Application of Bifidobacterium bifidum in anti-atherosclerosis and promoting energy metabolism

Abstract

This invention discloses the application of Bifidobacterium bifidum in anti-atherosclerosis and promoting energy metabolism, belonging to the field of microbial technology. Bifidobacterium bifidum (… Bifidobacterium bifidum NHNK-618 was deposited at the China Center for Type Culture Collection on August 9, 2024, with accession number CCTCCNO:M20241761. NHNK-618 possesses functions such as inhibiting atherosclerosis-related pathogens, promoting vascular endothelial cell relaxation, reducing vascular endothelial adhesion and oxidative damage, improving energy metabolism in hepatic and renal cells, reducing fat accumulation in hepatocytes, and inhibiting high-energy-accumulation-related pathogens. It can enhance the body's overall energy utilization efficiency through multi-organ synergy and multi-target regulation, and thus can be used to prepare products for anti-atherosclerosis and energy metabolism promotion.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Bifidobacterium bifidum in anti-atherosclerosis and promoting energy metabolism. Background Technology

[0002] Atherosclerosis is a chronic, progressive vascular disease caused by the abnormal accumulation of lipids, inflammatory cells, and connective tissue in the arterial walls. These processes trigger chronic inflammation, eventually leading to narrowing or thrombosis. Atherosclerotic lesions can increase vascular fragility and narrow blood vessels, obstructing blood flow and causing myocardial ischemia, cerebral ischemia, etc. In severe cases, plaque rupture can lead to myocardial infarction, stroke, and limb gangrene.

[0003] When Candida albicans infects the organism, the hyphae of Candida albicans are more invasive, making it easier to penetrate the mucosal barrier and enter the bloodstream. Simultaneously, it stimulates the immune system to release large amounts of pro-inflammatory cytokines, triggering persistent low-grade inflammation. These inflammatory factors, once in the bloodstream, damage vascular endothelial cells, increase vascular permeability, and thus accelerate lipid deposition and plaque formation. Studies have shown that Candida albicans in the gut and circulatory system can directly or indirectly participate in the process of atherosclerosis through the "microbiota-immune-vascular axis": its sporophytes mediate chronic low-grade inflammation and endothelial damage, and its culture supernatant can induce coronary artery injury, myocardial inflammation, and inflammatory infiltration of arterial tissue in mice.

[0004] AMPK is a key regulator of cellular energy metabolism. Increased AMPK expression and activation prevent endothelial cells from undergoing apoptosis or necrosis due to severe oxidative damage, maintaining the integrity of the vascular intima. Endothelial nitric oxide synthase (eNOS) catalyzes the production of nitric oxide (NO), which not only dilates blood vessels but also inhibits platelet aggregation, smooth muscle cell proliferation, prevents vascular wall thickening, and inhibits leukocyte adhesion. A crucial step in atherosclerotic plaque formation is the migration of immune cells from the blood through adhesion molecules VCAM-1 and ICAM-1, which are highly expressed on the surface of vascular endothelial cells, to the subendothelial layer, where they transform into foam cells, becoming the core component of the plaque. Downregulation of VCAM-1 and ICAM-1 gene expression can fundamentally inhibit the formation of lipid streaks and plaques.

[0005] Imbalances in the body's energy metabolism, particularly reduced energy expenditure efficiency, are key intrinsic factors driving the development and progression of atherosclerosis. Besides the metabolic regulation of host cells, the composition of the gut microbiome also profoundly influences the host's energy acquisition efficiency.

[0006] Enterobacter cloacae ( Enterobacter cloacaeThis process produces endotoxins, inhibiting the expression of fasting-induced adipokines in the gut, thus reducing the body's ability to utilize fat. Furthermore, studies have found that in individuals experiencing weight rebound after dieting, active rumenococci (…) Ruminococcus gnavus Their abundance is significantly higher due to adaptation to highly volatile diets. When diets are irregular or lacking in dietary fiber, they can instead break down intestinal mucoproteins and the protective mucus layer of the intestinal lining. These specific bacterial groups often have highly efficient energy extraction capabilities or can disrupt the intestinal barrier, leading to metabolic endotoxemia.

[0007] The liver is the central hub of energy metabolism in the human body. Upregulation of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) expression, the master switch for mitochondrial biosynthesis, significantly increases the number and function of mitochondria in hepatocytes, increasing basal energy expenditure. Upregulation of coupling protein 2 (UCP2) helps decouple the mitochondrial oxidative respiratory chain to generate heat and energy, and can also directly inhibit intracellular fat synthesis, reducing the formation of fat droplets and blocking fatty liver, a co-risk factor for atherosclerosis, at its source. Upregulation of growth differentiation factor 15 (GDF15), a stress-metabolic hormone, transmits signals to the central nervous system, regulating appetite and energy balance, while improving insulin sensitivity.

[0008] The kidneys are vital excretory and endocrine organs, and their energy metabolism directly affects the body's fluid and electrolyte balance. Upregulating GDF15 expression in renal tubular cells enhances the kidneys' ability to adapt to metabolic stress. This helps maintain the kidneys' efficient filtration function, ensuring the timely excretion of metabolic waste and excess water, preventing the accumulation that leads to increased vascular pressure and endothelial damage.

[0009] Current clinical interventions for metabolic cardiovascular diseases often focus on single-dimensional regulation, such as using lipid-lowering drugs to lower blood cholesterol or using hypoglycemic drugs to control blood sugar. However, these methods often neglect the remodeling of energy metabolism in the body as a whole. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to provide the application of Bifidobacterium bifidum in anti-atherosclerosis and promoting energy metabolism.

[0011] This invention provides Bifidobacterium bifidum ( Bifidobacterium bifidum The application of [the product] in the preparation of products for anti-atherosclerosis and promotion of energy metabolism is characterized in that the Bifidobacterium bifidum is [Bifidobacterium bifidum]. Bifidobacterium bifidum NHNK-618 was deposited at the China Center for Type Culture Collection on August 9, 2024, with accession number CCTCC NO: M 20241761.

[0012] Furthermore, the anti-atherosclerosis and energy metabolism promotion includes inhibiting atherosclerosis-related pathogens, which includes inhibiting the formation of Candida albicans biofilm and inhibiting the formation of Candida albicans hyphae.

[0013] Furthermore, the anti-atherosclerosis and energy metabolism promotion also include promoting vascular endothelial cell relaxation, which includes upregulating the expression of the endothelial nitric oxide synthase gene eNOS in oxidatively damaged human vascular endothelial cells EA.hy926.

[0014] Furthermore, the anti-atherosclerosis and energy metabolism promotion also include reducing vascular endothelial cell adhesion, which includes downregulating the type I vascular cell adhesion protein gene in oxidatively damaged human vascular endothelial cells. VACM-1 and intercellular adhesion molecule-1 gene ICAM-1 The expression.

[0015] Furthermore, the anti-atherosclerosis and energy metabolism promotion also include reducing oxidative damage to vascular endothelial cells, which includes upregulating the adenosine monophosphate-activated protein kinase gene in the AMPK signaling pathway. AMPK The expression of [a substance] and the improvement of the survival rate of oxidatively damaged vascular endothelial cells.

[0016] Furthermore, the anti-atherosclerosis and energy metabolism promotion also includes enhancing hepatocyte energy metabolism. This enhancement includes upregulating the expression of HepG2 energy consumption-related genes in hepatocytes, including the peroxisome proliferator-activated receptor gamma coactivator 1α gene. PGC-1α Growth differentiation factor 15 gene GDF15 and uncoupling linker 2 gene UCP2 .

[0017] Furthermore, the anti-atherosclerosis and energy metabolism promotion also includes enhancing renal cell energy metabolism, which includes upregulating the growth differentiation factor 15 gene, an energy consumption-related gene in renal tubular cells (HKC). GDF15 The expression.

[0018] Furthermore, the anti-atherosclerosis and energy metabolism promotion also include reducing hepatocyte fat accumulation, which includes inhibiting the formation of lipid droplets within hepatocytes and upregulating the uncoupling protein 2 gene, a gene related to the inhibition of lipid synthesis in hepatocytes. UCP2 The expression.

[0019] Furthermore, the anti-atherosclerosis and energy metabolism promotion also includes inhibiting high-energy accumulation-related pathogens, including inhibiting the growth of Enterobacter cloacae and active Ruminococcus.

[0020] Furthermore, the product is prepared from an article, which is one of the following: live Bifidobacterium bifidum, fermentation / secretion products, or inactivated bacterial cells.

[0021] Bifidobacterium bifidum in this application ( Bifidobacterium bifidum NHNK-618, with accession number CCTCC NO: M 20241761, demonstrates that it inhibits atherosclerosis-related pathogens, promotes endothelial cell relaxation, reduces endothelial adhesion and oxidative damage, enhances energy metabolism in liver and kidney cells, reduces fat accumulation in liver cells, and inhibits high-energy-accumulation-related pathogens. This allows it to improve overall energy utilization efficiency through multi-organ synergy and multi-target regulation, making it suitable for developing products that combat atherosclerosis and promote energy metabolism.

[0022] Biological Preservation Instructions Bifidobacterium bifidum ( Bifidobacterium bifidum NHNK-618 was deposited on August 9, 2024, at the China Center for Type Culture Collection (CCTCC, No. 299 Bayi Road, Wuchang District, Wuhan, 430072, China), with accession number CCTCC NO: M 20241761. Attached Figure Description

[0023] Figure 1 This is the phylogenetic tree of Bifidobacterium bifidum NHNK-618 in Example 1 of the present invention; Figure 2 This is a diagram showing the results of Bifidobacterium bifidum NHNK-618 inhibiting Candida albicans biofilm formation in Example 2 of the present invention; Figure 3 This is a diagram showing the results of Bifidobacterium bifidum NHNK-618 inhibiting Candida albicans hyphal formation in Example 3 of the present invention; Figure 4 This is a graph showing the results of Bifidobacterium bifidum NHNK-618 increasing the survival rate of vascular endothelial cells after oxidative damage in Example 4 of the present invention. Figure 5 This is a diagram showing the results of Bifidobacterium bifidum NHNK-618 inhibiting the formation of lipid droplets in hepatocytes in Example 9 of the present invention; The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0024] This invention provides the application of Bifidobacterium bifidum in anti-atherosclerosis and promoting energy metabolism. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0025] The *Bifidobacterium bifidum* NHNK-618 described in this application is derived from the intestines of *Abalone variegata* and identified as *Bifidobacterium bifidum* by 16S rDNA analysis. Bifidobacterium bifidum This strain is Gram-positive and exhibits highly pleomorphic cell morphology under a microscope, displaying rod-shaped, V-shaped, Y-shaped, or forked shapes. The cells are predominantly arranged singly or in pairs. When grown on MRS plates containing 0.1% (m / v) cysteine, it forms smooth, opaque, circular, milky-white colonies with neat edges. In MRS liquid medium containing 0.1% (m / v) cysteine, it grows in a uniformly turbid medium; upon prolonged static incubation, a white precipitate forms. The optimal growth temperature is 37°C.

[0026] Bifidobacterium bifidum ( Bifidobacterium bifidum NHNK-618, deposited by China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan, Wuhan University, on August 9, 2024, with accession number CCTCCNO: M20241761.

[0027] Furthermore, the Bifidobacterium bifidum NHNK-618 provided by this invention exists in the following forms in the application described in this invention: unsterilized live bacteria or sterilized inactivated bacterial cells, or in the form of fermentation / secretion products (i.e., supernatant), or in the form of biofilm, or in the form of derivatives. The derivative forms are preferably selected from: metabolites, metabolic biological products, probiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, and are preferably selected from: fermentation / secretion products, live bacteria, and inactivated bacterial cells.

[0028] The method for preparing the live bacteria and the inactivated bacterial cells is as follows: The Bifidobacterium bifidum (… Bifidobacterium bifidum The culture medium was inoculated into MRS liquid medium containing 0.1% (m / v) cysteine ​​and incubated at 37°C for 24 h to obtain the fermentation broth. The precipitate was collected by centrifugation at 5000 rpm for 10 min, and washed with PBS to obtain the live bacteria. A portion of the live bacteria was inactivated at 121°C for 15 min to obtain the inactivated bacterial cells.

[0029] The method for preparing the fermentation / secretion product is as follows: The Bifidobacterium bifidum (… Bifidobacterium bifidum The culture medium was inoculated into MRS liquid medium containing 0.1% (m / v) cysteine ​​and cultured at 37°C for 24 h to obtain the fermentation broth. The supernatant was collected by centrifugation and filtered with a precision of 0.22 μm. The obtained filtrate was the fermentation / secretion product.

[0030] It should be noted that, unless otherwise specified, all reagents, consumables, and biological products used in this invention are commercially available products. In the following examples, the MRS solid and liquid culture media and related reagents used were all from Qingdao Haibo Biotechnology Co., Ltd., the DMEM culture medium was from Beijing Solarbio Technology Co., Ltd., the fetal bovine serum (FBS) was from Biological Industries (BI) in Israel, and the sterile PBS used was from Hefei Baisha Biotechnology Co., Ltd. (0.01 M, pH=7.2). The invention is further illustrated below with reference to the examples.

[0031] Example 1: Nucleic acid identification of NHNK-618 1. 16S rDNA gene sequence analysis Single colonies were picked and incubated overnight at 37°C in MRS liquid medium containing 0.1 m / v cysteine. The cells were then collected by centrifugation at 8000 rpm for 1 min and processed according to the instructions of the Gram-positive bacterial DNA extraction kit. The universal primers for bacterial 16S sequencing, 27F and 1492R, were used. The PCR amplification volume was 20 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min, 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, 35 cycles, followed by a 72°C extension for 10 min.

[0032] 2. Results Please see Figure 1 The sequencing results of the PCR product are shown in SEQ ID NO.1. After homology comparison (BLASTN) between the sequencing results of the PCR product and the published standard sequence in GenBank, it was determined that strain NHNK-618 is Bifidobacterium bifidum. Bifidobacterium bifidum Sequences similar to Bifidobacterium bifidum NHNK-618 were selected in the BLASTN interface, and a phylogenetic tree was constructed using MEGA software. The results showed that although NHNK-618 and strains such as FB7, 2733, and FMT707W are all Bifidobacterium bifidum, they are distantly related and therefore have strain-specific characteristics.

[0033] Example 2: NHNK-618 inhibits Candida albicans biofilm formation 1. Preparation of NHNK-618 fermentation products Single colonies were picked and incubated in MRS liquid medium containing 0.1% (m / v) cysteine, and cultured statically at 37°C for 48 h. The culture time was then adjusted to OD using PBS. 600 =1.0, then centrifuged at 5000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the sterile fermentation product.

[0034] 2. Preparation of Candida albicans culture Single colonies of *Candida albicans* (CGMCC 1.592) were picked and inoculated into Sabouraud dextrose agar (Qingdao Haibo Biotechnology Co., Ltd.). After static incubation at 30°C for 48 hours, the culture temperature was adjusted to OD using Sabouraud dextrose agar. 600 =0.3.

[0035] 3. NHNK-618 fermentation products inhibit Candida albicans biofilm formation. Please see Figure 2 100 μL of *Candida albicans* culture was added to a 96-well plate. The experimental group received 100 μL of NHNK-618 fermentation product, while the control group received an equal volume of PBS. Each group was divided into three replicates. The plates were incubated at 37°C for 24 h. After incubation, the supernatant was discarded, and each well was washed twice with 100 μL of sterile PBS. Then, 100 μL of 4% (m / v) paraformaldehyde fixative (Wuhan Saive Biotechnology Co., Ltd.) was added to each well, and the plates were fixed at room temperature for 30 min. The fixative was discarded, and 100 μL of crystal violet solution (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and the plates were stained at room temperature for 30 min. After staining, the plates were washed twice with sterile PBS and air-dried. 100 μL of anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.) was added to each well, and the absorbance at 600 nm was measured after standing for 1 min. The inhibition rate of the biofilm was calculated using the formula shown in Table 1. Table 1 , The results showed that the fermentation product of NHNK-618 could inhibit the formation of Candida albicans biofilm, with an inhibition rate of 45.73%~47.91%.

[0036] Example 3: NHNK-618 inhibits Candida albicans hyphae formation. 1. Preparation of NHNK-618 fermentation products and inactivated bacterial cells Single colonies of Bifidobacterium bifidum NHNK-618 were picked and incubated in MRS liquid medium containing 0.1% (m / v) cysteine ​​at 37°C for 48 h. The culture time was adjusted to OD500 with PBS. 600 =1.0, then centrifuged at 5000 rpm for 10 min, collected the supernatant, and filtered through a 0.22 μm sterile filter membrane to obtain the sterile fermentation product. The precipitated bacterial sludge was resuspended in sterile PBS and adjusted to OD. 600=1.0, sterilize at 121℃ for 15 minutes to obtain inactivated bacteria.

[0037] 2. Preparation of Candida albicans culture Refer to Example 2.

[0038] 3. NHNK-618 inhibits the formation of Candida albicans hyphae. Please see Figure 3 100 μL of Candida albicans culture was added to each well of a 96-well plate. The experimental group received 100 μL of NHNK-618 fermentation product or inactivated cells, while the control group received an equal volume of PBS. Each group was divided into three replicates, and the plates were incubated at 37°C for 3 hours. After incubation, the old culture medium in the 96-well plates was discarded, and the plates were washed once with 70% (v / v) ethanol (Sinopharm Chemical Reagent Co., Ltd.), once with 0.25% (w / v) SDS solution, and three times with sterile water. The plates were then stained with 0.1% (w / v) crystal violet for 10 min, followed by washing once with 0.25% (w / v) SDS solution and three times with sterile water. After the 96-well plates were air-dried, 100 μL of anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.) was added to each well. After standing for 1 min, the absorbance at OD=600 nm was measured. The inhibition rate of Candida albicans mycelial formation was calculated using the formula shown in Table 2. Table 2 , The results showed that both the fermentation product of NHNK-618 and the inactivated cells could inhibit the formation of Candida albicans hyphae, with an inhibition rate of 75.40% to 85.81%.

[0039] Example 4: NHNK-618 improves the survival rate of vascular endothelial cells after oxidative damage. 1. Preparation of NHNK-618 fermentation products and live bacteria Single colonies of NHNK-618 were picked and incubated in MRS liquid medium containing 0.1% (m / v) cysteine ​​at 37°C for 48 h. The culture temperature was then adjusted to OD using DMEM medium. 600 =1.0, then centrifuged at 5000 rpm for 10 min, collected the supernatant, and filtered through a 0.22 μm sterile filter membrane to obtain the fermentation product. The precipitated sludge was resuspended in DMEM medium and adjusted to OD. 600 =1.0, which means that live bacteria are obtained.

[0040] 2. Culture of human vascular endothelial cells Human vascular endothelial cells EA.hy926 (BNCC342387, Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and then cultured at 37℃ and 5% CO2. After the cells reached 80%~90% confluence, they were passaged or plated. 3. NHNK-618 improves the survival rate of vascular endothelial cells after oxidative damage. Please see Figure 4 EA.hy926 cells were seeded at a rate of 5 × 10^4 cells / well in 96-well cell culture plates and cultured for 24 h until cell adhesion. After culture, the supernatant was discarded, and 100 μL of DMEM medium containing 1.5 mM H2O2 was added to each well. The cells were then cultured at 37°C for 4 h. After the second culture, the supernatant was discarded, and 100 μL of DMEM medium containing 0.1% (v / v) NHNK-618 fermentation product or live bacteria was added to each well in the experimental group, while the control group received an equal volume of DMEM medium. The cells were cultured at 37°C and 5% CO2 for 20 h. After the third culture, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for another 2 h. The absorbance at 450 nm was measured. The calculation formulas and results are shown in Table 3. Table 3 , The results showed that both the fermentation product and live bacteria of NHNK-618 improved the survival rate of human vascular endothelial cells EA.hy926 under oxidative damage, with a relative survival rate of 141.51%~240.19%.

[0041] Example 5: NHNK-618 regulates the expression of genes related to oxidative damage in vascular endothelial cells. 1. Preparation of NHNK-618 fermentation products and live bacteria Refer to Example 4.

[0042] 2. Culture of human vascular endothelial cells Refer to Example 4.

[0043] 3. NHNK-618 reduces oxidative damage to vascular endothelial cells. EA.hy926 cells were seeded at a rate of 1×10^6 cells / well in 6-well cell culture plates and cultured for 24 h until cell adhesion. After culture, the supernatant was discarded, and 2 mL of DMEM medium containing 1.5 mM H2O2 was added to each well. The cells were then cultured at 37°C for 4 h. After culture, the supernatant was discarded again. For the experimental groups, 2 mL of DMEM medium containing 0.1% (v / v) NHNK-618 fermentation product or live bacteria was added, while for the control group, an equal volume of DMEM medium was added. Cells were cultured at 37°C and 5% CO2 for 20 h.

[0044] After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the reagent instructions. The concentration and purity were then determined. After extraction, the RNA was reverse transcribed into cDNA. Using GADPH as an internal reference gene, qPCR was used to measure the RNA. VCAM-1, ICAM-1, eNOS and AMPK Gene expression levels.

[0045] The relative gene expression fold of the control group was F=1, using 2 -ΔΔCT The F-value for each sample is calculated using the following method: F = 2. -ΔΔCT ,in: ΔCT 实验 =CT 实验 -CT 内参（实验） ; ΔCT 对照 =CT 对照 -CT 内参（对照） ; ΔΔCT=ΔCT 实验 -ΔCT 对照 .

[0046] The calculation results are shown in Tables 4 and 5: Table 4 , Table 5 , The results showed that NHNK-618 can upregulate the endothelial nitric oxide synthase gene. eNOS and adenosine-activated protein kinase gene AMPK Expression of [something], downregulation of vascular adhesion factors VCAM-1 and intercellular adhesion factors ICAM-1 The expression of this substance helps protect the integrity of the vascular endothelial barrier and reduce oxidative damage.

[0047] Example 6: NHNK-618 reduces fat accumulation in hepatocytes 1. Preparation of NHNK-618 fermentation products Refer to Example 4.

[0048] 2. Culture of human hepatocytes HepG2 Human hepatocytes HepG2 (BNCC338070, Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and then cultured at 37℃ and 5% CO2. After the cells reached 80%~90% confluence, they were passaged or plated.

[0049] 3. NHNK-618 inhibits fat accumulation in hepatocytes. HepG2 cells were seeded at 1×10^6 cells / well in 6-well cell culture plates and cultured for 24 h until cell adhesion. After culture, the supernatant was discarded, and 2 mL of fatty acid medium was added to each well for 12 h of culture. After culture, the supernatant was discarded again. The experimental group was cultured in 2 mL of DMEM medium containing 1% (v / v) NHNK-618 fermentation product, while the control group was cultured in an equal volume of DMEM medium for another 24 h. After culture, the supernatant was discarded, and each well was washed twice with 2 mL of sterile PBS. Then, 2 mL of 4% (m / v) paraformaldehyde fixative (Wuhan Saive Biotechnology Co., Ltd.) was added to each well for 30 min at room temperature. After the fixative was discarded, the cells were washed with PBS, and 500 μL of Oil Red O solution (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well for 30 min at room temperature. After staining, the cells were washed once with 60% isopropanol and twice with sterile PBS. The absorbance at OD=540 nm was measured after dissolving Oil Red O in isopropanol. The calculation formula and results are shown in Table 6. Table 6 , The results showed that the fermentation product of NHNK-618 could inhibit the formation of lipid droplets in hepatocytes, with an inhibition rate of 20.10%~26.54%.

[0050] Example 7: NHNK-618 enhances the expression of genes related to energy metabolism in hepatocytes. 1. Preparation of NHNK-618 fermentation products and live bacteria Refer to Example 4.

[0051] 2. Culture of human hepatocytes HepG2 Refer to Example 6.

[0052] 3. NHNK-618 increases the expression of genes related to energy metabolism in hepatocytes. HepG2 cells were seeded at a rate of 1×10^6 cells / well in 6-well cell culture plates and cultured for 24 h until cell adhesion. After culture, the supernatant was discarded. 2 mL of DMEM medium containing 0.1% (v / v) NHNK-618 fermentation product or live bacteria was added to each well in the experimental groups, while an equal volume of DMEM medium was added to the control group. Cells were cultured at 37℃ and 5% CO2 for 12 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. 2 mL of fatty acid medium was added to each well, and the cells were cultured for another 24 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the reagent instructions. The concentration and purity were then determined. After extraction, the RNA was reverse transcribed into cDNA. Using GADPH as an internal reference gene, the expression levels of related genes were determined by qPCR.

[0053] The relative gene expression fold of the control group was F=1, using 2 -ΔΔCT The F-value of each sample was calculated using the method described above.

[0054] The calculation results are shown in Tables 7 and 8: Table 7 , Table 8 , The results showed that NHNK-618 could upregulate the peroxisome proliferator-activated receptor γ coactivator 1α gene. PGC-1α Growth differentiation factor 15 gene GDF15 and uncoupling protein 2 gene UCP2 It enhances energy metabolism and utilization while inhibiting fat synthesis.

[0055] Example 8: NHNK-618 enhances the expression of renal cell energy metabolism-related genes. 1. Preparation of NHNK-618 fermentation products Refer to Example 4.

[0056] 2. Renal tubular cell culture Human renal tubular cells HKC (BNCC338628, Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and then cultured at 37°C and 5% CO2. After the cells reached 80%~90% confluence, they were passaged or plated.

[0057] 3. NHNK-618 increases the expression of genes related to energy metabolism in renal cells. HKC cells were seeded at a rate of 1×10^6 cells / well in 6-well cell culture plates and cultured for 24 h until cell adhesion. After culture, the supernatant was discarded. 2 mL of DMEM medium containing 0.1% (v / v) NHNK-618 fermentation product was added to each well in the experimental group, while the control group received an equal volume of DMEM medium. Cells were cultured at 37℃ and 5% CO2 for 12 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. 2 mL of fatty acid medium was added to each well, and the cells were cultured for another 24 h. After culture, the medium was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the reagent instructions. The concentration and purity were then determined. After extraction, the RNA was reverse transcribed into cDNA. Using GADPH as an internal control gene, qPCR was used to measure the RNA. GDF15 Gene expression levels.

[0058] The relative gene expression fold of the control group was F=1, using 2 -ΔΔCTThe F-value of each sample was calculated using the method described above.

[0059] The calculation results are shown in Table 9: Table 9 , The results showed that NHNK-618 could upregulate renal tubular cells. GDF15 Gene expression, thereby promoting energy consumption.

[0060] Example 9: NHNK-618 fermentation product inhibits the growth of obesity pathogens. 1. Preparation of NHNK-618 fermentation products Refer to Example 2.

[0061] 2. Preparation of pathogens Special peptone medium: Special peptone: 23.0 g / L, soluble starch: 1.0 g / L, sodium chloride: 5.0 g / L.

[0062] Single colonies of Enterobacter cloacae BNCC185928 (Beijing Beina Chuanglian Biotechnology Research Institute) were picked and inoculated into nutrient broth liquid medium (Qingdao Haibo Biotechnology Co., Ltd.), and cultured at 37℃ with shaking for 48 h. After the culture was completed, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min, and the OD600 was adjusted to 0.5 with nutrient broth liquid medium for later use. A single colony of active rumenococcus BNCC372807 (Beijing Beina Chuanglian Biotechnology Research Institute) was inoculated into a special peptone medium containing 5% (v / v) defibrinated sheep blood and cultured anaerobically at 37°C for 3 days. After the culture was completed, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min and the OD600 of the bacterial culture was adjusted to 0.3 with special peptone liquid medium for later use.

[0063] 3. Fermentation products of NHNK-618 inhibit the growth of obesity pathogens. Please see Figure 5 4 mL of nutrient broth and 1 mL of *Enterobacter cloacae* were added to a 10 mL centrifuge tube. The experimental group received 1 mL of NHNK-613 fermentation product, while the control group received an equal volume of MRS medium. The mixtures were incubated at 37°C with shaking for 24 h, and the absorbance at 600 nm was measured after incubation. Alternatively, 4 mL of special peptone medium and 1 mL of active *Ruminococcus* were added to a centrifuge tube. The experimental group received 1 mL of NHNK-613 fermentation product, while the control group received an equal volume of MRS medium. The mixtures were incubated anaerobically at 37°C for 3 days, and the absorbance at 600 nm was measured after incubation. The calculation formulas and results are shown in Table 10. Table 10 , The results showed that NHNK-618 inhibited the growth of both Enterobacter cloacae and active rumenococci, with inhibition rates ranging from 36.24% to 54.43%.

[0064] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bifidobacterium bifidum ( Bifidobacterium bifidum Its application in the preparation of products that combat atherosclerosis and promote energy metabolism is characterized by, The Bifidobacterium bifidum is Bifidobacterium bifidum ( Bifidobacterium bifidum NHNK-618 was deposited at the China Center for Type Culture Collection on August 9, 2024, with accession number CCTCC NO: M 20241761.

2. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion includes inhibiting atherosclerosis-related pathogens, which includes inhibiting the formation of Candida albicans biofilm and the formation of Candida albicans hyphae.

3. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include promoting vascular endothelial cell relaxation, which includes upregulating the expression of the endothelial nitric oxide synthase gene eNOS in oxidatively damaged human vascular endothelial cells EA.hy926.

4. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include reducing vascular endothelial cell adhesion, which includes downregulating the type I vascular cell adhesion protein gene in oxidatively damaged human vascular endothelial cells. VACM-1 and intercellular adhesion molecule-1 gene ICAM-1 The expression.

5. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include reducing oxidative damage to vascular endothelial cells, which includes upregulating the adenosine monophosphate-activated protein kinase gene in the AMPK signaling pathway. AMPK The expression of [a substance] and the improvement of the survival rate of oxidatively damaged vascular endothelial cells.

6. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include enhancing hepatocyte energy metabolism. This enhancement includes upregulating the expression of HepG2 energy consumption-related genes in hepatocytes, including the peroxisome proliferator-activated receptor gamma coactivator 1α gene. PGC-1α Growth differentiation factor 15 gene GDF15 and uncoupling linker 2 gene UCP2 .

7. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include enhancing renal cell energy metabolism, which includes upregulating the growth differentiation factor 15 gene, a gene related to HKC energy consumption in renal tubular cells. GDF15 The expression.

8. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include reducing hepatocyte fat accumulation, which includes inhibiting the formation of lipid droplets in hepatocytes and upregulating the uncoupling protein 2 gene, a gene related to the inhibition of lipid synthesis in hepatocytes. UCP2 The expression.

9. The application according to claim 1, characterized in that, The anti-atherosclerosis and energy metabolism promotion also include inhibiting high-energy accumulation-related pathogens, including inhibiting the growth of Enterobacter cloacae and active Ruminococcus.

10. The application according to claim 1, characterized in that, The product is prepared from an article, which is one of the following: live Bifidobacterium bifidum, fermentation / secretion products, or inactivated bacterial cells.

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