Application of bifidobacterium breve in reducing weight of obese people and weight of white fat and beige fat in body

Abstract

The invention discloses application of bifidobacterium breve in reducing the weight of obese people and the weight of white fat and beige fat in bodies, and relates to the technical field of probiotics. The invention provides application of bifidobacterium breve in preparation of a product for relieving or improving obesity. The preservation number of the bifidobacterium breve is CGMCC No. 22242. The bifidobacterium breve B2798 can reduce the weight of white fat and liver tissues, reduce the area of each fat cell and liver fat vacuole and lipid droplet leaching caused by high fat diet, change the diversity of intestinal flora, regulate the proportion of the intestinal flora, increase the abundance of beneficial bacteria, and recover a plurality of key metabolites to a normal level; the expression quantities of mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and mRNA regulated and controlled by the mmu-miR-203-3p, the mmu-miR-193a-3p and the mmu-miR-378b are regulated and controlled, so that white fat, beige fat and brown fat are regulated and controlled, and the obesity

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and specifically relates to the application of Bifidobacterium breve in reducing the body weight, visceral white fat and subcutaneous white fat weight of obese people. Background Art

[0002] Obesity is a metabolic disease caused by multiple factors, manifested as abnormal accumulation of fat in the body and an increase in body weight. Obesity is caused by a long-term imbalance between energy intake and energy consumption, which is the result of the interaction between genetic factors and environmental factors. Obesity is harmful to physical health and is closely related to various chronic diseases such as cardiovascular diseases and diabetes. In lipid metabolism, adipose tissue plays a very important role, and its main function is to store triglycerides and secrete fatty acids. As an important place for the body to store and utilize energy, adipose tissue is traditionally classified into white adipose tissue (WAT) and brown adipose tissue (BAT), and the two play opposite roles in regulating energy balance. White adipose tissue mainly stores energy in the form of triglycerides, while brown adipose tissue specifically dissipates energy as heat through the uncoupling of oxidative phosphorylation mediated by uncoupling protein 1 (UCP1) and adenosine triphosphate (ATP) synthesis. Beige adipocytes have the characteristics of both white and brown adipocytes. Moreover, after beige adipocytes are activated, a large amount of uncoupling protein 1 will be expressed in the body, which helps to promote its heat production and energy consumption.

[0003] Restricting energy intake and promoting energy consumption are the main goals for alleviating or improving obesity. Currently, the intervention strategies for obesity mainly include bariatric surgery, lifestyle intervention, drug intervention, and probiotic intervention, etc. Weight loss is one of the more effective methods, but patients may experience mental health problems after surgery. A healthy diet and regular physical exercise are the main lifestyle interventions for alleviating or improving obesity and related diseases, but their compliance is poor and it is difficult to adhere to for a long time. Existing weight loss drugs include phentermine, semaglutide, and orlistat, etc., but weight loss drugs have certain side effects.

[0004] Studies have shown that supplementing probiotics can alleviate obesity and its related diseases. Probiotics are active microorganisms that colonize the human body and change the composition of the flora in a certain part of the host, and can improve the microecological balance of the host and play a beneficial role. Some studies have found that probiotics have a positive effect on fat metabolism and can promote the browning process of white fat. Probiotics can promote the browning of white fat and increase energy consumption, which is expected to become a new way to improve obesity diseases.

[0005] Chinese Patent CN116396905A discloses a Bifidobacterium breve HC2953, a bacterial agent and its application. The Bifidobacterium breve described in the invention ( Bifidobacterium breveHC2953 can reduce the concentrations of total cholesterol, triglyceride, low-density lipoprotein cholesterol and alanine aminotransferase in the body, increase the concentration of high-density lipoprotein cholesterol, increase the excretion of bile acids in the obese body, improve the state of lipid metabolism disorder in the body, and at the same time has extremely strong colonization and adhesion abilities, and thus can achieve the technical effect of lipid-lowering.

[0006] At present, developing functional probiotics that can effectively reduce the weight of obese people is still an objective that requires efforts. Summary of the Invention

[0007] The object of the present invention is to provide the application of Bifidobacterium breve in reducing the weight of obese people, the weight of white fat and beige fat in the body.

[0008] To achieve the above-mentioned invention object, the technical solution of the present invention is as follows: On the one hand, the present invention provides the application of Bifidobacterium breve in preparing a product for relieving or improving obesity, and the Bifidobacterium breve is Bifidobacterium breve B2798, and the preservation number is CGMCC No. 22242.

[0009] Specifically, the Bifidobacterium breve B2798 ( Bifidobacterium breve B2798) is isolated from the intestine of a healthy infant, and has been preserved in the China General Microbiological Culture Collection Center on April 27, 2021. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its microorganism preservation number is CGMCC No. 22242. The detection result of this strain is viable.

[0010] Specifically, the obesity is obesity caused by a high-fat diet.

[0011] Specifically, the product helps to control the fat in the body.

[0012] Specifically, the viable count of Bifidobacterium breve in the product is 1×10 6 CFU / g - 1×10 11 CFU / g or 1×10 6 CFU / ml - 1×10 11 CFU / ml.

[0013] Specifically, such as 1×10 6 CFU / ml (CFU / g), 1×10 7 CFU / ml (CFU / g), 1×10 8 CFU / ml (CFU / g), 1×10 9 CFU / ml (CFU / g), 1×10 10 CFU / ml (CFU / g), 1×10 11CFU / ml (CFU / g), etc., and other point values within this numerical range can be selected.

[0014] Further, the viable count of Bifidobacterium breve in the product is 1×10 9 CFU / g or 1×10 9 CFU / ml.

[0015] Specifically, the product includes drugs, foods, and health products.

[0016] Further, the product is a functional probiotic drug, a functional food, or a functional health product.

[0017] Specifically, the Bifidobacterium breve includes, but is not limited to, the fermentation broth of Bifidobacterium breve, the supernatant of the fermentation broth of Bifidobacterium breve, viable Bifidobacterium breve, and the freeze-dried powder of Bifidobacterium breve.

[0018] Further, the supernatant of the fermentation broth refers to the liquid after inoculating the strain into the culture medium and culturing.

[0019] Further, the supernatant of the fermentation broth refers to the clear liquid on the upper layer after centrifuging the fermentation broth; it contains rich metabolites and a part of bacterial cell debris during the process of bacterial growth and reproduction.

[0020] Specifically, the preparation of the freeze-dried powder of Bifidobacterium breve includes the following steps: (1) Activation of the strain; (2) Preparation of the seed liquid; (3) Inoculation and fermentation; (4) Termination of fermentation; (5) Freeze-drying.

[0021] According to some embodiments of the present invention, the activation of the strain in step (1) is to inoculate Bifidobacterium breve into RCM liquid medium and anaerobically culture at 37°C for 18 - 24 h, and subculture 1 - 2 times to obtain the activated strain.

[0022] According to some embodiments of the present invention, the preparation of the seed liquid in step (2) is to inoculate the activated strain into RCM liquid medium and anaerobically culture at 37°C until the pH value reaches 4.5 - 4.8.

[0023] According to some embodiments of the present invention, the inoculation and fermentation in step (3) is to inoculate the seed liquid into RCM liquid medium at an inoculation amount of 1‰ and ferment for 18 h under controlled fermentation conditions.

[0024] Specifically, the fermentation conditions are: incubate at a constant temperature of 30°C in the early stage of fermentation, and naturally ferment until the pH reaches 5.0; then adjust the fermentation temperature to 37°C for constant temperature culture, and control the pH to maintain at 6.0, and maintain anaerobic fermentation.

[0025] During the above process, the pH is controlled by the method of dropwise adding a neutralizing agent, and the neutralizing agent is NaOH.

[0026] Among them, the anaerobic condition is achieved by the method of purging nitrogen once every two hours.

[0027] According to some embodiments of the present invention, the termination of fermentation in step (4) is: when the acid production of the bacteria stops, the fermentation is terminated to obtain the high-density fermentation broth of each strain, and the viable count of the fermentation broth reaches 2×10 10 CFU / ml or more.

[0028] According to some embodiments of the present invention, the freeze-drying described in step (5) includes the following steps: 1) Bacterial cell concentration: The high-density fermentation broth is centrifuged at 12,000g to concentrate the bacterial cells; 2) Adding a cryoprotectant: 5 times of the cryoprotectant solution is added to the concentrated bacterial cell solution; The composition of the cryoprotectant solution is as follows: 14 Kg of skim milk powder, 10 Kg of lactose, 1.1 Kg of vitamin C, 0.8 Kg of sodium glutamate, and 1000 L of distilled water; 3) Drying: The above-mentioned bacterial cell suspension after adding the cryoprotectant is freeze-dried to obtain freeze-dried bacterial powder, and the total viable count in the bacterial powder is controlled to reach 1.0×10 11 CFU / g or more.

[0029] Furthermore, when the product is a drug, the drug further includes a pharmaceutically acceptable carrier.

[0030] Still further, the pharmaceutically acceptable carrier is selected from one or more of excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0031] Furthermore, the food includes but is not limited to compressed candies, yogurt, canned foods, biscuits, chocolates, pastries, creams, cheeses, milk powders, ice creams, ice lollies, jams, purees, candied fruits, preserved fruits, breads, egg rolls, protein drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee, and puffed foods.

[0032] Furthermore, the health product contains 1×10 6 CFU / g - 1×10 11 CFU / g or 1×10 6 CFU / ml - 1×10 11 CFU / ml of Bifidobacterium breve.

[0033] Furthermore, the product has any one of the following functions: (1) Reducing the weight of white fat and liver tissue; (2) Reduce the area of adipocytes and hepatic fat vacuoles, and leach out lipid droplets; (3) Regulate the proportion of intestinal flora and increase the abundance of beneficial bacteria; (4) Regulate metabolites to return to normal levels; (5) Regulate the expression levels of mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and the mRNAs they regulate.

[0034] Further, the metabolites described in step (4) include 2-oxoadipic acid, 3-oxoadipic acid, 4-piperidinepropionic acid, 9-oxoadipic acid, choline, and nicotinamide.

[0035] Specifically, the product can be taken directly orally, diluted with cold water, or mixed with warm water for consumption.

[0036] On the other hand, the present invention provides the use of Bifidobacterium breve in the preparation of a drug for treating obesity-related diseases, and the preservation number of the Bifidobacterium breve is CGMCC No. 22242.

[0037] Specifically, the obesity-related diseases include but are not limited to cardiovascular diseases, metabolic syndrome, diabetes, hyperglycemia, insulin resistance, dyslipidemia, and hypolipidemia.

[0038] On the other hand, the present invention provides a product for alleviating or improving obesity, and the preservation number of the Bifidobacterium breve is CGMCC No. 22242.

[0039] Specifically, the product includes drugs, foods, and health products.

[0040] Specifically, the product has any one of the following effects: (1) Reduce the weight of white adipose and liver tissues; (2) Reduce the area of adipocytes and hepatic fat vacuoles, and leach out lipid droplets; (3) Regulate the proportion of intestinal flora and increase the abundance of beneficial bacteria; (4) Regulate metabolites to return to normal levels; (5) Regulate the expression levels of mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and the mRNAs they regulate.

[0041] Specifically, the viable count of Bifidobacterium breve in the product is 1×10 6 CFU / g - 1×10 11 CFU / g or 1×10 6 CFU / ml - 1×10 11 CFU / ml.

[0042] Furthermore, the viable count of Bifidobacterium breve in the product is 1×10 9 CFU / g or 1×10 9 CFU / ml.

[0043] The beneficial effects of the present invention are as follows: The probiotic Bifidobacterium breve B2798 of the present invention can reduce the weights of white adipose and liver tissues, and significantly reduce the areas of various adipocytes and problems such as liver fat vacuoles and lipid droplet leaching caused by high-fat diet; meanwhile, the probiotic Bifidobacterium breve B2798 can restore a variety of key metabolites to normal levels by changing the diversity of intestinal flora, regulating the proportion of intestinal flora, and increasing the abundance of beneficial bacteria; regulating the expression levels of mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and their regulated mRNAs to regulate white adipose, beige adipose and brown adipose, thereby effectively alleviating obesity caused by high-fat diet. Description of the Drawings

[0044] Figure 1 is the experimental flow chart.

[0045] Figure 2 is the graph of the changes in the body weight of mice and the weights of various adipose and liver tissues during the experiment.

[0046] Figure 3 is the graph of the results of HE staining and Oil Red O staining.

[0047] Figure 4 is the graph of the results of metagenomic flora analysis.

[0048] Figure 5 is the graph of the results of metabolomics.

[0049] Figure 6 is the graph of the results of combined analysis.

[0050] Figure 7 is the graph of the results of mRNA and microRNA target gene prediction. Detailed Embodiments

[0051] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further clarified below with reference to specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention and not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are all conventional operating methods, and the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0052] Bifidobacterium breve B2798 with the preservation number of CGMCC No. 22242 has been disclosed in Patent CN116731891A.

[0053] Basic implementation example A probiotic Bifidobacterium breve B2798 powder, wherein the microbial preservation number of Bifidobacterium breve B2798 ( Bifidobacterium breve B2798) is CGMCC No. 22242; the viable count in the probiotic powder is greater than or equal to 1.0×10 9 CFU / g.

[0054] The preparation method of the probiotic powder comprises the following steps: 1) Activation of the strain: The strains stored at -40°C are respectively inoculated into the corresponding liquid medium sterilized at 121°C for 15 min, and anaerobically cultured at 37°C for 18 - 24 h, and passaged and cultured 1 - 2 times in this way to obtain the activated strain; the Bifidobacterium breve is the RCM liquid medium.

[0055] 2) Preparation of the seed liquid: Take the activated strains in step 1) and inoculate them into the RCM liquid medium, and anaerobically culture at 37°C until the pH value reaches 4.5 - 4.8 and then stop; 3) Inoculation and fermentation: Inoculate the seed liquid in step 2) into the RCM liquid medium at 1‰, and ferment for 18 h under the controlled fermentation conditions; the fermentation conditions are controlled as follows: keep the temperature at 30°C for constant culture in the early stage of fermentation, and naturally ferment until the pH reaches 5.0; then adjust the fermentation temperature to 37°C for constant culture, and control the pH to remain at 6.0, and keep anaerobic fermentation.

[0056] In the above process, the pH is controlled by the method of adding a neutralizing agent dropwise, and the neutralizing agent is NaOH.

[0057] Among them, the anaerobic condition is achieved by the method of passing nitrogen once every two hours.

[0058] 4) Termination of fermentation: When the acid production of the bacteria stops, terminate the fermentation to obtain the high-density fermentation broth of each strain, and the viable count in the fermentation broth reaches 2×10 10 CFU / ml or more.

[0059] Among them, it can be judged whether the acid production stops according to the fact that the pH no longer decreases and the addition of the neutralizing agent stops.

[0060] 5) Freeze-drying: a. Concentration of the bacteria: Concentrate the bacteria in the high-density fermentation broth by centrifugation at 12000g; b. Adding a cryoprotectant: Add 5 times the amount of cryoprotectant solution to the concentrated bacterial solution. The composition of the cryoprotectant solution is as follows: 14 Kg of skim milk, 10 Kg of lactose, 1.1 Kg of vitamin C, 0.8 Kg of sodium glutamate, and 1000 L of distilled water.

[0061] c. Drying: Freeze-dry the bacterial suspension after adding the cryoprotectant above to obtain freeze-dried bacterial powder, and control the total viable count in the bacterial powder to reach 1.0×10 11 CFU / g or more.

[0062] Example 1 1 Experimental method 1.1 Strains contained in the probiotic bacterial powder Bifidobacterium breve B2798 ( Bifidobacterium breve B2798), provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd.

[0063] 1.2 Experimental method Six-week-old male C57 mice were divided into 3 groups, namely the control group, the model group, and the intervention group (administered B2798 probiotics), with 8 mice in each group. High-fat diet was used to induce obesity, and then Bifidobacterium breve B2798 (1×10 9 CFU / mouse / day, dissolved in 0.1 mL of physiological saline) was intragastrically administered for 12 weeks, and the other groups were intragastrically administered the same dose of physiological saline ( Figure 1 ).

[0064] 1.3 Detection method Weigh the body weight at weeks 0 and 12 respectively, and collect fecal samples to analyze the intestinal flora of the mice.

[0065] Obtaining eWAT, iWAT, and BAT: eWAT (epididymal white adipose tissue): After the mice were anesthetized, the abdominal skin was incised with a scalpel to expose the abdominal cavity. According to the anatomical landmarks, the epididymal white adipose tissue was accurately separated. The separated eWAT tissue was immediately placed in a sterile centrifuge tube containing 4% paraformaldehyde.

[0066] iWAT (inguinal beige adipose tissue): Through the same anesthesia and surgical method, the adipose tissue was found at the inguinal area of the mice, and the iWAT was anatomically separated along both sides of the abdomen. Pay attention to avoiding damage to adjacent organs during resection, and after sampling, it was also placed in a sterile centrifuge tube containing 4% paraformaldehyde.

[0067] BAT (scapular brown adipose tissue): Under anesthesia, the tissue was separated at the scapular area of the back, and after sampling, it was also placed in a sterile centrifuge tube containing 4% paraformaldehyde. All the obtained adipose tissues were immediately subjected to subsequent processing.

[0068] HE staining includes the following steps: 1. Tissue fixation: The excised tissue sample is placed in 10% neutral formalin solution for fixation for 24 hours to ensure complete infiltration of the tissue.

[0069] 2. Dehydration: The fixed tissue is dehydrated with a series of ethanol solutions with increasing concentrations, and each concentration of ethanol is soaked for 15 - 30 minutes.

[0070] 3. Clearing: The dehydrated tissue is placed in xylene for clearing for 30 minutes.

[0071] 4. Impregnation with wax: The cleared tissue is immersed in molten paraffin at a temperature of 60 - 65 °C for 4 hours.

[0072] 5. Paraffin embedding: The tissue sample is taken out and placed in a paraffin block, waiting for cooling and solidification. After the paraffin is completely hardened, a microtome is used for sectioning.

[0073] 6. Sectioning: The paraffin - embedded tissue is sectioned into thin slices with a thickness of 4 - 6 μm using a microtome and placed on a glass slide.

[0074] 7. Dewaxing: The sections are successively placed in xylene for dewaxing for 5 - 10 minutes.

[0075] 8. Hydration: The dewaxed sections are successively soaked in ethanol solutions with different concentrations (100%, 90%, 80%, 70%) and finally washed with distilled water.

[0076] 9. HE staining: The tissue sections are placed in hematoxylin solution for staining for 5 - 10 minutes.

[0077] The sections are rinsed with running water to remove excess hematoxylin.

[0078] The sections are placed in eosin solution for staining for 2 - 5 minutes.

[0079] The sections are washed with running water to remove excess eosin.

[0080] 10. Dehydration: The stained sections are successively placed in ethanol solutions with different concentrations for dehydration.

[0081] 11. Clearing and mounting: The sections are placed in xylene for clearing, and a neutral balsam is dropped on them for mounting, and a coverslip is covered.

[0082] Microscopic observation: Use a microscope to observe the HE - stained sections and evaluate the morphological structure of the tissue.

[0083] Oil Red O staining includes the following steps: 1. Tissue section: Prepare tissue sections according to the HE staining procedure.

[0084] 2. Deparaffinization and hydration: As in the deparaffinization and hydration steps of HE staining, the sections need to be deparaffinized and hydrated to distilled water.

[0085] 3. Staining: Immerse the hydrated tissue sections in Oil Red O staining solution (Oil Red O dissolved in 50% isopropanol) and stain for 30 - 60 minutes. Oil Red O can bind to lipid droplets and appear red.

[0086] 4. Rinsing: Gently rinse the sections with distilled water to remove excess Oil Red O dye.

[0087] 5. Dehydration and clearing: Dehydrate with ethanol solutions of different concentrations and then clear with xylene.

[0088] 6. Mounting: Drop neutral balsam and cover with a coverslip, ensuring no air bubbles.

[0089] 7. Microscopic observation: Observe the staining results under a microscope. Lipid droplets will appear red, while other cells and structures will appear in different colors.

[0090] Method for metagenomic microbiota analysis: Extract metagenomic DNA from mouse fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Construct a DNA library using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) to generate DNA fragments with a length of approximately 300 bp. Generate paired-end reads in both the forward and reverse directions through the Illumina NovaSeq6000 sequencing platform. Use the KneadData pipeline (http: / / huttenhower.sph.harvard.edu / kneaddata; v0.7.5) for quality control, and then use Bowtie2 to align it with the mouse genome to remove host DNA sequences (v2.3.5.1). Use MEGAHIT to assemble the sequences into contigs, Kraken2 for species annotation, and perform functional annotation and corresponding metabolic pathway annotation based on the UniRef 90 database (https: / / www.uniprot.org / help / uniref) through the HUMAnN2 pipeline.

[0091] Metabolomics detection: After the sample was slowly thawed at 4°C, an appropriate amount of the sample was added to pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortex-mixed, ultrasonically treated at low temperature for 30 min, allowed to stand at -20°C for 10 min, centrifuged at 14000 g at 4°C for 20 min, the supernatant was taken and dried under vacuum. When performing mass spectrometry analysis, 100 μL of acetonitrile-water solution (acetonitrile:water = 1:1, v / v) was added for reconstitution, vortexed, centrifuged at 14000 g at 4°C for 15 min, and the supernatant was taken for injection analysis. The Q Exactive series mass spectrometers were used to collect the first-level and second-level spectra of the samples. After the samples were separated by the Vanquish LC ultra-high performance liquid chromatography system (UHPLC), mass spectrometry analysis was performed using the Q Exactive series mass spectrometers (Thermo), and electrospray ionization (ESI) positive and negative ion modes were used for detection respectively. The original data was converted into the.mzXML format by ProteoWizard, and then the XCMS software was used for peak alignment, retention time correction, and extraction of peak areas. The data extracted by XCMS was first subjected to metabolite structure identification and data preprocessing, then the quality of the experimental data was evaluated, and finally data analysis was performed.

[0092] Methods for mRNA and microRNA transcriptome sequencing: 1. RNA extraction and detection Total RNA quality detection: The concentration and purity were detected using nanodrop <Thermo Scientific NanoDrop2000 (Thermo Scientific, Waltham, Massachusetts, USA)>, and the integrity was detected using RNA-specific agarose gel electrophoresis or 2100 detection <Agilent 2100 Bioanalyzer, RNA 6000 Nano kit 5067-1511 (Agilent Technologies Inc, California, USA)>.

[0093] 2. Library construction and quality inspection Select total RNA with a total amount ≥ 1 μg and use the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc; Ipswich, Massachusetts, USA) (strand-specific library construction kit NEBNext Ultra Directional RNA Library Prep Kit for Illumina). Enrich mRNA with polyA tails using Oligo(dT) magnetic beads, and then randomly fragment the mRNA using divalent cations by ion fragmentation. Using the fragmented mRNA as a template and random oligonucleotides as primers, synthesize cDNA. Purify the double-stranded cDNA, then perform double-end repair, introduce "A" bases at the 3' end, and ligate sequencing adapters. Screen cDNA around 400 - 500 bp using AMPure XP beads, perform PCR amplification, and purify the PCR products again using AMPure XP beads to finally obtain the library. Use the Agilent 2100 Bioanalyzer (Agilent Technologies Inc, California, USA) and the Agilent High Sensitivity DNA Kit (Agilent Technologies Inc, California, USA, 5067 - 4626) to detect the library quality. Detect the total library concentration using Pico green (Quantifluor-ST fluorometer, Promega, Madison, Wisconsin, USA, E6090; Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, California, USA, P7589), and quantitatively detect the effective library concentration by QPCR (StepOnePlus Real-Time PCR Systems, Thermo Scientific, Waltham, Massachusetts, USA)). Normalize and mix equal volumes of the multiplexed DNA libraries. Gradually dilute and quantify the mixed library and perform PE150 mode sequencing on the Illumina sequencer.

[0094] 1.4 Experimental Results 1.4.1 Phenotypic Results Compared with the control group, a high-fat diet significantly increased the body weight of mice, indicating the success of the model. p<0.0001); Compared with the model group, Bifidobacterium breve B2798 can effectively alleviate obesity in mice caused by a high-fat diet ( p <0.01). The weights of white adipose and liver tissues were significantly reduced under the action of the probiotic (there was no significant difference in the weight of brown adipose tissue) ( Figure 2 ).

[0095] Furthermore, through HE staining and Oil Red O staining results, it was found that Bifidobacterium breve B2798 could significantly reduce the area of each adipocyte and significantly alleviate problems such as liver fat vacuoles and lipid droplet leaching caused by a high-fat diet ( Figure 3 ).

[0096] 1.4.2 Metagenomic microbiota analysis From the results of the Shannon index and Simpson index, a high-fat diet significantly affected intestinal diversity ( p <0.0001), while the intake of probiotics had a certain alleviating effect, but not significantly. The β-diversity results showed that under the action of the probiotic, the results of some individuals were separated from the model group at the 1 / 2 principal component.

[0097] From the results at the phylum level, under the action of a high-fat diet, the abundance of Actinobacteria decreased significantly, and the abundance of Bacteroidetes increased significantly. Under the action of the probiotic, the abundance of Bacteroidetes decreased significantly. From the results at the genus level, compared with the control group, the dominant genera unique to the model group were: Enterococcus, Vibrio, and the dominant genera unique to the intervention group were: Akkermansia muciniphila, Anaerostipes, Roseburia ( Figure 4 ).

[0098] 1.4.3 Metabolomics results In the results of non-targeted metabolomics, the principal component analysis results showed that under the action of a high-fat diet, the model group, intervention group, and control group were completely separated at the 1 / 2 principal component; the metabolic results of the model group and the intervention group were partially separated. Partial least squares discriminant analysis (PLS-DA) showed that the control group, model group, and intervention group were completely separated in space.

[0099] A total of 438 differential metabolites were identified between the control group and the model group, with 71 unique differential metabolites, of which 206 differential metabolites were up-regulated and 232 differential metabolites were down-regulated; 107 were identified between the model group and the intervention group, with 39 unique differential metabolites, of which 19 differential metabolites were up-regulated and 88 differential metabolites were down-regulated. Among them, various metabolites such as 2-oxoadipic acid, 3-oxoadipic acid, 4-piperidinepropionic acid, 9-oxoadipic acid, choline, and nicotinamide returned to normal levels under the action of the probiotic ( Figure 5 ).

[0100] 1.4.4 Results of combined analysis Correlation analysis was performed on the metagenomic results, non-targeted metabolomic results, and phenotypic results. The abundances of Lactobacillus johnsonii, Mucilaginibacter ruminantium, Enterobacter sp. P55, etc. were significantly negatively correlated with the body weight change rate, liver weight, and white fat weight, and significantly positively correlated with the brown fat weight ( Figure 6 ).

[0101] 1.4.5 Prediction Results of mRNA and microRNA Target Genes mRNA and microRNA transcriptome sequencing were performed on the white adipose tissue of each group and compared with the differentially expressed genes of mRNA we measured. It was found that the expression levels of 23 mRNAs such as mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and their regulated Nrbp2, Zfp970, Eef1a2, etc. were abnormally expressed with the intake of high-fat diet and gradually returned to normal levels with the intake of probiotics. This indicates that mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and their regulated mRNAs may be one of the key targets for probiotics to relieve obesity ( Figure 7 ).

[0102] In summary, the probiotic Bifidobacterium breve B2798 can reduce the weights of white adipose and liver tissues, and significantly reduce the areas of each adipocyte and problems such as liver fat vacuoles and lipid droplet leaching caused by high-fat diet; at the same time, probiotic B2798 can restore various key metabolites such as 2-oxoadipic acid, 3-oxoadipic acid, 4-piperidinepropionic acid, 9-oxoadipic acid, choline, and nicotinamide to normal levels by changing the intestinal flora diversity, regulating the intestinal flora ratio, and increasing the abundance of beneficial bacteria; regulating the expression levels of mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and their regulated mRNAs to regulate white adipose, beige adipose, and brown adipose, thereby effectively relieving obesity caused by high-fat diet.

[0103] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Bifidobacterium breve in preparing a product for alleviating or improving obesity, characterized in that: The Bifidobacterium breve is Bifidobacterium breve B2798, and its preservation number is CGMCC No. 22242.

2. The use according to claim 1, characterized in that: The obesity is the obesity caused by a high-fat diet.

3. The use according to claim 1, characterized in that: The viable count of Bifidobacterium breve in the product is 1×10 6 CFU / g-1×10 11 CFU / g or 1×10 6 CFU / ml-1×10 11 CFU / ml.

4. The use according to claim 3, characterized in that: The viable count of Bifidobacterium breve in the product is 1×10 9 CFU / g or 1×10 9 CFU / ml.

5. The use according to claim 1, characterized in that: The products include medicines, foods and health products.

6. The use according to claim 5, characterized in that: The product is a medicine, which also includes a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

8. The use according to any one of claims 1 to 7, characterized in that: The product described has any of the following effects: (1) Reduce white fat and liver tissue weight; (2) Reduce the area of ​​adipocytes and liver fat vacuoles and lipid droplet leaching; (3) Regulate the proportion of intestinal flora and increase the abundance of beneficial bacteria; (4) Regulate metabolites to restore normal levels; (5) Regulate the expression levels of mmu-miR-203-3p, mmu-miR-193a-3p, mmu-miR-378b and their regulated mRNAs.

9. Use of Bifidobacterium breve in the preparation of a drug for treating obesity-related diseases, characterized in that: The Bifidobacterium breve is the Bifidobacterium breve B2798 in claim 1, and its deposit number is CGMCC No. 22242.

10. A product for alleviating or improving obesity, characterized in that: The Bifidobacterium breve is the Bifidobacterium breve B2798 in claim 1, and its deposit number is CGMCC No. 22242.

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