Bifidobacterium bifidum SX-P824 and application of bifidobacterium bifidum SX-P824 in preparation of lipid-lowering and / or antioxidant products

Abstract

The invention discloses a strain of bifidobacterium bifidum SX-P824 and an application of the strain of bifidobacterium bifidum SX-P824 in preparation of a product for lowering lipid and / or resisting oxidation. The preservation number of the bifidobacterium bifidum SX-P824 is CCTCC (China Center for Type Culture Collection) No: M 2025034, and the bifidobacterium bifidum SX-P824 is preserved in the China Center for Type Culture Collection on January 6, 2025. The bifidobacterium bifidum SX-P824 provided by the invention can obviously reduce the fat content in a hyperlipidemia animal model body, can degrade cholesterol and triglyceride, has good antioxidant and anti-inflammatory effects, and has good application prospects in the aspects of expanding the application field of bifidobacterium bifidum and developing probiotic preparations for preventing and treating hyperlipidemia and resisting oxidative stress. Wide popularization and application values are realized.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of Bifidobacterium bifidum SX-P824 and application thereof in preparing lipid-lowering and / or anti-oxidation products. Background Art

[0002] Hyperlipidemia has become a common metabolic disease. Typical symptoms include dizziness, fatigue, limb numbness, and possible xanthomas. In severe cases, it may lead to serious complications such as coronary heart disease and cerebral stroke. Hyperlipidemia can easily 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), which produces a large number of free radicals, attacks biological macromolecules, destroys cells, and activates inflammatory pathways. Under oxidative stress, the body's antioxidant defense system is unbalanced and cannot effectively remove excessive free radicals, which will affect the metabolism and regulation of lipids by organs such as the liver, and aggravate lipid metabolism disorders in patients with hyperlipidemia.

[0003] At present, there are many kinds of drugs used clinically for lipid-lowering. Commonly used Western medicines include statins, such as atorvastatin calcium, which can lower lipids and have certain antioxidant effects, but may cause side effects such as muscle pain, increased creatine kinase, abnormal liver function, and possible digestive tract discomfort. Therefore, it is necessary to develop safer and more effective lipid-lowering and antioxidant methods.

[0004] Probiotics are a class of active microorganisms that are beneficial to the human body and have been widely used in the development of functional foods or dietary supplements. Bifidobacterium bifidum ) is a facultative anaerobic, non-motile, non-spore-forming bacillus or long rod, Gram-positive, catalase-negative, and widely found 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 multiple physiological functions such as regulating the immune system and promoting the absorption of nutrients, but there are few research and application developments in lipid-lowering and anti-oxidation. It is urgent to further explore the application value of Bifidobacterium bifidum in the field of lipid-lowering and anti-oxidation, and develop related products with better lipid-lowering and anti-oxidation 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 use in preparing lipid-lowering and / or anti-oxidation products.

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

[0007] A second object of the present invention is to provide the Bifidobacterium bifidum ( Bifidobacterium bifidum )Application of SX-P824 in the preparation of lipid-lowering products.

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

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

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

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

[0012] The seventh object of the present invention is to provide the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of products having any two or three functions of lipid-lowering, anti-oxidation or anti-inflammatory.

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

[0014] In order to achieve the above object, the present invention is implemented by the following scheme: The invention provides a newly isolated Bifidobacterium bifidum, which is derived from fresh feces of centenarians in Suixi County, Zhanjiang City, Guangdong Province, has the functions of lowering cholesterol, lowering triglycerides, resisting oxidation and lowering blood lipids, and has good adhesion.

[0015] Therefore, the present invention requests protection for the following contents: A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824, with the deposit number of CCTCCNO: M 2025034, was deposited in China Center for Type Culture Collection (CCTCC) on January 6, 2025, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School, China Center for Type Culture Collection, Postal Code: 430072, and the classification name is Bifidobacterium bifidum ( Bifidobacterium bifidum ), deposited under the name Bifidobacterium bifidum SX-P824.

[0016] The Bifidobacterium bifidum ( Bifidobacterium bifidum) Application of SX-P824 in the preparation of lipid-lowering products. The lipid-lowering involved in the present invention refers to lowering blood lipid levels, and the Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824 is used for, including but not limited to, lowering the level of cholesterol and / or triglycerides in the body or blood.

[0017] The Bifidobacterium bifidum ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of products for degrading cholesterol and / or triglycerides.

[0018] The Bifidobacterium bifidum ( Bifidobacterium bifidum ) Use of SX-P824 in the preparation of products for preventing and / or treating hyperlipidemia.

[0019] The Bifidobacterium bifidum ( Bifidobacterium bifidum )Application of SX-P824 in the preparation of antioxidant products.

[0020] The Bifidobacterium bifidum ( Bifidobacterium bifidum )Application of SX-P824 in the preparation of lipid-lowering and antioxidant products.

[0021] The Bifidobacterium bifidum ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of a product for scavenging free radicals. The Bifidobacterium bifidum described in the application ( Bifidobacterium bifidum ) Free radicals used for scavenging include but are not limited to DPPH and hydroxyl radicals.

[0022] The Bifidobacterium bifidum ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of anti-inflammatory products.

[0023] The Bifidobacterium bifidum ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of products having any two or three functions of lipid-lowering, anti-oxidation or anti-inflammatory.

[0024] Preferably, the Bifidobacterium bifidum ( Bifidobacterium bifidum ) The 16S rDNA sequence of SX-P824 is shown in SEQ ID NO:3.

[0025] Preferably, the Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824 includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) at least one of live bacteria, culture solution or culture supernatant of SX-P824.

[0026] More preferably, it also includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Lyophilized powder of SX-P824 bacteria.

[0027] More preferably, it also includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Lyophilized powder of culture supernatant of SX-P824.

[0028] More preferably, it also includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Bacterial suspension of SX-P824.

[0029] A microbial agent comprising the Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824.

[0030] Preferably, the Bifidobacterium bifidum ( Bifidobacterium bifidum ) The 16S rDNA sequence of SX-P824 is shown in SEQ ID NO:3.

[0031] Preferably, the Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824 includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) at least one of live bacteria, culture solution or culture supernatant of SX-P824.

[0032] More preferably, it also includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Lyophilized powder of SX-P824 bacteria.

[0033] More preferably, it also includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Lyophilized powder of culture supernatant of SX-P824.

[0034] More preferably, it also includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) Bacterial suspension of SX-P824.

[0035] Compared with the prior art, the present invention has the following beneficial effects: 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 broad promotion and application value in expanding the application field of Bifidobacterium bifidum and developing probiotic preparations for preventing and treating hyperlipidemia and resisting oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the colony morphology of Bifidobacterium bifidum SX-P824.

[0037] Figure 2 This is a microscopic morphological observation of Bifidobacterium bifidum SX-P824 after Gram staining, with a magnification of 10×100.

[0038] Figure 3This is the comparison and identification result of the 16SrDNA sequence of Bifidobacterium bifidum SX-P824 at NCBI.

[0039] Figure 4 This is a statistical graph of the in vitro cholesterol degradation rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the cholesterol degradation rate is 50%.

[0040] Figure 5 This is a statistical graph of the in vitro triglyceride degradation rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the triglyceride degradation rate is 50%.

[0041] Figure 6 The total antioxidant capacity of Bifidobacterium bifidum SX-P824 in vitro is statistically analyzed. The dotted line in the figure indicates the same Fe 2+ The antioxidant concentration was 1 nM.

[0042] Figure 7 This is a statistical result diagram of the 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%.

[0043] Figure 8 This is a statistical result diagram of the in vitro hydroxyl radical scavenging rate of Bifidobacterium bifidum SX-P824. The dotted line in the figure indicates that the hydroxyl radical scavenging rate is 50%.

[0044] Figure 9 Figure 3 is the effect of in vitro stimulation of PBMC by Bifidobacterium bifidum SX-P824 on the secretion level of proinflammatory cytokines. A is the statistical result of IL-6 secretion level of PBMC in each group, and B is the statistical result of TNF-α secretion level of PBMC in each group.

[0045] Figure 10 This is the Gram staining image of Bifidobacterium bifidum SX-P824 after adhesion to Caco-2 cells, with a magnification of 10×40.

[0046] Figure 11 The figure shows the therapeutic effect of Bifidobacterium bifidum SX-P824 on the zebrafish hyperlipidemia model. A shows the Oil Red O staining results of each group of zebrafish hyperlipidemia models. B shows the statistical diagram of the relative concentration of body fat in the zebrafish hyperlipidemia model. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0048] Example 1 Screening and identification of Bifidobacterium bifidum SX-P824 1. Isolation of strains Fresh stool samples from centenarians in Suixi County, Zhanjiang City, Guangdong Province were diluted in gradients and inoculated into MRS solid medium, cultured anaerobically at 37°C for 48 hours, and single colonies on the plates were streaked. The streaking culture was performed three times to obtain purified monoclonal strains. The monoclonal strains obtained by three generations of streaking were inoculated into MRS liquid medium, cultured anaerobically at 37°C for 48 hours, and 30v / v% glycerol (containing 0.5w / v% cysteine) was added. The bacterial liquid was mixed with 30% glycerol in equal volumes and stored in a -80°C refrigerator.

[0049] The method for streaking the strain plate is as follows: in the anaerobic workstation, after mixing the bacterial solution, immerse the disposable inoculation loop into the bottom of the bacterial solution and scrape it repeatedly three times to ensure that the bacterial solution is smeared on the inoculation loop, use the inoculation loop to streak three areas on the culture medium plate, and record the label.

[0050] 2. Morphological identification of strains (1) Colony morphology observation The isolated monoclonal strain was streaked on a plate and its colony morphology was as follows: Figure 1 As shown, it is round, with a smooth convex surface, milky white and opaque.

[0051] (2) Gram staining and microscopic morphological observation After the isolated monoclonal strains were subjected to Gram staining and examined under an oil immersion microscope, Gram-positive strains appeared purple and Gram-negative strains appeared red. Figure 2 As shown, the strain is purple and is a Gram-positive bacillus.

[0052] 3. Molecular biological identification of strains The isolated monoclonal strain was subjected to full-length 16S rDNA amplification by PCR, and 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)), and then the amplified product was subjected to electrophoresis, and the electrophoresis product was collected for sequencing, and the 16S rDNA sequence of the monoclonal strain was obtained as follows:

[0053] The 16S rDNA sequence of the monoclonal strain (SEQ ID NO: 3) was compared with the standard bacterial species sequence at NCBI for identification. Figure 3 As shown, the comparison results showed that the monoclonal strain was Bifidobacterium bifidum, and was named Bifidobacterium bifidum SX-P824.

[0054] 4. Strain preservation The above-obtained Bifidobacterium bifidum SX-P824 has been deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School, zip code: 430072), with a preservation date of January 6, 2025, and is classified and named Bifidobacterium bifidum ( Bifidobacterium bifidum ), the deposit number is CCTCC NO: M 2025034, the deposit name is Bifidobacterium bifidum SX-P824.

[0055] Example 2 In vitro functional assay of Bifidobacterium bifidum SX-P824 1. Preparation of bacterial suspension, culture supernatant and lyophilized powder of Bifidobacterium bifidum SX-P824 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, and then the bacterial solution was inoculated into the high-pressure sterilized MRS medium at a ratio of 2v / v%, and anaerobically cultured at 37°C for 24 hours; the cultured bacterial solution was collected and centrifuged at 8000 r / min for 8 minutes, the supernatant after centrifugation was collected, and passed through a 0.22 µm microporous filter membrane to obtain the culture supernatant of Bifidobacterium bifidum SX-P824, which was placed in a freeze dryer and freeze-dried for 72 hours to obtain a culture supernatant freeze-dried powder of Bifidobacterium bifidum SX-P824; the bacterial precipitate after centrifugation was collected and resuspended in PBS with an equal volume of the bacterial solution before centrifugation to obtain a bacterial suspension of Bifidobacterium bifidum SX-P824, and then placed in a freeze dryer and freeze-dried for 72 hours to obtain a bacterial freeze-dried powder of Bifidobacterium bifidum SX-P824.

[0056] 2. Lipid-lowering ability determination (1) Cholesterol-lowering ability 1) Experimental methods 10 mg of the lyophilized powder of Bifidobacterium bifidum SX-P824 prepared in this example was inoculated into 1 mL of MRS liquid culture medium containing 1 g / L cholesterol as the experimental group, and anaerobically cultured at 37°C, and the cultured bacterial liquid was collected at 0 h and 48 h of culture, and the supernatant was collected after centrifugation to obtain the culture supernatant (i.e., corresponding to 10 mg / mL bacterial liquid) as the test sample. 10 mg of distilled water was added into 1 mL of MRS liquid culture medium containing 1 g / L triglyceride as the blank group, and cultured and sampled in the same manner.

[0057] The total cholesterol (T-CHO) test kit (Nanjing Jiancheng Biological, A111-1-1) was used to determine the triglyceride content (C, mmol / L) in the test sample. The specific steps are as follows: first, the test sample was centrifuged at 10,000 rpm for 10 minutes, and the supernatant after centrifugation was collected as the test sample; 2.5 μL of distilled water was added to 250 μL of working solution as a blank well; 2.5 μL of calibrator (cholesterol, 6.55 mmol / L) was added to 250 μL of working solution as a calibration well; 2.5 μL of the test sample was added to 250 μL of working solution as a sample well; after the solution in each well was fully mixed, 100 μL of the solution was pipetted into each 96-well plate, and then incubated at 37°C for 10 minutes, and the OD value at a wavelength of 500 nm was read by an enzyme reader.

[0058] 2) Result determination Formula (1): Cholesterol content C (mmol / L) = [(A 样本 -A 空白 ) / (A 标准 -A 空白 )]×C 标准 ; Formula (2): Cholesterol degradation rate (%) = (C0-C 48 ) / C0×100%; Among them, A 样本 , A 标准 and A 空白 These are the OD values ​​of the sample well, calibration well, and blank well in this experiment. 500 Value; C 标准 is the concentration of the calibrator, 6.55mmol / L; C0 is the cholesterol content in the culture medium at 0h of culture; C 48 Cholesterol content in the culture medium after 48 h of culture.

[0059] According to the OD values ​​of the culture solution at 0h and 48h, the cholesterol content C (C, mmol / L) was calculated using formula (1), and the cholesterol degradation rate (%) was calculated using formula (2). A cholesterol degradation rate of more than 50% was considered to be good in cholesterol-lowering ability.

[0060] 3) Experimental results like Figure 4 As shown, compared with the blank group, the cholesterol degradation rate in the culture solution with freeze-dried powder of Bifidobacterium bifidum SX-P824 was 77.208%, which was higher than 50%, indicating that Bifidobacterium bifidum SX-P824 had a good cholesterol-lowering ability.

[0061] (2) Triglyceride-lowering ability 1) Experimental methods 10 mg of the lyophilized powder of Bifidobacterium bifidum SX-P824 prepared in this example was inoculated into 1 mL of MRS liquid culture medium containing 1 g / L triglyceride as the experimental group, and anaerobically cultured at 37°C, and the culture solution was collected at 0 h and 48 h of culture, and the supernatant was collected after centrifugation to obtain the culture supernatant (i.e., corresponding to 10 mg / mL of bacteria) as the test sample. 10 mg of distilled water was added into 1 mL of MRS liquid culture medium containing 1 g / L triglyceride as the blank group, and cultured and sampled in the same manner.

[0062] The triglyceride (TG) test kit (Nanjing Jiancheng Biological, A110-1-1) was used to determine the triglyceride content (C, mmol / L) in the test sample. The specific steps are as follows: first, the test sample was centrifuged at 10,000 rpm for 10 minutes, and the supernatant after centrifugation was collected as the test sample; 2.5 μL of distilled water was added to 250 μL of working solution as a blank well; 2.5 μL of calibration substance (triglyceride, 2.68 mmol / L) was added to 250 μL of working solution as a calibration well; 2.5 μL of the test sample was added to 250 μL of working solution as a sample well; after the solution in each well was fully mixed, 100 μL of the solution was pipetted into each 96-well plate, and then incubated at 37°C for 10 minutes, and the OD value at a wavelength of 500 nm was read by an enzyme reader.

[0063] 2) Result determination Formula (3): Triglyceride content C' (mmol / L) = [(A' 样本 -A' 空白 ) / (A' 标准 -A' 空白 )]×C' 标准 ; Formula (4): Triglyceride degradation rate % = (C'0-C' 48 ) / C'0×100% Among them, A' 样本 , A' 标准 and A' 空白 These are the OD values ​​of the sample well, calibration well, and blank well in this experiment. 500 Value; C' 标准is the concentration of the calibrant, 2.68mmol / L; C'0 is the triglyceride content in the culture solution at 0h of culture; C' 48 It is the triglyceride content in the culture medium after 48 hours of cultivation.

[0064] According to the OD values ​​of the culture solution at 0h and 48h, the triglyceride content (C, mmol / L) was calculated using formula (3), and the triglyceride degradation rate (%) was calculated using formula (4). A triglyceride degradation rate of more than 50% was considered to be good in the ability to reduce triglycerides.

[0065] 3) Experimental results like Figure 5 As shown, compared with the blank group, the triglyceride degradation rate in the culture solution with freeze-dried powder of Bifidobacterium bifidum SX-P824 was 58.13%, which was higher than 50%, indicating that Bifidobacterium bifidum SX-P824 had a good triglyceride-lowering ability.

[0066] 3. Antioxidant Capacity (1) Total antioxidant capacity test - equivalent to Fe 2+ Antioxidant capacity concentration Under acidic conditions, antioxidants can reduce Fe 3+ -TPTZ produces blue Fe 2+ -TPTZ, Fe can be calculated based on the absorbance reading at 593 nm 2+ The antioxidant concentration was regarded as the total antioxidant capacity of the sample.

[0067] 1) Experimental methods The total antioxidant capacity (T-AOC) assay kit (FRAP method) (Legen Biotechnology, TO1005-100T) was used to detect the antioxidant capacity of Bifidobacterium bifidum SX-P824. The specific method is as follows: First, prepare the FRAP working solution with a volume ratio of FRAP Assay Buffer: TPTZ solution: ferric chloride solution = 10:1:1. Then, 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 concentration gradient of Fe 2+ Standard solution.

[0068] Add 30 μL of distilled water to 264 μL of FRAP working solution as a blank well; add 30 μL of 8 different concentrations of FeSO4 standard solutions to 264 μL of FRAP working solution as standard wells; dissolve the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Bifidobacterium bifidum SX-P824 obtained in this example with sterile water to obtain a sample to be tested with a concentration of 10 mg / mL, and add 30 μL of the sample to be tested to 264 μL of FRAP working solution as a measurement well.

[0069] After the solutions in each well were thoroughly mixed, 100 μL of the solution was pipetted into each 96-well plate, which was then placed in a 37°C water bath for 30 min, and the OD value of each well at a wavelength of 593 nm was read using an enzyme reader.

[0070] 2) Result determination After subtracting the OD value of the blank well from each well, the OD calibration value of each well was obtained. The OD calibration value of the standard well was used as the horizontal coordinate, and the concentration of the standard substance corresponding to the OD calibration value of each standard well was used as the vertical coordinate. The standard curve and the corresponding linear regression equation were constructed. The OD calibration value measured in the measurement well was substituted into the linear regression equation to obtain the Fe concentration corresponding to the sample concentration of 10 mg / mL. 2+ Antioxidant capacity of Fe 2+ Concentrations greater than 1 nM are considered to have good overall antioxidant capacity.

[0071] 3) Experimental results The linear regression equation constructed in this embodiment is y=1.5927x+0.2777 (R 2 =0.9759). After substituting into the calculation, Figure 6 As shown in the figure, compared with the blank group, the Fe 2+ The concentrations were 0.565 nM and 1.855 nM, respectively, and the Fe 2+ The concentration is higher than 1 nM, indicating that the culture supernatant freeze-dried powder of Bifidobacterium bifidum SX-P824 contains antioxidant substances and has good antioxidant capacity.

[0072] (2) DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) free radical scavenging rate experiment 1) Experimental methods The DPPH free radical scavenging ability kit (Nanjing Jiancheng Biological, A153-1-1) was used to detect the DPPH free radical scavenging ability of Bifidobacterium bifidum SX-P824. The specific method is as follows: 400 μL of 80 v / v% methanol solution and 600 μL of working solution were added to 2 mL sterile EP tube 1 as a blank tube; the lyophilized powder of the cells of Bifidobacterium bifidum SX-P824 or the lyophilized powder of the culture supernatant obtained in this example was used as the sample to be tested, and 400 μL of 10 mg / mL sample solution and 600 μL of 80 v / v% methanol solution were added to 2 mL sterile EP tube 2 as the control tube of the sample group; 400 μL of 10 mg / mL sample solution and 600 μL of working solution were added to 2 mL sterile EP tube 3 as the measuring tube of the sample group; 400 μL of 1×PBS and 600 μL of working solution were added to 2 mL sterile EP tube 4 as the measuring tube of the blank group; 400 μL of 1×PBS and 600 μL of 80 v / v% methanol solution were added to 2 mL sterile EP tube 5 as the control tube of the blank group.

[0073] Mix the solutions in the above sterile EP tubes 1 to 5, keep them at room temperature 25°C in the dark for 30 min, centrifuge them at 4000 rpm for 5 min, pipette 800 μL of the supernatant after centrifugation into a cuvette, adjust to zero with 80% methanol solution, and read the OD value at a wavelength of 517 nm using a visible light spectrophotometer.

[0074] 2) Result determination Formula (5): DPPH free radical scavenging rate (%) = (1-(A'' 测定 -A'' 对照 )÷A'' 空白 )×100%; Among them, A'' 对照 、A'' 测定 and A'' 空白 are the OD values ​​of the control tube, assay tube, and blank tube in this experiment. 517 value.

[0075] According to OD 517 The DPPH free radical scavenging rate of the test tube was calculated using formula (5); if the DPPH free radical scavenging rate was above 50%, it was considered that the strain had good antioxidant capacity.

[0076] 3) Experimental results like Figure 7 As shown, compared with the blank group, the DPPH radical scavenging rates of the freeze-dried powder of the bacteria and the freeze-dried powder of the culture supernatant of Bifidobacterium bifidum SX-P824 were 31.46% and 92.49%, respectively. The DPPH radical scavenging rate of the freeze-dried powder of the culture supernatant of Bifidobacterium bifidum SX-P824 was higher than 50%, indicating that it has good antioxidant ability.

[0077] (3) Hydroxyl free radical scavenging rate experiment 1) Experimental methods Using hydroxyl radicals (OH- ) assay kit (Nanjing Jiancheng Biological, A018-1-1) was used to detect the hydroxyl radical scavenging ability of Bifidobacterium bifidum SX-P824. The specific method is as follows: Add 400 μL of distilled water to a 5 mL sterile centrifuge tube 1 as a blank well; add 200 μL of distilled water and 200 μL of 0.03% H2O2 standard application solution to a 5 mL sterile centrifuge tube 2 as a standard well; add 200 μL of distilled water and 200 μL of substrate application solution to a 5 mL sterile centrifuge tube 3 as a control well; use the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Bifidobacterium bifidum SX-P824 obtained in this example as the sample to be tested, 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 a sample well.

[0078] Add 400 μL of reagent three application solution to each of the above sterile centrifuge tubes 1 to 4, mix quickly, start timing at the same time, react at 37°C for 1 minute, then immediately add 2 mL of color developer to terminate the reaction, mix, let stand at room temperature for 20 minutes, aspirate 100 μL of the solution and add it to a 96-well plate, and read the OD value of each well at a wavelength of 550 nm using an enzyme reader.

[0079] 2) Result determination Formula (6): Hydroxyl free radical inhibition ability (U / mL) = (A''' 对照 -A''' 测定 ) / (A'' 标准 -A''' 空白 )×C'' 标准 ×1 / V 样 ×N; Among them, A''' 对照 、A''' 测定 、A'' 标准 and A''' 空白 These are the OD values ​​of the control well, sample well, standard well, and blank well in this experiment. 550 Value; C'' 标准 is the standard concentration, 8.824mmol / L; V 样 is the sampling volume, 0.2 mL; N is the dilution multiple of the sample before testing.

[0080] According to OD 550 The hydroxyl radical scavenging rate of the sample well was calculated using formula (6); a hydroxyl radical scavenging rate of more than 50% was considered to have good antioxidant capacity.

[0081] 3) Experimental results like Figure 8As shown, compared with the blank group, the hydroxyl radical scavenging rates of the freeze-dried powder of Bifidobacterium bifidum SX-P824 and the freeze-dried powder of the culture supernatant were 64.09% and 61.71%, respectively, both higher than 50%, indicating that Bifidobacterium bifidum SX-P824 has good antioxidant capacity.

[0082] 4. Determination of anti-inflammatory ability (1) Experimental methods 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. The bacterial solution was then inoculated into the autoclaved MRS medium at a ratio of 2 v / v% and anaerobically cultured at 37°C for 24 h. The bacterial precipitate after centrifugation was collected and resuspended in RPMI-1640 medium (containing 10% fetal bovine serum) to obtain a SX-P824 bacterial suspension. The concentration of the SX-P824 bacterial suspension was adjusted to 1×10 8 CFU / mL.

[0083] Fresh blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were separated using human peripheral blood lymphocyte separation medium (Solarbio, P8610 / P8900). After 4 h of static culture in a carbon dioxide incubator (37°C, 5% CO2), the cells were centrifuged at 1000 rpm for 5 min, and resuspended in RPMI-1640 medium (containing 10% fetal bovine serum) to obtain PBMC suspension, and the cell density was adjusted to 2×10 6 The PBMC suspension was added into a 96-well cell culture plate at 100 μL / well.

[0084] PBMC were divided into a blank control group and an experimental group, with 3 replicates in each group. The blank control group was added with 100 μL of RPMI-1640 medium, and the experimental group was added with the above 100 μL of SX-P824 bacterial suspension (the final concentration of Bifidobacterium bifidum SX-P824 was 5×10 7 CFU / mL), mixed well and placed in a carbon dioxide incubator (37°C, 5% CO2) for 72 hours. After the culture, centrifuge at 4000r / min for 5 minutes to collect the cell culture supernatant.

[0085] The concentrations of proinflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) in the cell culture supernatants collected above were detected using Human IL-6 ELISA KIT (Ruixin Bio, RX106126H) and Human TNF-α ELISA KIT (Ruixin Bio, RX104793H).

[0086] (2) Data analysis: All data were statistically analyzed using GraphPad Prism 8, and experimental data were expressed as mean ± SEM. t-test analysis was used, and significant differences compared with the blank control group were expressed as: *P<0.05, **P<0.01, ***P<0.001.

[0087] Experimental results: like Figure 9 As shown in A and B, compared with the blank control group, the secretion levels of IL-6 and TNF-α in the experimental group were significantly reduced, indicating that Bifidobacterium bifidum SX-P824 has anti-inflammatory ability.

[0088] 5. Adhesion ability Adhesion ability is the first step for probiotics to colonize in the intestine. In probiotic research, adhesion ability is one of the important indicators for evaluating the potential of probiotics. Probiotics can competitively inhibit the adhesion of pathogens by adhering to intestinal epithelial cells, thereby exerting a probiotic effect. The Caco-2 cell model is a human cloned colon adenocarcinoma cell, which is similar in structure and function to differentiated small intestinal epithelial cells; the intestinal mucosal environment can be simulated by the Caco-2 cell monolayer to test the ability of probiotics to adhere to intestinal epithelial cells. This example uses a strain adhesion Caco-2 cell experiment to determine the adhesion ability of Bifidobacterium bifidum SX-P824.

[0089] (1) Experimental methods On the first day of the experiment, Caco-2 cells were cultured at 4×10 4 The cells were plated at a density of 100 μg / well in a 24-well plate and cultured in DMEM complete medium containing 10 v / v% fetal bovine serum (FBS). The next day, the medium was replaced with DMEM medium without dual antibodies and cultured until polarized. The cells were then incubated with a suspension of Bifidobacterium bifidum SX-P824 (0.5 mL, 10 8 CFU / mL) for 2 h. Gram staining was then used to observe whether the bacteria adhered to the Caco-2 cells.

[0090] At the same time, the bacterial solutions before and after co-culture were diluted in a gradient manner and then cultured on bacterial plates. The adhesion rate was calculated according to formula (7).

[0091] Formula (7): Adhesion rate (%) = (bacterial concentration after adhesion / bacterial concentration before adhesion) × 100%.

[0092] (2) Experimental results According to statistics, the bacterial concentration of Bifidobacterium bifidum SX-P824 before adhering to Caco-2 cells was 1.56×10 8CFU / mL, the bacterial concentration after adhesion to Caco-2 cells was 7.00×10 7 CFU / mL, so the adhesion rate of Bifidobacterium bifidum SX-P824 to Caco-2 cells was 44.87%.

[0093] like Figure 10 As shown, it can be seen that Bifidobacterium bifidum SX-P824 exists on the edge and surface of Caco-2 cells, indicating that Bifidobacterium bifidum SX-P824 has the ability to adhere to intestinal epithelial cells, which is consistent with the adhesion rate results.

[0094] The above results show that the Bifidobacterium bifidum SX-P824 of the present invention has excellent lipid-lowering, anti-oxidation and adhesion abilities.

[0095] Example 3 In vivo functional assay of Bifidobacterium bifidum SX-P824 1. Preparation of freeze-dried powder and bacterial suspension of Bifidobacterium bifidum SX-P824 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. The bacterial solution was then inoculated into an autoclaved MRS liquid medium at a ratio of 2v / v% and anaerobically cultured at 37°C for 24 h. The supernatant was collected after centrifugation at 8000 r / min for 8 min and filtered through a 0.22 µm microporous filter membrane to obtain the culture supernatant of Bifidobacterium bifidum SX-P824. The supernatant was then placed in a freeze dryer and freeze-dried for 72 h to obtain a culture supernatant lyophilized powder of Bifidobacterium bifidum SX-P824. The culture supernatant lyophilized powder was dissolved in physiological saline to obtain a lyophilized powder solution. The bacterial precipitate after centrifugation was collected and resuspended in physiological saline to adjust the concentration to 1×10 6 CFU / mL to obtain bacterial suspension.

[0096] 2. Construction and treatment of zebrafish hyperlipidemia model (1) Construction of zebrafish hyperlipidemia model Twenty-five normally developed 5 dpf (days-post fertilization) zebrafish were placed in a cell culture plate and randomly divided into five groups, 5 in each group, namely the normal group, model group, positive group, culture supernatant freeze-dried powder group and bacterial group.

[0097] E3 culture water was prepared according to the formula shown in Table 1. 5 mL of E3 culture water was added to the normal group, and 1 mg of ordinary feed was fed; 5 mL of 1% glucose solution was added to the other 4 groups, and 1 mg of high-fat feed was fed to construct a high-fat model; the 5 groups of zebrafish were placed in a 28.5℃ incubator and cultured for 5 days.

[0098] Table 1E3 Aquaculture water formula (1L)

[0099] (2) Treatment of zebrafish hyperlipidemia model After the high-fat model was established, 5 mL of E3 culture water was added to the normal group, and 1 mg of ordinary feed (Shanghai Haisheng Biological Experiment Equipment Co., Ltd.) was fed; 5 mL of E3 culture water was added to the model group, and 1 mg of high-fat feed (Shanghai Feixi Biotechnology Co., Ltd.) was fed; 5 mL of atorvastatin calcium solution was added to the positive group, and the final concentration of atorvastatin calcium was 2.5 μg / mL, and 1 mg of high-fat feed was fed; 5 mL of freeze-dried powder solution was added to the culture supernatant freeze-dried powder group, and the final concentration of culture supernatant freeze-dried powder was 0.125 mg / mL, and 1 mg of high-fat feed was fed; 5 mL of 1×10 6 CFU / mL bacterial suspension, and fed with high-fat diet. Placed in a 28.5℃ incubator for a total of 4 days.

[0100] (3) Determination of body fat content in zebrafish hyperlipidemia model - Oil Red O staining After the treatment, the solution in each well was discarded and the zebrafish were washed twice with E3 culture water, fixed with 4% paraformaldehyde solution at 4°C, rinsed with PBS twice, dehydrated with 1,2-propylene glycol gradient, and then placed in a cell culture plate and added with Oil Red O dye solution. After staining, it was decolorized with 1,2-propylene glycol, placed under a stereo microscope for observation and photographed. Image J software was used to analyze the oil red O staining of zebrafish fat in each group, and the corresponding gray value (S) was calculated. The ratio of the gray value of each group to the normal group was used 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%.

[0101] All data were statistically analyzed using GraphPad Prism 8, and experimental data were expressed as mean ± SEM. Compared with the normal group, the results were analyzed by t-test: #P<0.05, ##P<0.01, ###P<0.001; compared with the model group, the results were analyzed by t-test: &P<0.05, &&P<0.01, &&&P<0.001; compared with the model group, the results were analyzed by one-way ANOVA: *P<0.05, **P<0.01, ***P<0.001.

[0102] (4) Experimental results like Figure 11As shown in A and B in the figure, the Oil Red O staining area in the zebrafish of the model group was significantly larger than that in the normal group, indicating that the hyperlipidemia model of zebrafish was successfully established; the Oil Red O staining area in the zebrafish of the positive group was significantly smaller than that in the model group, indicating that atorvastatin calcium effectively reduced the fat content in the zebrafish; the Oil Red O staining area in the zebrafish of the lyophilized powder group and the bacterial group was significantly smaller than that in the model group, indicating that Bifidobacterium bifidum SX-P824 can reduce the fat content in the zebrafish and has a lipid-lowering effect.

[0103] Example 4 Safety evaluation of Bifidobacterium bifidum SX-P824 1. Experimental methods Twelve male mice (strain ICR) were randomly divided into a saline group and a SX-P824 group, with 6 mice in each group. The mice in the saline group were gavaged with 0.1 mL / 10 g of 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 organs (heart, liver, spleen, lung and kidney) of each group of mice were collected to calculate the organ index. The organ index calculation formula is: organ weight / mouse body weight. Twelve female mice were treated and the organ index was calculated using the above method.

[0104] 2. Experimental results Table 2 Statistical results of mouse organ index

[0105] As shown in Table 2, there was no significant difference in organ index between the saline group and the SX-P824 group for both male and female mice, indicating that the Bifidobacterium bifidum SX-P824 of the present invention has substantially no negative impact on organs and has good safety.

[0106] 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 ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should 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 lipid-lowering products.

3. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of products for degrading cholesterol and / or triglycerides.

4. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum ) Use of SX-P824 in the preparation of products for preventing and / or treating hyperlipidemia.

5. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum )Application of SX-P824 in the preparation of antioxidant products.

6. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of anti-inflammatory products.

7. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum ) Application of SX-P824 in the preparation of products having any two or three functions of lipid-lowering, anti-oxidation or anti-inflammatory.

8. The use according to any one of claims 2 to 7, characterized in that: The Bifidobacterium bifidum ( Bifidobacterium bifidum ) The 16S rDNA sequence of SX-P824 is shown in SEQ ID NO:

3.

9. The use according to any one of claims 2 to 7, characterized in that: The Bifidobacterium bifidum ( Bifidobacterium bifidum )SX-P824 includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) at least one of live bacteria, culture solution or culture supernatant of SX-P824.

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

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