Use of leuconostoc mesenteroides and / or metabolites thereof

Abstract

Provided is use of leuconostoc mesenteroides and / or metabolites thereof in preparing a composition for reducing body fat, suppressing appetite, promoting the ketone body level or regulating fasting blood glucose, wherein the leuconostoc mesenteroides is leuconostoc mesenteroides subsp. mesenteroides with the preservation number of DSM34443.

Description

Use of leuconostoc mesenteroides and / or metabolites thereof TECHNICAL FIELD

[0001] The present disclosure relates to a leuconostoc mesenteroides, in particular to a use of leuconostoc mesenteroides subspecies mesenteroides and / or metabolites thereof for preparing a body fat-reducing, appetite-reducing, ketone body amount-promoting, or fasting blood glucose-regulating composition. BACKGROUND

[0002] Leuconostoc mesenteroides is an important species of Leuconostoc, a genus of lactic acid bacteria, which grows well under anaerobic culture and has an optimal growth temperature of 30-40°C. Leuconostoc mesenteroides has antagonistic effects on common pathogenic bacteria such as Shigella, Salmonella, and Staphylococcus aureus, and is a probiotic with development potential. SUMMARY

[0003] Therefore, the present disclosure provides a leuconostoc mesenteroides and / or metabolites thereof, wherein the leuconostoc mesenteroides is leuconostoc mesenteroides subspecies mesenteroides with accession number DSM34443.

[0004] In some embodiments, the leuconostoc mesenteroides and / or metabolites thereof are used for preparing a body fat-reducing composition.

[0005] In some embodiments, the leuconostoc mesenteroides can promote fat metabolism.

[0006] In some embodiments, the leuconostoc mesenteroides can reduce fat cell oil droplet accumulation.

[0007] In some embodiments, the leuconostoc mesenteroides can promote cholesterol metabolism.

[0008] In some embodiments, the leuconostoc mesenteroides can increase the content of high-density cholesterol in blood.

[0009] In some embodiments, the leuconostoc mesenteroides can promote high-density cholesterol production. In some embodiments, the leuconostoc mesenteroides can promote an increase in the expression amount of a CETP gene, a SCARB1 gene, or a LDLR gene.

[0010] In some embodiments, the leuconostoc mesenteroides and / or metabolites thereof are used for preparing an appetite-reducing composition.

[0011] In some embodiments, the leuconostoc mesenteroides can promote the secretion of incretins.

[0012] In some embodiments, the leuconostoc mesenteroides can promote the secretion of GLP-1 by intestinal cells.

[0013] In some embodiments, the leuconostoc mesenteroides and / or metabolites thereof are used for preparing a ketone body amount-promoting composition. In some embodiments, the leuconostoc mesenteroides and / or metabolites thereof are used for preparing a fasting blood glucose-regulating composition.

[0014] In some embodiments, Leuconostoc mesenteroides can produce ketone bodies.

[0015] In some embodiments, Leuconostoc mesenteroides is used to produce beta-hydroxybutyric acid.

[0016] In some embodiments, Leuconostoc mesenteroides can increase serum ketone body concentration.

[0017] In some embodiments, Leuconostoc mesenteroides can increase urine ketone body content.

[0018] In some embodiments, Leuconostoc mesenteroides or its metabolite is used for preparing a composition for regulating fasting blood glucose.

[0019] In some embodiments, Leuconostoc mesenteroides can reduce insulin resistance index.

[0020] In some embodiments, Leuconostoc mesenteroides can promote incretin secretion.

[0021] In some embodiments, Leuconostoc mesenteroides can promote GLP-1 secretion by intestinal cells.

[0022] In some embodiments, Leuconostoc mesenteroides can reduce glycated albumin concentration.

[0023] In summary, the Leuconostoc mesenteroides and / or its metabolite of any of the embodiments can promote fat metabolism, reduce fat cell oil droplet accumulation, promote cholesterol metabolism, increase high-density cholesterol content in blood, promote high-density cholesterol production, promote CETP gene, SCARB1 gene, or LDLR gene expression, thereby achieving the purpose of reducing body fat. The Leuconostoc mesenteroides and / or its metabolite of any of the embodiments can promote incretin secretion and / or promote GLP-1 secretion by intestinal cells, thereby achieving the purpose of reducing appetite. The Leuconostoc mesenteroides and / or its metabolite of any of the embodiments can produce ketone bodies, produce beta-hydroxybutyric acid, increase serum ketone body concentration, and increase urine ketone body content, thereby achieving the purpose of promoting ketone body content. The Leuconostoc mesenteroides and / or its metabolite of any of the embodiments can reduce insulin resistance index, promote incretin secretion and / or promote GLP-1 secretion by intestinal cells, and reduce glycated albumin concentration, thereby achieving the purpose of regulating fasting blood glucose. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a phylogenetic tree of Leuconostoc mesenteroides TCI818.

[0025] Figure 2 is a cell state diagram of the fat accumulation inhibition test of Leuconostoc mesenteroides.

[0026] Figure 3 is a result diagram of the fat accumulation inhibition test of Leuconostoc mesenteroides.

[0027] Figure 4 is a graph of the results of the test of Leuconostoc mesenteroides for promoting liver cholesterol metabolism.

[0028] Figure 5 is a graph of the results of the test of Leuconostoc mesenteroides for cholesterol-related gene expression.

[0029] Figure 6 is a graph of the results of the test of Leuconostoc mesenteroides for promoting enterokinin.

[0030] Figure 7 is a graph of the results of the test of Leuconostoc mesenteroides for promoting BHB secretion.

[0031] Figure 8 is a graph of the results of the change in serum ketone body concentration in a human experiment.

[0032] Figure 9 is a graph of the results of the change in urine ketone body concentration in a human experiment.

[0033] Figure 10 is a graph of the results of the change in high-density cholesterol content in a human experiment.

[0034] Figure 11 is a graph of the results of the change in average body fat rate in a human experiment.

[0035] Figure 12 is a graph of the results of the change in average trunk fat weight in a human experiment.

[0036] Figure 13 is a graph of the results of the change in average visceral fat area in a human experiment.

[0037] Figure 14 is a graph of the results of the change in average skeletal muscle weight in a human experiment.

[0038] Figure 15 is a graph of the results of the change in average basal metabolic rate in a human experiment.

[0039] Preservation of biological material

[0040] Leuconostoc mesenteroides TCI818, deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany); date of deposit: September 7, 2022; accession number: DSM 34443. DETAILED DESCRIPTION

[0041] The Leuconostoc mesenteroides of the present application is a strain isolated from leeks. The Leuconostoc mesenteroides is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession number DSM 34443.

[0042] The Leuconostoc mesenteroides of the present application, which can also be referred to as Leuconostoc mesenteroides TCI818, is a Gram-positive bacterium of the genus Leuconostoc, is a facultative anaerobe, and is Leuconostoc mesenteroides subsp. mesenteroides.

[0043] In some embodiments, Leuconostoc mesenteroides or metabolites thereof are used for the preparation of a muscle building and fat reducing composition, wherein the Leuconostoc mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM 34443.

[0044] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof are used for the preparation of a body fat reducing composition.

[0045] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof are used for the preparation of an appetite reducing composition.

[0046] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof are used for the preparation of a ketone body amount promoting composition.

[0047] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof are used for the preparation of a fasting blood glucose regulating composition.

[0048] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof can promote fat metabolism.

[0049] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof can reduce fat cell oil droplet accumulation.

[0050] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof can promote cholesterol metabolism.

[0051] In some embodiments, Leuconostoc mesenteroides and / or metabolites thereof can increase high-density cholesterol content in blood.

[0052] In some embodiments, Leuconostoc mesenteroides can promote high-density cholesterol production. In some embodiments, Leuconostoc mesenteroides can promote an increase in the expression of a CETP gene, a SCARB1 gene, or a LDLR gene.

[0053] The protein encoded by the CETP gene (Gene ID: 1071) is plasma cholesteryl ester transfer protein, which is mainly involved in the transport of cholesteryl esters and triglycerides in plasma. It helps to regulate cholesterol levels, and when CETP expression rises, cholesteryl esters in HDL can be more effectively transferred to LDL or VLDL. In the case of simultaneous up-regulation of the low-density lipoprotein receptor (LDLR), the cholesteryl esters transferred to LDL / VLDL are more easily cleared by the liver. Promoting overall reverse cholesterol transport (i.e., transporting cholesterol from the periphery to the liver), ultimately helps to reduce the burden of cholesterol in the blood and cardiovascular risk.

[0054] Among them, the SCARB1 gene (Gene ID: 949) encodes a scavenger receptor class BI (SR-BI), which is mainly responsible for the internalization and transport of high-density lipoprotein (HDL) cholesterol. This protein helps cholesterol to be transferred from the blood to the liver, and is a receptor for high-density lipoprotein, which regulates the excretion of high-density lipoprotein. Enhanced SR-BI function means that reverse cholesterol transport can be more efficient, helping to remove excess cholesterol in the periphery, reducing the risk of atherosclerosis.

[0055] Among them, the LDLR gene (Gene ID: 3949) encodes a low-density lipoprotein receptor. Its main role is to recognize and remove low-density lipoprotein (LDL) cholesterol in the blood by internalizing LDL into the liver, helping to regulate blood lipid levels. When the expression of LDLR increases, the uptake capacity of hepatocytes to LDL is enhanced, thereby reducing the concentration of LDL in the blood. Removing excess LDL cholesterol in the blood helps to reduce the accumulation of cholesterol in the arterial wall, reducing the risk of cardiovascular disease. At the same time, when CETP transfers cholesterol from HDL to LDL, up-regulated LDLR can more effectively remove this part of LDL, further improving the overall cholesterol balance.

[0056] In some embodiments, Leuconostoc intestinalis can promote the secretion of incretins. There are two common incretins, including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Among them, GLP-1 can inhibit glucagon secretion, and GIP can increase glucagon secretion, both in a glucose-dependent manner. GIP promotes energy storage by directly acting on adipose tissue, and enhances bone formation by stimulating osteoblast proliferation and inhibiting cell apoptosis. In contrast, GLP-1 plays a role in glucose regulation by slowing gastric emptying and glucose-dependent glucagon secretion inhibition. At the same time, GLP-1 also has the function of promoting fat metabolism, converting white fat into brown fat.

[0057] In some embodiments, Leuconostoc intestinalis can promote the secretion of GLP-1 by intestinal cells.

[0058] In some embodiments, Leuconostoc intestinalis can produce ketone bodies. Ketogenesis is a metabolic reaction triggered when blood glucose concentration is low, or when carbohydrate reserves (such as glycogen) in cells are depleted. This process converts energy from fatty acids into ketone bodies to provide an alternative energy source.

[0059] In some embodiments, Leuconostoc mesenteroides is used to produce beta-hydroxybutyrate. Beta-hydroxybutyrate (BHB) is one of the ketone bodies, which is usually synthesized in the liver. Here, BHB can directly act on L cells in the intestine, prompting L cells to secrete more GLP-1, because BHB can bind to receptors on the surface of L cells, activate intracellular signaling pathways, and thus promote the synthesis and secretion of GLP-1. BHB can also indirectly promote the secretion of GLP-1 by changing the composition of the intestinal microbiota, that is, changes in the intestinal microbiota affect the intestinal environment and the function of L cells, and thus affect the secretion of GLP-1. BHB can activate specific metabolic sensing molecules such as AMPK (AMP-activated protein kinase), which play an important role in energy metabolism and nutrient sensing, ultimately affecting the secretion of GLP-1.

[0060] In some embodiments, Leuconostoc mesenteroides can increase serum ketone body concentration. In some embodiments, Leuconostoc mesenteroides can increase urine ketone body content. Ketone bodies can promote the secretion of GLP-1 in the intestine.

[0061] In some embodiments, Leuconostoc mesenteroides can reduce insulin resistance index.

[0062] In some embodiments, Leuconostoc mesenteroides can reduce glycated albumin concentration. Glycated albumin is important for blood glucose monitoring in diabetic patients. This test can be performed without fasting, and because the half-life of albumin is about two to four weeks, it can reflect short-term blood glucose control.

[0063] In some embodiments, the effective dose of Leuconostoc mesenteroides is 100 mg / day.

[0064] In some embodiments, the aforementioned composition contains a specific amount of Leuconostoc mesenteroides or its metabolites.

[0065] In some embodiments, the aforementioned composition can be a health product, a food product, or a food additive for non-medical purposes. In other words, the health product, food product, or food additive contains a specific amount of Leuconostoc mesenteroides.

[0066] In some embodiments, the aforementioned health product, food product, or food additive can further comprise a food industry acceptable carrier. For example, the food industry acceptable carrier can comprise one or more of the following agents: a solvent, a buffer, an emulsifier, a suspending agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the ordinary skill and routine techniques of one of ordinary skill in the art.

[0067] In some embodiments, the aforementioned food industry acceptable carrier of the health product, food product, or food additive comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), and an aqueous solution containing alcohol.

[0068] In some embodiments, the food product can be, but is not limited to, beverages, fermented foods, bakery products, health foods for non-medical purposes, and dietary supplements.

[0069] In some embodiments, the composition has a specific amount of Leuconostoc mesenteroides. That is, the effective amount of Leuconostoc mesenteroides TCI818 is 100 mg per day. Specifically, assuming that one serving of the composition is administered per day and the Leuconostoc mesenteroides is in a dry powder form, the composition contains at least 100 mg of Leuconostoc mesenteroides.

[0070] Example 1: Strain identification and phylogenetic tree

[0071] First, the isolated strain from leek was subjected to strain identification. The 16S ribosomal gene (16S rDNA) sequence of the isolated strain (i.e., SEQ ID NO: 1) was obtained by polymerase chain reaction (PCR), and the total length of the gene sequence was 1,996,682. In the same manner, the 16S ribosomal gene (16S rDNA) sequence of strain L337 (i.e., SEQ ID NO: 2), the 16S ribosomal gene (16S rDNA) sequence of strain L407 (i.e., SEQ ID NO: 3), the 16S ribosomal gene (16S rDNA) sequence of strain L347 (i.e., SEQ ID NO: 4), the 16S ribosomal gene (16S rDNA) sequence of strain L350 (i.e., SEQ ID NO: 5), the 16S ribosomal gene (16S rDNA) sequence of strain L345 (i.e., SEQ ID NO: 6), the 16S ribosomal gene (16S rDNA) sequence of strain L340 (i.e., SEQ ID NO: 7), and the 16S ribosomal gene (16S rDNA) sequence of strain L330 (i.e., SEQ ID NO: 8) were obtained for comparison. Then, each of the above sequences was analyzed using the analysis platform of the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https: / / www.bv-brc.org / ), which compared the 16S ribosomal gene (16S rDNA) sequences of other Leuconostoc strains (as shown in Table 1) and determined the correlation of the target strain with a specific taxon based on the similarity score in the comparison results. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method (as shown in FIG. 1), and based on the phylogenetic tree, it was determined that the 16S rDNA sequence of the isolated strain had the highest similarity to Leuconostoc mesenteroides subsp. mesenteroides (ATCC 8293). Therefore, the isolated strain was named Leuconostoc mesenteroides subsp. mesenteroides strain TCI818.

[0072] Table 1

[0073] Here, some of the strains in Table 1 above were purchased as comparison strains and were named strain L407, strain L377, strain L345, strain L347, strain L350, strain L340, and strain L330 for subsequent comparison experiments.

[0074] Example 2: Preservation and cultivation of Leuconostoc mesenteroides

[0075] The isolated Leuconostoc mesenteroides was inoculated into MRS medium (BD Difco 4 CFU / mL) at a 1% inoculation amount (about 1 x 10 TM Lactobacilli MRS Broth, 1% (v / v)) and cultured at 37°C in an anaerobic environment for 24 hours to form TCI818 bacterial liquid.

[0076] The TCI818 bacterial liquid was centrifuged at 5000 rpm for 20 minutes to separate the supernatant and Leuconostoc mesenteroides cells. The supernatant was filtered through a 0.2 μm filter membrane, and the obtained filtrate was the TCI818 sample (i.e., the TCI818 sample contains the metabolic products of Leuconostoc mesenteroides).

[0077] Example 3: Simulated gastrointestinal digestion experiment

[0078] Leuconostoc mesenteroides was tested in simulated gastric juice (pH 3) and simulated intestinal juice (pH 7) to confirm the acid-base tolerance of Leuconostoc mesenteroides TCI818 in the digestive tract of organisms.

[0079] 3-1. Preparation of reagents:

[0080] 0.5% w / v saline (PBS) was prepared and autoclaved at high temperature and high pressure.

[0081] Simulated gastric juice was prepared: an appropriate amount of pepsin was dissolved in 0.5% w / v sterile saline to give a final concentration of 3 g / L, and the pH was adjusted to 3.0 ± 0.2. Then, the solution was filtered through a 0.22 μm filter membrane in a sterile operation table to obtain sterile artificial simulated gastric juice.

[0082] Simulated intestinal juice was prepared: an appropriate amount of pancreatin was weighed and dissolved in 0.5% w / v sterile saline to give a final concentration of 1 g / L, and 0.45% choline salt was added (not added as a control group), and the pH was adjusted to 8.0 ± 0.2. Then, the solution was filtered through a 0.22 μm filter membrane in a sterile operation table to obtain sterile artificial simulated intestinal juice.

[0083] 3-2. Operation steps:

[0084] Step one: the TCI818 bacterial liquid prepared in Example 2 was taken as the first sample for bacterial count testing.

[0085] Step two: 200 ul of the TCI818 bacterial liquid prepared in Example 2 was added to a 1.5 ml microcentrifuge tube containing 300 ul of 0.5% w / v sterile saline and 1 ml of simulated gastric juice. After mixing well, it was placed in an anaerobic incubator for incubation at 37°C for 2 hours to form the second sample for the simulated gastric acid test, and a sample was taken for bacterial count testing.

[0086] Step three: After repeating step two, centrifuge the tube, remove the supernatant, add 1 ml of artificial intestinal fluid, and place it in an anaerobic incubator at 37°C for 2 hours to form the third sample of the intestinal fluid simulation test, and take a sample for bacterial count test.

[0087] Step four: Dilute the above-mentioned first, second and third samples by 10 times, take 100 ul of the diluent and spread it on MRS agar plates, and incubate in an anaerobic incubator (37°C) for 2 days. After 2 days, calculate the bacterial count in each group by plate count method, and back-calculate the viable bacteria content per gram.

[0088] 3-3. Experimental results:

[0089] The viable bacteria count of the first sample was 11.34 log CFU / mL, the viable bacteria count of the second sample was 10.88 log CFU / mL, and the viable bacteria count of the third sample was 10.23 log CFU / mL. Among them, log CFU / mL represents the colony-forming unit (CFU, colony-forming unit) contained in each milliliter of bacterial solution and is expressed in logarithm (log).

[0090] Therefore, Leuconostoc mesenteroides TCI818 has the function of resisting gastric acid and bile salts.

[0091] Example 4: Lipid oil droplet accumulation test

[0092] Fatty acids are stored in the form of oil droplets (Lipid droplet) in adipocytes. Therefore, this test analyzes the oil droplets after staining to observe the number of oil droplets in the cells, thereby confirming the state of fat accumulation. Subsequently, the dye is eluted and analyzed as a quantitative numerical index.

[0093] 4-1. Test materials and equipment description:

[0094] Cell strain: Mouse bone marrow stromal cells (hereinafter referred to as OP9 cells) were used. OP9 cells were purchased from OP9 cell strain (CRL-2749) of American Type Culture Collection. TM ).

[0095] ​Pre-adipocyte expansion medium: 90% Minimum Essential Medium Alpha (MEMa, brand: Gibco), 20% Fetal Bovine Serum (brand: Gibco), and 1% Penicillin-streptomycin (brand: Gibco).

[0096] Differentiation medium: 86% Minimum Essential Medium Alpha (MEMa, brand: Gibco), 10% Fetal Bovine Serum (brand: Gibco), 1% Penicillin-streptomycin (brand: Gibco), 0.1% Dexamethasone (brand: Sigma-Aldrich), 1% Insulin (brand: Sigma-Aldrich), 0.5% Indomethacin (brand: Sigma-Aldrich), and 1.4% 3-Isobutyl-1-methylxanthine (IBMX, brand: Sigma-Aldrich).

[0097] Reagents: Oil-red O staining reagent (brand Sigma), 10% Formaldehyde (brand ECHO), 100% Isopropanol (brand ECHO), Dulbecco’s phosphate buffered saline (brand Gibco, hereafter PBS).

[0098] Equipment: Microscope (brand zeiss), Full spectrum optical analyzer (brand: BioTek Epoch).

[0099] 4-2. Test procedure

[0100] First, 8 x 10 4The OP9 cells were seeded in each well of a 24-well culture plate containing 500 μL of preadipocyte proliferation medium and incubated at 37°C for 7 days. During the 7-day incubation period, the fresh 500 μL of differentiation medium was replaced every 3 days. After 7 days of incubation, the formation of lipid droplets in the cells in each well was observed using a microscope (brand: ZEISS) to confirm that the cells were completely differentiated into adipocytes for use in subsequent experiments.

[0101] Experimental group: 0.5 μL of the TCI818 sample incubated in Example 2 was added to each well of 500 μL of the differentiation medium as a test sample (i.e., a concentration of 0.1%) and added to the medium containing the differentiated adipocytes, which were incubated at 37°C for 7 days. The medium was replaced every 3 days during the 7-day cell treatment period.

[0102] Control group: 0.5 μL of the MRS medium was added to each well of 500 μL of the differentiation medium as a control sample (i.e., a concentration of 0.1%) and added to the medium containing the differentiated adipocytes, which were incubated at 37°C for 7 days. The medium was replaced every 3 days during the 7-day cell treatment period.

[0103] Blank group: No treatment was performed, i.e., no additional compounds were added to the differentiation medium containing the differentiated adipocytes, which were incubated at 37°C for 7 days. The medium was replaced every 3 days during the 7-day cell treatment period.

[0104] Next, the oil red O staining was performed according to the following procedure. After 7 days of cell treatment, the medium was removed, the adipocytes were washed twice with 1 mL of PBS, 1 mL of 10% formaldehyde was added and reacted with the adipocytes at room temperature for 30 minutes to fix the adipocytes. After removing the formaldehyde, the adipocytes were gently washed twice with 1 mL of PBS, 1 mL of 60% isopropanol was then added to each well of the cells, reacted for 1 minute, and then the isopropanol was removed and 1 mL of an oil red O working solution was added to react with the adipocytes, which were reacted at room temperature for 1 hour. Then, the oil red O working solution was removed and 1 mL of 60% isopropanol was immediately added to the adipocytes to decolorize for 5 seconds. The cells were observed and photographed using a microscope (magnification: 400X), and the results are shown in FIG. 2.

[0105] Subsequently, the stained groups were subjected to oil red O quantification according to the following procedure. 100% isopropanol was added to each well, and the wells were placed on a shaker to react for 10 minutes to dissolve the lipid droplets. Then, 100 μL of the solution was taken from each well to a 96-well culture plate, and the absorbance value (OD510nm) of each well was read using a full-spectrum optical analyzer at a wavelength of 510 nm.

[0106] After measurement, the lipid oil droplet accumulation (%) was calculated by substituting the measured absorbance value into the following equation I. In other words, the lipid oil droplet accumulation (%) of each group was calculated by taking the lipid oil droplet accumulation of the blank group as 100%, and the conversion results are shown in Figure 2.

[0107] Lipid oil droplet accumulation (%) = (OD 510 sample / OD 510 control) x 100% (1) Equation I

[0108] wherein OD 510 sample represents the absorbance value of the group to be converted, and OD 510 control represents the absorbance value of the blank group.

[0109] 4-3. Experimental results:

[0110] Referring to Figure 2, it can be observed that the number of red (dark) oil droplets in the cells of the blank group or control group is significantly more than that of the experimental group, and the cell size is also larger. It can be seen that Leuconostoc mesenteroides can effectively inhibit the size of fat cells, thereby achieving the function of weight loss.

[0111] Referring to Figure 3, under the condition that the lipid oil droplet accumulation of the blank group is 100%, the lipid oil droplet accumulation of the control group is only 94.8%, while the lipid oil droplet accumulation of the experimental group is significantly reduced to only 82.3%. It can be seen that Leuconostoc mesenteroides can effectively inhibit fat accumulation, has the function of reducing fat formation in the recipient, and thereby achieves the function of reducing body fat.

[0112] Example 5: Cholesterol metabolism test of liver

[0113] The liver plays a key role in cholesterol metabolism, as it is responsible for the synthesis and breakdown of cholesterol and its conversion into bile acids for excretion. The liver also regulates the storage and release of cholesterol, affecting the level of cholesterol in the blood. This test uses NBD cholesterol (NBD-Cholesterol), a fluorescently labeled cholesterol derivative, to observe the effect of Leuconostoc mesenteroides on the liver's cholesterol metabolism.

[0114] In this test, U18666A is used as a cholesterol-binding agent, which inhibits the transport of cholesterol within cells. This inhibition leads to the accumulation of intracellular cholesterol, thereby affecting the metabolic pathways of cholesterol. Therefore, in this test, the cholesterol-binding agent serves as a positive control to verify the sensitivity and responsiveness of the test system to changes in cholesterol metabolism.

[0115] 5-1. Test materials and equipment description:

[0116] Cells: Human hepatoma cell line Hep G2 (hereinafter referred to as hepatocytes) were purchased from the American Type Culture Collection. ) Hep G2 cell line (ATCC HB-8065 TM ), hereinafter referred to as liver cells.

[0117] Culture medium: 90% DMEM (Dulbecco's Modified Eagle Medium, Gibco; Cat. 11965-092), 10% fetal bovine serum (Fetal Bovine Serum) and 1% penicillin-streptomycin (Gibco; Cat. 15140122).

[0118] Reagents: DPBS Dulbecco's Phosphate Buffer (Gibco; Cat. 14200-075), cell lysis buffer (Lysis Buffer), and cholesterol binder U18666A (Sigma-Aldrich (MilliporeSigma)).

[0119] Equipment: Cholesterol Uptake Cell-based Assay Kit (Cayman brand) includes NBD cholesterol and flow cytometer (brand: BD).

[0120] 5-2. Testing Process

[0121] First, 4 × 10 5 The liver cells were seeded into each well of a 6-well culture plate and cultured at 37°C overnight for subsequent experiments.

[0122] Experimental group: 500 μL of culture medium per well contained 0.5 μL of the TCI818 sample cultured in Example 2 as a test sample (i.e., a concentration of 0.1%), and the test sample was added to a serum-free culture medium containing 20 ug / ml NBD cholesterol and cultured at 37°C for 24 hours.

[0123] Positive control group: 500 μL of culture medium per well contained 0.5 μL of cholesterol binder control sample (i.e., concentration was 0.1%), and the control sample was added to serum-free culture medium containing 20 ug / ml NBD cholesterol and cultured at 37°C for 24 hours.

[0124] Blank group: without any treatment, i.e. without additional compounds added to the serum-free medium containing 20 ug / ml of NBD cholesterol, incubated at 37°C for 24 hours. To simulate the state of cholesterol absorption by liver cells under normal metabolism.

[0125] After the supernatant of each group of liver cells after incubation was removed, it was washed once with DPBS buffer, and then cell lysis buffer was added for 3 to 5 minutes to lyse the cell membrane to form a liver cell lysate. The liver cell lysate was then suspended by tapping, and after adding the medium to neutralize it, centrifugation was performed, and the supernatant was removed after centrifugation.

[0126] Next, the cholesterol detection kit was used to process the fluorescence signal read by the flow cytometer to analyze and convert the cholesterol content in the liver cells.

[0127] 5-3. Experimental results:

[0128] It should be particularly noted that the results shown in Figure 4 are presented in relative multiples, i.e. the quantitative results of the experimental groups are converted into performance quantities relative to the blank group by taking the quantitative results of the blank group as 100. Among them, the GraphPad Prism One-way ANOVA formula is used for calculation and analysis of whether there is a statistically significant difference. In the figure, "*" represents p value less than 0.05 relative to the blank group, "**" represents p value less than 0.01 relative to the blank group, and "***" represents p value less than 0.001 relative to the blank group, "#" represents p value less than 0.05 relative to the control group, "##" represents p value less than 0.01 relative to the control group, and "###" represents p value less than 0.001 relative to the control group.

[0129] Referring to Figure 4, the relative cholesterol content of the positive control is 10024% when the cholesterol content of the blank group is taken as 100%.

[0130] The relative cholesterol content of the experimental group is as high as 12488% when the cholesterol content of the blank group is taken as 100%, which is also higher than that of the positive control group. That is, Leuconostoc mesenteroides can effectively improve the metabolic rate of cholesterol, has better efficiency than known cholesterol binders, promotes the absorption of cholesterol by the liver, and thus reduces the content of blood lipids.

[0131] Example 6: Cholesterol-related gene expression

[0132] 6-1. Detection target and solution configuration

[0133] Cells: human liver cancer cells Hep G2, purchased from American Type Culture Collection, ) Hep G2 cell line (ATCC HB-8065 TM ), hereinafter referred to as liver cells.

[0134] Culture medium: 90% DMEM (Dulbecco's Modified Eagle Medium, Gibco; Cat. 11965-092), 10% fetal bovine serum (Fetal Bovine Serum, Gibco), and 1% penicillin-streptomycin (Gibco; Cat. 15140122).

[0135] Reagents: DPBS Dulbecco's Phosphate Buffered Saline (purchased from Gibco; Cat. 14200-075), cell lysis buffer.

[0136] Equipment: RNA extraction kit (purchased from Geneaid, Taiwan, China, Lot No. FC24015-G), reverse transcriptase ( III Reverse Transcriptase purchased from Invitrogene, USA, No. 18080-051), Primer set, qPCR kit (KAPA CYBR FAST qPCR Kits (2x) (KAPA Biosystems)), real-time polymerase chain reaction system ABI StepOnePlus TM Real-Time PCR system (Thermo Fisher Scientific, USA).

[0137] 6-2. Operation process

[0138] First, take a 6-well culture plate and inoculate 1×10 6 The liver cells were cultured overnight in 2 mL of culture medium. The cultured liver cells were divided into three groups: blank group, control group and experimental group.

[0139] Blank group: No treatment was performed, i.e., no additional compounds were added to the culture medium to simulate the normal metabolic state of the cells, and then cultured at 37°C for 24 hours.

[0140] Control group: 500 μL of culture medium per well contained 0.5 μL of the MRS culture medium in Example 2 as the test sample (i.e., concentration was 0.1%), and then cultured at 37° C. for 24 hours to further observe whether the MRS culture medium had any effect on the test.

[0141] Experimental group: 0.5 μL of the TCI818 sample incubated in Example 2 was added to each well of 500 μL of medium as a test sample (i.e., a concentration of 0.1%), and then incubated at 37°C for 24 hours.

[0142] After removing the supernatant from the incubated liver cells of each group, the cells were washed once with DPBS buffer, and then 600 μL of cell lysis buffer was added to lyse the cell membranes to form liver cell lysates.

[0143] Next, RNA extraction kits were used to collect the RNA in the liver cell lysates of each group. Next, the extracted RNA of each group was taken as a template, and reverse transcription was performed using a reverse transcriptase with a primer to produce corresponding cDNA. Subsequently, a real-time polymerase chain reaction system and a qPCR kit were used to perform quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) on the reverse transcription products of each group using the primers in Table 2 to observe the expression levels of the genes of the liver cells of each group.

[0144] Table 2

[0145] In Table 2, F is a forward primer, and R is a reverse primer.

[0146] In this case, the instrument settings for the qRT-PCR were 95°C for 1 second, 60°C for 20 seconds, for a total of 40 cycles, and the relative gene quantification was performed using the 2-ΔΔCt method. In this case, the qRT-PCR performed using the cDNA can indirectly quantify the mRNA expression levels of each gene, and thus infer the expression levels of the proteins encoded by each gene, as shown in FIG. 5.

[0147] 6-3. Results and Discussion

[0148] It should be particularly noted that the results shown in FIG. 5 are presented in relative fold, i.e., the quantification results of the experimental group are converted into expression levels relative to the blank group by taking the quantification results of the blank group as 1. In this case, the standard deviation was calculated using the STDEV formula of the Excel software, and a one-tailed Student t-test was used in the Excel software to analyze whether there was a statistically significant difference. In the figure, “*” represents a p-value less than 0.05 relative to the blank group, “**” represents a p-value less than 0.01 relative to the blank group, and “***” represents a p-value less than 0.001 relative to the blank group.

[0149] Referring to FIG. 5. When the expression amount of the CETP gene of the blank group is regarded as 1, the expression amount of the CETP gene of the control group relative to the blank group is 1.06, and the relative expression amount of the CETP gene of the experimental group is 1.45. That is, the expression amount of the CETP gene of the experimental group is significantly improved compared to the blank group.

[0150] When the expression amount of the SCARB1 gene of the blank group is regarded as 1, the expression amount of the SCARB1 gene of the control group relative to the blank group is 1.06, and the relative expression amount of the SCARB1 gene of the experimental group is 1.67. That is, the expression amount of the SCARB1 gene of the experimental group is significantly improved compared to the blank group.

[0151] When the expression amount of the LDLR gene of the blank group is regarded as 1, the expression amount of the LDLR gene of the control group relative to the blank group is 1.39, and the relative expression amount of the LDLR gene of the experimental group is 1.93. That is, the expression amount of the LDLR gene of the experimental group is significantly improved compared to the blank group.

[0152] Here, the expression amount of the three genes of the experimental group is significantly improved compared to the blank group, and only one gene of the control group is improved, and the other two genes are not improved. It can be known that Leuconostoc citreum TCI818 can significantly improve the expression amount of the cholesterol-related genes, and the control group does not have such an effect.

[0153] Therefore, it can be known that the liver cells can promote the generation of high-density cholesterol with the assistance of Leuconostoc citreum, and improve the metabolic efficiency of cholesterol, thereby reducing the content of blood lipids.

[0154] Example 7: Test for promoting secretion of enterocrines

[0155] Glucagon-like peptide-1 (hereinafter referred to as GLP-1) is a glucagon-like peptide secreted by intestinal endocrine cells (L cells) on the wall of the small intestine, which has the effects of promoting insulin secretion, inhibiting glucagon secretion, thereby regulating blood glucose and inhibiting appetite, to achieve the effect of controlling weight.

[0156] This test observes the secretion of GLP-1 by intestinal endocrine cells in the experimental group (adding sample), the positive control group (adding glucose), and the blank group (no addition).

[0157] 7-1. Test materials and equipment description:

[0158] Cell strain: human colorectal adenocarcinoma cells were purchased from American Type Culture Collection (ATCC), USA, NCL-H716 cell line (CCL-251) of the colon, hereinafter referred to as colon cells.

[0159] Culture medium:

[0160] 1. RPMI-1640 medium (purchased from Gibco) added with 2 mM L-glutamine (Gibco), 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco; Cat. 15140122). 2. High glucose medium (DMEM high glucose, Gibco). 3. Glucose-free medium (DMEM without glucose, Gibco).

[0161] Reagents: phosphodiesterase inhibitor (IBMX, purchased from Sigma), forskolin (brand Sigma), phosphate buffer (Dulbecco's phosphate buffered saline brand Gibco, hereinafter referred to as PBS).

[0162] Equipment: full-spectrum optical analyzer (brand: BioTek Epoch), enterostatin detection kit (GLP-1 ELISA KIT, model CEA804Mi) for detection.

[0163] 7-2. Test procedure:

[0164] The colon cells were cultured using RPMI-1640 medium, and the cultured colon cells were subcultured using Matrix gel substrate glue into 24-well plates (3x10 5 / well). After the substrate glue solidified, the cells were cultured with high glucose medium overnight.

[0165] After the medium was replaced with glucose-free medium containing 10 mM forskolin and 10 mM IBMX, the colon cells were treated for 4 hours, and then the cells were divided into three groups: blank group, control group and experimental group. Here, forskolin is an activator of adenylyl cyclase that can increase the intracellular cAMP level; IBMX is a phosphodiesterase inhibitor that can prevent the degradation of cAMP. The combination of the two can significantly increase the intracellular cAMP concentration, thereby affecting the signal transduction and function of the cells. The use of glucose-free medium can avoid the interference of glucose on the experimental results, ensuring that the observed effects are mainly from the effects of forskolin and IBMX.

[0166] Blank group: After removing the glucose-free culture solution, 500 μL / well of PBS containing 1 mM CaCl2was added, and 10 μM of Forskolin and 10 μM of IBMX were added, and left overnight.

[0167] Positive control group: After removing the glucose-free culture solution, 500 μL / well of PBS containing 1 mM CaCl2was added, and 30 mM glucose, 10 μM of Forskolin and 10 μM of IBMX were added, and left overnight.

[0168] Experimental group: After removing the glucose-free culture solution, 500 μL / well of PBS containing 1 mM CaCl2was added, and 10 μM of Forskolin and 10 μM of IBMX were added, and left overnight.

[0169] The PBS of each group was aspirated, and the GLP-1 content secreted by the rectal cells into the culture solution was detected using the enterokinin detection kit.

[0170] Finally, the absorbance value of each group at OD450nm was read by the ELISA reader. By comparing the absorbance values, the relative GLP-1 content of each group was calculated relative to the blank group (i.e., its relative GLP-1 content was considered to be 100%), and the results are shown in Figure 6.

[0171] 7-3. Experimental results:

[0172] The results were analyzed using student t-test with Excel software to determine whether there was a statistically significant difference between the two sample populations, as shown in Figure 6 (in the figure, "*" represents a p value less than 0.05, "**" represents a p value less than 0.01, and "***" represents a p value less than 0.001, "#" represents a p value less than 0.05 relative to the control group, "##" represents a p value less than 0.01 relative to the control group, and "###" represents a p value less than 0.001 relative to the control group. The more "*" represents, the more statistically significant the difference is.

[0173] Referring to Figure 6, the relative GLP-1 content of the control group was 118.2% compared to the blank group (normal cell metabolism). In other words, in a high-sugar environment, rectal cells secrete more GLP-1, which is significantly increased by 18.2% relative to the blank group.

[0174] Compared to the blank group, the relative GLP-1 content in the experimental group was 125.9%. In other words, the relative GLP-1 content in the experimental group was higher than in both the blank and control groups. This suggests that Leuconostoc mesenteroides TCI818 effectively promotes GLP-1 secretion in rectal cells. This, in turn, promotes insulin secretion and inhibits glucagon secretion, thereby regulating blood sugar and suppressing appetite, achieving weight control.

[0175] Example 8: BHB secretion promotion test

[0176] β-hydroxybutyrate (hereinafter referred to as BHB) is a type of ketone body, usually synthesized in the liver. As we age, our metabolic reactions deteriorate, the liver's synthesis efficiency gradually deteriorates, and the amount of BHB synthesized gradually becomes insufficient.

[0177] 8-1. Test materials and equipment description:

[0178] Cells: Human hepatocellular carcinoma cell line Hep G2, purchased from the American Type Culture Collection. ) Hep G2 cell line (ATCC HB-8065 TM ), hereinafter referred to as liver cells.

[0179] Culture medium: 90% DMEM (Dulbecco's Modified Eagle Medium, Gibco; Cat. 11965-092), 10% fetal bovine serum (Fetal Bovine Serum, Gibco), and 1% penicillin-streptomycin (Gibco; Cat. 15140122).

[0180] β-Hydroxybutyrate Assay Kit, beta Hydroxybutyrate Assay Kit (Colorimetric), model ab83390.

[0181] 8-2.Testing process:

[0182] Here, the TCI818 sample cultured in Example 2 was used as a test sample, and the comparison strains L407, L377, L345, L347, L350, L340, and L330 listed in Table 1 were cultured in the same manner as in Example 2 as control samples to test whether the BHB content of liver cells could be increased after treatment with the test sample or the control sample.

[0183] First, a standard curve was prepared using BHB standard samples.

[0184] Cells were plated at 1×10 4 Cells were seeded into 24-well plates with 2 ml of pretreatment medium per well and cultured at 37°C until the cells attached.

[0185] Next, the cells were grouped. The blank group had no test sample added, the experimental group had 0.125% of the TCI818 sample from Example 2 added, the control group L407 had 0.125% of the L407 strain from Example 1 added, the control group L377 had 0.125% of the L377 strain from Example 1 added, the control group L345 had 0.125% of the L345 strain from Example 1 added, the control group L347 had 0.125% of the L347 strain from Example 1 added, the control group L350 had 0.125% of the L350 strain from Example 1 added, the control group L340 had 0.125% of the L340 strain from Example 1 added, and the control group L330 had 0.125% of the L330 strain from Example 1 added. The cells were cultured for 24 hours to obtain culture fluids for each group.

[0186] The culture supernatant of each group was obtained, and the absorbance of the supernatant at 450 nm was measured using a spectrophotometer. The absorbance of the supernatant was converted into BHB content using a standard curve.

[0187] 8-3. Experimental results:

[0188] See Figure 7. The BHB secretion levels of the control strains L407, L377, and L345 were 3.33 mg / L, 3.46 mg / L, 4.60 mg / L, 4.60 mg / L, 6.66 mg / L, 6.72 mg / L, and 7.73 mg / L, respectively. In contrast, the experimental strains secreted 15.88 mg / L, achieving a significant BHB-boosting effect that was two to five times greater than that of the other strains.

[0189] Example 9: Human testing

[0190] 9-1. Sample: Capsules made with the Leuconostoc mesenteroides prepared in Example 2, containing 100 mg of bacterial powder. The bacterial powder was prepared by adding 2.2% (w / w) trehalose, 4.6% (w / w) lactose, and 10% (w / w) skim milk powder to the Leuconostoc mesenteroides liquid prepared in Example 2, mixing thoroughly, freeze-drying, and then grinding.

[0191] 9-2. Subjects: 10 subjects. Each subject was 20 years of age or older and had elevated body fat. Elevated body fat refers to greater than 25% body fat for men and greater than 30% body fat for women.

[0192] 9-3. Test items: serum ketone body concentration, urine ketone body content, blood glycated albumin concentration, insulin resistance index (HOMA-IR), blood high-density lipoprotein cholesterol (HDL-C), whole body fat rate, trunk fat weight, visceral fat area, whole body skeletal muscle weight, and basal metabolic rate.

[0193] Among them, the serum ketone body concentration, blood glycated albumin concentration, insulin resistance index (HOMA-IR), and blood high-density lipoprotein cholesterol (HDL-C) were measured by a commissioned person after blood sampling.

[0194] Among them, the urine ketone body content was measured by colorimetry using ketone urine test paper (Our Health), and the urine ketone body content was determined according to the color of the test paper.

[0195] Among them, the whole body fat rate (%), trunk fat weight (kg), visceral fat area (cm 2 ), whole body skeletal muscle weight (kg), and basal metabolic rate (kcal) were measured using a body composition analyzer (model: InBody770, Biospace Co. Ltd., Seoul, Republic of Korea).

[0196] 9-4. Test procedure:

[0197] Ten subjects were instructed to ingest 100 mg of the capsules prepared from the Leuconostoc mesenteroides cell of Example 2 per day for four weeks. Measurements were taken before the start of ingestion (i.e., week 0, also referred to as the blank group), after two weeks of ingestion (i.e., week 2, also referred to as experimental group A), and after four weeks of ingestion (i.e., week 4, also referred to as experimental group B).

[0198] 9-5. Test results:

[0199] Referring to FIG. 8, after four weeks of daily ingestion of 100 mg of Leuconostoc mesenteroides, the average serum ketone body concentration of the 10 subjects was 0.095 mmol / L (blank group) and increased to 0.135 mmol / L (experimental group B) while maintaining daily diet and routine. Among them, the average serum ketone body concentration of 8 of the 10 subjects increased, which means that the proportion of the number of people improved was 80%, and the difference in the average serum ketone body concentration before and after the use of the Leuconostoc mesenteroides of the present application for only four weeks was 42.1%. That is, daily ingestion of 100 mg of Leuconostoc mesenteroides can effectively increase the concentration of ketone bodies in the blood.

[0200] Referring to FIG. 9, the urine of the 10 subjects before the test was negative for ketone bodies. After the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for four weeks, the urine of 8 of the 10 subjects (week 0) was positive for ketone bodies, and the proportion of subjects who improved was 80% (week 4). Here, the trace amount refers to 0.5 to 1.5 mg / dl. That is, taking 100 mg of Leuconostoc mesenteroides every day can effectively increase the amount of ketone bodies in the urine under normal dietary and exercise conditions.

[0201] Referring to FIG. 10, the average HDL cholesterol level of the 10 subjects before the test was 56.2 mg / dl (blank group). After the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for two weeks under normal dietary and exercise conditions, the average HDL cholesterol level was 56.5 mg / dl (experimental group A), and after the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for four weeks, the average HDL cholesterol level was 59.3 mg / dl (experimental group B), showing a clear increasing trend. Among the 10 subjects, the average HDL cholesterol level of 8 subjects increased, and the proportion of subjects who improved was 80%. In addition, the average HDL cholesterol level increased by 5.5% after four weeks of taking Leuconostoc mesenteroides. That is, taking 100 mg of Leuconostoc mesenteroides every day can effectively increase the HDL cholesterol level in the blood.

[0202] Referring to FIG. 11, the average body fat ratio of the 10 subjects before the test was 32.8% (blank group). After the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for two weeks under normal dietary and exercise conditions, the average body fat ratio was 32.6% (experimental group A), and after the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for four weeks, the average body fat ratio was 32.2% (experimental group B), showing a clear decreasing trend. Among the 10 subjects, the average body fat ratio of 7 subjects decreased, and the proportion of subjects who improved was 70%. In addition, the average body fat ratio decreased by 0.6% after four weeks of taking Leuconostoc mesenteroides. That is, taking 100 mg of Leuconostoc mesenteroides every day can effectively reduce body fat.

[0203] Referring to FIG. 12, the average trunk fat weight of the 10 subjects before the test was 11.73 kg (blank group). When the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for two weeks while maintaining their daily diet and routine, the average trunk fat weight was 11.68 kg (test group A). When the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for four weeks, the average trunk fat weight was 11.57 kg (test group B), showing a gradual decrease. Among the 10 subjects, the average trunk fat weight of 7 subjects decreased, meaning that the improvement rate was 70%. The average trunk fat weight of the subjects decreased by 0.16 kg after taking Leuconostoc mesenteroides for four weeks. This means that taking 100 mg of Leuconostoc mesenteroides every day can effectively reduce the trunk fat weight.

[0204] Referring to FIG. 13, the average visceral fat area of the 10 subjects before the test was 107.7 cm 2 (Blank group). When the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for two weeks while maintaining their daily diet and routine, the average visceral fat area was 106.6 cm 2 (test group A). When the 10 subjects took 100 mg of Leuconostoc mesenteroides every day for four weeks, the average visceral fat area was 106.1 cm 2 (test group B), showing a gradual decrease. Among the 10 subjects, the average visceral fat area of 8 subjects decreased, meaning that the improvement rate was 80%. The average visceral fat area of the subjects decreased by 1.6 cm after taking Leuconostoc mesenteroides for four weeks. This means that taking 100 mg of Leuconostoc mesenteroides every day can effectively reduce the visceral fat area. 2

[0205] The normal range of the visceral fat area of a male according to the current medical standard is 50 cm 2 to 100 cm 2 , and the normal range of the visceral fat area of a female is 40 cm 2 to 80 cm 2 . Among the 10 subjects, 3 subjects had an excessive visceral fat area. The average visceral fat area of the 3 subjects before the test was 146.9 cm 2 . When the 3 subjects took 100 mg of Leuconostoc mesenteroides every day for two weeks while maintaining their daily diet and routine, the average visceral fat area decreased to 142.6 cm 2 , meaning that the improvement rate was 100%. The actual decrease in the visceral fat area was 4.3 cm 2 .

[0206] ​Referring to FIG. 14, the average skeletal muscle weight of the 10 subjects before the test was 25.16 kg (blank group), and after the 10 subjects ingested 100 mg of Leuconostoc mesenteroides per day for two weeks while maintaining their daily diet and routine, the average skeletal muscle weight was 25.24 kg (test group A), and after the 10 subjects ingested 100 mg of Leuconostoc mesenteroides per day for four weeks, the average skeletal muscle weight was 25.35 kg (test group B), showing a gradual increasing tendency. Among the 10 subjects, the average skeletal muscle weight of 7 subjects increased, which means that the improvement ratio was 70%, and the average skeletal muscle weight of the subjects increased by 0.19 kg after the 10 subjects ingested Leuconostoc mesenteroides for four weeks. That is, the ingestion of 100 mg of Leuconostoc mesenteroides per day can effectively increase the muscle mass of the human body.

[0207] Referring to FIG. 15, the average basal metabolic rate of the 10 subjects before the test was 1360.4 kcal (blank group), and after the 10 subjects ingested 100 mg of Leuconostoc mesenteroides per day for two weeks while maintaining their daily diet and routine, the average basal metabolic rate was 1360.3 kcal (test group A), and after the 10 subjects ingested 100 mg of Leuconostoc mesenteroides per day for four weeks, the average basal metabolic rate was 1367.0 kcal (test group B), showing a rapid increasing tendency. Among the 10 subjects, the average basal metabolic rate of 6 subjects increased, which means that the improvement ratio was 60%, and the average basal metabolic rate of the subjects increased by 6.6 kcal after the 10 subjects ingested Leuconostoc mesenteroides for four weeks. That is, the ingestion of 100 mg of Leuconostoc mesenteroides per day can effectively increase the basal metabolic rate of the human body.

[0208] Of course, the present application can have other various embodiments, and those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application, and these corresponding changes and modifications should all belong to the protection scope of the claims of the present application.

[0209] Industrial applicability

[0210] In summary, the Leuconostoc intestini of any embodiment and / or metabolites thereof can promote fat metabolism, reduce fat cell oil droplet accumulation, promote cholesterol metabolism, increase high-density cholesterol content in blood, promote high-density cholesterol production, promote the expression of CETP gene, SCARB1 gene or LDLR gene, thereby achieving the purpose of reducing body fat. The Leuconostoc intestini of any embodiment and / or metabolites thereof can promote the secretion of incretins and / or promote the secretion of GLP-1 by intestinal cells, thereby achieving the purpose of reducing appetite. The Leuconostoc intestini of any embodiment and / or metabolites thereof can produce ketone bodies, produce β-hydroxybutyric acid, increase serum ketone body concentration, and increase urine ketone content, thereby achieving the purpose of promoting ketone body content. The Leuconostoc intestini of any embodiment and / or metabolites thereof can reduce insulin resistance index, promote the secretion of incretins and / or promote the secretion of GLP-1 by intestinal cells, and reduce glycated albumin concentration, thereby achieving the purpose of regulating fasting blood glucose.

Claims

1. Use of Leuconostoc mesenteroides and / or metabolites thereof for the preparation of a body fat reducing composition, wherein the Leuconostoc mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides having the accession number DSM 34443.

2. Use according to claim 1, wherein the Leuconostoc mesenteroides is used to promote fat metabolism.

3. Use according to claim 2, wherein the Leuconostoc mesenteroides is used to reduce fat cell oil droplet accumulation.

4. Use according to claim 1, wherein the Leuconostoc mesenteroides is used to promote cholesterol metabolism.

5. Use according to claim 4, wherein the Leuconostoc mesenteroides is used to increase high density cholesterol levels in blood.

6. Use according to claim 4, wherein the Leuconostoc mesenteroides is used to promote high density cholesterol production.

7. Use according to claim 5, wherein the Leuconostoc mesenteroides is used to promote increased expression of the CETP gene, the SCARB1 gene or the LDLR gene.

8. Use of Leuconostoc mesenteroides and / or metabolites thereof for the preparation of an appetite reducing composition, wherein the Leuconostoc mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides having the accession number DSM 34443.

9. Use according to claim 8, wherein the Leuconostoc mesenteroides is used to promote incretin secretion.

10. Use according to claim 9, wherein the Leuconostoc mesenteroides is used to promote GLP-1 secretion by intestinal cells.

11. Use of Leuconostoc mesenteroides or metabolites thereof for the preparation of a composition for promoting ketone body levels, wherein the Leuconostoc mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides having the accession number DSM 34443.

12. Use according to claim 11, wherein the Leuconostoc mesenteroides is used to produce ketone bodies.

13. Use according to claim 12, wherein the Leuconostoc mesenteroides is used to produce beta-hydroxybutyric acid.

14. Use according to claim 11, wherein the Leuconostoc mesenteroides is used to increase serum ketone body concentration.

15. Use according to claim 11, wherein the Leuconostoc mesenteroides is used to increase urine ketone body content.

16. Use of Leuconostoc mesenteroides or metabolites thereof for the preparation of a composition for modulating fasting blood glucose, wherein the Leuconostoc mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides having the accession number DSM 34443.

17. Use according to claim 16, wherein the Leuconostoc mesenteroides is used to decrease insulin resistance index.

18. Use according to claim 16, wherein the Leuconostoc mesenteroides is used to promote incretin secretion.

19. Use according to claim 18, wherein the Leuconostoc mesenteroides is used to promote GLP-1 secretion by intestinal cells.

20. Use according to claim 16, wherein the Leuconostoc mesenteroides is used to decrease glycated albumin concentration.

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