Lactobacillus fermentum for improving heart rhythm, sclerotin and blood pressure as well as product and application of lactobacillus fermentum

Abstract

The invention discloses lactobacillus fermentum for improving heart rhythm, sclerotin and blood pressure as well as a product and application thereof, and relates to the technical field of microorganisms. The lactobacillus fermentum NHNK-623 disclosed by the invention is preserved in the China Center for Type Culture Collection on November 18, 2024, and the preservation number of the lactobacillus fermentum NHNK-623 is CCTCC (China Center for Type Culture Collection) NO: M 20242574. NHNK-623 has the effects of adsorbing calcium ions, up-regulating expression of intestinal epithelial cell calcium and vitamin D absorption and transport related genes, promoting intestinal absorption and transport of calcium ions, promoting intestinal transformation of 1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1 The composition has the functions of promoting myocardial cell repair, regulating bone metabolism related genes, promoting osteoblast repair, promoting vascular endothelial cell repair and promoting blood vessels to generate nitric oxide.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a fermented Lactobacillus mucosae for improving heart rhythm, bone mass and blood pressure, as well as products and applications thereof. Background Art

[0002] Arrhythmia can affect the heart's pumping function, leading to an increase in myocardial oxygen consumption. For example, during ventricular tachycardia, the rapid and uncoordinated contraction of ventricular muscle will cause a significant increase in myocardial oxygen consumption. At the same time, due to the heart pumping function disorder caused by arrhythmia, coronary perfusion will be insufficient, and in severe cases, it can lead to myocardial infarction.

[0003] Calcium plays a crucial role in the normal electrophysiological activities and contractile functions of the heart. The action potential of normal myocardial cells depends on the activities of multiple ion channels, among which calcium ions (Ca²⁺) play a key role. Calcium deficiency will cause imbalance among other ion channels in myocardial cells, leading to abnormal repolarization of myocardial cells. In addition, the contractility of myocardial cells will weaken. This change in contractility will affect the heart's pumping function, resulting in a decrease in cardiac output. To compensate for this decline in pumping function, arrhythmia may occur. Experimental results show that the incidence of arrhythmia in animals in the low-calcium feeding group is significantly higher than that in the normal-calcium group. The specific manifestations are various types of arrhythmias, such as ventricular premature beats, tachycardia, etc.

[0004] Intestinal flora dysbiosis is closely related to cardiovascular diseases. Dysregulation of the expression of genes related to calcium absorption in intestinal epithelial cells may lead to heart problems such as arrhythmia. Arrhythmia is one of the most common complications in patients with hypertension. The incidence of atrial fibrillation in hypertensive patients is 2-3 times that in non-hypertensive populations, and about 30%-50% of atrial fibrillation patients are accompanied by hypertension.

[0005] Endothelial cells regulate vascular smooth muscle relaxation by releasing nitric oxide (NO), thereby dilating blood vessels and reducing blood pressure. In hypertension, the ability of endothelial cells to synthesize NO decreases, resulting in enhanced vasoconstriction and further elevation of blood pressure.

[0006] The relationship between vitamin D and hypertension is a research field that has received extensive attention in recent years. Animal experiments show that vitamin D deficiency causes vasoconstriction and elevated blood pressure; supplementing vitamin D can improve vascular elasticity. At the same time, it reduces the level of oxidized low-density lipoprotein (ox-LDL) and slows down vascular endothelial dysfunction. Active vitamin D activates eNOS (endothelial nitric oxide synthase) through VDR, increasing NO release and dilating blood vessels. Multiple epidemiological studies have found that the prevalence of hypertension is relatively high in populations with vitamin D deficiency, and it has been found that low vitamin D levels are associated with both elevated systolic and diastolic blood pressures.

[0007] Vitamin D (VD) is a fat-soluble vitamin, which consists of two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The two forms cannot be transported to each other, and the main source of human vitamin D is vitamin D3. Active vitamin D can directly reduce inflammatory reactions, improve endothelial cell function, and delay vascular calcification. The conversion of vitamin D into the active form of 1,25-dihydroxyvitamin D3 requires the catalysis of two key enzymes. Vitamin D is first catalyzed by 25-hydroxylase to convert into 25-hydroxyvitamin D, and then catalyzed by 1α-hydroxylase to convert into 1,25-dihydroxyvitamin D3. Intestinal epithelial cells contain vitamin D receptors (VDRs) and have both 25-hydroxylase and 1α-hydroxylase activities. Therefore, they can absorb and convert vitamin D into physiologically active 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 is the main storage form of vitamin D in the human body and a metabolite that can be detected in the blood. By detecting it, the total vitamin D situation can be determined.

[0008] Currently, the commonly used drugs for treating osteoporosis mainly include bisphosphonates, denosumab, teriparatide, etc. However, the above drugs have side effects such as gastrointestinal reactions and a sharp drop in bone density after drug withdrawal, and even increase the risk of osteosarcoma. The commonly used antihypertensive drugs mainly include enalapril, amlodipine, metoprolol, etc. However, long-term use will cause risks such as edema, headache, and bradycardia. The anti-arrhythmic drugs mainly include amiodarone, propafenone, verapamil, etc. However, most of these drugs have organ toxicity and may exacerbate heart diseases. In comparison, functional probiotics are more acceptable to the public due to their high safety and effectiveness.

[0009] Marine microorganisms have developed complex molecular adaptation abilities to cope with these harsh conditions, which affect their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes. Marine microorganisms are more likely to synthesize enzymes and secondary metabolites with unique structures than terrestrial microorganisms. Exploring the application of marine biological intestinal microorganisms has important practical significance in expanding the value of the marine industry. Summary of the Invention

[0010] In view of this, the present invention provides a fermented Lactobacillus mucosae and its products and applications for improving heart rhythm, bone mass, and blood pressure.

[0011] The present invention provides a fermented Lactobacillus mucosae for improving heart rhythm, bone mass, and blood pressure ( Limosilactobacillus fermentum), the strain is Lactobacillus mucosae NHNK-623, which was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, postal code 430072) on November 18, 2024, and its deposit number is Lactobacillus mucosae CCTCC NO: M 20242574.

[0012] The present invention also provides the above-mentioned Lactobacillus mucosae ( Limosilactobacillus fermentum ) for use in the preparation of a product for improving heart rhythm, bone mass and blood pressure.

[0013] Furthermore, the calcium and vitamin D3 absorption includes at least one of the following a)-j): a) Adsorbing calcium ions; b) Up-regulating the expression of at least one of the transient receptor potential cation channel subfamily V member 6 gene and the tight junction protein 2 gene related to calcium absorption and transport in human colon epithelial cells TRPV6 and CLD2 the tight junction protein 2 gene; c) Up-regulating the expression of the vitamin D receptor gene related to promoting the physiological action of vitamin D in human colon epithelial cells VDR ; d) Promoting intestinal absorption and transport of calcium ions; e) Promoting the conversion of vitamin D3 to active vitamin D3 in the intestine; f) Promoting cardiomyocyte repair; g) Regulating bone metabolism-related genes, up-regulating the expression of the Runt-related transcription factor 2 gene related to osteoblast bone formation Runx2 and down-regulating the expression of at least one of the homeobox genes related to bone resorption Msx2 ; h) Promoting osteoblast repair; i) Promoting vascular endothelial cell repair; j) Promoting the production of nitric oxide by blood vessels.

[0014] The present invention also provides a product for improving heart rhythm, bone mass and blood pressure, which is characterized by comprising the above-mentioned Lactobacillus mucosae ( Limosilactobacillus fermentum ), and acceptable excipients and / or adjuvants.

[0015] The Lactobacillus mucosae disclosed in the present invention ( Limosilactobacillus fermentum), and its preservation number is CCTCC NO: M 20242574. Experiments have shown that NHNK-623 has the functions of adsorbing calcium ions, upregulating the expression of genes related to calcium and vitamin D absorption and transport in intestinal epithelial cells, promoting intestinal absorption and transport of calcium ions, promoting the conversion of 1,25-dihydroxyvitamin D3 in the intestine, promoting myocardial cell repair, regulating bone metabolism-related genes, promoting osteoblast repair, promoting vascular endothelial cell repair, and promoting blood vessels to produce nitric oxide.

[0016] Biological preservation description Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum ), NHNK-623, was deposited on November 18, 2024, at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, postal code 430072), and its preservation number is CCTCC NO: M 20242574. Description of the drawings

[0017] Figure 1 This is the MRS plate colony map and Gram stain map of Lactobacillus mucosae fermentum NHNK-623 in the present invention; Figure 2 This is the experimental result map of Lactobacillus mucosae fermentum NHNK-623 increasing the survival rate of myocardial cells with oxidative damage in the present invention; Figure 3 This is the experimental result map of Lactobacillus mucosae fermentum NHNK-623 increasing the survival rate of osteoblasts with oxidative damage in the present invention; Figure 4 This is the experimental result map of Lactobacillus mucosae fermentum NHNK-623 increasing the survival rate of vascular endothelial cells with oxidative damage in the present invention.

[0018] The following specific embodiments will further illustrate the present invention in combination with the above-mentioned drawings. Specific embodiments

[0019] The present invention provides Lactobacillus mucosae fermentum and its applications. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0020] The Lactobacillus mucosae fermentum strain NHNK-623 of the present invention is derived from naturally fermented shrimp paste and is identified as Lactobacillus mucosae fermentum by 16S rDNA Limosilactobacillus fermentumThis strain is Gram-positive and appears as rod-shaped and curved under the microscope; it grows on MRS plates and can form smooth, semi-transparent, round colonies that are white and have neat edges; it grows uniformly turbid in MRS liquid medium, and after standing for a long time, the bacteria form a white precipitate. The optimum growth temperature is 37 °C.

[0021] Furthermore, in the applications described in the present invention, the Lactobacillus mucosae NHNK-623 provided by the present invention exists in the form of live bacteria, inactivated bacteria, or as a fermentation product (i.e., supernatant), and the preferred derivative forms are selected from: metabolites, metabolic bioproducts, probiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, live bacteria, and inactivated bacteria.

[0022] Example 1 Isolation of NHNK-623 The shrimp paste fermented naturally in Qingdao was shaken and mixed evenly, then diluted 10 times with physiological saline. After mixing evenly again, 100 μL of the diluted solution was spread on MRS medium and placed in an anaerobic bag. After culturing at 37 °C for 48 h, white colonies were picked and repeatedly streaked for purification until regular and uniform single colonies were obtained, which were named NHNK-623.

[0023] Gram staining and microscopic examination: The strain NHNK-623 is a Gram-positive colony, which appears as rod-shaped and curved under the microscope; it grows on MRS plates and can form white, smooth, round, semi-transparent colonies with neat edges; it can grow uniformly turbid in MRS liquid medium, and after standing for a long time, the bacteria form a white precipitate. As Figure 1 shown.

[0024] Example 2 Nucleic acid identification of NHNK-623 1. 16S rDNA gene sequence analysis: Single colonies were picked and inoculated into MRS liquid medium, cultured overnight at 37 °C, and the bacteria were collected by centrifugation at 8000 rpm for 1 min. The operation was carried out according to the instructions of the Gram-positive bacteria DNA extraction kit (bacterial DNA extraction kit, Solarbio). The primers used were the universal primers 27F and 1492R for bacterial 16S sequencing, and the PCR amplification system was 20 μL. The PCR amplification program was pre-denaturation at 95 °C for 5 min, 94 °C for 15 s, 57 °C for 15 s, 72 °C for 1 min, for 35 cycles; extension at 72 °C for 10 min.

[0025] 2. Results After the PCR products were sequenced and compared with the published standard sequences in the GenBank database (BLASTN), it was found that the NHNK-623 strain was Lactobacillus mucosae Limosilactobacillus fermentum .

[0026] Example 3: Experiment on the Adsorption of Calcium Ions by NHNK-623 1. Preparation of viable and inactivated cells of NHNK-623 Pick a single colony of Lactobacillus mucosae NHNK-623 and inoculate it into MRS liquid medium. Incubate statically at 37 °C for 48 h, centrifuge at 5000 rpm for 10 min to obtain the precipitate, wash it twice with PBS, and then resuspend the cells with PBS and adjust the OD 600 = 1.0 to obtain viable cells. Autoclave at 121 °C for 15 min to obtain inactivated cells.

[0027] 2. Experiment on the Adsorption of Calcium Ions by NHNK-623 In the experimental group, add 1 mL of 2 mmol / L CaCl2 solution and 1 mL of viable or inactivated cell suspension of NHNK-623 to the centrifuge tube. In the control group, add 1 mL of CaCl2 solution and 1 mL of PBS. After incubating at room temperature for 3 h, use a calcium ion assay kit (microplate method for calcium assay kit, Nanjing Jiancheng) to measure the absorbance value of the supernatant at 600 nm, and convert it to the concentration of calcium ions according to the kit instructions.

[0028] The calculation formula and results are shown in Table 1 below: Table 1 Adsorption of Calcium Ions by NHNK-623

[0029] The results showed that both viable and inactivated cells of NHNK-623 could adsorb calcium ions, and the adsorption rate was between 5.49% and 16.28%.

[0030] Example 4: Regulation of the Expression of Genes Related to Calcium Ion and Vitamin D Absorption and Transport in Caco-2 Cells by NHNK-623 1. Preparation of NHNK-623 Pick a single colony of Lactobacillus mucosae NHNK-623 and inoculate it into fresh MRS medium. Incubate at 37 °C for 24 h. Adjust to OD 600 = 0.5 with DMEM medium (Solarbio), take the supernatant at 5000 rpm, and then filter it through a 0.22 μm filter membrane to obtain a sterile fermentation product. Collect the centrifuged precipitate of cells, wash it twice with sterile PBS, resuspend the cells with DMEM medium and adjust the OD 600 = 0.5 to obtain a viable cell suspension. Wash a part of the precipitated viable cells twice with sterile PBS and then autoclave at 121 °C for 15 min, resuspend with DMEM medium and adjust to OD 600 = 0.5 to obtain inactivated cells.

[0031] 2. Culture of human colon epithelial cells Caco-2 After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, Caco-2 cells (BNCC350769, Beina Bio) were cultured at 37°C and 5% CO2. After the cell confluence reached 80 - 90%, subculture or plating operations were carried out.

[0032] 3. NHNK-623 regulates the expression of genes related to calcium and vitamin D absorption and transport in Caco-2 cells Caco-2 cells were seeded in 6-well cell culture plates at a density of 1×10^6 cells / well and cultured for 12 h until the cells adhered. The cell culture medium was removed and the cells were washed twice with sterile PBS. In the experimental group, 1.9 mL of DMEM medium and 100 μL of NHNK-623 live bacteria / fermentation product / inactivated bacteria suspension were added, and in the control group, an equal volume of DMEM medium was added. The cells were cultured at 37°C and 5% CO2 for 24 h. After the culture, the supernatant was discarded and the cells were washed twice with sterile PBS. Subsequently, 1 mL of cell RNA extraction reagent was added to each well, and total RNA was extracted and its concentration and purity were measured according to the reagent instructions (Trizol total RNA extraction reagent, Solarbio). After extraction, it was reverse transcribed into cDNA, and GADPH was used as the internal reference gene, and the expression levels of cation channel protein genes TRPV6, vitamin D receptor protein genes VDR and tight junction protein genes CLD2 were measured by qPCR. The relative expression fold of the control group gene F = 1, and the F value of each sample was calculated using the 2 -ΔΔCT method.

[0033] Formula: F = 2 -ΔΔCT , where: △CT 实验 = CT 实验 - CT 内参（实验）; △CT 对照 = CT 对照 - CT 内参（对照）; △△CT = △CT 实验 - △CT 对照。

[0034] The results are shown in Tables 2 to 4 below: Table 2 Regulation of genes related to calcium and vitamin D absorption and transport by NHNK-623 live bacteria

[0035] Table 3 Regulation of genes related to calcium and vitamin D absorption and transport by NHNK-623 inactivated bacteria

[0036] Table 4 Regulation of gene expression related to calcium and vitamin D absorption and transport by the fermentation product of NHNK-623

[0037] The results showed that NHNK-623 up-regulated the genes related to calcium ion absorption TRPV6 、 CLD2 and the VD receptor gene VDR , thus promoting the absorption and transport of calcium and VD by intestinal epithelial cells.

[0038] Example 5 Experiment on promoting calcium ion transport in Caco-2 cells by NHNK-623 1. Preparation of viable and inactivated cells of NHNK-623 The preparation methods of viable and inactivated cells of NHNK-623 refer to Example 4.

[0039] 2. Culture of human colon cells Caco-2 After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, Caco-2 cells were cultured at 37 °C and 5% CO2. After the cells were fused to 80-90%, passage or plating operations were carried out.

[0040] 3. Experiment on promoting calcium ion transport in Caco-2 cells by NHNK-623 When Caco-2 cells were fused to 80%, the cells were collected and adjusted to a concentration of 1×10^5 cells / mL with DMEM medium. Add 0.5 mL of cell suspension to the upper chamber of the transwell plate, and add 1.5 mL of DMEM medium to the lower chamber. Culture at 37 °C and 5% CO2, and change the medium every other day. Measure the transmembrane resistance value (TEER) of Caco-2 cells daily. When the TEER value is greater than 300 Ω / cm 2 , it can be used for the calcium ion transport experiment. Prepare a 2 mmol / L CaCl2 solution with D-Hanks solution, and add 200 μL to each well in the upper chamber of the transwell plate; add 200 μL of viable or inactivated cell suspension of NHNK-623 to the upper chamber of the transwell plate in the experimental group, and add an equal volume of DMEM medium to the control group. Culture at 37 °C and 5% CO2 for 3 h, measure the absorbance value of the lower chamber liquid at 600 nm with a calcium ion assay kit (microplate method for calcium test kit, Nanjing Jiancheng), and convert the calcium ion concentration according to the kit instructions.

[0041] The calculation formula and results are as shown in Table 5 below: Table 5 Promotion of calcium ion transport in Caco-2 cells by NHNK-623

[0042] The results showed that both viable and inactivated NHNK-623 bacteria could increase the calcium ion transport of intestinal epithelial cells, with relative growth rates ranging from 108.27% to 113.63%.

[0043] Example 6 Experiment on NHNK-623 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells 1. Preparation of viable and inactivated NHNK-623 bacteria The preparation methods of viable and inactivated NHNK-623 bacteria refer to Example 4.

[0044] 2. Culture of human colon cells Caco-2 The culture method of Caco-2 cells refers to Example 4.

[0045] 3. Experiment on NHNK-623 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells When the Caco-2 cells reached 80% confluence, the cells were collected and adjusted to a concentration of 1×10^5 cells / mL with DMEM medium. Add 0.5 mL of the cell suspension to the upper chamber of the transwell plate and 1.5 mL of DMEM medium to the lower chamber, and culture at 37°C and 5% CO2, changing the medium every other day. Measure the transmembrane resistance value (TEER) of Caco-2 cells daily. When the TEER value is greater than 300 Ω / cm 2 it can be used for the vitamin D3 transport experiment. Prepare a 100 μg / mL vitamin D3 solution with D-Hanks solution and add 200 μL to each well in the upper chamber of the transwell plate; add 200 μL of the viable or inactivated NHNK-623 bacteria suspension to the upper chamber of the transwell plate in the experimental group, and add an equal volume of DMEM medium to the control group. Culture at 37°C and 5% CO2 for 3 h, and measure the concentration of DHVD3 in the lower chamber liquid with a 1,25-dihydroxyvitamin D3 (DHVD3) enzyme-linked immunosorbent assay kit (Nanjing Jiancheng).

[0046] Table 6 NHNK-623 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells

[0047] Intestinal epithelial cells can absorb and convert vitamin D3 into 1,25-dihydroxyvitamin D3 by themselves. The results showed that both viable and inactivated NHNK-623 bacteria could further promote the absorption and conversion of vitamin D3 by intestinal epithelial cells Caco-2, increase the content of 1,25-dihydroxyvitamin D3 in the lower chamber, and the growth rate was between 7.85% and 89.42%. Therefore, NHNK-623 can promote the intestinal absorption and transport and utilization of vitamin D3.

[0048] Example 7: NHNK-623 promotes the repair of cardiomyocyte injury 1. Preparation of NHNK-623 viable bacteria suspension The preparation method of NHNK-623 viable bacteria suspension refers to Example 4.

[0049] 2. Culture of cardiomyocyte H9C2 H9C2 cells (BNCC337726, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37 °C and 5% CO2. After the cells were fused to 80-90%, passage or plating was performed.

[0050] 3. Experiment on NHNK-623 promoting the repair of cardiomyocyte injury H9C2 cells were seeded in a 96-well cell culture plate at a density of 1×10^4 cells / well and cultured for 24 h until the cells adhered. The medium was removed, and 100 μL of DMEM medium containing 1 mmol / L H2O2 was added to each well. Incubate at 37 °C and 5% CO2 for 2 h. After incubation, the medium was discarded. In the experimental group, 100 μL of DMEM medium containing 1% (v / v) NHNK-623 viable bacteria was added, and in the control group, an equal volume of DMEM medium was added. Incubate at 37 °C and 5% CO2 for 18 h, as Figure 2 shown. 10 μL of cck-8 reagent was added to each well, and the absorbance at 450 nm was measured after culturing at 37 °C for 4 h. The calculation formula and results are shown in Table 7 below: Table 7: NHNK-623 viable bacteria increase the survival rate of cardiomyocytes with oxidative injury

[0051] It can be seen from the data that NHNK-623 viable bacteria can increase the survival rate of H9C2 under oxidative injury, and the growth rate is 17.83% - 25.08%.

[0052] Example 8: NHNK-623 regulates the expression of bone metabolism-related genes in osteoblast ROS17 / 2.8 1. Preparation of NHNK-623 fermentation product and viable bacteria suspension The preparation methods of the fermentation product and viable bacteria suspension refer to Example 4.

[0053] 2. Culture of osteoblast ROS17 / 2.8 ROS17 / 2.8 cells (BNCC359280, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37 °C and 5% CO2. After the cells were fused to 80-90%, passage or plating was performed.

[0054] 3. NHNK-623 Regulates the Expression of ROS17 / 2.8 Bone Metabolism-related Genes Inoculate ROS17 / 2.8 cells at a density of 1×10^6 cells / well into a 6-well cell culture plate and incubate for 12 h until the cells adhere. Remove the cell culture medium, wash twice with sterile PBS, add 1.9 mL of DMEM medium, 100 μL of NHNK-623 fermentation product or live bacteria suspension to the experimental group, add an equal volume of DMEM medium to the control group, and culture at 37 °C and 5% CO2 for 24 h. After the culture, discard the supernatant, wash twice with sterile PBS, then add 1 mL of cell RNA extraction reagent to each well, and extract total RNA and measure its concentration and purity according to the reagent instructions (Trizol total RNA extraction reagent, Solarbio). After extraction, reverse transcribe it into cDNA, use GADPH as the internal reference gene, and measure the expression levels of bone formation-related genes Runx2 and bone resorption-related genes Msx2 by qPCR. The relative expression fold of the control group gene F = 1, and the 2 -ΔΔCT method is used to calculate the F value of each sample.

[0055] The results are shown in Tables 8 to 9 below: Table 8 NHNK-623 Fermentation Product Regulates the Expression of ROS17 / 2.8 Bone Metabolism-related Genes

[0056] Table 9 NHNK-623 Live Bacteria Regulates the Expression of ROS17 / 2.8 Bone Metabolism-related Genes

[0057] As can be seen from the data, NHNK-623 fermentation product and live bacteria can regulate the expression of Runt-related transcription factor 2 gene related to bone formation Runx2 and homeobox gene related to bone resorption Msx2 to promote bone formation and reduce bone resorption.

[0058] Example 9 NHNK-623 Promotes the Repair of Osteoblast Injury 1. Preparation of NHNK-623 Live Bacteria Suspension The preparation method of NHNK-623 live bacteria suspension refers to Example 4.

[0059] 2. Culture of Osteoblast ROS17 / 2.8 After activating ROS17 / 2.8 cells with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, culture them at 37 °C and 5% CO2. After the cells reach 80-90% confluence, perform subculture or plating operations.

[0060] 3. NHNK-623 Promotes the Repair of Osteoblast Injury Experiment Inoculate ROS17 / 2.8 cells at a density of 1×10^4 cells / well in a 96-well cell culture plate and incubate for 24 h until the cells adhere to the plate. Remove the culture medium and add 100 μL of DMEM medium containing 1 mmol / L H2O2 to each well. Incubate at 37 °C and 5% CO2 for 2 h. After incubation, discard the culture medium. Add 100 μL of DMEM medium containing 1% (v / v) viable NHNK-623 bacteria to the experimental group and an equal volume of DMEM medium to the control group. Incubate at 37 °C and 5% CO2 for 18 h, as Figure 3 shown. Add 10 μL of cck-8 reagent to each well and measure the absorbance at 450 nm after culturing at 37 °C for 4 h. The calculation formula and results are shown in Table 10 below: Table 10 Viable NHNK-623 Bacteria Increase the Survival Rate of Oxidatively Damaged Osteoblasts

[0061] The results showed that viable NHNK-623 bacteria could increase the survival rate of ROS17 / 2.8 under oxidative damage, and the growth rate was 17.61% - 31.31%.

[0062] Example 10 NHNK-623 Reduces Oxidative Damage to Vascular Endothelial Cells 1. Preparation of NHNK-623 Fermentation Product and Viable Bacteria Suspension The preparation method of NHNK-623 fermentation product and viable bacteria suspension refers to Example 4.

[0063] 2. Vascular Endothelial Cell Culture After activating EA.hy926 cells with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, culture them under the conditions of 37 °C and 5% CO2. After the cells reach 80 - 90% confluence, perform passage or plating operations.

[0064] 3. NHNK-623 Reduces Oxidative Damage to Vascular Endothelial Cells Experiment Inoculate EA.hy926 cells at a density of 1×10^4 cells / well in a 96-well cell culture plate and incubate for 24 h until the cells adhere to the plate. Remove the culture medium and add 100 μL of DMEM medium containing 1 mmol / L H2O2 to each well. Incubate at 37 °C and 5% CO2 for 2 h. After incubation, discard the culture medium. Add 100 μL of DMEM medium containing 1% (v / v) viable NHNK-623 bacteria or fermentation product to the experimental group and an equal volume of DMEM medium to the control group. Incubate at 37 °C and 5% CO2 for 18 h, as Figure 4As shown, add 10 μL of CCK-8 reagent to each well, and measure the absorbance at 450 nm after culturing at 37 °C for 4 h. The calculation formula and results are shown in Table 11 below: Table 11 NHNK-623 increases the survival rate of vascular endothelial cells with oxidative damage

[0065] The results showed that both the fermentation product and viable bacteria of NHNK-623 could improve the survival rate of vascular endothelial cells with oxidative damage, and the relative growth rate was 104.85% - 135.58%.

[0066] Example 11 NHNK-623 increases the NO content in vascular endothelial cells 1. Preparation of NHNK-623 fermentation product and viable bacteria suspension The preparation method of NHNK-623 fermentation product and viable bacteria suspension refers to Example 4.

[0067] 2. Culture of vascular endothelial cells The vascular endothelial cell line EA.hy926 (BNCC342387, Beina Biotech) was activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and then cultured at 37 °C and 5% CO2. After the cells reached 80 - 90% confluence, subculture or plating was performed.

[0068] 3. Seed 1×10^5 EA.hy926 cells per well in a 6-well cell culture plate and culture for 24 h until the cells adhered to the plate. Remove the medium, add 2 mL of DMEM medium containing 1 mmol / L H2O2 to each well, and incubate at 37 °C and 5% CO2 for 2 h. After incubation, discard the medium. Add 2 mL of DMEM medium containing 1% (v / v) NHNK-623 viable bacteria or fermentation product to the experimental group, and add an equal volume of DMEM medium to the control group. Incubate at 37 °C and 5% CO2 for 18 h. After the culture, collect the cells, wash the cells twice with PBS, collect the cell pellet, add 0.3 mL of normal saline, and ultrasonically lyse the cells under ice bath conditions. Use a NO detection kit to measure the nitric oxide content in each group. The calculation formula and results are shown in Table 12 below: Calculation formula: Table 12 NHNK-623 increases the NO content in vascular endothelial cells

[0069] The results showed that both the viable bacteria and fermentation product of NHNK-623 could increase the NO content in EA.hy926 cells.

[0070] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fermented Lactobacillus mucosae that improves heart rhythm, bone mass, and blood pressure ( Limosilactobacillus fermentum ), the strain is Lactobacillus mucosae NHNK-623, which was deposited at the China Center for Type Culture Collection on November 18, 2024, with the deposit number CCTCC NO: M 20242574.

2. The use of Lactobacillus mucosae fermentum as claimed in claim 1 Limosilactobacillus fermentum in the preparation of a product for improving heart rhythm, bone mass and blood pressure.

3. The application according to claim 2, characterized in that, The improvement of heart rhythm, bone mass, and blood pressure includes at least one of the following a)-j): a) Adsorbing calcium ions; b) Up-regulating at least one of the expressions of the transient receptor potential cation channel subfamily V member 6 gene related to calcium absorption and transport in human colon epithelial cells TRPV6 and the tight junction protein 2 gene CLD2 ; c) Up-regulating the expression of vitamin D receptor genes related to the physiological function of vitamin D in human colon epithelial cells VDR ; d) Promoting intestinal absorption and transport of calcium ions; e) Promoting the conversion of vitamin D3 to active vitamin D3 in the intestine; f) Promoting cardiomyocyte repair; g) Regulating bone metabolism-related genes, up-regulating the expression of Runt-related transcription factor 2 gene related to osteoblast bone formation, and down-regulating the expression of at least one of the homeobox genes related to bone resorption; Runx2 ; Msx2 ; h) Promoting osteoblast repair; i) Promoting vascular endothelial cell repair; j) Promoting the production of nitric oxide by blood vessels.

4. A product for improving heart rhythm, bone mass and blood pressure, characterized in that, Comprising the fermented Lactobacillus mucosae as described in claim 1 ( Limosilactobacillus fermentum ), and acceptable excipients and / or adjuvants.

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