in Table 2, the survival rate of the strain of this application reached 12.3% after culturing in a medium with a bile salt concentration of 0.2% for 2 hours; the survival rate of the strain of this application reached 1.22% after culturing in a medium with a bile salt concentration of 0.5% for 2 hours, indicating that the strain of this application has a certain bile salt tolerance.

[0080] 4. LC-MS non-targeted metabolomics detection

[0081] 4.1 Sample Collection and Processing

[0082] Bacillus sabolicus ZHT-9 was inoculated onto TSA medium and cultured at 37℃ for 24 h to activate the strain. Single colonies were picked and inoculated into TSB medium and cultured with shaking at 37℃ and 180 rpm for 16 h to prepare seed culture. The fresh seed culture was inoculated into the fermentation medium TSB at an inoculation rate of 1% (v / v) and fermented at 37℃ and 150 rpm for 24 h.

[0083] 4.2 Collection of Supernatant

[0084] The fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C to remove the bacterial cells. The supernatant was collected, and the above steps were repeated twice to obtain a crude extract of ZHT-9 sterile metabolites. The collected supernatant was stored at -80°C to prevent degradation of the metabolites.

[0085] 4.3 Selection of Detection Technology and Parameter Setting

[0086] The crude extract of ZHT-9 metabolites was sent to Sangon Biotech Co., Ltd. for LC-MS non-targeted metabolomics analysis to obtain information such as metabolite number, integral value and corresponding metabolite name.

[0087] 5. Results and Analysis of LC-MS Non-targeted Metabolomics Detection

[0088] Table 3 lists the types of metabolites detected in the extracellular supernatant of ZHT-9, including their names, molecular formulas, molecular weights, retention times, mass spectrometry characteristics, and possible metabolic pathways.

[0089] 5.1 Free fatty acids

[0090] The detected metabolites of free fatty acids were oleic acid and linoleic acid.

[0091] Oleic acid: molecular formula is C 18 H 34 O2; molecular weight 282.2559 Da; retention time 8.4285 min; mass spectrometry characteristic [MH]-; peak area 45799.28; metabolic pathways involved include ko00061, ko00073, and ko01040 in the KEGG database.

[0092] Linoleic acid: molecular formula is C 18 H 32 O2; molecular weight 280.2402 Da; retention time 7.9136 min; mass spectrometry characteristic [MH]-; peak area 14319.31; metabolic pathways involved include ko00591, ko01040, and ko01100 in the KEGG database.

[0093] Oleic acid, secreted by bacteria, is a monounsaturated fatty acid that participates in major metabolic pathways including fatty acid biosynthesis and the biosynthesis of unsaturated fatty acids. Linoleic acid, a polyunsaturated fatty acid, cannot be synthesized endogenously in animals and must be obtained through diet. It participates in metabolic pathways including the linoleic acid metabolic pathway and the biosynthesis of unsaturated fatty acids. When oleic acid and linoleic acid are ingested, they may affect animal weight and regulate serum cholesterol levels.

[0094] 5.2 Amino Acids

[0095] The detected amino acid metabolites include L-carnitine, L-glutamine, L-valine, and L-leucine.

[0096] L-carnitine: molecular formula C7H 15 NO3; molecular weight 161.1052 Da; retention time 0.8116 min; mass spectrometry characteristics [M+H]+; peak area 3219.97; metabolic pathway involved is unclear.

[0097] L-Glutamine: Molecular formula is C5H 10N2O3; molecular weight 146.0691 Da; retention time 1.236 min; mass spectrometry characteristics [M+H]+; peak area 32520.28; involved in metabolic pathways including ko00220, ko00230, ko00240, ko00250, ko00470, ko00630, ko00750, ko00910, ko00970, ko01100, ko01120, ko01230, ko01232, ko01240, ko02010, ko02020 from the KEGG database.

[0098] L-valine: Molecular formula is C5H 11 NO2; molecular weight 117.079 Da; retention time 1.162 min; mass spectrometry characteristics [M+H]+; peak area 143715.06; involved in metabolic pathways including ko00280, ko00290, ko00311, ko00460, ko00770, ko00970, ko01100, ko01110, ko01210, ko01230, ko01240, and ko02010 from the KEGG database.

[0099] L-Leucine: Molecular formula C6H 13 NO2; molecular weight 131.0946 Da; retention time 1.356 min; mass spectrometry characteristics [M+H]+; peak area 5356715.89; involved in metabolic pathways including ko00280, ko00290, ko00970, ko01100, ko01110, ko01210, ko01230, ko02010, and ko05131 from the KEGG database.

[0100] These amino acid metabolites can affect body weight and blood lipid levels by regulating gut microbiota, energy metabolism, and lipid metabolism. Studies have shown that dietary supplementation with L-carnitine can effectively reduce body weight and cholesterol and triglyceride levels in blood lipids. Supplementation with L-glutamine in a high-fat diet can reduce body weight and improve lipid metabolism in mice, lowering serum cholesterol levels. L-glutamine, L-proline, L-valine, and L-leucine participate in various metabolic processes in the body and can indirectly affect body weight and lipid metabolism. Valine and leucine belong to branched-chain amino acids (BCAAs). BCAAs may help control body weight by improving energy metabolism, regulating insulin sensitivity, and reducing inflammation. Furthermore, dietary BCAA intake is negatively correlated with the risk of dyslipidemia, including total cholesterol (TC) and triglycerides (TG).

[0101] 5.3 Organic acids and their derivatives

[0102] The detected organic acids and their derivatives include metabolites such as 3-hydroxybutyric acid and propionic acid.

[0103] 3-Hydroxybutyric acid (3-HB): molecular formula C4H8O3; molecular weight 104.0473 Da; retention time 1.1981 min; mass spectrometry characteristics [M+H]+; peak area 750.21; metabolic pathways involved include ko00650 and ko01100 in the KEGG database.

[0104] Propionic acid: molecular formula C3H6O2; molecular weight 74.0368 Da; retention time 1.4078 min; mass spectrometry characteristic [MH]-; peak area 14754.11; involved in metabolic pathways including ko00640, ko00642, ko00760, ko01100, ko01120, and ko01220 in the KEGG database.

[0105] 3-HB may affect body weight by promoting fat metabolism or energy utilization, and may also improve blood lipid levels by regulating lipid metabolism. Propionate can reduce body weight gain, lower blood lipid and insulin levels in obese rats, and may have the effect of increasing the body's energy expenditure. In addition, propionate reduces cholesterol synthesis by inhibiting the activity of acetyl-CoA synthase and serves as a precursor for hepatic glucose production, thereby reducing cholesterol synthesis in the liver.

[0106] 5.4 Glycerol phospholipids

[0107] The detected metabolites of glycerophospholipids included glycerol-3-phosphate choline.

[0108] Glycerol-3-phosphate choline: Molecular formula C8H 21 NO6P + The molecular weight is 258.1106 Da; the retention time is 0.7823 min; the mass spectrometry characteristics are [M+K]+; the peak area is 94965.63; the metabolic pathways involved are ko00564 and ko00565 in the KEGG database.

[0109] The effects of glycerol-3-phosphate choline on body weight and blood lipids have been explored in several studies. Firstly, glycerol-3-phosphate choline is a key intermediate in phospholipid synthesis, participating in the synthesis of triglycerides (TAGs). In animal experiments, glycerol-3-phosphate choline supplementation significantly reduced cholesterol and triglyceride levels in the liver. However, glycerol-3-phosphate choline may exert complex regulatory effects on body weight and blood lipids in certain situations, requiring further investigation.

[0110] Table 3 Metabolites of strain ZHT-9

[0111]

[0112] Experimental Example 1: Feeding Experiment in Monogastric Animals

[0113] 1. Laboratory animals

[0114] Thirty-six 7-week-old SPF-grade female Kunming rats were randomly divided into three groups of 12 rats each: a control group, an experimental group of ZHT-9 whole-cell fermentation broth (ZQ), and an experimental group of ZHT-9 metabolites (ZDX).

[0115] The control group was fed a standard SPF-grade diet (purchased from Chengdu Dashuo Experimental Animal Co., Ltd., with the following nutritional composition: moisture ≤10%, crude protein ≥18%, crude fat ≥4%, crude fiber ≤5%, crude ash ≤8%, 1.0%≤calcium≤1.8%, 0.6%≤phosphorus≤1.2%, lysine ≥0.82%, methionine + cystine ≥0.53%). The experimental group ZQ was fed a standard SPF-grade diet supplemented with ZHT-9 whole bacteria, resulting in a live bacteria count of 5 x 10^6 bacteria per gram of feed. 8 CFU. The experimental group (ZDX) was fed a standard SPF-grade diet supplemented with a crude extract of ZHT-9 bacteria metabolites, resulting in 30 µl of sterile crude metabolite extract per gram of feed. The preliminary trial lasted 7 days, and the formal trial lasted 30 days. After the trial, whole blood was collected from mice (with anticoagulant) to measure total cholesterol, triglycerides, and other blood parameters.

[0116] 2. Test Methods

[0117] 2.1 Collection of mouse intestinal samples

[0118] Six 7-week-old mice from each of the Control, ZQ, and ZDX groups were randomly selected and euthanized by cervical dislocation. The desired intestinal tract was wiped with 75% alcohol, and the cecum was ligated. Cecal contents from each group were squeezed into 2 mL sterile EP tubes, sealed, and immediately placed in liquid nitrogen. Cecal fecal samples from each group were carefully transferred to the laboratory and stored at -80 °C for subsequent intestinal diversity analysis.

[0119] 2.2 Analysis of gut bacterial diversity

[0120] Fecal samples from the cecum were sent to Sangon Biotech Co., Ltd. for microbial diversity analysis. Total DNA was extracted from the Control, ZQ, and ZDX groups, and conserved 16S rDNA regions were amplified using V3 + V4 primers. The products were then purified, quantified, and homogenized to form sequencing libraries. The constructed libraries underwent quality control, and those that passed were sequenced using an Illumina MISEQ03 instrument. Sequencing yielded paired-end sequences containing barcode sequences, as well as primer and adapter sequences added during sequencing. After removing primer and adapter sequences, paired reads were merged into a single sequence based on the overlap relationship between PE reads. Samples were then identified and differentiated according to the barcode tag sequences to obtain data for each sample. Finally, quality control filtering was performed on each sample's data to obtain valid data. Clustering operations were used to divide the data into Operational Taxonomic Units (OTUs), and species annotation and abundance analysis were performed to reveal the species composition of the samples. A sequence with a similarity greater than 97% is defined as an OTU, and each OTU corresponds to a representative sequence. Representative OTU sequences are compared with a microbial reference database to obtain species classification information for each OTU and community composition of samples at the phylum to genus level.

[0121] 2 Results and Analysis

[0122] Animal serum indicators

[0123] Table 4 shows that after 37 days of feeding, the total cholesterol concentrations in the whole blood of mice in the ZQ and ZDX groups were 2.12 ± 0.22 mmol / L and 1.74 ± 0.52 mmol / L, respectively, both significantly lower than the control group (2.18 ± 0.37 mmol / L). The triglyceride concentrations in the whole blood of mice in the ZQ and ZDX groups were 1.35 ± 0.51 mmol / L and 1.01 ± 0.25 mmol / L, respectively, both significantly lower than the control group (1.805 ± 0.27 mmol / L). These results indicate that the ZHT-9 bacterium and its metabolites have a significant lipid-lowering function in animal blood.

[0124] Table 4 Effects of ZHT-9 on blood lipid levels in mice

[0125]

[0126] Note: * indicates a significant difference from the control group (p≤0.05).

[0127] Animal growth

[0128] Table 5 shows that there was no significant difference in initial weight among the control group, ZQ group, and ZDX group. After 37 days of feeding, the final weight, average daily food intake, and average daily weight gain of the control group mice were 77.37 ± 1.97 g, 11.79 ± 0.65 g, and 1.12 ± 0.41 g, respectively; the final weights of the ZQ and ZDX groups were 76.33 ± 2.03 g and 74.72 ± 0.70 g, respectively, both significantly lower than those of the control group; the average daily food intake of the ZQ and ZDX groups were 12.37 ± 1.03 g and 12.09 ± 0.50 g, respectively, both significantly higher than those of the control group. These results indicate that the "eat more but don't gain weight" phenomenon in mice is not solely due to the effects of the ZHT-9 whole bacteria, but also because the metabolites of this bacteria have the same effect.

[0129] Table 5. Effects of ZH-9 whole bacteria and its metabolites on mouse growth performance.

[0130]

[0131] Note: * indicates a significant difference from the control group (p≤0.05).

[0132] 2.3 Effects of ZHT-9 on the diversity of animal gut microbiota

[0133] 2.3.1 OTU Clustering

[0134] The average conserved fragment lengths of the cecal microorganism 16S rDNA in the control, ZQ, and ZDX groups were 421.92 bp, 424.53 bp, and 418.85 bp, respectively. The control, ZQ, and ZDX groups contained 696 OTUs, 570 OTUs, and 841 OTUs, respectively. The classification of OTU numbers in the control, ZQ, and ZDX groups is shown in the table below. Figure 4 See Table 6.

[0135] Table 6. Classification and percentage of OTU numbers in the control group, ZQ group, and ZDX group

[0136]

[0137]

[0138]

[0139]

[0140] Note: The single letter preceding the taxonomic name is an abbreviation of the taxonomic rank, separated by "__". Taxonomic databases may contain intermediate ranks within taxonomic lineages that lack scientific names; these are marked as "norank". After taxonomic alignment, some lineages below the confidence threshold may lack classification information; these are marked as "Unclassified" in statistical analysis.

[0141] 2.4 Relative Species Abundance

[0142] At the phylum-level composition, Bacillota and Bacteroidetes were the two major bacterial groups, dominating in the control, ZQ, and ZDX groups, followed by Actinomycetota. In the control group, the relative abundances of Bacillota, Bacteroidetes, and Actinomycetota were approximately 42.12%, 52.94%, and 1.18%, respectively; in the ZQ group, they were approximately 61.33%, 33.63%, and 1.86%; and in the ZDX group, they were approximately 54.15%, 35.97%, and 1.27%. Figure 5 As shown in A, the relative abundance of Firmicutes / Bacillota in the ZQ and ZDX groups was increased compared to the control group, while the abundance of Bacteroidetes was decreased.

[0143] At the genus taxonomic level, the relative abundance of bacterial groups such as norank_Muribaculaceae, Lactobacillus, Ligilactobacillus, norank_Lachnospiraceae, HT002, Alistipes, and Bacteroide occupied the dominant positions in the control group, ZQ group, and ZDX group. The relative abundances of norank_Muribaculaceae in the control group, ZQ group, and ZDX group were 42.00%, 24.66%, and 17.70%, respectively; the relative abundances of Lactobacillus were 20.50%, 31.42%, and 15.68%, respectively; the relative abundances of Ligilactobacillus were 3.17%, 10.76%, and 5.28%, respectively; the relative abundances of norank_Lachnospiraceae were 6.80%, 3.26%, and 19.11%, respectively; the relative abundances of HT002 were 5.39%, 11.04%, and 4.28%, respectively; the relative abundances of Alistipes were 2.70%, 3.62%, and 12.86%, respectively; and the relative abundances of Bacteroides were 3.43%, 1.22%, and 1.45%, respectively. Figure 5 As shown in B, the relative abundance of *Muribaculaceae* and *Alistipes* species in groups ZQ and ZDX was lower than that in the control group, while the abundance of *Ligilactobacillus* and *Bacteroides* species was also lower. In group ZQ, the relative abundance of *Lactobacillus* species was higher than that in the control group, while the abundance of *Lachnospiraceae* was lower. In group ZDX, the relative abundance of *Lactobacillus* species was lower than that in the control group, while the abundance of *Lachnospiraceae* was higher.

[0144] At the species taxonomic level, the relative abundances of *Muribaculaceae* sp. in the control group, ZQ group, and ZDX group were 41.98%, 24.65%, and 17.69%, respectively; the relative abundances of *Lactobacillus johnsonii* were 18.20%, 28.74%, and 14.29%, respectively; the relative abundances of *Ligilactobacillus* sp. were 3.17%, 10.76%, and 5.30%, respectively; and the relative abundances of *HT002* sp. were... The relative abundances of *Lachnospiraceae* sp. were 5.39%, 11.04%, and 4.28%, respectively; the relative abundances of *Alistipes* sp. were 5.48%, 2.16%, and 12.78%, respectively; the relative abundances of *Alistipes* sp. were 0.71%, 1.39%, and 9.01%, respectively; the relative abundances of *Alistipes* inops were 1.69%, 1.71%, and 3.77%, respectively; and the relative abundances of *Prevotellaceae* sp. were 2.49%, 0.75%, and 1.4%, respectively. 1%; the relative abundances of Saccharimonadaceae_sp. were 1.36%, 0.69%, and 0.96%, respectively; the relative abundances of Rikenellaceae_sp. were 0.25%, 1.61%, and 0.64%, respectively; the relative abundances of Clostridia_sp. were 0.97%, 0.29%, and 1.03%, respectively; the relative abundances of Odoribacter_sp. were 0.25%, 1.61%, and 0.64%, respectively; the relative abundances of Clostridia_s The relative abundances of *P.* were 0.52%, 0.54%, and 1.00%, respectively; the relative abundances of *Lachnospiraceae_bacterium_10-1* were 0.01%, 0.003%, and 1.52%, respectively; the relative abundances of *unclassified_Lachnospiraceae* were 1.71%, 1.32%, and 5.89%, respectively; and the relative abundances of *unclassified_Alistipes* were 0.20%, 0.48%, and 1.07%, respectively (see [link to relevant documentation]). Figure 5 C).

[0145] Integrating phylum-genus-species data on cecal microbiota diversity in mice, we found that the relative abundance of *Muribaculaceae* sp. in the ZQ and ZDX groups was significantly downregulated compared to the control group. This bacterium belongs to the phylum *Bacteroidota* and colonizes the intestinal mucosa, interacting with the host's immune system to remodel the gut microbiota. *Muribaculaceae* is typically associated with carbohydrate metabolism, and its reduced abundance may decrease energy absorption, thus contributing to weight control. The relative abundance of *Ligilactobacillus* bacteria was increased in both the ZQ and ZDX groups, potentially contributing to weight control by regulating gut microbiota balance and reducing the growth of harmful bacteria. *Lactobacillus johnsonii* was specifically enriched in the ZQ group; this bacterium has the ability to regulate lipid and bile acid metabolism, thereby improving blood lipid levels. Additionally, *Lactobacillus acidophilus* is a common probiotic that can help regulate gut microbiota and reduce fat accumulation. The abundance of *Lachnospiraceae* species was significantly increased in the ZDX group, with *norank_Lachnospiraceae* upregulated by 181% at the genus level and *Lachnospiraceae_sp.* upregulated by 133% at the species level. *Lachnospiraceae* is one of the major producers of short-chain fatty acids (SCFAs), especially butyrate. SCFAs play an important role in regulating host metabolism, energy balance, and inflammatory responses. For example, butyrate can promote lipidolysis in adipose tissue, reduce fat accumulation, and improve insulin sensitivity. Therefore, *Lachnospiraceae* may have beneficial effects on body weight and blood lipids by producing SCFAs. The abundance of *Bacteroides* species (potentially pro-inflammatory bacteria) was reduced in both the ZQ and ZDX groups compared to the control group, which may reduce lipopolysaccharide (LPS) load and alleviate endotoxin-induced chronic inflammation. Additionally, although the *Alistipes* genus level was increased in the ZQ and ZDX groups, these may be non-pathogenic strains (such as *Alistipes_inops*), requiring further functional verification. Prevotellaceae sp. was decreased in both the ZQ and ZDX groups. Bacteria of this genus are typically associated with high-fiber diets, and the reduced abundance may have reduced energy absorption. Saccharimonadaceae sp. was also decreased in both the ZQ and ZDX groups. Bacteria of this genus are typically associated with carbohydrate metabolism, and the reduced abundance may have contributed to reduced energy intake.Odoribacter sp. was increased in both the ZQ and ZDX groups. Bacteria of the Odoribacter genus are usually associated with the production of medium- and short-chain fatty acids, and changes in its abundance may affect energy metabolism.

[0146] In summary, the ZQ and ZDX groups showed significant differences in cecal microbiota composition compared to the control group. These differences may contribute to weight loss and lipid reduction by regulating energy metabolism, reducing fat absorption, and improving gut microbiota balance. Furthermore, the metabolite group (ZDX) was superior to the whole-microbiota group (ZQ) in both weight loss and lipid reduction.

[0147] Since mice and humans share biological and genomic similarities, we can infer from the mouse experiment in Example 1 that this strain also has the effects of reducing weight and blood lipids in humans.

[0148] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. The application of metabolites of Bacillus salsa ZHT-9 in the preparation of drugs for treating lipid-lowering and weight-regulating diseases; the preservation number of Bacillus salsa is CCTCC NO: M20241011; The method for preparing the metabolites of Bacillus salsa ZHT-9 includes the following steps: ① Inoculate Bacillus sabolicus ZHT-9 onto TSA medium and culture at 37±1℃ for 24±1h to activate the strain. Pick a single colony and inoculate it into TSB medium. Culture with shaking at 37±1℃ and 180±5 rpm for 16±1h to prepare seed culture. Inoculate the fresh seed culture into fermentation medium at an inoculation rate of (1±0.1)%v / v and ferment at 37±1℃ and 150±5 rpm for 16±1h to obtain Bacillus sabolicus ZHT-9 fermentation broth. ② Centrifuge the fermentation broth at 8000±100 rpm for 10±1 min at 4±0.5℃ to remove the cells, collect the supernatant, and repeat the above steps 1-3 times to obtain a crude extract of sterile metabolites.

2. Use according to claim 1, characterized in that, The fermentation medium is either TSB medium or NB medium.

3. Use according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

4. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, water injections, powder injections, sprays, suppositories, or pills.

5. The application according to claim 1, characterized in that, The drug can be administered orally, by injection, by inhalation spray, or by rectal administration.

Images