Bifidobacterium bifidum SX-P824 and its application in the preparation of products for reducing lipid and / or antioxidation

Abstract

The present invention discloses a Bifidobacterium bifidum SX-P824 and its application in preparing products for reducing lipid and / or antioxidation. The Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 has a deposit number of CCTCC No: M 2025034 and was deposited at the China Center for Type Culture Collection on January 6, 2025. The Bifidobacterium bifidum SX-P824 provided by the present invention can significantly reduce the fat content in hyperlipidemic animal models, degrade cholesterol and triglycerides, and has good antioxidant and anti-inflammatory effects. It has broad popularization and application value in expanding the application fields of Bifidobacterium bifidum and developing probiotic preparations for preventing and treating hyperlipidemia and antioxidative stress.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically, to a strain of Bifidobacterium bifidum SX-P824 and its application in the preparation of lipid-lowering and / or antioxidant products. Background Art

[0002] Hyperlipidemia has become a common metabolic disease. Typical symptoms include dizziness, fatigue, numbness of limbs, and possible xanthomas, etc. In severe cases, it may lead to serious complications such as coronary heart disease and stroke. Hyperlipidemia is prone to cause oxidative stress. When low-density lipoprotein cholesterol (LDL-C) in the blood increases, it is easily oxidized and modified to form oxidized low-density lipoprotein (ox-LDL), generating a large number of free radicals, attacking biological macromolecules, damaging cells, and also activating the inflammatory pathway. Under the state of oxidative stress, the body's antioxidant defense system is out of balance and cannot effectively scavenge excessive free radicals, which will affect the lipid metabolism and regulation functions of organs such as the liver, and exacerbate the lipid metabolism disorder of hyperlipidemia patients.

[0003] Currently, there are a variety of drugs used for lipid-lowering clinically. Common western medicines include statin drugs. For example, atorvastatin calcium can lower lipids and has certain antioxidant effects, but it may cause side effects such as muscle pain, elevated creatine kinase, abnormal liver function, and possible gastrointestinal discomfort. Therefore, it is necessary to develop safer and more effective lipid-lowering and antioxidant methods.

[0004] Probiotics are a class of beneficial active microorganisms to the human body and have been widely used in the development of functional foods or dietary supplements. Bifidobacterium bifidum ( Bifidobacterium bifidum ) is a facultative anaerobic, non-motile, sporeless bacillus or long bacillus, Gram-positive, catalase-negative, and widely exists in fermented dairy products, yogurt and other foods. As a probiotic, Bifidobacterium bifidum can regulate the proportion of intestinal flora, reduce the number of harmful bacteria, and improve the intestinal environment. Bifidobacterium bifidum also has various physiological functions such as regulating the immune system and promoting the absorption of nutrients, but there is less research and application development in the aspects of lipid-lowering and antioxidant. It is urgent to further explore the application value of Bifidobacterium bifidum in the field of lipid-lowering and antioxidant, and develop related products with better lipid-lowering and antioxidant effects to provide more effective protection for people's health. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a strain of Bifidobacterium bifidum SX-P824 and its application in the preparation of lipid-lowering and / or antioxidant products.

[0006] The first object of the present invention is to provide a strain of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0007] The second object of the present invention is to provide the application of the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in the preparation of products for reducing blood lipid.

[0008] The third object of the present invention is to provide the application of the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in the preparation of products for degrading cholesterol and / or triglyceride.

[0009] The fourth object of the present invention is to provide the application of the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in the preparation of products for preventing and / or treating hyperlipidemia.

[0010] The fifth object of the present invention is to provide the application of the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in the preparation of antioxidant products.

[0011] The sixth object of the present invention is to provide the application of the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in the preparation of anti-inflammatory products.

[0012] The seventh object of the present invention is to provide the application of the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in the preparation of products with any two or three functions of reducing blood lipid, antioxidant or anti-inflammatory.

[0013] The eighth object of the present invention is to provide a microbial inoculum.

[0014] In order to achieve the above object, the present invention is realized by the following scheme:

[0015] The present invention provides a newly isolated Bifidobacterium bifidum strain, which is derived from the fresh feces of centenarians in Suixi County, Zhanjiang City, Guangdong Province, and has the functions of reducing cholesterol, reducing triglyceride, antioxidant and reducing blood lipid, and has good adhesion.

[0016] Therefore, the present invention claims the following:

[0017] A Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824, with the preservation number of CCTCC NO: M 2025034, was preserved in the China Center for Type Culture Collection on January 6, 2025, abbreviated as CCTCC, and the preservation address is: Opposite the First Affiliated Primary School, within Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postcode: 430072, and the taxonomic name is Bifidobacterium bifidum ( Bifidobacterium bifidum ), and the preservation name is Bifidobacterium bifidum SX-P824.

[0018] The Bifidobacterium bifidum (Bifidobacterium bifidum 1) Application of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in preparing products for reducing blood lipid. The reduction of blood lipid involved in the present invention refers to reducing the blood lipid level. In the said application, the Bifidobacterium bifidum (

[0019] ) SX-P824 is used for reducing the content of cholesterol and / or triglyceride in vivo or in blood, including but not limited to. Bifidobacterium bifidum Application of Bifidobacterium bifidum (

[0020] ) SX-P824 in preparing products for degrading cholesterol and / or triglyceride. Bifidobacterium bifidum Application of Bifidobacterium bifidum (

[0021] ) SX-P824 in preparing products for preventing and / or treating hyperlipidemia. Bifidobacterium bifidum Application of Bifidobacterium bifidum (

[0022] ) SX-P824 in preparing products for antioxidation. Bifidobacterium bifidum Application of Bifidobacterium bifidum (

[0023] ) SX-P824 in preparing products for reducing blood lipid and antioxidation. Bifidobacterium bifidum Application of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 in preparing products for scavenging free radicals. The free radicals scavenged by the Bifidobacterium bifidum (

[0024] ) in the said application include but not limited to DPPH and hydroxyl free radicals. Bifidobacterium bifidum Application of Bifidobacterium bifidum (

[0025] ) SX-P824 in preparing products for anti-inflammation. Bifidobacterium bifidum Application of Bifidobacterium bifidum (

[0026] ) SX-P824 in preparing products with any two or three functions of reducing blood lipid, antioxidation or anti-inflammation. Bifidobacterium bifidum Preferably, the 16S rDNA sequence of the Bifidobacterium bifidum (

[0027] ) SX-P824 is as shown in SEQ ID NO:3. Bifidobacterium bifidum Preferably, the Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 includes at least one of viable bacteria, culture broth or culture supernatant of the Bifidobacterium bifidum (

[0028] ) SX-P824. Bifidobacterium bifidum More preferably, it further includes freeze-dried powder of the cells of the Bifidobacterium bifidum (

[0029] More preferably, it further includes the freeze-dried powder of the culture supernatant of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0030] More preferably, it further includes the bacterial suspension of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0031] A microbial inoculum contains Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0032] Preferably, the 16S rDNA sequence of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 is as shown in SEQ ID NO:3.

[0033] Preferably, Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824 includes at least one of viable bacteria, culture broth or culture supernatant of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0034] More preferably, it further includes the freeze-dried powder of the bacterial cells of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0035] More preferably, it further includes the freeze-dried powder of the culture supernatant of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0036] More preferably, it further includes the bacterial suspension of Bifidobacterium bifidum ( Bifidobacterium bifidum ) SX-P824.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The Bifidobacterium bifidum SX-P824 provided by the present invention can significantly reduce the fat content in the hyperlipidemia animal model, and can degrade cholesterol and triglycerides, and has good antioxidant and anti-inflammatory effects. It has wide popularization and application value in expanding the application fields of Bifidobacterium bifidum and developing probiotic preparations for preventing and treating hyperlipidemia and antioxidant stress. Description of the Drawings

[0039] Figure 1 It is the colony morphology diagram of Bifidobacterium bifidum SX-P824.

[0040] Figure 2 It is the microscopic morphology observation diagram of Bifidobacterium bifidum SX-P824 after Gram staining, and the magnification is 10×100.

[0041] Figure 3Alignment and identification results of the 16S rDNA sequence of Bifidobacterium bifidum SX-P824 on NCBI.

[0042] Figure 4 Statistical result chart of in vitro cholesterol degradation rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the cholesterol degradation rate is 50%.

[0043] Figure 5 Statistical result chart of in vitro triglyceride degradation rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the triglyceride degradation rate is 50%.

[0044] Figure 6 Statistical result chart of in vitro total antioxidant capacity of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the equivalent Fe 2+ Antioxidant concentration is 1 nM.

[0045] Figure 7 Statistical result chart of in vitro DPPH free radical scavenging rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the DPPH free radical scavenging rate is 50%.

[0046] Figure 8 Statistical result chart of in vitro hydroxyl free radical scavenging rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the hydroxyl free radical scavenging rate is 50%.

[0047] Figure 9 Effect of Bifidobacterium bifidum SX-P824 on the secretion level of pro-inflammatory cytokines by in vitro stimulation of PBMC. A is the statistical result of the IL-6 secretion level of PBMC in each group, and B is the statistical result of the TNF-α secretion level of PBMC in each group.

[0048] Figure 10 Gram staining map of Bifidobacterium bifidum SX-P824 after adhesion to Caco-2 cells, magnification is 10×40.

[0049] Figure 11 Therapeutic effect of Bifidobacterium bifidum SX-P824 on zebrafish hyperlipidemia model. A is the Oil Red O staining result map of zebrafish hyperlipidemia model in each group, and B is the statistical chart of the relative concentration of body fat in zebrafish hyperlipidemia model. Detailed implementation mode

[0050] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0051] Screening and Identification of Bifidobacterium bifidum SX-P824 in Example 1

[0052] 1. Isolation of Strains

[0053] The fresh fecal samples of centenarians in Suixi County, Zhanjiang City, Guangdong Province were gradient-diluted and then inoculated into MRS solid medium respectively, and anaerobically cultured at 37°C for 48 h. Single colonies on the plate were picked for streak plating, and streak culture was carried out three times to obtain purified monoclonal strains. The monoclonal strains obtained from the third-generation streak were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 48 h. 30 v / v% glycerol (containing 0.5 w / v% cysteine) was added, and the bacterial solution was mixed with 30% glycerol in equal volume and stored in a -80°C refrigerator.

[0054] The method of streak plating of strains is as follows: In the anaerobic workstation, after mixing the bacterial solution, dip a disposable inoculation loop into the bottom of the bacterial solution and scrape it repeatedly 3 times to ensure that the bacterial solution is smeared on the inoculation loop. Then streak the inoculation loop on the culture medium plate in three zones and record the label.

[0055] 2. Morphological Identification of Strains

[0056] (1) Observation of Colony Morphology

[0057] The isolated monoclonal strains were cultured by streak plating, and their colony morphology is as Figure 1 shown, presenting a circular shape, with a convex and smooth surface, milky white, and opaque.

[0058] (2) Gram Staining and Microscopic Morphology Observation

[0059] After Gram staining the isolated monoclonal strains, microscopic examination was carried out using an oil immersion lens. Gram-positive bacteria appear purple and Gram-negative bacteria appear red. As Figure 2 shown, this strain appears purple and is a Gram-positive bacillus.

[0060] 3. Molecular Biology Identification of Strains

[0061] The full-length 16S rDNA of the isolated monoclonal strains was amplified by PCR. The amplification primers used were 16S universal primers: 1492R (5’-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO:1)) and 27F (5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO:2)). Then, electrophoresis was carried out on the amplification products, and the electrophoresis products were collected for sequencing. The 16S rDNA sequence of this monoclonal strain was obtained as follows:

[0062]

[0063] The 16S rDNA sequence (SEQ ID NO: 3) of this monoclonal strain was compared and identified with the standard strain sequences on NCBI. As Figure 3 shown, the comparison result showed that this monoclonal strain was Bifidobacterium bifidum, named Bifidobacterium bifidum SX-P824.

[0064] 4. Strain preservation

[0065] The obtained Bifidobacterium bifidum SX-P824 has been preserved in the China Center for Type Culture Collection (abbreviated as CCTCC, address: Inside Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, opposite the First Affiliated Primary School, postal code: 430072). The preservation date was January 6, 2025. The taxonomic name is Bifidobacterium bifidum ( Bifidobacterium bifidum ), the preservation number is CCTCC NO: M 2025034, and the preservation name is Bifidobacterium bifidum SX-P824.

[0066] Example 2 In vitro functional determination of Bifidobacterium bifidum SX-P824

[0067] 1. Preparation of the bacterial suspension, culture supernatant and freeze-dried powder of Bifidobacterium bifidum SX-P824

[0068] The Bifidobacterium bifidum SX-P824 obtained in Example 1 was resuspended in MRS medium and anaerobically cultured at 37 °C until the OD600 value of the bacterial solution was measured to be 1.6 by an ultraviolet spectrophotometer. Then, the bacterial solution was inoculated into the autoclaved MRS medium at a ratio of 2 v / v%, and anaerobically cultured at 37 °C for 24 h; the cultured bacterial solution was collected and centrifuged at 8000 r / min for 8 min, and the supernatant after centrifugation was collected and filtered through a 0.22 µm microporous filter membrane to obtain the culture supernatant of Bifidobacterium bifidum SX-P824, which was put into a freeze-dryer and freeze-dried for 72 h to obtain the freeze-dried powder of the culture supernatant of Bifidobacterium bifidum SX-P824; the bacterial cell precipitate after centrifugation was collected and resuspended in PBS with the same volume as the bacterial solution before centrifugation to obtain the bacterial suspension of Bifidobacterium bifidum SX-P824, which was then put into a freeze-dryer and freeze-dried for 72 h to obtain the freeze-dried powder of the bacterial cells of Bifidobacterium bifidum SX-P824.

[0069] 2. Determination of lipid-lowering ability

[0070] (1) Cholesterol-lowering ability

[0071] 1) Experimental method

[0072] 10 mg of the freeze-dried cells of Bifidobacterium bifidum SX-P824 prepared in this example were inoculated into 1 mL of MRS liquid medium containing 1 g / L cholesterol as the experimental group, and anaerobically cultured at 37°C. The culture broth was collected at 0 h and 48 h of culture respectively, and the supernatant was collected after centrifugation to obtain the culture supernatant (i.e., the corresponding 10 mg / mL bacterial solution) as the test sample. 10 mg of distilled water was added to 1 mL of MRS liquid medium containing 1 g / L triglyceride as the blank group, and cultured and sampled in the same way.

[0073] The content of triglyceride (C, mmol / L) in the test sample was determined using a total cholesterol (T-CHO) test kit (Nanjing Jiancheng Bioengineering Institute, A111-1-1). The specific steps were as follows: First, the test sample was centrifuged at 10000 rpm for 10 minutes, and the supernatant after centrifugation was collected as the sample to be tested; 2.5 μL of distilled water was added to 250 μL of the working solution as the blank well; 2.5 μL of the calibrator (cholesterol, 6.55 mmol / L) was added to 250 μL of the working solution as the calibration well; 2.5 μL of the sample to be tested was added to 250 μL of the working solution as the sample well; after the solutions in each well were thoroughly mixed, 100 μL of the solution was pipetted into a 96-well plate, and then incubated at 37°C for 10 min, and the OD value at a wavelength of 500 nm was read with an enzyme-linked immunosorbent assay reader.

[0074] 2) Result determination

[0075] Formula (1): Cholesterol content C (mmol / L) = [(A 样本 -A 空白 ) / (A 标准 -A 空白 )] × C 标准 ;

[0076] Formula (2): Cholesterol degradation rate (%) = (C 0 -C 48 ) / C 0 × 100%;

[0077] Among them, A 样本 、A 标准 and A 空白 are the OD 500 values of the sample well, calibration well and blank well in this experiment in sequence; C 标准 is the calibrator concentration, 6.55 mmol / L; C 0 is the cholesterol content in the medium at 0 h of culture; C 48 is the cholesterol content in the medium at 48 h of culture.

[0078] According to the OD values of the cultured bacterial solutions at 0 h and 48 h of culture, the content of cholesterol C (C, mmol / L) was calculated using formula (1), and the cholesterol degradation rate (%) was calculated using formula (2). When the cholesterol degradation rate is above 50%, it is considered that the cholesterol-lowering ability is good.

[0079] 3) Experimental results

[0080] As Figure 4 shown, compared with the blank group, the cholesterol degradation rate in the cultured bacterial solution added with the freeze-dried powder of Bifidobacterium bifidum SX-P824 was 77.208%, which was higher than 50%, indicating that Bifidobacterium bifidum SX-P824 has good cholesterol-lowering ability.

[0081] (2) Triglyceride-lowering ability

[0082] 1) Experimental method

[0083] The freeze-dried powder of 10 mg of Bifidobacterium bifidum SX-P824 prepared in this example was inoculated into 1 mL of MRS liquid medium containing 1 g / L triglyceride as the experimental group, and anaerobically cultured at 37°C. The cultured bacterial solutions were collected at 0 h and 48 h of culture respectively, and the supernatant was collected after centrifugation to obtain the culture supernatant (i.e., corresponding to 10 mg / mL of bacteria), which was used as the test sample. 10 mg of distilled water was added to 1 mL of MRS liquid medium containing 1 g / L triglyceride as the blank group, and cultured and sampled in the same way.

[0084] The content of triglyceride (TG) in the test sample was measured using a triglyceride (TG) test kit (Nanjing Jiancheng Bio, A110-1-1). The specific steps were as follows: First, the test sample was centrifuged at 10000 rpm for 10 minutes, and the supernatant after centrifugation was collected as the sample to be tested; 2.5 μL of distilled water was added to 250 μL of the working solution as the blank hole; 2.5 μL of the calibrator (triglyceride, 2.68 mmol / L) was added to 250 μL of the working solution as the calibration hole; 2.5 μL of the sample to be tested was added to 250 μL of the working solution as the sample hole; after the solutions in each hole were thoroughly mixed, 100 μL of the solution was pipetted into a 96-well plate, and then incubated at 37°C for 10 min, and the OD value at a wavelength of 500 nm was read with an enzyme-labeled instrument.

[0085] 2) Result determination

[0086] Formula (3): The content of triglyceride C' (mmol / L) = [(A' 样本 - A' 空白 ) / (A' 标准 - A' 空白 )] × C' 标准 ;

[0087] Formula (4): Triglyceride degradation rate % = (C’ 0 - C’ 48 ) / C’ 0 × 100%

[0088] Among them, A’ 样本 , A’ 标准 and A’ 空白 are the OD 500 values of the sample well, calibration well and blank well in this experiment in sequence; C’ 标准 is the calibration product concentration, 2.68 mmol / L; C’ 0 is the triglyceride content in the cultured bacterial liquid at 0 h of culture; C’ 48 is the triglyceride content in the cultured bacterial liquid at 48 h of culture.

[0089] According to the OD values of the cultured bacterial liquid at 0 h and 48 h of culture, calculate the triglyceride content (C, mmol / L) using formula (3), and calculate the triglyceride degradation rate (%) using formula (4). If the triglyceride degradation rate is above 50%, it is considered that the ability to reduce triglyceride is good.

[0090] 3) Experimental results

[0091] As Figure 5 shown, compared with the blank group, the triglyceride degradation rate in the cultured bacterial liquid with the freeze-dried powder of Bifidobacterium bifidum SX-P824 was 58.13%, higher than 50%, indicating that Bifidobacterium bifidum SX-P824 has good triglyceride-lowering ability.

[0092] 3. Antioxidant capacity

[0093] (1) Total antioxidant capacity experiment - equivalent to the concentration of Fe 2+ antioxidant capacity

[0094] Under acidic conditions, antioxidant substances can reduce Fe 3+ -TPTZ to produce blue Fe 2+ -TPTZ. According to the absorbance read at 593 nm, the concentration of Fe 2+ antioxidation can be calculated, which is regarded as the total antioxidant capacity of the sample.

[0095] 1) Experimental method

[0096] Use the total antioxidant capacity (T-AOC) assay kit (FRAP method) (Regen Biotech, TO1005-100T) to detect the antioxidant capacity of Bifidobacterium bifidum SX-P824. The specific method is as follows:

[0097] First, prepare the FRAP working solution with a volume ratio of FRAP Assay Buffer: TPTZ solution: ferric chloride solution = 10:1:1. Secondly, dilute the ferrous standard solution (10 mM) with distilled water to concentrations of 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.2, and 1.5 mM to prepare a series of Fe 2+ standard solutions.

[0098] Add 30 μL of distilled water to 264 μL of the FRAP working solution as the blank well; add 30 μL of 8 different concentrations of FeSO 4 standard solutions to 264 μL of the FRAP working solution as the standard wells; dissolve the freeze-dried culture supernatant or freeze-dried cells of Bifidobacterium bifidum SX-P824 obtained in this example with sterile water to obtain a test sample with a concentration of 10 mg / mL, and add 30 μL of the test sample to 264 μL of the FRAP working solution as the measurement well.

[0099] After thoroughly mixing the solutions in each well, pipette 100 μL of the solution into a 96-well plate, then incubate it in a water bath at 37 °C for 30 min, and read the OD value of each well at a wavelength of 593 nm using a microplate reader.

[0100] 2) Result determination

[0101] After subtracting the OD value of the blank well from the OD value of each well, the OD calibration value of each well is obtained. Using the OD calibration value of the standard wells as the abscissa and the concentration of the standard product corresponding to the OD calibration value of each standard well as the ordinate, a standard curve and the corresponding linear regression equation are constructed. Substitute the OD calibration value measured in the measurement well into the linear regression equation to obtain the antioxidant capacity of Fe 2+ corresponding to the test sample concentration of 10 mg / mL. If the Fe 2+ concentration is greater than 1 nM, it is considered that the total antioxidant capacity is good.

[0102] 3) Experimental results

[0103] The linear regression equation constructed in this example is y = 1.5927x + 0.2777 (R 2 = 0.9759). After substitution and calculation, as Figure 6 shown, compared with the blank group, the Fe 2+ concentrations of the freeze-dried cells and freeze-dried culture supernatant of Bifidobacterium bifidum SX-P824 are 0.565 nM and 1.855 nM respectively. The Fe 2+ concentration of the culture supernatant of Bifidobacterium bifidum SX-P824 is higher than 1 nM, indicating that the freeze-dried culture supernatant of Bifidobacterium bifidum SX-P824 contains antioxidant substances and has good antioxidant capacity.

[0104] (2)DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) Radical Scavenging Rate Experiment

[0105] 1) Experimental Method

[0106] The DPPH radical scavenging ability of Bifidobacterium bifidum SX-P824 was detected using a DPPH radical scavenging ability kit (Nanjing Jiancheng Bioengineering Institute, A153-1-1). The specific method is as follows:

[0107] Add 400 μL of 80 v / v% methanol solution and 600 μL of working solution to a 2 mL sterile EP tube 1 as the blank tube; use the freeze-dried cell powder or freeze-dried culture supernatant of Bifidobacterium bifidum SX-P824 obtained in this example as the test sample, and add 400 μL of 10 mg / mL sample solution and 600 μL of 80 v / v% methanol solution to a 2 mL sterile EP tube 2 as the control tube of the sample group; add 400 μL of 10 mg / mL sample solution and 600 μL of working solution to a 2 mL sterile EP tube 3 as the measurement tube of the sample group; add 400 μL of 1×PBS and 600 μL of working solution to a 2 mL sterile EP tube 4 as the measurement tube of the blank group; add 400 μL of 1×PBS and 600 μL of 80 v / v% methanol solution to a 2 mL sterile EP tube 5 as the control tube of the blank group.

[0108] Mix the solutions in the above sterile EP tubes 1-5, let them stand in the dark at room temperature (25 °C) for 30 min, then centrifuge at a speed of 4000 revolutions per minute for 5 minutes. Pipette 800 μL of the centrifuged supernatant into a cuvette, zero with 80% methanol solution, and use a visible light spectrophotometer to read the OD value at a wavelength of 517 nm.

[0109] 2) Result Judgment

[0110] Formula (5): DPPH radical scavenging rate (%) = (1 - (A’’ 测定 - A’’ 对照 ) ÷ A’’ 空白 ) × 100%;

[0111] Among them, A’’ 对照 , A’’ 测定 and A’’ 空白 are the OD 517 values of the control tube, measurement tube, and blank tube in this experiment, respectively.

[0112] According to the OD 517 value, calculate the DPPH radical scavenging rate of the measurement tube using formula (5); if the DPPH radical scavenging rate is above 50%, it is considered that the antioxidant ability of the strain is good.

[0113] 3) Experimental results

[0114] As Figure 7 shown, compared with the blank group, the DPPH radical scavenging rates of the freeze-dried cell powder and the freeze-dried culture supernatant of Bifidobacterium bifidum SX-P824 were 31.46% and 92.49% respectively. The DPPH radical scavenging rate of the freeze-dried culture supernatant of Bifidobacterium bifidum SX-P824 was higher than 50%, indicating its good antioxidant ability.

[0115] (3) Hydroxyl radical scavenging rate experiment

[0116] 1) Experimental method

[0117] The hydroxyl radical (OH - ) scavenging ability of Bifidobacterium bifidum SX-P824 was detected using a hydroxyl radical (OH

[0118] ) assay kit (Nanjing Jiancheng Bioengineering Institute, A018-1-1). The specific method is as follows: 2 O 2 Add 400 μL of distilled water to a 5 mL sterile centrifuge tube 1 as the blank well; add 200 μL of distilled water and 200 μL of 0.03% H 2 O 2 standard application solution to a 5 mL sterile centrifuge tube 2 as the standard well; add 200 μL of distilled water and 200 μL of substrate application solution to a 5 mL sterile centrifuge tube 3 as the control well; use the freeze-dried culture supernatant or the freeze-dried cell powder of Bifidobacterium bifidum SX-P824 obtained in this example as the test sample, and add 200 μL of substrate application solution and 200 μL of 10 mg / mL sample solution to a 5 mL sterile centrifuge tube 4 as the sample well.

[0119] Add 400 μL of reagent III application solution to each of the above sterile centrifuge tubes 1-4 respectively, mix well quickly, start timing at the same time, react at 37 °C for 1 minute, then immediately add 2 mL of color reagent to terminate the reaction, mix well, place at room temperature for 20 minutes, pipette 100 μL of the solution into a 96-well plate, and read the OD value of each well at a wavelength of 550 nm using an enzyme-linked immunosorbent assay reader.

[0120] 2) Result determination

[0121] Formula (6): Hydroxyl radical inhibition ability (U / mL) = (A''' 对照 - A''' 测定 ) / (A'' 标准 - A''' 空白 ) × C'' 标准 × 1 / V 样 × N;

[0122] Among them, A''' 对照 , A''' 测定 , A''标准 and A''' 空白 are the OD values of the control well, sample well, standard well, and blank well in this experiment in sequence; 550 C'' 标准 is the standard product concentration, 8.824 mmol / L; V 样 is the sampling volume, 0.2 mL; N is the dilution factor before sample testing.

[0123] According to the OD 550 value, the hydroxyl radical scavenging rate of the sample well is calculated using formula (6); if the hydroxyl radical scavenging rate is above 50%, it is considered that the antioxidant capacity of the strain is good.

[0124] 3) Experimental results

[0125] As Figure 8 shown, compared with the blank group, the hydroxyl radical scavenging rates of the freeze-dried cell powder and freeze-dried culture supernatant of Bifidobacterium bifidum SX-P824 are 64.09% and 61.71% respectively, both higher than 50%, indicating that Bifidobacterium bifidum SX-P824 has good antioxidant capacity.

[0126] 4. Determination of anti-inflammatory ability

[0127] (1) Experimental method

[0128] The Bifidobacterium bifidum SX-P824 obtained in Example 1 was resuspended in MRS medium and anaerobically cultured at 37°C until the OD600 value of the bacterial solution measured by an ultraviolet spectrophotometer was 1.6. Then, the bacterial solution was inoculated into the autoclaved MRS medium at a ratio of 2 v / v%, and anaerobically cultured at 37°C for 24 h; the cell precipitate after centrifugation was collected and resuspended in RPMI-1640 medium (containing 10% fetal bovine serum) to obtain the SX-P824 cell suspension, and the concentration of the SX-P824 cell suspension was adjusted to 1×10 8 CFU / mL.

[0129] Fresh blood of healthy volunteers was collected, and peripheral blood mononuclear cells (PBMC) were separated using human peripheral blood lymphocyte separation solution (Solarbio, P8610 / P8900). After static culture in a carbon dioxide incubator (37°C, 5% CO 2 ) for 4 hours, centrifuged at 1000 r / min for 5 min, and resuspended using RPMI-1640 medium (containing 10% fetal bovine serum) to obtain the PBMC suspension, which was adjusted to a cell density of 2×10 6 cells / mL. The PBMC suspension was added to a 96-well cell culture plate at 100 μL / well.

[0130] PBMCs were divided into a blank control group and an experimental group, with 3 replicate wells in each group. Among them, 100 μL of RPMI-1640 medium was added to the blank control group, and 100 μL of the above-mentioned SX-P824 bacterial suspension (the final concentration of Bifidobacterium bifidum SX-P824 was 5×10 7 CFU / mL) was added to the experimental group. After mixing, it was placed in a carbon dioxide incubator (37 °C, 5% CO 2 ) and cultured for 72 h. After the culture, the cell culture supernatant was collected by centrifugation at 4000 r / min for 5 min.

[0131] The concentrations of pro-inflammatory cytokines (interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α)) in the above-mentioned collected cell culture supernatant were detected using Human IL-6 ELISA KIT (Ruixin Bio, RX106126H) and Human TNF-α ELISA KIT (Ruixin Bio, RX104793H).

[0132] (2) Data analysis:

[0133] All data were statistically analyzed using GraphPad Prism 8, and the experimental data were expressed as mean ± SEM. Analyzed by t-test, the significant differences compared with the blank control group were expressed as: *P<0.05, **P<0.01, ***P<0.001.

[0134] Experimental results:

[0135] As Figure 9 shown in A and B of

[0136] compared with the blank control group, the secretion levels of IL-6 and TNF-α in the experimental group were significantly decreased. It indicates that Bifidobacterium bifidum SX-P824 has anti-inflammatory ability.

[0137] Adhesion ability is the first step for probiotics to colonize in the intestine. In probiotic research, adhesion ability is one of the important indicators to evaluate the potential of probiotics. Probiotics can competitively inhibit the adhesion of pathogenic bacteria by adhering to intestinal epithelial cells, thereby exerting probiotic effects. The Caco-2 cell model is a human cloned colon adenocarcinoma cell, and its structure and function are similar to those of differentiated small intestinal epithelial cells; the intestinal mucosal environment can be simulated through a monolayer of Caco-2 cells to test the ability of probiotics to adhere to intestinal epithelial cells. In this example, the adhesion ability of Bifidobacterium bifidum SX-P824 was determined by the experiment of the strain adhering to Caco-2 cells.

[0138] (1) Experimental method

[0139] On the first day of the experiment, Caco-2 cells were seeded at 4×104 Plate the cells at a density of [number of cells] per well into a 24-well plate and culture them in complete DMEM medium containing 10 v / v% fetal bovine serum (FBS). The next day, change to DMEM medium without antibiotics and culture until polarization. Then, co-culture with the Bifidobacterium bifidum SX-P824 bacterial suspension (0.5 mL, 10 8 CFU / mL) for 2 h. After that, observe whether the bacteria adhere to Caco-2 cells by Gram staining.

[0140] Meanwhile, after gradient dilution of the bacterial solution before and after co-culture, culture by spreading on a bacterial plate and calculate the adhesion rate according to formula (7).

[0141] Formula (7): Adhesion rate (%) = (bacterial concentration after adhesion / bacterial concentration before adhesion) × 100%.

[0142] (2) Experimental results

[0143] After statistics, the bacterial concentration of Bifidobacterium bifidum SX-P824 before adhering to Caco-2 cells is 1.56×10 8 CFU / mL, and the bacterial concentration after adhering to Caco-2 cells is 7.00×10 7 CFU / mL. Therefore, the adhesion rate of Bifidobacterium bifidum SX-P824 to Caco-2 cells is 44.87%.

[0144] As Figure 10 shown, it can be seen that Bifidobacterium bifidum SX-P824 exists on the edges and surfaces of Caco-2 cells, indicating that Bifidobacterium bifidum SX-P824 has the ability to adhere to intestinal epithelial cells and is consistent with the adhesion rate results.

[0145] Based on the above results, it is shown that the Bifidobacterium bifidum SX-P824 of the present invention has excellent lipid-lowering, antioxidant and adhesion abilities.

[0146] Example 3 In vivo function determination of Bifidobacterium bifidum SX-P824

[0147] 1. Preparation of freeze-dried powder and bacterial suspension of Bifidobacterium bifidum SX-P824

[0148] The Bifidobacterium bifidum SX-P824 obtained in Example 1 was resuspended in MRS medium and anaerobically cultured at 37 °C until the OD600 value of the bacterial solution was 1.6 as measured by an ultraviolet spectrophotometer. Then, the bacterial solution was inoculated into the autoclaved MRS liquid medium at a ratio of 2 v / v%, and anaerobically cultured at 37 °C for 24 h; centrifuged at 8000 r / min for 8 min, the supernatant after centrifugation was collected, passed through a 0.22 µm microporous filter membrane, and the culture supernatant of Bifidobacterium bifidum SX-P824 was obtained. Then, it was placed in a freeze dryer and freeze-dried for 72 h to obtain the freeze-dried powder of the culture supernatant of Bifidobacterium bifidum SX-P824. The freeze-dried powder of the culture supernatant was dissolved in physiological saline to obtain a freeze-dried powder solution; the cell precipitate after centrifugation was collected and resuspended in physiological saline, and the concentration was adjusted to 1×10 6 CFU / mL to obtain a bacterial suspension.

[0149] 2. Construction and treatment of zebrafish hyperlipidemia model

[0150] (1) Construction of zebrafish hyperlipidemia model

[0151] Twenty-five normally developed 5 dpf (days-post fertilization) zebrafish were placed in a cell culture plate. According to 5 fish per group, they were randomly divided into 5 groups, which were respectively recorded as the normal group, the model group, the positive group, the freeze-dried powder group of the culture supernatant, and the bacterial body group.

[0152] Prepare E3 culture water according to the formula shown in Table 1. Add 5 mL of E3 culture water to the normal group and feed 1 mg of ordinary feed; add 5 mL of 1% glucose solution to the remaining 4 groups and feed 1 mg of high-fat feed to construct a hyperlipidemia model; the 5 groups of zebrafish were placed in an incubator at 28.5 °C and cultured for 5 d.

[0153] Table 1 Formula of E3 culture water (1L)

[0154]

[0155] (2) Treatment of zebrafish hyperlipidemia model

[0156] After the construction of the hyperlipidemia model, add 5 mL of E3 culture water to the normal group and feed 1 mg of ordinary feed (Shanghai Haisheng Biological Experimental Equipment Co., Ltd.); add 5 mL of E3 culture water to the model group and feed 1 mg of high-fat feed (Shanghai Feixi Biotechnology Co., Ltd.); add 5 mL of atorvastatin calcium solution to the positive group, and the final concentration of atorvastatin calcium is 2.5 μg / mL, and feed 1 mg of high-fat feed; add 5 mL of freeze-dried powder solution to the freeze-dried powder group of the culture supernatant, and the final concentration of the freeze-dried powder of the culture supernatant is 0.125 mg / mL, and feed 1 mg of high-fat feed; add 5 mL of 1×106 CFU / mL bacterial suspension, fed with 1 mg of high-fat diet. Placed in an incubator at 28.5 °C for a total of 4 days of treatment.

[0157] (3)Determination of body fat content in zebrafish hyperlipidemia model - Oil Red O staining

[0158] After the treatment, discard the solution in each well, wash the zebrafish twice with E3 culture water, add 4% paraformaldehyde solution and fix at 4 °C, wash twice with PBS, dehydrate with 1,2-propanediol gradient, then place the zebrafish in a cell culture plate and add Oil Red O staining solution. After staining, decolorize with 1,2-propanediol, observe under a stereomicroscope and take pictures for recording. Use Image J software to analyze the Oil Red O staining of body fat in zebrafish of each group, count the corresponding gray value (S), and take the ratio of the gray value of each group to that of the normal group as the relative fat level (%) of zebrafish in each group. Taking the model group as an example: zebrafish relative fat level (%) = (S 模型组 / S 正常组 ) × 100%.

[0159] All data were statistically analyzed using GraphPad Prism 8, and the experimental data were expressed as mean ± SEM. Analyzed by t-test, compared with the normal group: #P < 0.05, ##P < 0.01, P < 0.001; analyzed by t-test, compared with the model group: &P < 0.05, &&P < 0.01, &&&P < 0.001; analyzed by one-way ANOVA, compared with the model group: *P < 0.05, **P < 0.01, ***P < 0.001.

[0160] (4)Experimental results

[0161] As Figure 11 shown in A and B of, the Oil Red O staining area in the body of zebrafish in the model group was significantly larger than that in the normal group, indicating that the zebrafish hyperlipidemia model was successfully constructed; the Oil Red O staining area in the body of zebrafish in the positive group was significantly smaller than that in the model group, indicating that atorvastatin calcium effectively reduced the body fat content in zebrafish; the Oil Red O staining areas in the bodies of zebrafish in the freeze-dried powder group and the bacterial body group were both significantly smaller than those in the model group, indicating that Bifidobacterium bifidum SX-P824 could reduce the body fat content in zebrafish and had a lipid-lowering effect.

[0162] Example 4 Safety evaluation of Bifidobacterium bifidum SX-P824

[0163] 1. Experimental method

[0164] Twelve male mice (ICR strain) were randomly divided into a normal saline group and an SX-P824 group, with 6 mice in each group. For 3 consecutive days, the mice in the normal saline group were gavaged with 0.1 mL / 10 g of normal saline, and the mice in the SX-P824 group were gavaged with 0.1 mL / 10 g of SX-P824 bacterial solution (1×10 8 CFU / mL). After 3 weeks of conventional feeding, the viscera (heart, liver, spleen, lung, and kidney) of the mice in each group were collected, and the organ index was calculated. The formula for calculating the organ index is: weight of internal organ / mouse body weight. The above method was used to treat 12 female mice and calculate the organ index.

[0165] 2. Experimental results

[0166] Table 2 Statistical results of mouse organ index

[0167]

[0168] As shown in Table 2, there were no significant differences in the organ indices between the normal saline group and the SX-P824 group, whether in male mice or female mice, indicating that the Bifidobacterium bifidum SX-P824 of the present invention has basically no negative impact on the viscera and has good safety.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. 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 claims of the present invention.

Claims

1. A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824, characterized in that, The Bifidobacterium bifidum ( Bifidobacterium bifidum ) The deposit number of SX-P824 is CCTCC No: M 2025034, and it was deposited in the China Center for Type Culture Collection on January 6, 2025.

2. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum )Application of SX-P824 in the preparation of microbial agents with anti-inflammatory function.

3. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum )Application of SX-P824 in the preparation of medicines for treating hyperlipidemia.

4. The use according to claim 2 or 3, characterized in that: The Bifidobacterium bifidum ( Bifidobacterium bifidum ) The 16S rDNA sequence of SX-P824 is shown in SEQ ID NO:

3.

5. A microbial agent, characterized in that: Comprising the Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum )SX-P824.

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