Bifidobacterium bifidum JYBB-322 for improving mitochondrial activity of myocardial cells, metagen preparation and application of metagen preparation

Abstract

The invention relates to the technical field of probiotics, in particular to bifidobacterium bifidum JYBB-322 for improving mitochondrial activity of myocardial cells, a metagen preparation and application of the bifidobacterium bifidum JYBB-322 and the metagen preparation. The bifidobacterium bifidum JYBB-322 is preserved in the China General Microbiological Culture Collection Center on September 20, 2023, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.28499. The invention also discloses a preparation method of the bifidobacterium bifidum JYBB-322. The bifidobacterium bifidum JYBB-322 provided by the invention has the effects of resisting oxidation and the like, and can reduce the apoptosis level of cells in a body and relieve oxidative damage of mitochondria in the body. By taking the metagen preparation provided by the invention, the mitochondrial activity of myocardial cells can be improved, no side effect exists, and the negative influence caused by the use of medicines is effectively avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a strain of Bifidobacterium bifidum JYBB-322 for improving the mitochondrial activity of cardiomyocytes, a postbiotic preparation and applications thereof. Background Art

[0002] The activity of mitochondria is of great significance to heart health. Mitochondria convert nutrients such as fatty acids and glucose into adenosine triphosphate (ATP) through the process of oxidative phosphorylation. Myocardial cells require a continuous and large amount of energy supply to maintain rhythmic contraction and relaxation to ensure the stability of blood circulation. If mitochondrial activity is impaired and adenosine triphosphate is insufficiently produced, myocardial contractility will be weakened, and the heart's pumping function will decline. If this continues for a long time, it is very likely to cause serious heart diseases such as heart failure.

[0003] At the same time, mitochondria also play a key role in maintaining the homeostasis of the internal environment of cardiomyocytes. They participate in regulating intracellular calcium homeostasis to ensure the normal excitation-contraction coupling of cardiomyocytes. Mitochondria also play an important role in the regulation of cell apoptosis. Appropriate mitochondrial activity can prevent excessive apoptosis of cardiomyocytes, maintain the stability of cardiomyocyte number and function, and play a strong protective role in the normal structure and function of the heart.

[0004] Given the important role of cardiomyocyte mitochondrial activity in maintaining heart health, researchers have been exploring various effective ways to improve it. Some drugs are used to improve cardiomyocyte mitochondrial activity, such as Levocarnitine. Levocarnitine can promote lipid metabolism, transport long-chain fatty acids to the mitochondrial matrix for oxidative decomposition, provide more energy for cells, and thus increase the activity of cardiomyocyte mitochondria and promote cardiomyocyte metabolism. However, the use of chemical drugs also has certain disadvantages. Some patients may experience adverse reactions such as gastrointestinal discomfort and allergies after taking Levocarnitine, and long-term use may also increase the burden on the liver and kidneys. Coenzyme Q10 is also often used to enhance mitochondrial function. It plays an important role in cellular respiration and metabolism and can help mitochondria produce energy more efficiently. However, the supplementation effect of coenzyme Q10 varies greatly from person to person, and long-term and large-scale use may have a certain inhibitory effect on the body's ability to synthesize coenzyme Q10.

[0005] Performing aerobic exercise is a natural and effective way to enhance mitochondrial activity. Exercise can increase the activity of enzymes involved in energy metabolism in mitochondria, improve the efficiency of mitochondrial oxidative phosphorylation, and enhance the energy supply capacity of cardiomyocytes. However, the effects of exercise do not occur overnight and require long-term adherence and appropriate exercise intensity and frequency. Moreover, excessive exercise may instead lead to increased oxidative stress and damage to mitochondria. For some people with severe heart diseases or poor physical conditions, there are certain limitations and risks in implementing exercise programs.

[0006] There are also studies indicating that certain specific probiotic strains, such as Lactobacillus plantarum ( Lactobacillus plantarum ) TCI999, have shown the ability to enhance mitochondrial activity in cell experiments (Chinese Invention Patent CN110734869 B). However, the effects of different probiotic strains may vary, and currently, there is a lack of Bifidobacterium bifidum with the function of improving mitochondrial activity in cardiomyocytes. Summary of the Invention

[0007] Aiming at the technical problem of the lack of Bifidobacterium bifidum with the function of improving mitochondrial activity in cardiomyocytes, the present invention provides a strain of Bifidobacterium bifidum JYBB-322 for improving mitochondrial activity in cardiomyocytes, a postbiotic preparation, and its applications.

[0008] The technical solution of the present invention is as follows: In the first aspect, the present invention provides a strain of Bifidobacterium bifidum JYBB-322 for improving mitochondrial activity in cardiomyocytes. Bifidobacterium bifidum ( Bifidobacterium bifidum ) JYBB-322 is deposited in the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being September 20, 2023, and the deposit number being CGMCC No. 28499. The 16S rDNA sequence of Bifidobacterium bifidum JYBB-322 is as shown in SEQ ID No: 1.

[0009] In the second aspect, the present invention provides a postbiotic preparation for improving mitochondrial activity in cardiomyocytes, and the postbiotic preparation contains the above-mentioned Bifidobacterium bifidum JYBB-322.

[0010] It should be further noted that the postbiotic preparation for improving mitochondrial activity in cardiomyocytes provided by the present invention also contains auxiliary substances allowed in the pharmaceutical field, such as carriers, fillers, diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, surfactants, etc.; preferably, the postbiotic preparation for improving mitochondrial activity in cardiomyocytes provided by the present invention also contains maltodextrin.

[0011] It should be further noted that the dosage form of the postbiotic preparation for improving myocardial cell mitochondrial activity provided by the present invention can be selected from one of tablets, powders, granules, capsules, dripping pills, sustained-release agents, suspensions, oral liquid preparations, injections, etc., and the administration method can be selected as oral administration or parenteral methods (intravenous injection, subcutaneous injection, etc.); preferably, the dosage form of the postbiotic preparation for improving myocardial cell mitochondrial activity provided by the present invention is powder, and the administration method is oral administration.

[0012] It should be further noted that the cell count of Bifidobacterium bifidum JYBB-322 in the postbiotic preparation for improving myocardial cell mitochondrial activity provided by the present invention is 1.0×10 7 cfu / g to 1.0×10 8 cfu / g; preferably, the cell count of Bifidobacterium bifidum JYBB-322 in the postbiotic preparation for improving myocardial cell mitochondrial activity provided by the present invention is 1.0×10 8 cfu / g.

[0013] It should be further noted that the postbiotic preparation for improving myocardial cell mitochondrial activity provided by the present invention is prepared according to the following preparation method: After heat-inactivating the bacterial solution of Bifidobacterium bifidum JYBB-322, it is concentrated and freeze-dried to obtain postbiotic powder; the postbiotic powder is mixed with maltodextrin to prepare a postbiotic preparation with a cell count of 1.0×10 7 cfu / g to 1.0×10 8 cfu / g.

[0014] It should be further noted that the bacterial solution of Bifidobacterium bifidum JYBB-322 is prepared according to the following preparation method: Transfer the activated single colony of Bifidobacterium bifidum JYBB-322 to MRS liquid medium, and culture it statically at a constant temperature of 37°C to obtain a seed solution; Inoculate the seed solution into MRS liquid medium at an inoculation amount of 1% by mass, and culture it statically at a constant temperature of 37°C to obtain a bacterial solution.

[0015] It should be further noted that the MRS liquid medium is prepared according to the following preparation method: Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate tribasic, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, dissolve with distilled water and make up the volume to 1 L, adjust the pH value of the solution to 6.8, and sterilize it at 121°C and 0.1 MPa for 20 min to obtain it.

[0016] Thirdly, the present invention provides an application of the above-mentioned Bifidobacterium bifidum JYBB-322 in the preparation of a postbiotic preparation for improving the mitochondrial activity of cardiomyocytes.

[0017] The beneficial effects of the present invention are as follows: The Bifidobacterium bifidum JYBB-322 provided by the present invention has effects such as antioxidation, and can reduce the level of apoptosis in vivo and alleviate the oxidative damage of mitochondria in the body. Taking the postbiotic preparation provided by the present invention can improve the mitochondrial activity of cardiomyocytes, has no side effects, and effectively avoids the negative effects caused by the use of drugs. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is a bar chart of the mitochondrial activity of cardiomyocytes of mice in each group in Example 3.

[0020] Figure 2 It is a bar chart of the ATP content of cardiomyocytes of mice in each group in Example 3.

[0021] Figure 3 It is a bar chart of the mitochondrial DNA integrity of cardiomyocytes of mice in each group in Example 4.

[0022] Figure 4 It is a bar chart of the reactive oxygen species level in cardiomyocytes of mice in each group in Example 5.

[0023] Figure 5 It is a bar chart of the Bcl-2 protein expression level in cardiomyocytes of mice in each group in Example 6.

[0024] Figure 6 It is a bar chart of the P53 protein expression level in cardiomyocytes of mice in each group in Example 6.

[0025] Figure 7 It is a bar chart of the Caspase-3 protein expression level in cardiomyocytes of mice in each group in Example 6.

[0026] Figure 8 It is a bar chart of the Caspase-9 protein expression level in cardiomyocytes of mice in each group in Example 6.

[0027] In the figure, different lowercase letters indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences. Detailed Embodiments

[0028] To enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the 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 protection scope of the present invention.

[0029] Example 1 Isolation and Identification of Bifidobacterium bifidum JYBB-322 1. Strain Collection and Purification (1) Sampling In February 2021, infant feces were collected from Zhangqiu District, Jinan City, Shandong Province, and transported back to the laboratory under cold chain for standby.

[0030] (2) Isolation and Purification of Candidate Strains ① Take 1 g of infant feces and add it to a conical flask containing sterile normal saline, stir and shake at 4°C for 30 min, and set aside.

[0031] ② Gradient dilute the solution in step ① with sterile normal saline, dilute it from 10 -1 to 10 -7 for a total of 7 gradients, and set aside.

[0032] ③ Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 5 g of calcium carbonate, 15 g of agar, dissolve with distilled water and make up to 1 L, adjust the pH value of the solution to 6.8, heat and mix evenly, sterilize at 121°C and 0.1 MPa for 20 min, pour the sterilized medium into petri dishes, and let it cool to obtain MRS plate medium.

[0033] ④ Use a spreader to spread the solutions with different dilution concentrations prepared in step ② onto the MRS plate medium respectively, place them in an anaerobic incubator, and incubate statically at 37°C for 48 h.

[0034] ⑤ Select 22 single colonies according to the characteristics of colony diameter of 1 mm - 2 mm, round colony, neat edge, slightly white, with a bulge in the middle and a relatively large calcium dissolution circle. Inoculate them onto the MRS plate medium by the streaking method, culture at 37°C under anaerobic conditions for 48 h, repeat the above operation 2 - 3 times, pick the single colonies, place them in glycerol tubes and store at -70°C as candidate strains.

[0035] (3) Strain Screening Myocardial cell mitochondrial dysfunction generates a large amount of reactive oxygen species (ROS), causing damage to the heart. Through the ability of strains to scavenge oxidative free radicals, strains with the potential to alleviate mitochondrial dysfunction were initially screened.

[0036] The specific screening method is as follows: ① Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate tribasic, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, dissolve with distilled water and make up to 1 L, adjust the pH value of the solution to 6.8, heat and mix well, sterilize at 121 °C and 0.1 MPa for 20 min, and obtain MRS liquid medium after cooling.

[0037] ② Pick one inoculation loop of single colonies of the above 22 candidate strains respectively and inoculate them into MRS liquid medium, culture at 37 °C for 24 h to obtain fermentation broth for standby.

[0038] ③ Transfer 400 μL of the fermentation broth prepared from 22 different strains into culture tubes, then take 1 mL of DPPH-ethanol solution with a concentration of 0.2 mM and add it to each culture tube respectively, place in the dark at room temperature for 60 min. Then use a visible light spectrophotometer to detect the absorbance value A of the solution at 517 nm.

[0039] Transfer 400 μL of distilled water into a culture tube, then take 1 mL of DPPH-ethanol solution with a concentration of 0.2 mM and add it to the culture tube, place in the dark at room temperature for 60 min. Then use a visible light spectrophotometer to detect the absorbance value A0 of the solution at 517 nm.

[0040] Calculate the scavenging rate of the candidate strains for reactive oxygen species according to the following formula.

[0041] Scavenging rate (%) = ((A0 - A) / A0) × 100%.

[0042] Compare the scavenging rates of each candidate strain, screen out a candidate strain with the highest scavenging rate for reactive oxygen species. The specific scavenging rate data of this candidate strain for reactive oxygen species is 88%. Identify, preserve this strain, and use it for subsequent experiments.

[0043] (4)Identification of the strain Send the candidate strain with the highest scavenging rate for reactive oxygen species screened above to Sangon Biotech (Shanghai) Co., Ltd. for identification.

[0044] After identification, the 16S rDNA sequence (SEQ ID No: 1) of this candidate strain is as follows:

[0045] The identification results were compared with the database by BLAST to confirm that the candidate strain was Bifidobacterium bifidum ( Bifidobacterium bifidum ). Based on this, the candidate strain was named Bifidobacterium bifidum JYBB-322.

[0046] (5) Preservation of the strain Bifidobacterium bifidum JYBB-322 was sent to the China General Microbiological Culture Collection Center for preservation. The preservation information is as follows: Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: September 20, 2023; Preservation number: CGMCC No. 28499; Taxonomic name: Bifidobacterium bifidum Bifidobacterium bifidum .

[0047] Example 2 Preparation of postbiotic preparation S1: Activate Bifidobacterium bifidum JYBB-322 preserved at -80°C on MRS plate medium. The preparation method of MRS plate medium is as follows: Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 5 g of calcium carbonate, 15 g of agar, dissolve with distilled water and make up the volume to 1 L, adjust the pH value of the solution to 6.8, heat and mix well, sterilize at 121°C and 0.1 MPa for 20 min, pour the sterilized medium into petri dishes, and let it cool to obtain.

[0048] S2: Then, pick 1 single colony after activation and transfer it to 100 mL of MRS liquid medium, and incubate it statically at 37°C for 12 h to obtain a seed solution. The MRS liquid medium is prepared as follows: Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, dissolve with distilled water and make up the volume to 1 L, adjust the pH value of the solution to 6.8, heat and mix well, sterilize at 121°C and 0.1 MPa for 20 min, and let it cool to obtain.

[0049] S3: Inoculate the seed solution into MRS liquid medium (prepared in the same way as in step S2) at an inoculation amount of 1% by mass, and then incubate it statically at 37°C under anaerobic conditions for 24 h to obtain a bacterial solution.

[0050] S4: Heat inactivate the bacterial solution at 115 °C for 30 min, and then obtain the postbiotic powder through concentration and freeze-drying; S5: Mix the postbiotic powder with maltodextrin (purchased from Baolingbao Biology Co., Ltd.) and prepare postbiotic preparations containing 1.0×10 7 cfu / g, 5.0×10 7 cfu / g and 1.0×10 8 cfu / g respectively.

[0051] Example 3 Effects of Bifidobacterium bifidum JYBB-322 on mitochondrial activity and adenosine triphosphate content of cardiomyocytes Purchase 10 male 6-month-old SPF-grade Kunming mice and 40 male 6-month-old senescence-accelerated mouse prone 8 (SAMP8) mice.

[0052] After 2 weeks of adaptive feeding, 10 SPF-grade Kunming mice were used as the control group and were only treated with intragastric administration of 100 μL of gelatin aqueous solution; Randomly divide the SAMP8 mice into 4 groups as follows: Model group: 10 SAMP8 mice, only treated with intragastric administration of 100 μL of gelatin aqueous solution; Group A: 10 SAMP8 mice, treated with intragastric administration of 0.02 g of the 1.0×10 8 cfu / g postbiotic preparation prepared in Example 2 + 100 μL of gelatin aqueous solution every day; Group B: 10 SAMP8 mice, treated with intragastric administration of 0.04 g of the 1.0×10 8 cfu / g postbiotic preparation prepared in Example 2 + 100 μL of gelatin aqueous solution every day; Group C: 10 SAMP8 mice, treated with intragastric administration of 0.06 g of the 1.0×10 8 cfu / g postbiotic preparation prepared in Example 2 + 100 μL of gelatin aqueous solution every day; Among them, the preparation method of the gelatin aqueous solution is: add 0.1 g of gelatin to 1000 mL of pure water, and then place it in a water bath at 60 °C and heat it to completely dissolve the gelatin, thus obtaining it.

[0053] Change the drinking water of each group of mice every two days, continue to feed maintenance feed during the treatment period, and the treatment time lasts for 2 weeks.

[0054] After 2 weeks of feeding, the mice in each group were dissected, and fresh heart tissues were taken. Mitochondria were extracted according to the operating steps of the mitochondrial extraction kit (Solarbio). Immunofluorescence staining of the heart tissues was performed using the fluorescent probe staining method (JC-1), and then the mitochondrial membrane potential of cardiomyocytes was detected by flow cytometry. The mitochondrial activity was calculated using the FlowJo analysis software.

[0055] The results are as Figure 1 shown. It can be seen that the mitochondrial activity of the model group was significantly lower than that of the control group, indicating that there was impaired mitochondrial activity in the rapid aging mouse model. Groups A, B, and C were all gavaged with the postbiotic preparation containing Bifidobacterium bifidum JYBB-322. Among them, Group A used a lower dose (0.02 g) of the postbiotic preparation, and its mitochondrial activity was higher than that of the model group, initially showing the promoting effect of this postbiotic preparation on mitochondrial activity. The mitochondrial activities of Group B (0.04 g of the postbiotic preparation) and Group C (0.06 g of the postbiotic preparation) were even more significantly higher than that of the model group and closer to the control group level, which strongly proved that the postbiotic preparation prepared in Example 2 could effectively improve the mitochondrial activity of cardiomyocytes in rapidly aging mice.

[0056] The fresh heart tissues of each group were ground, and then according to the instructions of the ATP content assay kit (Nanjing Jiancheng), the adenosine triphosphate content in cardiomyocytes of each group was measured by colorimetry.

[0057] The results are as Figure 2 shown. It can be seen that the adenosine triphosphate content of the model group was significantly lower than that of the control group, indicating that there was insufficient adenosine triphosphate production in the rapid aging mouse model. Group A was gavaged with a lower dose (0.02 g) of the postbiotic preparation, and its adenosine triphosphate content was higher than that of the model group, indicating that this postbiotic preparation had a certain effect on increasing the adenosine triphosphate content. Groups B and C were gavaged with 0.04 g and 0.06 g of the postbiotic preparation respectively, and the adenosine triphosphate contents of both were significantly higher than that of the model group and were relatively close to the control group. This fully proved that the postbiotic preparation prepared in Example 2 could effectively increase the adenosine triphosphate content of cardiomyocytes in rapidly aging mice.

[0058] Overall, Bifidobacterium bifidum JYBB-322 could effectively improve the mitochondrial activity of cardiomyocytes in rapidly aging mice by reducing mitochondrial oxidative damage and other mechanisms, which had a positive significance for maintaining the energy supply and internal environment homeostasis of heart health. At the same time, Bifidobacterium bifidum JYBB-322 could also effectively improve the adenosine triphosphate production level of cardiomyocytes in rapidly aging mice, which had a positive effect on maintaining the energy supply of cardiomyocytes.

[0059] Example 4 Effect of Bifidobacterium bifidum JYBB-322 on the integrity of mitochondrial DNA in cardiomyocytes Mitochondrial DNA is particularly vulnerable to oxidative damage because it is close to the electron transport chain, which produces reactive oxygen species as a by-product. When mitochondrial DNA is damaged, mitochondrial DNA fragments are released into the cytoplasm, triggering an immune response and leading to the development of various diseases. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a biomarker of oxidative stress, and its detection is commonly used as an indicator of mitochondrial DNA (mtDNA) damage.

[0060] Therefore, fluorescence staining analysis of 8-hydroxy-2'-deoxyguanosine in cardiomyocytes of each group of mice in Example 3 was performed to detect the integrity of mitochondrial DNA in cardiomyocytes. The specific steps are as follows: (1) Prepare the sample: Centrifuge the sample to be tested and transfer the precipitate to a new centrifuge tube. Wash the precipitate once with PBS, and then resuspend the cell pellet in an appropriate buffer.

[0061] (2) Prepare the slide: Immerse the slide in 95% alcohol for 10 min, then wash it 3 times with sterile deionized water for 10 min each time. Air-dry the slide and then coat the slide with a layer of poly-L-lysine.

[0062] (3) Fix the cardiomyocytes: Drop the suspended cardiomyocytes onto the poly-L-lysine-coated slide and let them stand for 20 min to allow the cardiomyocytes to adhere to the slide. Then fix the cells with methanol at room temperature and wash them 3 times with PBS after 10 min.

[0063] (4) Permeabilization: Use 3% Triton X-100 permeabilizer (PBST) to rupture the cell membrane for 1 h to make the cell membrane permeable and facilitate the entry of antibodies into the cells.

[0064] (5) Blocking: Immerse the slide in 10% goat serum for 30 min to block the binding of non-specific antibodies to cardiomyocytes.

[0065] (6) Add the primary antibody: Add the 8-OHdG antibody to bind to the immunogen of the cardiomyocytes to be tested. Place the slide in a box with high humidity and incubate it overnight in a 4°C refrigerator.

[0066] (7) Washing: Wash the slide with PBS buffer to wash away the unbound antibodies.

[0067] (8) Add the secondary antibody: Add the fluorescently labeled secondary antibody to bind to the primary antibody and form a complex.

[0068] (9) Washing: Wash the slide with PBS buffer to wash away the unbound secondary antibody.

[0069] (10)Nuclear staining: Add DAPI nuclear stain, stain the cell nuclei for 5 min, and wash off the floating stain with PBS buffer.

[0070] (11)Mounting: Mount the slides using an anti-fluorescence quenching mounting medium.

[0071] (12)Microscopic observation: Observe the slides under a fluorescence microscope, detect the immunofluorescence signals in the samples, and calculate the mitochondrial DNA damage rate according to the formula: damage rate = number of cells emitting red fluorescence in the image / total number of cells in the image × 100%.

[0072] The results are as Figure 3 shown. It can be seen that the mitochondrial DNA damage rate of the control group is relatively low, indicating that the mitochondrial DNA of normal mouse cardiomyocytes is less damaged by oxidation. The mitochondrial DNA damage rate of the model group is significantly higher than that of the control group, indicating that the mitochondrial DNA of the rapid aging mouse model has suffered relatively severe oxidative damage, which is consistent with the fact that mitochondria are close to the electron transport chain and are vulnerable to attack by reactive oxygen species. Group A was gavaged with 0.02 g of postbiotic preparation, and its mitochondrial DNA damage rate was lower than that of the model group, showing that the postbiotic preparation has a certain effect of reducing mitochondrial DNA damage. The mitochondrial DNA damage rates of Group B (0.04 g of postbiotic preparation) and Group C (0.06 g of postbiotic preparation) are even lower, and the damage rate of Group B is the lowest, which fully proves that the Bifidobacterium bifidum JYBB-322 provided by the present invention can significantly reduce the oxidative damage of mitochondrial DNA in cardiomyocytes of rapid aging mice by exerting antioxidant effects, which has a positive significance for maintaining the integrity of mitochondrial DNA and heart health. The postbiotic preparation prepared in Example 2 can effectively reduce the damage rate of mitochondrial DNA in cardiomyocytes of rapid aging mice.

[0073] Example 5 Effect of Bifidobacterium bifidum JYBB-322 on the level of reactive oxygen species in cardiomyocytes Continue to use the mice in each group in Example 3 as experimental subjects. Take the freshly dissected heart tissues, and detect the level of reactive oxygen species in the myocardial tissues of each group of mice according to the instructions of the ROS detection kit (Nanjing Jiancheng).

[0074] The results are as Figure 4As shown, it can be seen that the level of reactive oxygen species (ROS) in the control group is relatively low, indicating that the oxidative stress in the myocardial cells of normal mice is at a relatively low level. The level of ROS in the model group is significantly higher than that in the control group, suggesting that there is a significant increase in oxidative stress in the rapid aging mouse model, which can damage cell structures such as mitochondria. Group A was gavaged with 0.02 g of postbiotics preparation, and its ROS level was lower than that in the model group, indicating that this postbiotics preparation has a certain effect of reducing the ROS level in myocardial cells. The ROS levels in Group B (0.04 g of postbiotics preparation) and Group C (0.06 g of postbiotics preparation) were further reduced, and the ROS level in Group C was relatively lower. This fully demonstrates that the postbiotics preparation prepared in Example 2 can effectively reduce the ROS level in the myocardial cells of rapid aging mice.

[0075] In summary, Bifidobacterium bifidum JYBB-322 significantly reduces the ROS level in the myocardial cells of rapid aging mice by exerting its antioxidant effect, alleviates the damage of oxidative stress to cell structures such as mitochondria, and has a positive significance for maintaining the homeostasis of the intracellular environment of myocardial cells and heart health.

[0076] Example 6 Effect of Bifidobacterium bifidum JYBB-322 on the expression levels of apoptosis-related proteins (Bcl-2, P53, Caspase-3, and Caspase-9) in myocardial cells Continuing with the mice in each group in Example 3 as experimental subjects, fresh heart tissues obtained from dissection were taken, and protein extraction was performed using a BCA protein extraction kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.). Then, the protein expression levels of Bcl-2, P53, Caspase-3, and Caspase-9 in mouse myocardial cells were detected according to the experimental method of Western Blot.

[0077] The specific operation steps are as follows: S1. Sample preparation: Protein samples are prepared by lysing cells or tissues and extracting proteins.

[0078] S2. Protein quantification: A BCA protein extraction kit is used to determine the protein concentration of the samples.

[0079] S3. Protein separation: A gel is prepared using an SDS-PAGE gel preparation kit (Shanghai Beyotime Biotechnology Co., Ltd.), and electrophoresis is performed for protein separation.

[0080] S4. Protein transfer: The separated proteins are transferred from the gel to a PVDF membrane through a semi-dry transfer membrane system device.

[0081] S5. Blocking: The non-specific binding sites on the membrane are incubated in a blocking agent solution containing 5% bovine serum albumin.

[0082] S6, Primary antibody incubation: Incubate with a specific primary antibody for the target protein.

[0083] S7, Secondary antibody incubation: Add a secondary antibody labeled with horseradish peroxidase for incubation, and the secondary antibody binds to the primary antibody.

[0084] S8, Signal detection: Detect the signal generated by the bound secondary antibody, and then use a gel imaging system (Thermo Fisher Scientific) to visually observe and analyze the signal.

[0085] S9, Data analysis: Perform grayscale analysis on the signal emitted by the target protein band in the image analysis software Image J to quantify the signal intensity of the target protein.

[0086] The expression levels of Bcl-2 protein in cardiomyocytes of mice in each group were as Figure 5 shown. It can be seen that the expression level of Bcl-2 protein in the control group was relatively high. Bcl-2 protein is an anti-apoptotic protein, indicating that cardiomyocytes of normal mice have a strong anti-apoptotic ability. The expression level of Bcl-2 protein in the model group was significantly lower than that in the control group, suggesting that the anti-apoptotic ability of cardiomyocytes in the rapid aging mouse model decreased, and cells were more prone to apoptosis. Group A was gavaged with 0.02 g of postbiotic preparation, and its Bcl-2 protein expression level was higher than that in the model group, indicating that this postbiotic preparation could increase the expression of Bcl-2 protein in cardiomyocytes to a certain extent and enhance the anti-apoptotic ability. The Bcl-2 protein expression levels in Group B (0.04 g of postbiotic preparation) and Group C (0.06 g of postbiotic preparation) were further increased, and the Bcl-2 protein expression level in Group C was close to that in the control group, fully demonstrating that the postbiotic preparation prepared in Example 2 could effectively increase the expression level of Bcl-2 protein in cardiomyocytes of rapid aging mice.

[0087] The expression levels of P53 protein in cardiomyocytes of mice in each group were as Figure 6 shown. It can be seen that the expression level of P53 protein in the model group was significantly higher than that in the control group. P53 protein is an important tumor suppressor protein and also plays a key role in the regulation of cell apoptosis. The high expression in the model group reflects that there may be an abnormal apoptosis regulation state in cardiomyocytes of rapid aging mice. Group A was gavaged with 0.02 g of postbiotic preparation, and its P53 protein expression level was lower than that in the model group, indicating that this postbiotic preparation could initially regulate the expression of P53 protein. The P53 protein expression levels in Group B (0.04 g of postbiotic preparation) and Group C (0.06 g of postbiotic preparation) were further decreased and were close to the expression level in the control group, fully demonstrating that the postbiotic preparation prepared in Example 2 could effectively reduce the expression level of P53 protein in cardiomyocytes of rapid aging mice.

[0088] The expression levels of Caspase-3 protein in cardiomyocytes of each group of mice were as Figure 7 shown. It can be seen that the expression level of Caspase-3 protein in the model group was significantly higher than that in the control group. Caspase-3 is a key execution protein in the process of apoptosis, and its high expression means a higher level of apoptosis in cardiomyocytes of the rapid aging mouse model. Group A was gavaged with 0.02 g of postbiotics preparation, and the expression level of Caspase-3 protein in it was lower than that in the model group, indicating that this postbiotics preparation could initially inhibit the expression of Caspase-3 protein in cardiomyocytes, thereby reducing the level of apoptosis. The expression levels of Caspase-3 protein in Group B (0.04 g of postbiotics preparation) and Group C (0.06 g of postbiotics preparation) were further reduced and closer to that in the control group, fully demonstrating that the postbiotics preparation prepared in Example 2 could effectively inhibit the expression of Caspase-3 protein in cardiomyocytes of rapid aging mice.

[0089] The expression levels of Caspase-9 protein in cardiomyocytes of each group of mice were as Figure 8 shown. It can be seen that the expression level of Caspase-9 protein in the model group was significantly higher than that in the control group. Caspase-9 is a key initiating protein in the endogenous apoptosis pathway, and its high expression indicates that the endogenous apoptosis pathway in cardiomyocytes of the rapid aging mouse model was strongly activated. Group A was gavaged with 0.02 g of postbiotics preparation, and the expression level of Caspase-9 protein in it was lower than that in the model group, indicating that this postbiotics preparation could inhibit the expression of Caspase-9 protein in cardiomyocytes to a certain extent, thereby inhibiting the activation of the endogenous apoptosis pathway. The expression levels of Caspase-9 protein in Group B (0.04 g of postbiotics preparation) and Group C (0.06 g of postbiotics preparation) were further reduced and closer to that in the control group, fully demonstrating that the postbiotics preparation prepared in Example 2 could effectively inhibit the expression of Caspase-9 protein in cardiomyocytes of rapid aging mice.

[0090] Generally speaking, after the intervention of Bifidobacterium bifidum JYBB-322, the expression level of Bcl-2 protein in cardiomyocytes of mice increased, the expression level of P53 protein in cardiomyocytes of mice decreased, the expressions of Caspase-3 protein and Caspase-9 protein were inhibited, and the anti-apoptotic ability of cardiomyocytes of rapid aging mice was enhanced, indicating that Bifidobacterium bifidum JYBB-322 could improve the mitochondrial activity of cardiomyocytes, avoid mitochondrial damage, effectively prevent the apoptosis condition caused by insufficient adenosine triphosphate production, and thus achieved the positive effects of protecting heart health, maintaining stable heart function, and delaying heart aging.

[0091] Although the present invention has been described in detail by reference to the accompanying drawings and in conjunction with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention.

Claims

1. A strain of Bifidobacterium bifidum JYBB-322 for improving mitochondrial activity of cardiomyocytes, characterized in that: Bifidobacterium bifidum ( Bifidobacterium bifidum ) JYBB-322 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is September 20, 2023, and the deposit number is CGMCC No.28499.

2. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes, characterized in that: The postbiotic preparation comprises the Bifidobacterium bifidum JYBB-322 as claimed in claim 1.

3. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 2, characterized in that: Postbiotic preparations also contain maltodextrin.

4. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 2 or 3, characterized in that: The dosage form of postbiotic preparations is powder.

5. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 2 or 3, characterized in that: The number of Bifidobacterium bifidum JYBB-322 in the postbiotic preparation was 1.0×10 7 cfu / g~1.0×10 8 cfu / g.

6. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 5, characterized in that: The number of Bifidobacterium bifidum JYBB-322 in the postbiotic preparation was 1.0×10 8 cfu / g.

7. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 3, characterized in that: The postbiotic preparation is prepared according to the following preparation method: The bacterial liquid of Bifidobacterium bifidum JYBB-322 was heat-inactivated, concentrated, and freeze-dried to obtain postbiotic powder, which was then mixed with maltodextrin to prepare a mixture containing 1.0×10 7 cfu / g~1.0×10 8 cfu / g of postbiotic preparations.

8. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 7, characterized in that: The bacterial solution of Bifidobacterium bifidum JYBB-322 was prepared according to the following preparation method: The activated single colony of Bifidobacterium bifidum JYBB-322 was transferred to MRS liquid culture medium and cultured at 37°C to obtain seed liquid; The seed liquid was inoculated into MRS liquid culture medium at an inoculum rate of 1% by mass, and cultured at a constant temperature of 37°C to obtain a bacterial liquid.

9. A postbiotic preparation for improving mitochondrial activity of cardiomyocytes according to claim 8, characterized in that: MRS liquid culture medium was prepared according to the following preparation method: Take 10 g of peptone, 5 g of beef extract powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate, dissolve them in distilled water and make up to 1 L, adjust the pH value of the solution to 6.8, and sterilize at 121°C and 0.1 MPa for 20 min.

10. Use of the Bifidobacterium bifidum JYBB-322 according to claim 1 in the preparation of a postbiotic preparation for improving mitochondrial activity of cardiomyocytes.

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