Bifidobacterium animalis and application thereof in preparation of products for improving heart rate, sclerotin and blood pressure

Abstract

The invention relates to the technical field of microorganisms, in particular to bifidobacterium animalis and application of the bifidobacterium animalis to preparation of products for improving heart rate, bone and blood pressure. The bifidobacterium animalis is bifidobacterium animalis subsp. Lactis NHNK-625, and is preserved in the China Center for Type Culture Collection on February 28, 2025, and the preservation number of the bifidobacterium animalis is CCTCC NO: M 2025325. The traditional Chinese medicine composition has the effects of adsorbing calcium ions, promoting calcium absorption of intestinal epithelial cells and physiological action of vitamin D, promoting calcium ion transport of the intestinal epithelial cells, promoting activation and utilization of vitamin D, promoting myocardial cell repair, regulating osteoblast bone metabolism, promoting osteoblast repair and promoting vascular endothelial cell repair. The composition can be used for preparing products for improving heart rate, sclerotin and blood pressure.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to animal bifidobacteria and use thereof in preparing products for improving heart rate, bone quality and blood pressure. Background Art

[0002] Arrhythmias can affect the heart's pumping function, leading to increased myocardial oxygen consumption. For example, in ventricular tachycardia, the rapid and uncoordinated contraction of the ventricular muscle can significantly increase myocardial oxygen consumption. Furthermore, the impaired pumping function caused by arrhythmias can lead to insufficient coronary artery perfusion, which can, in severe cases, cause myocardial infarction.

[0003] Calcium plays a vital role in the normal electrophysiological activity and contractile function of the heart. The action potential of normal myocardial cells depends on the activity of multiple ion channels, among which calcium ions (Ca² + ) plays a key role. Calcium deficiency can cause an imbalance between other ion channels in myocardial cells, leading to abnormal myocardial repolarization. In addition, the contractile force of myocardial cells will be weakened. This change in contractile force will affect the heart's pumping function, resulting in a decrease in cardiac output. In order to compensate for this decrease in pumping function, arrhythmias may occur. Experimental results show that the incidence of arrhythmias in animals fed with low calcium was significantly higher than that in the normal calcium group. Specifically, it manifests as various types of arrhythmias, such as premature ventricular beats and tachycardia.

[0004] Gut microbiome imbalance is closely linked to cardiovascular disease. Dysregulation of genes involved in calcium absorption in intestinal epithelial cells may lead to heart problems such as arrhythmias. Arrhythmias are one of the most common complications in patients with hypertension. The incidence of atrial fibrillation in hypertensive patients is two to three times higher than in non-hypertensive individuals, and approximately 30% to 50% of patients with atrial fibrillation also have hypertension.

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

[0006] The relationship between vitamin D and hypertension has garnered significant research attention in recent years. Animal studies have shown that vitamin D deficiency causes vasoconstriction and elevated blood pressure; vitamin D supplementation can improve vascular elasticity. It also reduces oxidized low-density lipoprotein (ox-LDL) levels and mitigates endothelial dysfunction. Active vitamin D activates eNOS (endothelial nitric oxide synthase) via the VDR, increasing NO release and dilating blood vessels. Multiple epidemiological studies have found a higher prevalence of hypertension in people with vitamin D deficiency, and low vitamin D levels have been associated with elevated both systolic and diastolic blood pressure.

[0007] Vitamin D (VD) is a fat-soluble vitamin composed of two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The two forms cannot be transported into each other, and vitamin D3 is the primary source of vitamin D in the human body. Active vitamin D can reduce inflammation and improve endothelial cell function. The conversion of vitamin D3 into its active form, 1,25-dihydroxyvitamin D3, requires the catalysis of two key enzymes. Vitamin D3 is first converted to 25-hydroxyvitamin D3 by 25-hydroxylase and then to 1,25-dihydroxyvitamin D3 by 1α-hydroxylase. Intestinal epithelial cells contain the vitamin D receptor (VDR), which possesses both 25-hydroxylase and 1α-hydroxylase activities, allowing vitamin D3 to be absorbed and converted into the physiologically active 1,25-dihydroxyvitamin D3. 1,25-dihydroxyvitamin D3 is the primary storage form of vitamin D in the body and a metabolite that can be detected in the blood. Its detection can be used to determine total vitamin D status.

[0008] Currently, commonly used drugs for the treatment of osteoporosis include bisphosphonates, denosumab, and teriparatide. However, these drugs have side effects such as gastrointestinal reactions and a sudden drop in bone density after discontinuation, and may even increase the risk of osteosarcoma. Commonly used antihypertensive drugs include enalapril, amlodipine, and metoprolol, but long-term use can cause edema, headaches, and bradycardia. Antiarrhythmic drugs include iodine aminophenazone, propafenone, and verapamil, but most of these drugs are organ toxic and may aggravate heart disease. In comparison, probiotics, due to their high safety and effectiveness, have been reported in recent years in regulating osteoporosis and improving blood pressure and heart rate.

[0009] Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological traits and metabolic processes, with marine microorganisms being more likely to synthesize structurally distinct enzymes and secondary metabolites than terrestrial microorganisms. Exploring the applications of marine gut microorganisms has important practical implications for expanding the value of marine industries. Summary of the Invention

[0010] In response to the problems that current treatment plans for heart rate, bone mass and blood pressure are high in risk and have potential risks such as large side effects and toxicity, the present invention provides animal bifidobacterium and its use in preparing products for improving heart rate, bone mass and blood pressure. The animal bifidobacterium is animal bifidobacterium lactis subspecies NHNK-625, which can produce adsorbed calcium ions, promote calcium absorption and the physiological effects of vitamin D in intestinal epithelial cells, promote calcium ion transport in intestinal epithelial cells, promote the activation and utilization of vitamin D, promote myocardial cell repair, regulate osteoblast bone metabolism, promote osteoblast repair, and promote vascular endothelial cell repair. It can be used to prepare products for improving heart rate, bone mass and blood pressure.

[0011] The present invention first provides an animal bifidobacterium, wherein the animal bifidobacterium is animal bifidobacterium lactis subspecies ( Bifidobacterium animalis subsp. lactis ) NHNK-625, was deposited in the China Center for Type Culture Collection on February 28, 2025, and its deposit number is CCTCC NO: M 2025325.

[0012] The present invention also provides a product of the animal bifidobacterium, which is one of the live bacteria, fermentation / secretion products, and inactivated bacteria of the animal bifidobacterium lactis subspecies NHNK-625.

[0013] The preparation method of the live bacteria comprises the following steps: inoculating the animal Bifidobacterium lactis subspecies NHNK-625 into a culture medium, anaerobic culturing at 37° C. for 24-48 hours to obtain a fermentation liquid, centrifuging to obtain a precipitate, and washing the precipitate to obtain the live bacteria.

[0014] Based on the above, the method for preparing the inactivated bacteria is as follows: inactivating the above live bacteria at 121° C. for 15-30 min to obtain the inactivated bacteria.

[0015] The preparation method of the fermentation / secretion product is as follows: inoculating the animal Bifidobacterium lactis subsp. NHNK-625 into a culture medium, anaerobic culturing at 37° C. for 24-48 hours to obtain a fermentation liquid, centrifuging the supernatant, and filtering at a precision of 0.22 μm to obtain the filtrate, which is the fermentation / secretion product.

[0016] The present invention further provides use of the animal bifidobacterium or its product in preparing products for improving heart rate, bone quality and blood pressure.

[0017] Preferably, the mechanism by which the product improves heart rate, bone mass, and blood pressure is achieved through any one or more of the following methods: 1) Adsorption of calcium ions; 2) Promote calcium absorption in intestinal epithelial cells and the physiological effects of vitamin D; 3) Promote calcium ion transport in intestinal epithelial cells; 4) Promote the activation and utilization of vitamin D.

[0018] The physiological effect of promoting calcium absorption and vitamin D in intestinal epithelial cells is to upregulate genes related to intestinal epithelial cell calcium ion and vitamin D absorption and transport, and the genes related to intestinal epithelial cell calcium ion and vitamin D absorption and transport include transient receptor potential coumarin type 6 channel protein gene TRPV6 , claudin 2 gene CLD2 and vitamin D receptor protein genes VDR Any one or more of .

[0019] The said promoting the activation and utilization of vitamin D is to promote intestinal epithelial cells to convert vitamin D3 into 1,25-dihydroxyvitamin D3.

[0020] Preferably, the mechanism by which the product improves heart rate, bone mass, and blood pressure is achieved through any one or more of the following methods: 1) Promote myocardial cell repair; 2) Promote osteoblast repair; 3) Promote the repair of vascular endothelial cells.

[0021] The promotion of myocardial cell repair, osteoblast repair and vascular endothelial cell repair respectively increases the survival rate of oxidatively damaged myocardial cells, osteoblasts and vascular endothelial cells.

[0022] Preferably, the product improves heart rate, bone mass and blood pressure by regulating osteoblast bone metabolism.

[0023] The regulation of osteoblast bone metabolism includes any one or more of the following methods: 1) Upregulation of Runt-related transcription factor 2 gene Runx2 expression; 2) Upregulation of osteoblast-specific transcription factor genes Osterix expression; 3) Upregulation of osteocalcin gene Osteocalcin expression; 4) Upregulation of bone sialoglycoprotein gene Bsp expression; 5) Downregulation of zinc finger transcription factor genes Aj18 expression.

[0024] The animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp. lactis The deposit information of NHNK-625 is as follows: Deposit date: February 28, 2025; Name of depository: China Center for Type Culture Collection; Deposit number: CCTCC NO: M 2025325; Address of the depository: Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, China; Classification name: Bifidobacterium animalis subsp. lactis NHNK-625 ( Bifidobacterium animalis subsp. lactis NHNK-625).

[0025] The technical effects of this application are as follows: Experiments show that the animal Bifidobacterium lactis subspecies provided by the present invention is animal Bifidobacterium lactis subspecies NHNK-625, which can produce adsorbed calcium ions, promote calcium absorption and the physiological effects of vitamin D in intestinal epithelial cells, promote calcium ion transport in intestinal epithelial cells, promote the activation and utilization of vitamin D, promote myocardial cell repair, regulate osteoblast bone metabolism, promote osteoblast repair, and promote vascular endothelial cell repair. It can be used to prepare products that improve heart rate, bone quality, and blood pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Graphs showing the experimental results of colonies grown on a solid plate (A) and Gram staining microscopy (B) of Bifidobacterium animalis subsp. lactis NHNK-625 in Example 1 of the present invention; Figure 2 This is a graph showing the experimental results of Bifidobacterium animalis subsp. lactis NHNK-625 promoting the repair of damaged cardiomyocytes in Example 7 of the present invention, wherein A represents normal cardiomyocytes, B represents cardiomyocytes damaged by H2O2 oxidation in the control group, and C represents cardiomyocytes damaged by H2O2 oxidation treated with NHNK-625 in the experimental group; Figure 3 This is a graph showing the experimental results of Bifidobacterium animalis subsp. lactis NHNK-625 promoting osteoblast repair in Example 9 of the present invention, wherein A represents osteoblasts in a normal state, B represents osteoblasts damaged by H2O2 oxidation in the control group, and C represents osteoblasts damaged by H2O2 oxidation treated with NHNK-625 in the experimental group; Figure 4 This is a graph showing the experimental results of animal Bifidobacterium lactis subsp. lactis NHNK-625 promoting the repair of vascular endothelial cell damage in Example 10 of the present invention, wherein A represents vascular endothelial cells in a normal state, B represents vascular endothelial cells damaged by H2O2 oxidation in the control group, C represents vascular endothelial cells damaged by H2O2 oxidation treated with NHNK-625 fermentation product in the experimental group, and D represents vascular endothelial cells damaged by H2O2 oxidation treated with NHNK-625 live bacterial suspension in the experimental group. DETAILED DESCRIPTION

[0027] The present invention provides animal bifidobacterium, particularly animal bifidobacterium lactis subspecies NHNK-625 and its preparation and improving heart rate, bone, blood pressure product purposes. Those skilled in the art can refer to the content of this article and appropriately improve process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0028] The animal Bifidobacterium lactis subsp. NHNK-625 described in the present invention is derived from the intestine of Abalone diversicolor and is identified as animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp.lactis This strain is Gram-positive and rod- or rod-shaped under a microscope. It forms smooth, opaque colonies with neat, milky white, rounded protrusions on MRS solid medium (Qingdao Haibo Biotechnology Co., Ltd.) containing 0.1% (m / v) cysteine. It grows uniformly turbid in MRS liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) containing 0.1% (m / v) cysteine, and forms a white precipitate upon prolonged storage. Its optimal growth temperature is 37°C.

[0029] The animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp. lactis The deposit information of NHNK-625 is as follows: Deposit date: February 28, 2025; Name of depository: China Center for Type Culture Collection; Deposit number: CCTCC NO: M 2025325; Address of the depository: Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, China; Classification name: Bifidobacterium animalis subsp. lactis NHNK-625 ( Bifidobacterium animalis subsp.lactis NHNK-625).

[0030] Furthermore, in the application described in the present invention, the animal Bifidobacterium lactis subspecies NHNK-625 provided by the present invention exists in the form of non-sterilized live bacteria or sterilized inactivated bacteria, or in the form of fermentation / secretory products (i.e., supernatant), or in the form of derivatives. The derivative forms are preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation / secretory products, live bacteria, and inactivated bacteria.

[0031] The preparation method of the live bacteria comprises: inoculating the animal Bifidobacterium lactis subspecies NHNK-625 into a culture medium, anaerobic culturing at 37° C. for 24-48 hours to obtain a fermentation liquid, centrifuging to obtain a precipitate, and washing the precipitate to obtain the live bacteria; Based on the above, the method for preparing the inactivated bacteria is as follows: inactivating the above live bacteria at 121° C. for 15-30 min to obtain the inactivated bacteria; The preparation method of the fermentation / secretion product is as follows: inoculating the animal Bifidobacterium lactis subsp. NHNK-625 into a culture medium, anaerobic culturing at 37° C. for 24-48 hours to obtain a fermentation liquid, centrifuging the supernatant, and filtering at a precision of 0.22 μm to obtain the filtrate, which is the fermentation / secretion product.

[0032] In the following examples, the MRS liquid culture medium used was from Qingdao Haibo Biotechnology Co., Ltd., the MRS solid culture medium used was from Qingdao Haibo Biotechnology Co., Ltd., DMEM culture medium was from Beijing Solebogen Technology Co., Ltd., PBS (0.01 M, pH 7.2) was from Hefei White Shark Biotechnology Co., Ltd., penicillin-streptomycin was from Beijing Solebogen Technology Co., Ltd., and fetal bovine serum (FBS) was from Shanghai Xiaopeng Biotechnology Co., Ltd.

[0033] It should be noted that, unless otherwise specified, the reagents and consumables used in the present invention are all common commercial products. The detection reagents or kits used are operated using conventional methods in the art or according to the instructions. The present invention is further described in conjunction with the examples.

[0034] Example 1 Separation of NHNK-625 The sample was obtained from an adult, 5 cm long, 3-day-fasted abalone from a farm in Qingdao. The digestive tract was dissected using sterile instruments. After three rinses with sterile saline, the intestinal tissue was homogenized and ground. The homogenate was collected in 1 ml of sterile saline and serially diluted. The supernatant was streaked onto MRS solid plates containing 0.1% (m / v) cysteine. After anaerobic incubation at 37°C for 24–48 h, white colonies were selected and repeatedly inoculated and screened until a uniform single colony was obtained, designated NHNK-625. Gram staining microscopy: NHNK-625 is Gram-positive and appears rod-shaped under a microscope. When grown on MRS plates containing 0.1% (m / v) cysteine, it forms small white, smooth, round, and opaque round colonies. In MRS liquid medium containing 0.1% (m / v) cysteine, it grows uniformly in a turbid state and forms a white precipitate after prolonged storage. Figure 1 shown.

[0035] Example 2 Nucleic Acid Identification of NHNK-625 1. 16S rDNA gene sequence analysis: Single colonies were picked and transferred to MRS liquid medium containing 0.1% (m / v) cysteine. After anaerobically incubating overnight at 37°C, the cells were collected by centrifugation at 12,000 rpm for 1 minute and the DNA extraction kit (Bacterial DNA Extraction Kit, Beijing Solebold Technology Co., Ltd.) was used. The PCR amplification system consisted of a 50 μL system with a 5-min initial denaturation at 95°C, 35 cycles of 15 s at 94°C, 15 s at 57°C, and 40 s at 72°C, and a 10-min extension at 72°C. 2. Sequence analysis results: The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN), and it was found that the NHNK-625 strain was Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp.lactis ).

[0036] Example 3 NHNK-625 calcium ion adsorption experiment 1. Preparation of Live NHNK-625 Bacteria A single colony of animal Bifidobacterium lactis subsp. NHNK-625 was picked and placed in MRS liquid medium containing 0.1% (m / v) cysteine, incubated anaerobically at 37°C for 48 h, centrifuged at 5000 rpm for 10 min, and the precipitate was washed twice with PBS. The cells were then resuspended in PBS and the OD was adjusted. 600 =1.0 to obtain live bacterial suspension; 2. Calcium Ion Adsorption Experiment of NHNK-625 In the experimental group, 1 mL of 2 mmol / L CaCl₂ solution and 1 mL of a live NHNK-625 bacterial suspension were added to the centrifuge tube. In the control group, 1 mL of 2 mmol / L CaCl₂ solution and 1 mL of PBS (1× PBS buffer, Hefei White Shark Biotechnology Co., Ltd.) were added. After incubation at 37°C for 3 h, the absorbance of the supernatant at 600 nm was measured using a calcium ion assay kit (Calcium Test Kit Microplate Method, Nanjing Jiancheng Bioengineering Institute). The absorbance was converted to calcium ion concentration according to the kit instructions. The results are shown in Table 1.

[0037] Table 1 Adsorption rate of calcium ions by NHNK-625 The results showed that NHNK-625 could adsorb calcium ions with an adsorption rate of 14.29%~15.28%.

[0038] Example 4 NHNK-625 regulates the expression of genes related to calcium ion and vitamin D absorption and transport in intestinal epithelial cells Caco-2 1. Preparation of NHNK-625 Fermentation Products, Live Bacterial Suspensions, and Inactivated Bacteria Pick a single colony of Bifidobacterium animalis subsp. lactis NHNK-625 in fresh MRS liquid medium containing 0.1% (m / v) cysteine ​​and culture anaerobically at 37°C for 24 h. Adjust the OD to 0. 600 = 0.5, centrifuged at 5000 rpm to obtain the supernatant, and then filtered through a 0.22 μm filter membrane to obtain the filtrate to obtain the fermentation product; Collect the centrifuged precipitated cells (i.e., live cells), wash them twice with sterile PBS, resuspend them in DMEM medium and adjust the OD 600 = 0.5, to obtain a live bacterial suspension; Take part of the precipitated live bacteria, wash them twice with sterile PBS, sterilize them by high pressure at 121℃ for 15 min, and resuspend them in DMEM medium to adjust the OD 600 =0.5, and inactivated bacteria were obtained.

[0039] 2. Cultivation of Human Colonic Epithelial Cells Caco-2 (BNCC350769, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.) Caco-2 cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, cultured at 37°C and 5% CO2, and passaged or plated after the cells reached 80-90% confluence.

[0040] 3. NHNK-625 regulates the expression of genes related to calcium ion and vitamin D absorption and transport in Caco-2 cells Caco-2 cells were cultured at a rate of 1×10 6 Cells were inoculated into 6-well cell culture plates and cultured at 37°C, 5% CO2 for 12 h until the cells adhered. The cell culture medium was removed and washed twice with sterile PBS. 1.9 mL of DMEM culture medium and 100 μL of NHNK-625 live bacterial suspension / fermentation product / inactivated bacteria were added to each well of the experimental group, and an equal volume (2 mL) of DMEM culture medium was added to each well of the control group. The cells were cultured at 37°C, 5% CO2 for 24 h. After the culture was completed, the supernatant was discarded and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions (Trizol total RNA extraction reagent, Beijing Solebow Technology Co., Ltd.) and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA. GADPH As an internal reference gene, the transient receptor potential coumarin type 6 channel protein gene was determined by qPCR TRPV6、Claudin 2 gene CLD2 and vitamin D receptor protein genes VDR The relative expression of the control gene is F=1, and 2 -ΔΔCT The F value of each sample was calculated by the method. The results are shown in Table 2, Table 3 and Table 4.

[0041] Formula: F=2 -ΔΔCT ,in: △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。

[0042] Table 2 NHNK-625 live bacterial suspension up-regulated genes related to calcium and vitamin D absorption and transport in Caco-2 cells Table 3 NHNK-625 inactivated bacteria upregulates genes related to calcium and vitamin D absorption and transport in Caco-2 cells Table 4 NHNK-625 fermentation products up-regulated genes related to calcium and vitamin D absorption and transport in Caco-2 cells The results showed that NHNK-625 upregulated the expression of genes related to calcium ion and vitamin D absorption and transport, specifically upregulated the transient receptor potential coumarin type 6 channel protein gene. TRPV6 , claudin 2 gene CLD2 and vitamin D receptor protein genes VDR , thus promoting the calcium absorption of intestinal epithelial cells and the physiological effects of vitamin VD.

[0043] Example 5 Experiment on NHNK-625 promoting calcium ion transport in Caco-2 cells 1. Preparation of Live and Inactivated NHNK-625 Bacteria The preparation methods of NHNK-625 live bacterial suspension and inactivated bacterial bodies refer to Example 4; 2. Culture of Human Colonic Epithelial Cells Caco-2 The culture method of Caco-2 cells refers to Example 4; 3. NHNK-625 promotes calcium transport in Caco-2 cells When Caco-2 cells reached 80% confluency, cells were harvested and the concentration was adjusted to 1 × 10^5 cells / mL with DMEM medium to obtain a cell suspension. 0.5 mL of cell suspension was added to each well of the upper chamber of the transwell plate, and 1.5 mL of DMEM medium was added to each well of the bottom chamber. The plates were incubated at 37°C and 5% CO2, with the upper and lower chambers replaced every other day. The transmembrane electrical resistance (TEER) of the Caco-2 cells was measured daily using a transmembrane electrical resistance meter. When the TEER value was greater than 300 Ω / cm 2 It can be used for calcium ion transport experiments; A 2 mmol / L CaCl₂ solution was prepared using D-Hanks solution (Beijing Solaibao Technology Co., Ltd.). The old culture medium in the upper and lower chambers of the transwell plate was discarded. 200 μL of 2 mmol / L CaCl₂ solution was added to each well of the upper chamber, and 1.5 mL of D-Hanks solution was added to each well of the lower chamber. For the experimental group, 200 μL of a live NHNK-625 bacterial suspension or inactivated bacteria was added to each well of the upper chamber of the transwell plate. For the control group, an equal volume (200 μL) of DMEM medium was added to each well. The cells were incubated at 37°C and 5% CO₂ for 3 h. The absorbance of the lower chamber fluid at 600 nm was measured using a calcium ion assay kit (Calcium Test Kit Microplate Method, Nanjing Jiancheng Bioengineering Institute). The calcium ion concentration was calculated according to the kit instructions. The results are shown in Table 5.

[0044] Table 5 NHNK-625 promotes calcium transport in Caco-2 cells The results showed that both NHNK-625 live bacterial suspension and inactivated bacteria could increase the transport of calcium ions by intestinal epithelial cells, with a relative growth rate of 114.63%~118.27%.

[0045] Example 6 Experiment on the promotion of Caco-2 cells to convert vitamin D3 into 1,25-dihydroxyvitamin D3 by NHNK-625 1. Preparation of Live and Inactivated NHNK-625 Bacteria The preparation methods of NHNK-625 live bacterial suspension and inactivated bacterial bodies refer to Example 4; 2. Culture of Human Colonic Epithelial Cells Caco-2 The culture method of Caco-2 cells refers to Example 4; 3. Experiment on NHNK-625 promoting the conversion of vitamin D3 to 1,25-dihydroxyvitamin D3 in Caco-2 cells When Caco-2 cells reached 80% confluency, cells were harvested and the concentration was adjusted to 1 × 10^5 cells / mL using DMEM medium to prepare a cell suspension. 0.5 mL of cell suspension was added to each well of the upper chamber of the transwell plate, and 1.5 mL of DMEM medium was added to each well of the bottom chamber. The plates were incubated at 37°C and 5% CO2, with the upper and lower chambers replaced every other day. The transmembrane electrical resistance (TEER) of the Caco-2 cells was measured daily using a transmembrane electrical resistance meter. When the TEER value was greater than 300 Ω / cm 2 It can be used for experiments. A 100 μg / mL vitamin D3 solution was prepared using D-Hanks solution (Beijing Solaibao Technology Co., Ltd.). The old culture medium in the upper and lower chambers of the transwell plate was discarded. 200 μL of the vitamin D3 solution was added to each well of the upper chamber, and 1.5 mL of D-Hanks solution was added to each well of the lower chamber. For the experimental group, 200 μL of a live NHNK-625 bacterial suspension or inactivated bacteria was added to each well of the upper chamber of the transwell plate. For the control group, an equal volume (200 μL) of DMEM medium was added to each well. The cells were incubated at 37°C and 5% CO2 for 3 h. The concentration of 1,25-dihydroxyvitamin D3 (DHVD3) in the lower chamber was determined using an enzyme-linked immunosorbent assay kit (Nanjing Jiancheng Bioengineering Institute). The results are shown in Table 6 below.

[0046] Table 6 NHNK-625 promotes the conversion of Caco-2 cells to 1,25-dihydroxyvitamin D3 Intestinal epithelial cells can naturally absorb and convert vitamin D3 into 1,25-dihydroxyvitamin D3. Results showed that both live and inactivated NHNK-625 bacterial suspensions further promoted vitamin D3 absorption and conversion by intestinal epithelial Caco-2 cells, increasing 1,25-dihydroxyvitamin D3 levels in the lower compartment by a relative increase rate of 135.47% to 138.06%. Therefore, NHNK-625 can promote the activation and utilization of vitamin D3.

[0047] Example 7 NHNK-625 increases the survival rate of H9C2 cardiomyocytes induced by oxidative damage 1. Preparation of NHNK-625 Live Bacterial Suspension The preparation method of NHNK-625 live bacterial suspension is as described in Example 4; 2. Cultivation of H9C2 Cardiomyocytes (BNCC337726, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.) H9C2 cells were activated with DMEM containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin and cultured at 37°C and 5% CO2. Cells were passaged or plated after reaching 80-90% confluence. 3. NHNK-625 increases the survival rate of H9C2 cardiomyocytes induced by oxidative damage H9C2 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate and cultured at 37°C, 5% CO2 for 24 h until the cells adhered. The culture medium was removed, and 100 μL of DMEM medium containing 1 mmol / L H2O2 was added to each well. The cells were incubated at 37°C, 5% CO2 for 2 h. After incubation, the culture medium was discarded, and 100 μL of DMEM medium containing 1% (v / v) NHNK-625 live bacterial suspension was added to each well in the experimental group. An equal volume (100 μL) of DMEM medium was added to each well in the control group. The cells were incubated at 37°C, 5% CO2 for 18 h. The results are shown in Figure 3. Figure 2 As shown, 10 μL of CCK-8 reagent was added to each well, and the absorbance at 450 nm was measured after incubation at 37°C for 4 h. The calculation formula and results are as follows.

[0048] Table 7 NHNK-625 live bacteria increase the survival rate of cardiomyocytes H9C2 oxidatively damaged The results showed that live bacteria NHNK-625 could increase the survival rate of cardiomyocytes H9C2 under oxidative damage, with a growth rate of 41.75%~52.94%.

[0049] Example 8 NHNK-625 regulates the expression of ROS17 / 2.8 metabolism-related genes in osteoblasts 1. Preparation of NHNK-625 Fermentation Products and Live Bacterial Suspensions The preparation methods of the fermentation product and the live bacterial suspension are as described in Example 4. 2. Cultivation of osteoblasts ROS17 / 2.8 (BNCC359280, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.) ROS17 / 2.8 cells were activated with DMEM containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin and cultured at 37°C and 5% CO2. Cells were passaged or plated after reaching 80-90% confluence. 3. NHNK-625 regulates the expression of ROS17 / 2.8 bone metabolism-related genes ROS17 / 2.8 cells were seeded in 6-well cell culture plates at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The cell culture medium was removed and washed twice with sterile PBS. 1.9 mL of DMEM medium and 100 μL of NHNK-625 fermentation product or live bacterial suspension were added to each well of the experimental group, and an equal volume (2 mL) of DMEM medium was added to each well of the control group. The cells were cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the supernatant was discarded and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Trizol total RNA extraction reagent, Beijing Solebold Technology Co., Ltd.) was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA. GADPH The Runt-related transcription factor 2 gene related to bone formation was determined by qPCR. Runx2 , osteoblast-specific transcription factor genes Osterix , osteocalcin gene Osteocalcin , bone sialoglycoprotein gene Bsp and bone resorption-related zinc finger transcription factor genes Aj18 The relative expression of the control gene is F=1, and 2 -ΔΔCT The F value of each sample was calculated by the method, and the results are shown in Tables 8 and 9.

[0050] Table 8 NHNK-625 fermentation products regulate bone metabolism-related genes in ROS17 / 2.8 cells Table 9 NHNK-625 live bacteria regulates bone metabolism-related genes in ROS17 / 2.8 cells The results showed that NHNK-625 could upregulate the Runt-related transcription factor 2 gene related to bone formation. Runx2 , osteoblast-specific transcription factor genes Osterix , osteocalcin gene Osteocalcin , bone sialoglycoprotein gene Bsp and downregulation of bone resorption-related zinc finger transcription factor genes Aj18 expression.

[0051] Example 9 NHNK-625 increases the survival rate of osteoblasts induced by oxidative damage 1. Preparation of NHNK-625 Live Bacterial Suspension The preparation method of NHNK-625 live bacterial suspension is as described in Example 4; 2. Cultivation of ROS17 / 2.8 Osteoblasts The culture of ROS17 / 2.8 cells was carried out according to Example 8; 3. NHNK-625 increases the survival rate of osteoblasts induced by oxidative damage ROS17 / 2.8 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate and cultured for 24 h until the cells adhered. The culture medium was removed, and 100 μL of DMEM medium containing 1 mmol / L H2O2 was added to each well. The cells were incubated at 37°C and 5% CO2 for 2 h. After incubation, the culture medium was discarded, and 100 μL of DMEM medium containing 1% (v / v) NHNK-625 live bacterial suspension was added to each well of the experimental group. The same volume (100 μL) of DMEM medium was added to each well of the control group. The cells were incubated at 37°C and 5% CO2 for 18 h. The results are shown in Figure 2. Figure 3 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 4 h before measuring the absorbance at 450 nm. The calculation formula and results are as follows.

[0052] Table 10 NHNK-625 live bacteria increase the survival rate of osteoblasts damaged by oxidative damage The results showed that live bacteria NHNK-625 could increase the survival rate under ROS17 / 2.8 oxidative damage, with a growth rate of 67.32%~85.63%.

[0053] Example 10 NHNK-625 increases the survival rate of endothelial cells EA.hy926 induced by oxidative damage 1. Preparation of NHNK-625 fermentation product and live bacterial suspension The preparation of NHNK-625 fermentation product and live bacterial suspension was carried out according to Example 4; 2. Culture of endothelial cells EA.hy926 (BNCC342387, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.) Endothelial cells EA.hy926 were activated with DMEM containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin and cultured at 37°C in 5% CO2. Cells were passaged or plated after reaching 80-90% confluence. 3. NHNK-625 improves the survival rate of vascular endothelial cells oxidatively damaged EA.hy926 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate and cultured for 12 h until the cells adhered. The cell culture medium was removed, and 100 μL of DMEM medium containing 1.5 mM H2O2 was added to each well. The cells were cultured at 37°C and 5% CO2 for 6 h. After the culture period, 100 μL of DMEM medium containing 1% (v / v) NHNK-625 fermentation product or live bacterial suspension was added to each well of the experimental group, and an equal volume (100 μL) of DMEM medium was added to each well of the control group. The cells were cultured at 37°C and 5% CO2 for 24 h. The results are shown in Figure 3. Figure 4 After the incubation period, 10 μL of CCK-8 reagent was added to each well and the cells were incubated at 37°C and 5% CO2 for 2 h. The absorbance at 450 nm was measured. The results are shown in Table 11.

[0054] Calculation formula: relative survival rate (%) = 100% × (OD 450 / control group OD 450 ); Table 11 NHNK-625 increases the survival rate of EA.hy926 oxidatively damaged The results showed that NHNK-625 could increase the survival rate of EA.hy926 under oxidative damage, with the relative survival rate ranging from 107.97% to 268.73%.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent replacement, modification, etc. made by technicians in this field without any creative work within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An animal Bifidobacterium, characterized in that The animal bifidobacterium is animal bifidobacterium lactis subsp. Bifidobacterium animalis subsp. lactis ) NHNK-625, was deposited in the China Center for Type Culture Collection on February 28, 2025, and its deposit number is CCTCC NO: M 2025325.

2. The animal Bifidobacterium product according to claim 1, which is one of the live bacteria, fermentation / secretory products, and inactivated bacteria of the animal Bifidobacterium lactis subsp. NHNK-625.

3. Use of the animal bifidobacterium according to claim 1 or the product according to claim 2 in preparing products for improving heart rate, bone quality and blood pressure.

4. The use of the animal bifidobacterium or the product according to claim 3 in preparing products for improving heart rate, bone quality and blood pressure, characterized in that: The mechanism includes any one or more of the following: 1) Adsorption of calcium ions; 2) Promote calcium absorption in intestinal epithelial cells and the physiological effects of vitamin D; 3) Promote calcium ion transport in intestinal epithelial cells; 4) Promote the activation and utilization of vitamin D.

5. Use of the animal bifidobacterium or product according to claim 4 in preparing products for improving heart rate, bone quality and blood pressure, characterized in that: The physiological effect of promoting calcium absorption and vitamin D in intestinal epithelial cells is to upregulate genes related to intestinal epithelial cell calcium ion and vitamin D absorption and transport, and the genes related to intestinal epithelial cell calcium ion and vitamin D absorption and transport include transient receptor potential coumarin type 6 channel protein gene TRPV6 , claudin 2 gene CLD2 and vitamin D receptor protein genes VDR Any one or more of the following; the promoting the activation and utilization of vitamin D is to promote intestinal epithelial cells to convert vitamin D3 into 1,25-dihydroxyvitamin D3.

6. Use of the animal bifidobacterium or its product according to claim 3 in preparing products for improving heart rate, bone quality and blood pressure, characterized in that: The mechanism includes any one or more of the following: 1) Promote myocardial cell repair; 2) Promote osteoblast repair; 3) Promote the repair of vascular endothelial cells.

7. Use of the animal bifidobacterium or the product according to claim 6 in preparing products for improving heart rate, bone quality and blood pressure, characterized in that: The promotion of myocardial cell repair, osteoblast repair and vascular endothelial cell repair respectively increases the survival rate of oxidatively damaged myocardial cells, osteoblasts and vascular endothelial cells.

8. Use of the animal bifidobacterium or its product according to claim 3 in preparing products for improving heart rate, bone quality and blood pressure, characterized in that: The mechanism includes regulating osteoblast bone metabolism, which may include any one or more of the following methods: 1) Upregulation of Runt-related transcription factor 2 gene Runx2 expression; 2) Upregulation of osteoblast-specific transcription factor genes Osterix expression; 3) Upregulation of osteocalcin gene Osteocalcin expression; 4) Upregulation of bone sialoglycoprotein gene Bsp expression; 5) Downregulation of zinc finger transcription factor genes Aj18 expression.

Images