Bifidobacterium bifidum and application thereof

Abstract

The invention provides bifidobacterium bifidum and application thereof, and belongs to the technical field of microorganisms. Specifically, the invention provides bifidobacterium bifidum, and the bifidobacterium bifidum has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.30446. The invention also provides a preparation method of the bifidobacterium bifidum. The bifidobacterium bifidum disclosed by the invention has the effects of relieving liver lipid deposition and lipid production related gene expression of an individual with the non-alcoholic fatty liver disease (such as non-alcoholic steatohepatitis induced by a high-glucose and high-fat feed); in addition, the traditional Chinese medicine composition can relieve the pathological characteristics (hepatic cell steatosis, hepatic cell ballooning and inflammatory cell infiltration) of the individual liver of the high non-alcoholic fatty liver disease, and has the effect of relieving the liver injury caused by non-alcoholic fatty hepatitis induced by high-glucose and high-fat diet.

Description

Technical Field

[0001] The present invention relates to a Bifidobacterium bifidum and its application. Specifically, it relates to a Bifidobacterium bifidum strain that can alleviate symptoms related to non-alcoholic fatty liver disease and its application in alleviating symptoms related to non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD), now also known as metabolic dysfunction-associated fatty liver disease (MAFLD or metabolic dysfunction-associated steatotic liver disease, MASLD). NAFLD is currently the most common chronic liver disease. The spectrum of NAFLD ranges from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). Since NASH has the risk of progressing to liver fibrosis, cirrhosis, and hepatocellular carcinoma, it has attracted particular attention in clinical practice. Although the US Food and Drug Administration (FDA) has approved the thyromimetic selective β-receptor agonist Resmetirom as the first drug for the intervention of NASH, its effect is limited.

[0003] Probiotics refer to live microorganisms that can produce beneficial effects on the human body when taken in sufficient amounts. Research has shown that probiotics can regulate glucose and lipid metabolism, modulate the immune system, and assist in the treatment of cancer and depression. In the treatment of NASH, probiotics also show certain alleviating abilities. For example, Bacteroides uniformis reduces NASH by generating metabolites to inhibit hepatocyte steatosis, hepatocyte injury, and inflammatory responses; Parabacteroides distasonis can produce pentadecanoic acid to inhibit the progression of NASH. However, the current effect of probiotics in treating NASH is strain-specific, and there is still a lack of functional strains that can be truly translated and applied. Summary of the Invention

[0004] The present invention discovers that a strain of Bifidobacterium bifidum can alleviate the symptoms related to non-alcoholic steatohepatitis, specifically including: it can relieve the liver lipid deposition and the expression of lipid generation-related genes in individuals with non-alcoholic fatty liver disease (such as non-alcoholic steatohepatitis induced by a high-sugar and high-fat diet), can relieve the liver pathological features (hepatocyte steatosis, hepatocyte ballooning degeneration, and inflammatory cell infiltration) in individuals with high non-alcoholic fatty liver disease, and can relieve the liver damage caused by non-alcoholic steatohepatitis induced by a high-sugar and high-fat diet. In the present invention, this strain is named BB478. This strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC) (address of the depository unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101), deposit date: April 25, 2024; deposit number: CGMCC No. 30446; taxonomic name: Bifidobacterium bifidum.

[0005] Thus, on the one hand, the present invention provides a Bifidobacterium bifidum with a deposit number of CGMCC No. 30446.

[0006] On the other hand, the present invention also provides a bacterial preparation containing the Bifidobacterium BB478 of the present invention (i.e., the strain with a deposit number of CGMCC No. 30446, or referred to as Bifidobacterium BB478).

[0007] According to the specific implementation scheme of the present invention, the bacterial preparation of the present invention can be a liquid or solid preparation in the form of live bacteria or inactivated form.

[0008] In some specific implementation schemes of the present invention, the bacterial preparation is a liquid preparation.

[0009] In some other specific implementation schemes of the present invention, the bacterial preparation is a solid preparation, such as freeze-dried powder.

[0010] On the other hand, the present invention also provides a postbiotic, which is a postbiotic containing bacterial cells and metabolites obtained by fermenting and culturing the Bifidobacterium of the present invention or the bacterial preparation in a culture medium, or a postbiotic containing fermentation products. Preferably, the fermentation product obtained by fermenting and culturing the Bifidobacterium of the present invention in a culture medium is a postbiotic containing bacterial cells and metabolites; or, the fermentation product is further centrifuged to collect the supernatant of the fermentation broth and filtered through a filter membrane to obtain a postbiotic containing metabolites after removing the bacterial cells; optionally, the postbiotic can be further dried to prepare a powder.

[0011] On the other hand, the present invention also provides a method for culturing the Bifidobacterium bifidum, and the method comprises:

[0012] Inoculating the Bifidobacterium bifidum into a culture medium for culturing.

[0013] In some specific embodiments of the present invention, the culture medium is BS medium;

[0014] In some specific embodiments of the present invention, the culturing is carried out under anaerobic conditions.

[0015] On the other hand, the present invention also provides the use of the Bifidobacterium bifidum, the bacterial preparation or the postbiotics in the preparation of a medicine or food.

[0016] On the other hand, the present invention also provides the use of the Bifidobacterium bifidum, the bacterial preparation or the postbiotics in the preparation of a medicine for preventing non-alcoholic fatty liver disease or related symptoms.

[0017] According to the specific embodiments of the present invention, the prevention of non-alcoholic fatty liver disease or related symptoms includes one or more of the following:

[0018] Preventing non-alcoholic steatohepatitis;

[0019] Preventing the deposition of fat in the liver caused by a high-sugar and / or high-fat diet;

[0020] Preventing the increase in the levels of triglyceride and total cholesterol caused by a high-sugar and / or high-fat diet;

[0021] Preventing the increase in the levels of alanine aminotransferase and / or aspartate aminotransferase caused by a high-sugar and / or high-fat diet.

[0022] On the other hand, the present invention also provides the use of the Bifidobacterium bifidum, the bacterial preparation or the postbiotics in the preparation of a medicine for treating non-alcoholic fatty liver disease or related symptoms.

[0023] According to the specific embodiments of the present invention, the treatment of non-alcoholic fatty liver disease or related symptoms includes one or more of the following:

[0024] Treating non-alcoholic steatohepatitis;

[0025] Relieving the deposition of fat in the liver;

[0026] Reducing the levels of triglyceride and total cholesterol;

[0027] Reducing the levels of alanine aminotransferase and / or aspartate aminotransferase;

[0028] Alleviate the pathological features of non-alcoholic steatohepatitis; preferably, the pathological features of non-alcoholic steatohepatitis include hepatic steatosis, hepatocyte ballooning, and / or lobular inflammation;

[0029] Prepare to inhibit the expression levels of lipidogenic genes SREBP1, PPAR-γ, and / or Fasn.

[0030] On the other hand, the present invention also provides a pharmaceutical composition for treating non-alcoholic fatty liver disease or related symptoms, which comprises: the Bifidobacterium bifidum of the present invention, the bacterial preparation or the postbiotic, and a pharmaceutically acceptable carrier.

[0031] According to a specific embodiment of the present invention, the pharmaceutical composition of the present invention can be formed into any feasible probiotic pharmaceutical preparation, such as oral liquid, powder, tablet, capsule, etc.

[0032] On the other hand, the present invention also provides a method for preventing and / or treating non-alcoholic fatty liver disease or related symptoms, which comprises administering an effective amount of the Bifidobacterium bifidum, the bacterial preparation, the postbiotic, or the pharmaceutical composition of the present invention to a subject in need thereof.

[0033] The Bifidobacterium bifidum BB478 provided by the present invention has the effect of alleviating liver lipid deposition and the expression of lipidogenesis-related genes in individuals with non-alcoholic steatohepatitis induced by a high-sugar and high-fat diet. In addition, it can alleviate the liver pathological features (hepatic steatosis, hepatocyte ballooning, and inflammatory cell infiltration) in individuals with high non-alcoholic steatohepatitis and has the effect of alleviating liver damage caused by non-alcoholic steatohepatitis induced by a high-sugar and high-fat diet. Description of the Drawings

[0034] Figure 1 It is the colony morphology of the Bifidobacterium bifidum BB478 of the present invention on a BS agar plate.

[0035] Figure 2 It is a direct-view diagram of the Bifidobacterium bifidum BB478 of the present invention reducing liver fat deposition in zebrafish with non-alcoholic steatohepatitis (NASH).

[0036] Figure 3 It is a statistical chart of the Bifidobacterium bifidum BB478 of the present invention reducing liver fat deposition in zebrafish with non-alcoholic steatohepatitis (NASH).

[0037] Figure 4 It is a paraffin section and H&E staining of the Bifidobacterium bifidum BB478 of the present invention reducing the pathological features of non-alcoholic steatohepatitis (NASH) in zebrafish; steatosis - blue arrow, ballooning - green arrow, lobular inflammation - red arrow; the scale bar in the first row is 200μm, and the scale bar in the second row is 50μm.

[0038] Figure 5 This is the visualization diagram of the inhibitory effect of Bifidobacterium bifidum BB478 of the present invention on the aggregation of liver macrophages in zebrafish with non-alcoholic steatohepatitis (NASH).

[0039] Figure 6 This is the statistical chart of the inhibitory effect of Bifidobacterium bifidum BB478 of the present invention on the aggregation of liver macrophages in zebrafish with non-alcoholic steatohepatitis (NASH).

[0040] Figure 7A and Figure 7B This is the statistical chart of the reduction of the content of triglyceride (TG) and total cholesterol (TC) in the liver of zebrafish with non-alcoholic steatohepatitis (NASH) by Bifidobacterium bifidum BB478 of the present invention.

[0041] Figure 8A and Figure 8B This is the statistical chart of the reduction of the activity of alanine aminotransferase (ALT) and the level of aspartate aminotransferase (AST) in the liver of zebrafish with non-alcoholic steatohepatitis (NASH) by Bifidobacterium bifidum BB478 of the present invention.

[0042] Figures 9A - 9C This is the quantification diagram of the inhibition of the expression of lipidogenesis-related genes SREBP1, PPAR-γ, and Fasn in the liver of zebrafish with non-alcoholic steatohepatitis (NASH) by Bifidobacterium bifidum BB478 of the present invention.

[0043] Deposit of biological materials for patent procedures:

[0044] Bifidobacterium bifidum strain BB478:

[0045] Date of deposit: April 25, 2024;

[0046] Depositary institution: China General Microbiological Culture Collection Center (CGMCC);

[0047] Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101;

[0048] Deposit number: CGMCC No. 30446;

[0049] Taxonomic name: Bifidobacterium bifidum. Detailed implementation manners

[0050] The technical solution of the present invention will be further clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0051] Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0052] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, either endpoint of each numerical range and any value between the two endpoints can be selected.

[0053] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art of this technology.

[0054] Except for the specific methods, equipment, and reagent materials used in the embodiments, according to the knowledge of those skilled in the art of the present technology and the records of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to the methods, equipment, and reagent materials described in the embodiments of the present invention can also be used to implement the present invention.

[0055] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the technical field of the present technology.

[0056] The zebrafish used in the embodiments of the present invention are AB-line zebrafish and green fluorescent protein-labeled macrophage transgenic zebrafish (Tg(mpeg:EGFP)), purchased from Nanjing Yishulihua Biotechnology Co., Ltd. and propagated in the laboratory of Guangdong Nanxin Medical Technology Co., Ltd.

[0057] The main reagents used in the embodiments of the present invention are shown in Table 1.

[0058] Table 1

[0059]

[0060]

[0061] The main equipment used in the embodiments of the present invention are shown in Table 2.

[0062] Table 2

[0063]

[0064] Example 1: Isolation, Identification, and Preservation of Bifidobacterium bifidum BB478

[0065] 1. Strain Isolation

[0066] 1) Dissolve the feces (about 0.1 g) of a 23-year-old healthy male volunteer from Guangzhou, Guangdong Province in a 1.5 mL centrifuge tube containing 1 mL of sterile physiological saline, and thoroughly pipette and mix with a 1 mL sterile pipette tip for later use.

[0067] 2) Add 900 μL of sterile physiological saline to each of the 6 sterile 1.5 mL centrifuge tubes.

[0068] 3) Using the method of serial dilution, dilute the sample to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 .

[0069] 4) Pipette 100 μL of the sample dilution from the centrifuge tube containing the 10 -4 sample dilution and inoculate it onto MRS solid medium, BHI solid medium, and BS solid medium respectively. Spread the 100 μL of bacterial liquid evenly and quickly, paying attention to the gentle spreading technique. The operation must be carried out near the alcohol lamp flame. After spreading, mark the side of the petri dish with information including name, sample number, medium name, culture time, dilution gradient, culture conditions (anaerobic / aerobic), etc.

[0070] 5) Referring to step 4) above, complete the spreading for the dilution gradients of 10 -5 , 10 -6 , 10 -7 .

[0071] 6) After spreading, place the petri dishes in an anaerobic culture condition at 37°C for incubation. Observation and recording can be carried out after 48 h.

[0072] 7) Use an inoculation loop to pick a single colony on the plate and streak it onto BS solid medium, and incubate it anaerobically at 37°C for 48 h to isolate pure colonies.

[0073] 8) Inoculate the pure colonies on the plate into BS liquid medium, incubate it anaerobically at 37°C for 12 - 16 h, add 20% glycerol, and store it in a -80°C refrigerator.

[0074] 2. Molecular Biological Identification of Strains

[0075] Extract genomic DNA from the obtained strains, amplify the full-length 16S rDNA fragment using the 16S rDNA universal primers 27F and 1492R by PCR technology, and then perform sequencing to identify the species of the strains.

[0076] Among them, the primer sequences of the universal primers 27F and 1492R are as follows:

[0077] 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO.1);

[0078] 1492R: 5’-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO.2).

[0079] Experimental results: Strains screened from the feces of a 23-year-old healthy male volunteer in Guangzhou, Guangdong Province were identified by morphological observation and 16S rDNA. One of the strains (named BB478) was identified as Bifidobacterium bifidum, and its 16S rDNA sequence is shown as SEQ ID NO.3.

[0080]

[0081] The single colony of strain BB478 was inoculated onto BS solid medium and grew well anaerobically at 37°C. The colonies were spherical and had a smooth surface ( Figure 1 ).

[0082] 3. Preservation of the strain

[0083] The strain BB478 of the present invention has been deposited in the China General Microbiological Culture Collection Center (CGMCC) (Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101), Date of deposit: April 25, 2024; Deposit number: CGMCC No. 30446; Taxonomic name: Bifidobacterium bifidum.

[0084] Example 2. Preparation of Bifidobacterium bifidum BB478 bacterial suspension

[0085] After the activation culture of Bifidobacterium bifidum BB478, it was inoculated into BS liquid medium. After culturing at 37°C for 24 h, it was centrifuged at 4°C and 6000 r / min for 10 min to obtain a cell precipitate. After the cell precipitate was washed twice with PBS, the cells were resuspended with E3 Water and the cell concentration was adjusted to 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL to obtain a bacterial suspension.

[0086] Example 3. Evaluation of the improvement effect of Bifidobacterium bifidum BB478 on liver fat deposition and pathological characteristics in zebrafish with non-alcoholic steatohepatitis (NASH)

[0087] 1) Selection of juvenile fish

[0088] Healthy AB-strain zebrafish at 5 dpf (days post fertilization) were selected and placed in a 90-mm Petri dish.

[0089] 2) Model construction

[0090] The experimental settings included a normal group, a model group, and a Bifidobacterium bifidum BB478 intervention group, with 100 fish in each group. In the normal group, 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) was added, and the fish were fed with ordinary feed (twice a day, at 10:00 and 13:30 respectively; 6 mg each time). In the model group and the Bifidobacterium bifidum BB478 group, zebrafish were exposed to 50 mL of E3 Water containing 4% fructose (w / v) and overfed with high-fat feed (twice a day, at 10:00 and 13:30 respectively; 18 mg each time). Fresh solution was changed every 24 h for 7 days.

[0091] 3) Intervention with Bifidobacterium bifidum BB478

[0092] Seven days after the model was constructed, zebrafish in the normal group were still cultured in 50 mL of E3 Water and fed with ordinary feed (twice a day, at 10:00 and 13:30 respectively; 6 mg each time). Zebrafish in the model group were still cultured in 50 mL of E3 Water containing 4% fructose and fed with excessive high-fat feed (twice a day, at 10:00 and 13:30 respectively; 18 mg each time). Zebrafish in the Bifidobacterium bifidum BB478 intervention group were cultured in 50 mL of E3 Water containing 4% fructose and fed with excessive high-fat feed (twice a day, at 10:00 and 13:30 respectively; 18 mg each time). At the same time, Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water) was added to the zebrafish culture environment to a bacterial concentration of 1×10 6 CFU / mL for feeding intervention. The solution in each group was changed every 24 h for 14 days.

[0093] 4) Oil Red O staining

[0094] After the intervention, 15 zebrafish were selected from each group, washed twice with E3 Water, fixed with 4% paraformaldehyde solution for 24 h, and dehydrated with 25%, 50%, 75%, and 100% 1,2-propanediol gradients for 25 min each. After dehydration, Oil Red O staining was performed for 48 h, then decolorized with 1,2-propanediol for 30 min, and observed and photographed under a stereomicroscope to record the liver fat accumulation in zebrafish. Image J software was used to analyze the Oil Red O staining in the zebrafish liver, and the corresponding gray value (S) was statistically analyzed. The formula for calculating the relative liver fat content of zebrafish is as follows:

[0095]

[0096] The intuitive diagram and statistical chart of the inhibition of liver fat deposition in zebrafish by Bifidobacterium bifidum BB478 are shown inFigure 2 and Figure 3 。

[0097] The Oil Red O staining results showed that compared with the normal group, there were a large number of lipid droplets deposited in the livers of zebrafish in the model group ( Figure 2 ). The relative fat content in the livers of zebrafish in the model group was 882.60 ± 48.03%, which was significantly different from that in the normal group (100.00 ± 15.75%) (p < 0.001) ( Figure 3 ), indicating that a high-sugar and high-fat diet could promote fat deposition in the livers of zebrafish.

[0098] From Figure 2 and Figure 3 it can be seen that compared with the model group, there was less lipid droplet deposition in the livers of zebrafish in the Bifidobacterium bifidum BB478 intervention group. The relative fat content in the livers of zebrafish in the Bifidobacterium bifidum BB478 group was 489.90 ± 50.18% respectively, which was statistically different from that in the model group (882.60 ± 48.03%) (p < 0.001), indicating that Bifidobacterium bifidum BB478 could significantly reduce the fat deposition in the livers of zebrafish ( Figure 3 ).

[0099] 5) Preparation of paraffin sections

[0100] ① Fixation and sampling: Under a stereomicroscope, dissect the livers of zebrafish in each group and immediately immerse them in the tissue fixative for internal fixation for more than 24 hours. Then, take out the tissue from the fixative, trim the liver flat with a scalpel in a fume hood, and stick the trimmed zebrafish tissue on a label and place it in an embedding frame.

[0101] ② Dehydration and wax infiltration: Put the dehydration box into the dehydrator and dehydrate it successively with gradient ethanol. 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, absolute ethanol I for 30 minutes, absolute ethanol II for 30 minutes, ethanol-benzene for 5 - 10 minutes, xylene I for 5 - 10 minutes, xylene II for 5 - 10 minutes, melt paraffin I at 65°C for 1 hour, melt paraffin II at 65°C for 1 hour, and melt paraffin III at 65°C for 1 hour.

[0102] ③ Paraffin embedding: Embed the wax-infiltrated tissue in an embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take out the tissue from the dehydration box, place it in the embedding frame according to the requirements of the embedding surface, and stick the corresponding label. Cool it on a -20°C freezing table. After the wax solidifies, take out the wax block from the embedding frame and trim the wax block.

[0103] ④ Paraffin sectioning: Place the trimmed wax block on a paraffin slicer and cut sections with a thickness of 4 μm. Float the sections on warm water at 40°C on a spreading machine to flatten the tissue, pick up the tissue with a glass slide, and bake the slide in an oven at 60°C. Take it out after the water is dried and the wax is melted and store it at room temperature for later use.

[0104] 6) H&E staining steps

[0105] ① Dewax the paraffin sections to water: Immerse the sections successively in environment-friendly dewaxing solution I for 20 min - environment-friendly dewaxing solution II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water.

[0106] ② Pretreatment: Immerse the sections in the high-definition constant staining pretreatment solution for 1 min.

[0107] ③ Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with the differentiation solution, wash with tap water, blue with the blueing solution, and rinse with running water.

[0108] ④ Eosin staining: Immerse the sections in 95% alcohol for dehydration for 1 min, and then immerse them in eosin staining solution for 15 s.

[0109] ⑤ Dehydration and mounting: Immerse the sections successively in absolute ethanol I for 2 min - absolute ethanol II for 2 min - absolute ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min for clearing, and mount with neutral balsam.

[0110] ⑥ Imaging: Examine under a microscope and collect and analyze images.

[0111] 7) Data statistics

[0112] Use GraphPad Prism 6.0 software to statistically process the data. The experimental data are all expressed as mean ± SEM, and analyzed by unpaired t-test. Compared with the normal group: # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the model group: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0113] From the pathological sections ( Figure 4) It can be seen that the hepatocytes of zebrafish in the normal group were arranged neatly, with clear cell boundaries, nuclei being round and located in the central cells, and the cytoplasm being relatively abundant; while the hepatocyte structure of zebrafish in the model group was severely damaged, with obvious swelling, showing a vacuolar shape, disordered arrangement, a large amount of fatty degeneration (blue arrow) and ballooning degeneration (green arrow), and obvious lobular inflammation (red arrow), indicating the successful construction of this non-alcoholic steatohepatitis model. Compared with the model group, the hepatocyte structure of zebrafish in the Bifidobacterium bifidum BB478 intervention group was less damaged, with less fatty degeneration (blue arrow) and ballooning degeneration (green arrow), and a lighter degree of lobular inflammation (red arrow), indicating that Bifidobacterium bifidum BB478 has the effect of alleviating non-alcoholic steatohepatitis in zebrafish.

[0114] Example 4: Bifidobacterium bifidum BB478 can inhibit the aggregation of macrophages in the liver of zebrafish with non-alcoholic steatohepatitis

[0115] 1) Selection of juvenile fish

[0116] Select healthy Tg(mpeg:EGFP) line zebrafish that have developed to 5 dpf (days post fertilization) and place them in a 90 mm culture dish.

[0117] 2) Model construction

[0118] Set up a normal group, a model group, and a Bifidobacterium bifidum BB478 intervention group in the experiment, with 100 fish in each group. Add 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) to the normal group, and feed with normal feed (fed 2 times a day, at 10:00 and 13:30 respectively; 3 mg each time); add 50 mL of E3 Water containing 4% fructose (w / v) to the model group and the Bifidobacterium bifidum BB478 group, and feed with high-fat feed (fed 2 times a day, at 10:00 and 13:30 respectively; 9 mg each time); replace the fresh solution every 24 h; continue for 7 days.

[0119] 3) Intervention with Bifidobacterium bifidum BB478

[0120] Seven days after the model was constructed, the zebrafish in the normal group were still cultured in 50 mL of E3 Water and fed with ordinary feed (fed twice a day, at 10:00 and 13:30 respectively; 6 mg each time); the zebrafish in the model group were still cultured in 50 mL of E3 Water containing 4% fructose and fed with excessive high-fat feed (fed twice a day, at 10:00 and 13:30 respectively; 18 mg each time); the zebrafish in the Bifidobacterium bifidum BB478 intervention group were cultured in 50 mL of E3 water containing 4% fructose and fed with excessive high-fat feed (fed twice a day, at 10:00 and 13:30 respectively; 18 mg each time), and at the same time, Bifidobacterium bifidum BB478 bacterial liquid (diluted with E3 Water) was added to the E3 water to a bacterial concentration of 1×10 6 CFU / mL for feeding intervention. The solution was changed every 24 h for each group for 14 days.

[0121] 4) Microscopic imaging recording

[0122] After the intervention, 20 zebrafish were selected from each group and placed under an electric inverted fluorescence microscope to observe and photograph the distribution of macrophages in the zebrafish liver.

[0123] 5) Data statistics

[0124] GraphPad Prism 6.0 software was used to statistically process the data. The experimental data were all expressed as mean±SEM and analyzed by unpaired t-test. Compared with the normal group: # p<0.05, ## p<0.01, ### p<0.001; compared with the model group: * p<0.05, ** p<0.01, *** p<0.001.

[0125] It can be seen from Figure 5 and Figure 6 that compared with the normal group (8.80±0.80), the number of macrophages aggregated in the liver of zebrafish in the model group (19.40±2.26) increased significantly (p<0.01), indicating that 21 days of high-sugar and high-fat feeding could induce inflammatory cell infiltration in the liver of zebrafish. In addition, the number of macrophages aggregated in the liver of zebrafish in the Bifidobacterium bifidum BB478 group was 9.40±1.08, which was statistically different from that in the model group (19.40±2.26) (p<0.01)( Figure 6 ). It shows that after 14 days of intervention with Bifidobacterium bifidum BB478 in zebrafish, it can significantly inhibit the infiltration of inflammatory cells in the liver of zebrafish with non-alcoholic steatohepatitis.

[0126] Example 5: Bifidobacterium bifidum BB478 reduces the levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) activity, and aspartate aminotransferase (AST) in the liver of zebrafish

[0127] 1) Selection of juvenile fish

[0128] Select healthy AB-strain zebrafish that have developed to 5 dpf (days post fertilization) and place them in a 90 mm Petri dish.

[0129] 2) Model construction

[0130] Set up normal group, model group, 1×10 4 CFU / mL Bifidobacterium bifidum BB478 intervention group, 1×10 5 CFU / mL Bifidobacterium bifidum BB478 intervention group, 1×10 6 CFU / mL Bifidobacterium bifidum BB478 intervention group, with 100 fish in each group. Add 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) to the normal group and feed with normal feed (fed twice a day, at 10:00 and 13:30 respectively; 6 mg each time); for the model group and Bifidobacterium bifidum BB478 intervention groups (1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL), add 50 mL of E3 Water containing 4% fructose (w / v) and feed with excessive high-fat feed (fed twice a day, at 10:00 and 13:30 respectively; 18 mg each time); replace the fresh solution every 24 h; continue for 7 days.

[0131] 3) Intervention with Bifidobacterium bifidum BB478

[0132] Seven days after model construction, the normal group is cultured with 50 mL of E3 Water and fed with normal feed (fed twice a day, at 10:00 and 13:30 respectively; 6 mg each time); the model group is cultured with 50 mL of E3 Water containing 4% fructose and fed with excessive high-fat feed (fed twice a day, at 10:00 and 13:30 respectively; 18 mg each time); the 1×10 4 CFU / mL Bifidobacterium bifidum BB478 intervention group is added with 50 mL of 1×10 4 CFU / mL Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water); 1×10 5The group with Bifidobacterium bifidum BB478 at 1×10 CFU / mL was added with 50 mL of 1×10 CFU / mL Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water) containing 4% fructose; 5 The intervention group with Bifidobacterium bifidum BB478 at 1×10 CFU / mL was added with 50 mL of 1×10 CFU / mL Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water) containing 4% fructose, and was fed with an excessive high-fat diet (fed 2 times a day, at 10:00 and 13:30 respectively; 18 mg each time). The solution in each group was changed every 24 h, and the intervention lasted for 14 days. 6 The intervention group with Bifidobacterium bifidum BB478 at 1×10 CFU / mL was added with 50 mL of 1×10 CFU / mL Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water) containing 4% fructose; 6 The intervention group with Bifidobacterium bifidum BB478 at 1×10 CFU / mL was added with 50 mL of 1×10 CFU / mL Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water), and was fed with an excessive high-fat diet (fed 2 times a day, at 10:00 and 13:30 respectively; 18 mg each time). The solution in each group was changed every 24 h, and the intervention lasted for 14 days.

[0133] 4) Detect the contents of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in zebrafish in each group

[0134] After the intervention, the zebrafish were washed 2 times with E3 Water, and the zebrafish were collected into 1.5 mL centrifuge tubes. 3 tubes were collected for each experimental group, with 30 zebrafish in each tube; after the water in the centrifuge tubes was blotted dry, 500 μL of ethanol was added, and the zebrafish were homogenized and broken using a tissue grinding homogenizer (TGrinder H24) until there were no obvious tissue fragments. Then, centrifugation was carried out at 15000×g at 4 °C for 15 min, and the supernatant was collected. The levels of triglyceride, total cholesterol, alanine aminotransferase, and aspartate aminotransferase in zebrafish in each group were detected using a triglyceride assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), a total cholesterol assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), alanine aminotransferase activity (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), and aspartate aminotransferase (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.).

[0135] 5) Data statistics

[0136] GraphPad Prism 6.0 software was used to statistically process the data. The experimental data were all expressed as mean±SD, and unpaired t-tests were used for analysis. Compared with the normal group: # p<0.05, ## p<0.01, ### p<0.001; one-way ANOVA was used for analysis. Compared with the model group: * p<0.05, ** p<0.01, *** p<0.001.

[0137] Statistical charts of the reduction of triglyceride (TG) and total cholesterol (TC) contents in zebrafish with non-alcoholic steatohepatitis (NASH) by Bifidobacterium bifidum BB478 are shown in Figure 7A andFigure 7B .

[0138] It can be seen from Figure 7A and Figure 7B that compared with the normal group (triglyceride: 1.61±0.02 mmol / g prot, total cholesterol: 1.47±0.14 mmol / g prot), the triglyceride and total cholesterol in the zebrafish of the model group (triglyceride: 3.78±0.02 mmol / g prot, total cholesterol: 2.69±0.14 mmol / g prot) increased significantly (P<0.001), indicating that a high-sugar and high-fat diet can promote fat deposition in zebrafish. When the intervention concentration of Bifidobacterium bifidum BB478 was 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, the triglyceride content in zebrafish was 2.79±0.05 mmol / g prot, 2.42±0.02 mmol / g prot, 1.69±0.02 mmol / g prot respectively, showing a significant difference compared with the model group (3.78±0.02 mmol / g prot) (P<0.001). In addition, when the intervention concentration of Bifidobacterium bifidum BB478 was 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, the total cholesterol content in zebrafish was 1.67±0.01 mmol / g prot, 1.68±0.04 mmol / g prot, 1.44±0.03 mmol / g prot respectively, showing a significant difference compared with the model group (2.69±0.14 mmol / g prot) (P<0.001). Therefore, Bifidobacterium bifidum BB478 can significantly reduce the fat level in zebrafish.

[0139] Statistical charts of the reduction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents in zebrafish by Bifidobacterium bifidum BB478 are shown in Figure 8A and Figure 8B .

[0140] It can be seen from Figure 8A and Figure 8BIt can be seen that compared with the normal group (alanine aminotransferase: 33.61±3.89 U / g prot, aspartate aminotransferase: 21.76±2.19 U / g prot), the alanine aminotransferase and alanine aminotransferase in the zebrafish of the model group (alanine aminotransferase: 48.80±0.83 U / g prot, alanine aminotransferase: 29.40±1.78 U / g prot) were both significantly increased (P<0.01), indicating that a high-sugar and high-fat diet can induce liver injury in zebrafish. The intervention concentrations of Bifidobacterium bifidum BB478 were 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL. The alanine aminotransferase activities in zebrafish were 41.43±0.80 U / g prot, 31.30±1.01 U / g prot, and 32.42±1.00 U / g prot respectively, showing significant differences (P<0.001) compared with the model group (48.80±0.83 U / g prot). In addition, when the intervention concentrations of Bifidobacterium bifidum BB478 were 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, the alanine aminotransferase activities in zebrafish were 21.58±0.20 U / g prot, 16.40±0.17 U / g prot, and 14.71±0.51 U / g prot respectively, showing significant differences (P<0.001) compared with the model group (21.76±2.19 U / g prot). Therefore, Bifidobacterium bifidum BB478 can alleviate the liver injury induced by a high-sugar and high-fat diet in zebrafish.

[0141] Example 6. Effects of Bifidobacterium bifidum BB478 on the expression of lipidogenic genes SREBP1, PPAR-γ and Fasn in zebrafish

[0142] 1) Selection of juvenile fish

[0143] Select healthy AB-strain zebrafish at 5 dpf (days post fertilization) and place them in a 90 mm petri dish.

[0144] 2) Model construction

[0145] The experimental setup included a normal group, a model group, and a Bifidobacterium bifidum BB478 intervention group, with 100 fish in each group. The normal group was added with 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) and fed with ordinary feed (fed 2 times a day, at 10:00 and 13:30 respectively; 6 mg each time); the model group and the Bifidobacterium bifidum BB478 intervention group were added with 50 mL of E3 Water containing 4% fructose (w / v) and fed with excessive high-fat feed (fed 2 times a day, at 10:00 and 13:30 respectively; 18 mg each time); fresh solution was changed every 24 h; for 7 days continuously.

[0146] 3) Intervention with Bifidobacterium bifidum BB478

[0147] Seven days after the model was constructed, the normal group was cultured with 50 mL of E3 Water and fed with ordinary feed (fed 2 times a day, at 10:00 and 13:30 respectively; 6 mg each time); the model group was cultured with 50 mL of E3 Water containing 4% fructose and fed with excessive high-fat feed (fed 2 times a day, at 10:00 and 13:30 respectively; 18 mg each time); the Bifidobacterium bifidum BB478 intervention group was added with 50 mL of 1×10 6 CFU / mL Bifidobacterium bifidum BB478 bacterial solution (diluted with E3 Water), and fed with excessive high-fat feed (fed 2 times a day, at 10:00 and 13:30 respectively; 18 mg each time). The solution of each group was changed every 24 h for 14 days continuously.

[0148] 4) qRT-PCR detection

[0149] Thirty zebrafish were selected from each group for total RNA extraction. The expression levels of SREBP1, PPAR-γ, and Fasn mRNA were quantified by qRT-PCR and the relative expression levels of cDNA of each sample were calculated using the ΔΔCt method with β-actin as the internal reference. The primer sequences of each gene are shown in Table 3.

[0150] Table 3. Primer sequences

[0151]

[0152] 5) Data statistics

[0153] Data were statistically processed using GraphPad Prism 6.0 software. Experimental data were all expressed as mean ± SEM and analyzed by unpaired t-test. Compared with the normal group: # p < 0.05, ## p < 0.01, ###p < 0.001; compared with the model group: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0154] Statistics on the inhibition of the expression of lipid - generating genes SREBP1, PPAR - γ, and Fasn in zebrafish by Bifidobacterium bifidum BB478 are shown in Figure 9A 、 Figure 9B 、 Figure 9C 。

[0155] From Figure 9A 、 Figure 9B 、 Figure 9C it can be seen that compared with the normal group (SREBP1: 1.00 ± 0.16, Fasn: 1.00 ± 0.04, PPAR - γ: 1.00 ± 0.29), the lipid - generating genes in the model group (SREBP1: 1.71 ± 0.12, Fasn: 1.83 ± 0.15, PPAR - γ: 24.32 ± 0.48) were significantly increased (p < 0.05), indicating that after 21 days of high - sugar and high - fat feeding, the expression of lipid - generating genes SREBP1, PPAR - γ, and Fasn in zebrafish can be promoted. Compared with the model group (SREBP1: 1.71 ± 0.12, Fasn: 1.83 ± 0.15, PPAR - γ: 24.32 ± 0.48), the lipid - generating genes in the Bifidobacterium bifidum BB478 intervention group (SREBP1: 0.59 ± 0.02, Fasn: 0.40 ± 0.03, PPAR - γ: 2.74 ± 0.18) were significantly decreased (p < 0.001). Therefore, Bifidobacterium bifidum BB478 can inhibit the expression of lipid - generating genes SREBP1, PPAR - γ, and Fasn in zebrafish induced by high - sugar and high - fat diet.

[0156] The above - mentioned examples can show that Bifidobacterium bifidum BB478 of the present invention has the effect of alleviating liver fat deposition and the expression of lipid - generating related genes induced by high - sugar and high - fat feed. Bifidobacterium bifidum BB478 of the present invention has the effect of inhibiting liver inflammatory cell infiltration induced by high - sugar and high - fat. Bifidobacterium bifidum BB478 of the present invention has the effect of alleviating the liver pathological characteristics of non - alcoholic fatty liver hepatitis zebrafish induced by high - sugar and high - fat diet.

[0157] It can be understood that the above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein.

Claims

1. A Bifidobacterium bifidum having a deposit number of CGMCC No. 30446.

2. A bacterial preparation containing the Bifidobacterium bifidum according to claim 1.

3. The bacterial preparation according to claim 2, which is a liquid or solid preparation in a live or inactivated form.

4. A postbiotic, which is a postbiotic containing bacterial cells and metabolites obtained by fermenting and culturing the Bifidobacterium bifidum according to claim 1 or the bacterial preparation according to claim 2 in a culture medium, or a postbiotic containing fermentation products; Preferably, the fermentation product obtained by fermenting and culturing the Bifidobacterium bifidum according to claim 1 in a culture medium is a postbiotic containing bacterial cells and metabolites; or, the fermentation product is further centrifuged to collect the supernatant of the fermentation broth and filtered through a filter membrane to obtain a postbiotic containing metabolites after removing the bacterial cells; optionally, the postbiotic can be further dried to prepare a powder.

5. A method for culturing the Bifidobacterium bifidum according to claim 1, the method comprising: Inoculating the Bifidobacterium bifidum according to claim 1 into a culture medium for culturing; Preferably, the culture medium is a BS medium; Preferably, the culturing is carried out under anaerobic conditions.

6. Use of the Bifidobacterium bifidum according to claim 1, the bacterial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in the preparation of a medicament or food.

7. Use of the Bifidobacterium bifidum according to claim 1, the bacterial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in the preparation of a medicament for preventing non-alcoholic fatty liver disease or related symptoms; Preferably, the prevention of non-alcoholic fatty liver disease or related symptoms includes one or more of the following: Preventing non-alcoholic steatohepatitis; Preventing fat deposition in the liver caused by a high-sugar and / or high-fat diet; Preventing an increase in triglyceride and total cholesterol levels caused by a high-sugar and / or high-fat diet; Preventing an increase in the levels of alanine aminotransferase and / or aspartate aminotransferase caused by a high-sugar and / or high-fat diet.

8. Use of the Bifidobacterium bifidum according to claim 1, the bacterial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in the preparation of a medicament for treating non-alcoholic fatty liver disease or related symptoms.

9. The application according to claim 8, wherein, The treatment of non-alcoholic fatty liver disease or related symptoms includes one or more of the following: Treating non-alcoholic steatohepatitis; Relieving fat deposition in the liver; Reducing triglyceride and total cholesterol levels; Reducing the levels of alanine aminotransferase and / or aspartate aminotransferase; Relieving the pathological features of non-alcoholic steatohepatitis; preferably, the pathological features of non-alcoholic steatohepatitis include hepatocyte steatosis, hepatocyte ballooning and / or lobular inflammation; Preparing to inhibit the expression levels of lipidogenesis genes SREBP1, PPAR-γ and / or Fasn.

10. A pharmaceutical composition for treating non-alcoholic fatty liver disease or related symptoms, comprising: The Bifidobacterium bifidum according to claim 1, the bacterial preparation according to claim 2 or 3, or the postbiotic according to claim 4, and a pharmaceutically acceptable carrier.

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