Bifidobacterium pseudominutus aFMT-201, probiotic composition and application of probiotic composition in metabolic syndrome

Abstract

The invention relates to the technical field of microorganisms, and provides a Bifidobacterium pseudocatenatum aFMT-201, which is classified and named as Bifidobacterium pseudocatenatum, is preserved in Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC No: 65182. The invention also provides a preparation method of the Bifidobacterium pseudocatenatum aFMT-201. The invention also provides a probiotic composition containing the bifidobacterium pseudominutus aFMT-201, and a preparation method of the probiotic composition containing the bifidobacterium pseudominutus aFMT-201. Meanwhile, the invention also provides an application of the bifidobacterium pseudominulatum aFMT-201 or the composition in preparation of a medicine for preventing or treating the metabolic syndrome, and provides a medicine for preventing / treating the metabolic syndrome. The bifidobacterium pseudomicrochain aFMT-201 strain is separated from faeces of healthy adults, and the strain has the effects of remarkably lowering lipid and improving insulin sensitivity and can play an effective role in improving metabolic syndromes.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Bifidobacterium pseudomicroorganismum aFMT-201, a probiotic composition and applications thereof in metabolic syndrome. Background Art

[0002] Metabolic syndrome (MetS) is a metabolic disorder characterized by obesity, hyperglycemia, dyslipidemia, and hypertension. Currently, drugs used to treat MetS have problems such as large side effects and limited efficacy. In this context, intestinal flora regulation has gradually developed into a new direction for MetS treatment. However, the screening of specific probiotic strains and their mechanisms of action still need further exploration.

[0003] At present, the research on Bifidobacterium pseudocatenulatum is mostly focused on intestinal health, but its specific strains and role in metabolic regulation have not yet been clarified. The present invention obtains a new strain of Bifidobacterium pseudocatenulatum with significant lipid-lowering and insulin sensitivity-improving effects through screening, and the emergence of this strain just fills the gap in the field of "intestinal flora regulation to treat metabolic syndrome". Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Bifidobacterium pseudomicrocystis (Bifidobacteriumpseudocatenulatum) aFMT-201, a probiotic composition containing the bacterium and an application in metabolic syndrome. The aFMT-201 strain is isolated from the feces of healthy adults, has significant lipid-lowering and insulin-sensitivity-improving effects, and can play an effective role in improving metabolic syndrome.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] A Bifidobacterium pseudocatenulatum aFMT-201 was isolated from the feces of healthy adults and classified as Bifidobacterium pseudocatenulatum. It was preserved in the Guangdong Provincial Microbiological Culture Collection on September 23, 2024 and showed survival. The preservation number is GDMCC No: 65182. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] As one of the preferred embodiments of the present invention, the full-length sequence of the 16S rRNA gene of Bifidobacterium pseudotruncatum aFMT-201 is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the Bifidobacterium pseudocatenulatum aFMT-201 has the effects of reducing lipid and improving insulin sensitivity.

[0009] A probiotic composition containing the above-mentioned Bifidobacterium pseudocatenulatum aFMT-201.

[0010] As one of the preferred embodiments of the present invention, it further comprises a pharmaceutically acceptable carrier.

[0011] As one of the preferred embodiments of the present invention, the pharmaceutically acceptable carrier is a lyoprotectant, including trehalose and skim milk.

[0012] Use of the above-mentioned Bifidobacterium pseudocatenulatum aFMT-201 or the probiotic composition in the preparation of a drug for preventing or treating metabolic syndrome.

[0013] A drug for preventing / treating metabolic syndrome containing the above-mentioned Bifidobacterium pseudocatenulatum aFMT-201 or the probiotic composition.

[0014] The advantages of the present invention compared with the prior art are as follows:

[0015] In the present invention, a strain of Bifidobacterium pseudocatenulatum aFMT-201 was isolated from the feces of healthy adults. This strain has the significant effects of reducing lipid and improving insulin sensitivity, and mainly inhibits adipocyte differentiation by regulating the lipid synthesis pathway (such as PPARγ signal).

[0016] The present invention fills the blank of "regulating intestinal flora to treat metabolic syndrome" and provides a new way for the preparation of drugs for treating metabolic syndrome. Description of the Drawings

[0017] Figure 1 It is a morphological characteristic diagram of the Bifidobacterium pseudocatenulatum aFMT-201 strain in Example 2 of the present invention. Detailed Embodiments

[0018] The following detailed description of the embodiments of the present invention is carried out on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0019] The culture medium formulations involved in the following examples and experimental examples are as follows:

[0020] MRS Medium: Peptone 10 g / L, Beef Extract 10 g / L, Yeast Extract 5 g / L, Glucose 20 g / L, Sodium Acetate Trihydrate 5 g / L, Ammonium Citrate 2 g / L, Dipotassium Phosphate, K2HPO4 2 g / L, Magnesium Sulfate Heptahydrate, MgSO4·7H2O 0.2 g / L, Manganese Sulfate Monohydrate, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L.

[0021] MRS Agar Medium: Peptone 10 g / L, Beef Extract 10 g / L, Yeast Extract 5 g / L, Glucose 20 g / L, Sodium Acetate Trihydrate 5 g / L, Ammonium Citrate 2 g / L, Dipotassium Phosphate, K2HPO4 2 g / L, Magnesium Sulfate Heptahydrate, MgSO4·7H2O 0.2 g / L, Manganese Sulfate Monohydrate, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L, Agar 15 g / L.

[0022] Modified MRS Agar Medium: MRS Agar Medium + 0.05% L-cysteine.

[0023] High-glucose DMEM Medium Containing 10% Fetal Bovine Serum (FBS): High-glucose DMEM Medium (containing 4.5 g / L glucose) + 10% fetal bovine serum + 1% penicillin-streptomycin (Sangon Biotech, E600053-0500).

[0024] Adipocyte Differentiation Induction Medium (MDI Induction Medium): High-glucose DMEM Medium (containing 4.5 g / L glucose) + 1 μM dexamethasone + 0.5 mM IBMX + 10 μg / mL insulin.

[0025] Maintenance culture medium: high-glucose DMEM medium (containing 4.5 g / L glucose) + 10% fetal bovine serum + 1 μg / mL insulin + 1% penicillin-streptomycin.

[0026] Example 1, screening, isolation and identification of aFMT-201 strain:

[0027] (1) Healthy adult stool samples (fecal suspension) (approved by ethical review) were inoculated into modified MRS agar medium (MRS agar medium + 0.05% L-cysteine) and cultured anaerobically at 37°C for 48 h.

[0028] (2) A single colony was picked and purified, and preliminarily identified as Bifidobacterium pseudocatenulatum by MALDI-TOF.

[0029] (3) Through WGS whole genome sequencing, the full-length sequence of the 16S rRNA gene of the aFMT-201 strain of the present invention is shown in SEQ ID NO.1, and the similarity with the standard strain of B. pseudocatenulatum is 99.8%, confirming that it is Bifidobacterium pseudocatenulatum.

[0030] (4) Based on the identification results, the strain was named Bifidobacteriumpseudocatenulatum aFMT-201 strain, and was preserved in the Guangdong Provincial Microbiological Culture Collection Center on September 23, 2024 and showed survival, with the preservation number GDMCC No: 65182, and the preservation address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0031] Example 2, colony morphology of aFMT-201 strain:

[0032] The morphological characteristics of the aFMT-201 strain of the present invention are: Gram-positive, no spores, milky white colonies, round protrusions (diameter 1-2 mm), such as Figure 1 shown.

[0033] Example 3, probiotic composition:

[0034] The probiotic composition of this embodiment includes a bacterial suspension (1×10^9 CFU / mL) of Bifidobacterium pseudotruncatum aFMT-201 of the present invention and a lyophilization protective agent (trehalose, skim milk).

[0035] Experimental Example 1: Verification of the in vitro lipid-lowering activity of aFMT-201 strain:

[0036] In this experimental example, the inhibitory effect of the lysate of strain aFMT-201 on lipid accumulation was verified by directly quantifying the triglyceride (TG) content during adipocyte differentiation, providing highly sensitive quantitative data.

[0037] I. Experimental methods

[0038] 1. Bacterial cell preparation

[0039] (1) Inoculate strain aFMT-201 into MRS medium and culture anaerobically at 37 °C for 24 h. Centrifuge (6000×g, 10 min) to collect the bacterial cells, wash them 3 times with PBS, disrupt them by ultrasonication (power 300 W, sonication for 3 s, interval 5 s, total duration 10 min), and filter through a 0.22-μm filter membrane to obtain the bacterial cell lysate.

[0040] (2) Determine the protein concentration of the lysate by the BCA method and adjust it to 1.0 mg / mL for standby.

[0041] 2. Cell experiment

[0042] (1) Take 3T3-L1 mouse preadipocytes (ATCC CL-173) and culture them in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. When the cells grow to confluence (confluence reaches 100%), culture them for another 2 days to make them enter the growth arrest phase (contact inhibition), which serves as the starting point for differentiation induction (recorded as day 0 of differentiation).

[0043] (2) Set up an experimental group, a control group, and a positive control group, and perform differentiation induction separately.

[0044] Experimental group: On day 0, replace the medium in the above step with adipocyte differentiation induction medium (MDI induction solution), and add the lysate of aFMT-201 bacteria (final concentration 1.0 mg / mL). Induce and culture for 48 h (days 0 - 2) to promote the initiation of the differentiation program of preadipocytes. On day 2, replace the medium with maintenance medium and continue to culture the cells. Thereafter, change the medium every 2 days until the cells are fully mature around day 8 of differentiation, accumulating a large number of lipid droplets, and endpoint detection can be performed at this time.

[0045] Control group: The difference from the experimental group is that on day 0, replace the medium with adipocyte differentiation induction medium (MDI induction solution), and add PBS (equal volume).

[0046] Positive control group: The difference from the experimental group is that on day 0, replace the medium with adipocyte differentiation induction medium (MDI induction solution), and add 10 μM rosiglitazone (PPARγ agonist).

[0047] 3. Quantitative detection of triglyceride

[0048] Each group of cells was cultured until about the 8th day of differentiation, the treatment was paused, and the intracellular triglyceride (TG) content was quantitatively measured separately:

[0049] ① Cell lysis: Discard the culture supernatant, gently wash the cells twice with PBS to remove residual culture medium and serum components. Add lysis buffer (PBS containing 1% Triton X-100), lyse the cells on ice for 30 min, and centrifuge (12000×g, 5 min) to collect the supernatant.

[0050] ② Detection with TG detection kit: Use the Sigma-Aldrich triglyceride colorimetric detection kit (MAK266) and operate according to the instructions: Take 50 μL of the supernatant and mix it with 150 μL of the working reagent (containing lipase, glycerol kinase, peroxidase), incubate at 37 °C for 15 min; measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader (wavelength 540 nm), and calculate the TG concentration according to the standard curve (unit: μmol / mg protein).

[0051] 4. Data statistics

[0052] Each group was set with 6 replicate wells, and the results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used, and p < 0.05 was considered a significant difference.

[0053] II. Experimental results

[0054] The quantitative analysis results of the triglyceride content in each group are shown in Table 1.

[0055] Table 1 Quantitative analysis results of triglyceride content in each group

[0056] Group TG concentration (μmol / mg protein) Inhibition rate (vs control group) Control group 12.5±1.2 - Experimental group 7.3±0.8* 41.6％ Positive control group 5.1±0.6* 59.2％

[0057] Note: (* indicates p < 0.01; data are mean ± SD, n = 10).

[0058] From the above results, it can be seen that the cell lysate of aFMT-20 of the present invention significantly reduces the TG accumulation during the differentiation of 3T3-L1 cells (inhibition rate 41.6%), approaching the 59.2% effect of the positive drug rosiglitazone; the data confirm that this strain can inhibit adipocyte differentiation by regulating the lipid synthesis pathway (such as PPARγ signal).

[0059] Experimental example 2. Verification with animal model:

[0060] This experimental example was used to verify the intervention effect of aFMT-20 strain on the metabolic syndrome (obesity, insulin resistance, dyslipidemia) induced by high-fat diet (HFD).

[0061] I. Experimental method

[0062] 1. Experimental animals and grouping scheme

[0063] Animal strain: Male C57BL / 6J mice (8 weeks old, body weight 20±2 g), SPF level, randomly divided into three groups: "normal diet group", "high-fat diet group", and "HFD + aFMT-201 intervention group" (n = 10 / group).

[0064] Normal diet group (ND): Regular maintenance feed (10% fat energy, D12450J, Research Diets), intragastrically administered with an equal volume of sterile normal saline daily.

[0065] High-fat diet group (HFD): High-fat feed (60% fat energy, D12492, Research Diets), intragastrically administered with an equal volume of sterile normal saline daily.

[0066] HFD + aFMT-201 intervention group: High-fat feed (60% fat energy, D12492, Research Diets) + intragastrically administered with aFMT-201 bacterial suspension (1×10^9 CFU / mL) daily, 0.2 mL was intragastrically administered to each mouse, equivalent to 2×10^8 CFU / mouse / day.

[0067] Feeding conditions: Temperature 22±2°C, humidity 50±10%, 12 h light / dark cycle, free access to food and water.

[0068] 2. Preparation of bacterial suspension

[0069] The aFMT-201 strain of the present invention was inoculated into MRS medium (containing 0.05% L-cysteine) and anaerobically cultured at 37°C for 24 h. The bacterial cells were collected by centrifugation (6000×g, 10 min), washed 3 times with PBS, and resuspended to a concentration of 5×10^9 CFU / mL to obtain the aFMT-201 bacterial suspension, which was stored at 4°C for later use (verified by viable cell counting before use).

[0070] 3. Intervention period

[0071] Pre-adaptation period: All mice were fed with regular feed for 1 week.

[0072] Formal experiment: The normal diet group (ND) continued with regular feed, and the high-fat diet group (HFD) and the HFD + aFMT-201 intervention group switched to high-fat feed for 12 weeks.

[0073] Gavage protocol: Mice in the intervention group were gavaged with aFMT-201 bacterial suspension at a fixed time every day, while mice in the ND and HFD groups were gavaged with an equal volume of sterile normal saline. The initial body weights of mice in each group were approximately 20 g before the intervention, ensuring consistent starting conditions. Body weight changes were monitored during the intervention. At the end of the 12th week, after fasting for 12 h, fasting blood samples were collected from the mice to measure fasting blood glucose and serum insulin levels, and the insulin resistance index (HOMA-IR) was calculated. At the same time, the levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured to evaluate the blood lipid status.

[0074] Specific detection indexes and methods:

[0075] ① Physiological index monitoring (at the end of the 12th week)

[0076] Body weight and food intake: Record the body weight and feed consumption, and calculate the energy intake.

[0077] Fasting blood glucose: Blood was taken from the tail vein and measured with a blood glucose meter (OneTouch Ultra).

[0078] ② Metabolic parameter detection (at the end of the 12th week)

[0079] Serum collection: After fasting for 12 h, the mice were anesthetized and sacrificed, blood was taken from the heart, and the serum was separated by centrifugation (3000×g, 15 min).

[0080] Blood lipid analysis:

[0081] Total cholesterol (TC), triglyceride (TG): Enzymatic colorimetric method (kits: Abcam ab65390, ab65336).

[0082] Low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C): Precipitation method (kit: Sigma MAK045).

[0083] Insulin sensitivity:

[0084] Fasting insulin (FINS): ELISA method (kit: Mercodia Mouse Insulin ELISA).

[0085] Insulin resistance index (HOMA-IR): Calculation formula: HOMA-IR = [fasting blood glucose (mmol / L) × fasting insulin (mIU / L)] / 22.5.

[0086] II. Experimental results

[0087] 1. Body weight and metabolic phenotypes

[0088] The weight and metabolic phenotype results at the 12th week are shown in Table 2.

[0089] Table 2 Weight and Metabolic Phenotype Results

[0090]

[0091] Note: (* indicates p < 0.01; data are mean ± SD, n = 10).

[0092] 2. Lipid Improvement Effect

[0093] The lipid results at the 12th week are shown in Table 3.

[0094] Table 3 Lipid Results

[0095]

[0096] Note: (* indicates p < 0.01; data are mean ± SD, n = 10).

[0097] From the above results, it can be seen that: (1) The weight of mice in the normal diet group (ND) increased slightly with age, but the amplitude was small; the weight of mice in the high-fat diet group (HFD) increased rapidly and was significantly higher than that in the normal diet group (ND); in the HFD + aFMT-201 intervention group, the weight gain of mice was significantly inhibited; indicating that aFMT-201 intervention effectively prevented excessive weight gain caused by high-fat diet. (2) The high-fat diet group (HFD) showed significant metabolic abnormalities in all indicators (significantly increased body weight, blood glucose and blood lipids, aggravated insulin resistance, and increased levels of inflammatory factors), while the indicators of mice in the HFD + aFMT-201 intervention group were improved to varying degrees and were close to the normal control group level. The above proves that the strain aFMT-201 of the present invention can effectively relieve obesity and related metabolic disorders induced by high-fat diet.

[0098] In summary, the present invention isolated a strain of Bifidobacterium pseudocatenulatum aFMT-201 from the feces of healthy adults. This strain has a significant effect on reducing lipids and improving insulin sensitivity, and mainly inhibits adipocyte differentiation by regulating the lipid synthesis pathway (such as PPARγ signal), and can be used to prepare drugs for preventing or treating metabolic syndrome.

[0099] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Bifidobacterium pseudocatenulatum aFMT-201, characterized in that, It is classified as Bifidobacterium pseudocatenulatum and is deposited in the Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 65182.

2. The Bifidobacterium pseudocatenulatum aFMT-201 according to claim 1, wherein The full-length sequence of the 16S rRNA gene of the Bifidobacterium pseudocatenulatum aFMT-201 is shown in SEQ ID NO.

1.

3. The Bifidobacterium pseudocatenulatum aFMT-201 according to claim 1, wherein The Bifidobacterium pseudocatenulatum aFMT-201 has the effects of reducing lipid and improving insulin sensitivity.

4. A probiotic composition containing the Bifidobacterium pseudocatenulatum aFMT-201 according to any one of claims 1 to 3.

5. The probiotic composition according to claim 4, wherein It also contains a pharmaceutically acceptable carrier.

6. The probiotic composition according to claim 5, characterized in that, The pharmaceutically acceptable carrier is a lyoprotectant, including trehalose and skim milk.

7. Use of the Bifidobacterium pseudocatenulatum aFMT-201 according to any one of claims 1 to 3, or the probiotic composition according to any one of claims 4 to 6, in the preparation of a drug for preventing or treating metabolic syndrome.

8. A drug for preventing / treating metabolic syndrome containing the Bifidobacterium pseudocatenulatum aFMT-201 according to any one of claims 1 to 3, or the probiotic composition according to any one of claims 4 to 6.

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