Bifidobacterium adolescentis for improving urticaria and Alzheimer as well as product and application thereof

Abstract

The invention provides bifidobacterium adolescentis for improving urticaria and Alzheimer as well as a product and application thereof. The bifidobacterium adolescentis NHNK-622 disclosed by the invention is preserved in the China Center for Type Culture Collection on December 05, 2024, and the preservation number of the bifidobacterium adolescentis NHNK-622 is CCTCC (China Center for Type Culture Collection) NO: M 20242734. The NHNK-622 has the effects of inhibiting the growth of klebsiella pneumonia and / or generating alcohol, agglutinating klebsiella pneumonia, tolerating 10% alcohol, reducing alcohol-induced damaged skin cutin barrier and inflammation, reducing alcohol-induced damaged brain microvascular barrier and inflammation, improving the klebsiella pneumonia-induced damaged survival rate of brain microvascular endothelial cells, and improving the activity of the brain microvascular endothelial cells. Klebsiella pneumonia induced damage to brain microvascular barriers and inflammation is reduced, and Klebsiella pneumonia induced damage to intestinal barriers and inflammation is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and particularly relates to Bifidobacterium adolescentis for improving urticaria and Alzheimer's disease, products thereof, and applications thereof. Background Art

[0002] The ocean is the largest biosphere on the earth and the largest repository of global species diversity. Marine microorganisms are one of the earliest known sources of marine bioactive substances, including microorganisms such as algae, bacteria, fungi, and protozoa. These bioactive substances have important application values in various fields such as food, medicine, and cosmetics.

[0003] Chronic urticaria is manifested as spontaneous wheals or angioedema with a course of more than 6 weeks, often accompanied by severe itching, which seriously affects the quality of life of patients. Some studies have explored the gut microbiome of patients with chronic urticaria and healthy controls through metagenomic sequencing and targeted metabolomics, and evaluated the differences in functional correlations. The results show that the gut flora of patients with chronic urticaria is significantly different from that of healthy controls, and chronic urticaria is significantly correlated with a decrease in the level of short-chain fatty acid (SCFA) producers and an increase in the pathogen Klebsiella pneumoniae ( Klebsiella pneumoniae ). After transplanting Klebsiella pneumoniae into the mouse intestine, the allergic reactions of the recipient mice increased, and the blood lipopolysaccharide level increased. Lipopolysaccharide promoted IgE-mediated mast cell degranulation to increase allergic and inflammatory reactions in the body.

[0004] Klebsiella pneumoniae is an important pathogenic bacterium that can cause nosocomial-acquired infections and community-acquired infections, and can trigger diseases such as liver abscess, pneumonia, and bacteremia, seriously threatening human life and health. In recent years, researchers have isolated Klebsiella pneumoniae with high ethanol-producing ability from the feces of patients with auto-brewery syndrome. This bacterium continuously produces alcohol in the human body, causing vasodilation and increased permeability, and causing continuous damage to the liver and intestine. The endogenous alcohol produced by Klebsiella pneumoniae in the intestine reduces the tight junction proteins ZO-1, claudin-1, and claudin-4, the adherens junction proteins β-catenin and E-cadherin, and desmoplakin in intestinal epithelial cells, destroys the integrity of the intestinal barrier, and increases the severity of chronic urticaria. The core of the skin barrier is the brick-wall structure formed by lipids in the stratum corneum. Due to its lipophilicity, alcohol can penetrate and dissolve these lipids, weaken the barrier function, resulting in water loss and infiltration of external irritants. Alcohol may loosen the connections between keratinocytes to accelerate desquamation and reduce the cohesion of keratinocytes.

[0005] Among them, Alzheimer's disease (AD) is the most common type of dementia, characterized by impaired cognitive function caused by neurodegeneration, neuronal loss, accumulation of β-amyloid (Aβ), and neurofibrillary tangles. Chronic neuroinflammation is considered an important factor in the progression of the disease. It has been found that the incidence of dementia in hospitalized patients infected with Klebsiella pneumoniae increased by 1.7 times and showed a dose-dependent relationship. Klebsiella pneumoniae can migrate from the intestine to the blood and eventually enter the brain. The infection of Klebsiella pneumoniae will exacerbate the neuroinflammation in the brain, causing patients to exhibit symptoms of Alzheimer's disease. Certain proteins of Klebsiella pneumoniae (such as OmpA) have a similar structure to Aβ and may trigger an autoimmune response through molecular mimicry, promoting the generation of Aβ or inhibiting its clearance.

[0006] Marine microorganisms have developed complex molecular adaptation capabilities to cope with harsh conditions, affecting their primary and secondary metabolic pathways. This has led to unique physiological characteristics and changes in metabolic processes. Marine microorganisms are more likely than terrestrial microorganisms to synthesize enzymes and secondary metabolites with unique structures. Therefore, it is of great practical significance to provide products related to the marine industry developed using microbial technology. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides Bifidobacterium adolescentis and its products and applications for improving urticaria and Alzheimer's disease, which are used to solve the technical problems in the prior art.

[0008] The present invention provides Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), and this strain is Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) NHNK-622, 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, postcode 430072) on December 05, 2024, and its deposit number is Bifidobacterium adolescentis CCTCC NO: M 20242734.

[0009] Furthermore, according to the above Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), the product prepared therefrom is a live bacterium, inactivated bacterium or fermentation product of Bifidobacterium adolescentis ( Bifidobacterium adolescentis ).

[0010] Furthermore, the product also includes excipients and / or adjuvants.

[0011] The present invention also provides the application of the above Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) or the product prepared from the above Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) in the preparation of products for improving senile retinal degeneration.

[0012] Furthermore, the improvement of urticaria and Alzheimer's disease includes at least one of the following a)-h): a), inhibiting the growth and / or alcohol production of Klebsiella pneumoniae ( Klebsiella pneumoniae ); b), agglutinating Klebsiella pneumoniae; c), tolerating growth in 10% alcohol; d), reducing alcohol-induced damage to the skin keratin barrier and inflammation, upregulating barrier-related genes ZO-1, CLDN4 and OCLD , and downregulating the expression of at least one of the inflammation-related genes TNF-α , IL-6 and IL-1β ; e), reducing alcohol-induced damage to the brain microvascular barrier and inflammation, upregulating barrier-related genes ZO-1, CLDN5 and OCLD , and downregulating the expression of at least one of the inflammation-related genes TNF-α , IL-6 and IL-1β ; f), increasing the survival rate of brain microvascular endothelial cells induced by Klebsiella pneumoniae; g), reducing Klebsiella pneumoniae-induced damage to the brain microvascular barrier and inflammation, upregulating the expression of barrier-related genes ZO-1 , CLDN5 and OCLD , and downregulating the expression of at least one of the inflammation factor-related genes TNF-α , IL-6 and IL-1β ; h), reducing Klebsiella pneumoniae-induced damage to the intestinal barrier and inflammation, upregulating the expression of barrier-related genes ZO-1 , CLDN4 , and downregulating the expression of at least one of the inflammation factor-related genes TNF-α , IL-6 and IL-1β .

[0013] Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) disclosed in the present invention has a preservation number of CCTCC NO: M 20242734. Experiments show that NHNK-622 has the functions of inhibiting the growth and / or alcohol production of Klebsiella pneumoniae ( Klebsiella pneumoniae ), agglutinating Klebsiella pneumoniae, tolerating growth in 10% alcohol, reducing alcohol-induced damage to the skin keratin barrier and inflammation, reducing alcohol-induced damage to the brain microvascular barrier and inflammation, increasing the survival rate of brain microvascular endothelial cells induced by Klebsiella pneumoniae, reducing Klebsiella pneumoniae-induced damage to the brain microvascular barrier and inflammation, and reducing Klebsiella pneumoniae-induced damage to the intestinal barrier and inflammation.

[0014] Biological Deposit Description Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) NHNK-622 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 December 05, 2024, and its deposit number is CCTCC NO: M 20242734. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 It is the MRS plate colony map and Gram staining map of Bifidobacterium adolescentis NHNK-622 in Embodiment 1 of the present invention; Figure 2 It is the experimental result map of the agglutination effect between heat-inactivated Bifidobacterium adolescentis NHNK-622 and Klebsiella pneumoniae in Embodiment 4 of the present invention.

[0017] The following will further illustrate the embodiments of the present invention in conjunction with the drawings. Detailed Embodiments

[0018] The present invention provides Bifidobacterium adolescentis, its products and their applications in improving urticaria and Alzheimer's disease. Those skilled in the art can draw on the content of this article and appropriately improve 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 modifications 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.

[0019] The Bifidobacterium adolescentis strain NHNK-622 of the present invention is derived from the intestine of Haliotis diversicolor supertexta and is identified as Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) by 16S rDNA. This strain is Gram-positive and shows a curved rod-shaped arrangement under the microscope; it grows on the MRS plate and can form smooth, semi-transparent circular colonies, white in color with neat edges; it grows uniformly turbid in the MRS liquid medium, and the bacteria form a white precipitate after standing for a long time. The optimum growth temperature is 37°C.

[0020] Bifidobacterium adolescentis ( Bifidobacterium adolescentis)NHNK-622, Preservation Institution: China Center for Type Culture Collection, Address: Inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Preservation Date: December 05, 2024, Preservation Number: CCTCC NO: M 20242734.

[0021] Furthermore, in the applications described in the present invention, the Bifidobacterium adolescentis NHNK-622 provided by the present invention exists in the form of live bacteria, inactivated bacteria, or in the form of fermentation products (i.e., supernatant). The preferred derivative forms are selected from: metabolites, metabiotic products, probiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, live bacteria, and inactivated bacteria.

[0022] In vitro experiments show that the fermentation product of the Bifidobacterium adolescentis NHNK-622 of the present invention has the effect of inhibiting the growth of Klebsiella pneumoniae ( Klebsiella pneumoniae ), and the inhibition rate reaches 47.83% - 55.32%.

[0023] In vitro experiments show that the Bifidobacterium adolescentis NHNK-622 of the present invention has the effect of agglutinating Klebsiella pneumoniae, and the agglutination rate is 10.14% - 18.92%.

[0024] In vitro experiments show that the Bifidobacterium adolescentis NHNK-622 of the present invention can grow in a medium containing 10% alcohol, and the growth rate is 125.00% - 142.86%.

[0025] In vitro experiments show that the Bifidobacterium adolescentis NHNK-622 of the present invention has the effect of regulating the expression of genes related to the barrier and inflammation of alcohol-induced damaged skin keratinocytes HaCaT, upregulating the expression of genes related to the barrier zonula occludens protein ZO-1 , tight junction protein-4 gene CLDN4 and tight junction protein gene OCLD , and the relative expression levels are 1.10 - fold to 9.51 - fold; downregulating the expression of genes related to inflammation tumor necrosis factor-α gene TNF-α , interleukin-6 gene IL-6 and interleukin-1β gene IL-1β , and the relative expression levels are 0.15 - fold to 0.87 - fold.

[0026] In vitro experiments show that the Bifidobacterium adolescentis NHNK-622 of the present invention has the effect of regulating the expression of genes related to the barrier and inflammation of alcohol-induced damaged brain microvascular endothelial cells BEND3, upregulating the expression of genes related to the barrier zonula occludens protein ZO-1 , tight junction protein-5 gene CLDN5 and tight junction protein gene OCLDThe expression, with the relative expression level being 1.10 - fold to 1.83 - fold; down - regulate the expression of the inflammation - related tumor necrosis factor - α gene TNF-α , interleukin - 6 gene IL-6 and interleukin - 1β gene IL-1β The expression, with the relative expression level being 0.02 - fold to 0.88 - fold.

[0027] In vitro experiments show that the Bifidobacterium adolescentis NHNK - 622 of the present invention can increase the survival of brain microvascular endothelial cells BEND3 under the injury induced by Klebsiella pneumoniae, with the relative survival rate being 110.90% - 125.11%.

[0028] In vitro experiments show that the Bifidobacterium adolescentis NHNK - 622 of the present invention has the effect of regulating the barrier and inflammation - related gene expression of brain microvascular endothelial cells BEND3 injured by Klebsiella pneumoniae, up - regulate the expression of the barrier - related zonula occludens protein gene ZO-1 , claudin - 5 gene CLDN5 and claudin gene OCLD The expression, with the relative expression level being 1.10 - fold to 3.39 - fold; down - regulate the expression of the inflammation - related tumor necrosis factor - α gene TNF-α , interleukin - 6 gene IL-6 and interleukin - 1β gene IL-1β The expression, with the relative expression level being 0.04 - fold to 0.79 - fold.

[0029] In vitro experiments show that the Bifidobacterium adolescentis NHNK - 622 of the present invention has the effect of regulating the barrier and inflammation - related gene expression of colon epithelial cells Caco - 2 injured by Klebsiella pneumoniae, up - regulate the expression of the barrier - related zonula occludens protein gene ZO-1 and claudin - 4 gene CLDN4 The expression, with the relative expression level being 1.02 - fold to 3.28 - fold, down - regulate the expression of the inflammation - related tumor necrosis factor - α gene TNF-α , interleukin - 6 gene IL-6 and interleukin - 1β gene IL-1β The expression, with the relative expression level being 0.18 - fold to 0.83 - fold.

[0030] The reagents and consumables used in the present invention are all ordinary commercially available products. The present invention will be further described below in conjunction with the examples: Example 1 Isolation of NHNK - 622 The samples were obtained from adult Haliotis diversicolor individuals with a shell length of 5 cm from a farm, and they were fasted for 3 days. The digestive tract was dissected using sterile tools, rinsed 3 times with sterile physiological saline, and then the intestinal tissue was disrupted by homogenization and grinding. The homogenate was collected in 1 ml of sterile physiological saline, serially diluted, and the supernatant was streaked on an MRS (Qingdao Haibo Biotechnology) solid plate. After incubation at 37 °C for 24 - 48 h, white colonies were picked and repeatedly inoculated and screened until a uniform single colony was obtained, which was named NHNK - 622.

[0031] Gram staining and microscopic examination: The strain NHNK - 622 was a Gram - positive colony, showing a rod - shaped and curved arrangement under the microscope; it grew on the MRS plate, forming white, smooth, round and translucent colonies with neat edges; it grew evenly and turbidly in the MRS liquid medium, and the bacteria formed a white precipitate after standing for a long time. As Figure 1 shown.

[0032] Example 2 Identification of the 16S rDNA gene sequence of NHNK - 622 1. 16S rDNA gene sequence analysis: A single colony was picked into MRS liquid medium and cultured overnight at 37 °C. The bacteria were collected by centrifugation at 8000 rpm for 1 min, and the operation was carried out according to the instructions of the Gram - positive bacteria DNA extraction kit (Beijing 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, denaturation at 94 °C for 15 s, annealing at 57 °C for 15 s, extension at 72 °C for 1 min, for 35 cycles; and extension at 72 °C for 10 min.

[0033] 2. Results After sequencing the PCR products and performing a homology comparison (BLASTN) with the published standard sequences in the GenBank database, it was found that the strain NHNK - 622 was Bifidobacterium adolescentis.

[0034] Example 3 Experiment on the inhibition of Klebsiella pneumoniae growth by the fermentation products of NHNK - 622 1. Preparation of the fermentation products of NHNK - 622 A single colony of NHNK - 622 was picked into MRS liquid medium and statically cultured at 37 °C for 48 h. Then it was adjusted to OD600 = 1.0 with MRS, and the supernatant was taken at 5000 rpm and then filtered through a 0.22 μm filter membrane to obtain sterile fermentation products.

[0035] 2. Preparation of the Klebsiella pneumoniae bacterial suspension Inoculate Klebsiella pneumoniae CICC 10870 into BHI (Qingdao Haibo Biotechnology) liquid medium at 1% (v / v), and culture it with shaking at 37°C for 24 h. After the culture is completed, centrifuge at 5000 rpm to obtain the bacterial cells, and adjust the OD600 to 0.3 with BHI for standby.

[0036] 3. Experiment on the inhibition of the proliferation of Klebsiella pneumoniae by the fermentation product of NHNK-622 Add 3 mL of BHI medium and 0.3 mL of the fermentation product of NHNK-622 into a centrifuge tube. Add an equal volume of MRS medium to the control group. Inoculate the Klebsiella pneumoniae suspension at 1% (v / v), and measure the absorbance at 600 nm after shaking culture at 37°C for 24 h.

[0037] The results are shown in the following table: Table 1 Inhibition of the growth of Klebsiella pneumoniae by the fermentation product of NHNK-622

[0038] The results show that NHNK-622 can reduce the proliferation of Klebsiella pneumoniae, and the inhibition rate is between 47.83% and 55.32%.

[0039] Example 4 Experiment on the agglutination of Klebsiella pneumoniae by NHNK-622 1. Preparation of heat-inactivated cells of NHNK-622 Pick a single colony of NHNK-622 into MRS liquid medium, culture it 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 bacterial cells with PBS (Beijing Solarbio) and adjust the OD600 to 0.3. Autoclave at 121°C for 15 min to obtain heat-inactivated cells.

[0040] 2. Preparation of Klebsiella pneumoniae suspension Inoculate Klebsiella pneumoniae CICC 10870 into BHI liquid medium at 1% (v / v), and culture it with shaking at 37°C for 24 h. After the culture is completed, centrifuge at 5000 rpm to obtain the bacterial cells, and adjust the OD600 to 0.3 with PBS for standby.

[0041] 3. Co-agglutination experiment Mix the suspensions of Klebsiella pneumoniae and heat-inactivated cells of NHNK-622 at a volume ratio of 1:1. Take samples of NHNK-622 alone, Klebsiella pneumoniae alone, and the mixed reaction solution of NHNK-622 and Klebsiella pneumoniae at 30 min of reaction. The sampling range is the top 50 μL of the liquid surface. After aspiration, transfer it to a 96-well plate and measure its absorbance at OD = 600 nm. At the same time, stain the agglutinated precipitate with a Gram stain and observe the agglutination state of the bacterial cells. As Figure 2As shown in the figure, the agglutination rate calculation formula is as follows: Agglutination rate (%) = [(Ax + Ay) - 2Amix] / (Ax + Ay)×100%; Note: Ax: OD600 value measured for NHNK-622 alone at the reaction time; Ay: OD600 value measured for Klebsiella pneumoniae alone at the reaction time; Amix: OD600 value measured after the combined action of NHNK-622 and Klebsiella pneumoniae at the reaction time. The results are shown in the following table: Table 2 Agglutination of Klebsiella pneumoniae by heat-inactivated cells of NHNK-622

[0042] The results showed that heat-inactivated cells of NHNK-622 could agglutinate Klebsiella pneumoniae after 30 min of reaction.

[0043] Example 5 Alcohol tolerance experiment of NHNK-622 Pick a single colony of NHNK-622 and inoculate it into fresh MRS medium, and culture it at 37 °C for 24 h. After the culture, inoculate it into MRS medium containing 10% alcohol at a ratio of 2% (v / v). Take 100 μL of the bacterial suspension, measure the absorbance at OD = 600 nm, and record the OD values before and after culturing at 37 °C for 48 h. Calculation formula: Growth rate (%) = (A1 - A0) / A0 * 100% (A1: absorbance value after culture; A0: absorbance value before culture). The results are shown in the following table: Table 3 Alcohol tolerance experiment of NHNK-622

[0044] The results showed that NHNK-622 had the ability to tolerate alcohol and could grow in the medium containing 10% alcohol, with a growth rate of 125.00% - 142.86%.

[0045] Example 6 NHNK-622 regulates the expression of genes related to alcohol-induced damage to keratinocyte barrier and inflammation 1. Preparation of NHNK-622 Pick a single colony of NHNK-622 and inoculate it into fresh MRS medium, and culture it at 37 °C for 24 h. Adjust it to OD600 = 1.0 with DMEM medium (Beijing 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 precipitated bacteria, wash them twice with sterile PBS, resuspend the bacteria with DMEM medium and adjust OD600 = 0.5 to obtain a live bacterial suspension. Part of the precipitated live bacteria were washed twice with sterile PBS and then autoclaved at 121 °C for 15 min, and resuspended with DMEM medium and adjusted to OD600 = 0.5 to obtain heat-inactivated bacteria.

[0046] 2. Culture of immortalized human keratinocytes HaCaT After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, HaCaT cells (BNCC339817, Beijing Beina Biotechnology) were cultured at 37 °C and 5% CO2. After the cells reached 80 - 90% confluence, subculture or plating was performed.

[0047] 3. NHNK-622 regulates the expression of genes related to alcohol-induced damage to the keratinocyte barrier and inflammation HaCaT cells were seeded at 1×10^6 cells / well in a 6-well cell culture plate and cultured for 12 h until the cells adhered. The cell culture medium was removed, and the cells were washed twice with sterile PBS. Then, 1.9 mL of DMEM medium and 100 μL of NHNK-622 fermentation product / inactivated cells were added. In the control group, an equal volume of DMEM medium was added. After 3 h of treatment, 6% alcohol was added to induce damage, and 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. Then, 1 mL of cell RNA extraction reagent (Beijing Solarbio) was added to each well, and total RNA was extracted according to the reagent instructions, and the concentration and purity were measured. After extraction, it was reverse transcribed into cDNA, and the expression levels of ZO-1, CLDN4, OCLD, TNF-α, IL-6, and IL-1β genes were determined 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. The results are shown in the following table: Table 4 NHNK-622 fermentation product regulates the expression of genes related to alcohol-induced damage to the keratinocyte barrier and inflammation

[0048] Table 5 NHNK-622 inactivated cells regulate the expression of genes related to alcohol-induced damage to the keratinocyte barrier and inflammation

[0049] The results showed that NHNK-622 upregulated the expression of the barrier-related genes ZO-1, CLDN4, and OCLD, and downregulated the expression of the pro-inflammatory factor-related genes TNF-α, IL-6, and IL-1β. NHNK-622 could relieve alcohol-induced keratinocyte barrier damage and reduce inflammation simultaneously.

[0050] Example 7 NHNK-622 regulates the expression of genes related to alcohol-induced damage to the brain microvascular endothelial cell barrier and inflammation 1. Preparation of NHNK-622 fermentation product The preparation method of the NHNK-622 fermentation product refers to Example 3.

[0051] 2. Culture of Brain Microvascular Endothelial Cells After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, brain microvascular endothelial cells BEND3 (BNCC339490, Beijing Beina Biotechnology) were cultured at 37°C and 5% CO2. After the cells reached 80-90% confluence, subculture or plating operations were performed.

[0052] 3. NHNK-622 Regulates the Expression of Genes Related to Alcohol-Induced Injury of Brain Microvascular Endothelial Cell Barrier and Inflammation BEND3 cells were seeded at 1×10^6 cells / well in a 6-well cell culture plate and cultured for 12 h until the cells adhered. The cell culture medium was removed, and the cells were washed twice with sterile PBS. Then, 1.9 mL of DMEM medium and 100 μL of NHNK-622 fermentation product were added respectively, and an equal volume of DMEM medium was added to the control group. After 3 h of treatment, 6% alcohol was added to induce injury, and the cells were cultured at 37°C and 5% CO2 for 24 h. After the culture was completed, 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 according to the reagent instructions, and the concentration and purity were measured. After extraction, it was reverse transcribed into cDNA, and the expression levels of ZO-1, CLDN5, OCLD, TNF-α, IL-6, and IL-1β genes were measured by qPCR. The relative expression fold of the control group gene F = 1, and the F value of each sample was calculated by the 2-ΔΔCT method. The results are shown in the following table: Table 6 NHNK-622 Fermentation Product Regulates the Expression of Genes Related to Alcohol-Induced Injury of Brain Microvascular Endothelial Cell Barrier and Inflammation

[0053] The results showed that the NHNK-622 fermentation product could up-regulate the expression of vascular barrier-related genes ZO-1, CLDN5, and OCLD, and down-regulate the expression of inflammation-related genes TNF-α, IL-6, and IL-1β. NHNK-622 could alleviate alcohol-induced injury of brain microvascular endothelial cell barrier and reduce inflammation.

[0054] Example 8 NHNK-622 Improves the Survival Rate of Brain Microvascular Endothelial Cells Induced by Klebsiella pneumoniae 1. Preparation of NHNK-622 Fermentation Product The preparation method of the NHNK-622 fermentation product was referred to Example 3.

[0055] 2. Preparation of Klebsiella pneumoniae Bacterial Suspension Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured with shaking at 37 °C for 24 h. After the culture, the cells were centrifuged at 5000 rpm to obtain the cell pellet, washed 3 times with PBS, and then resuspended with DMEM and adjusted to OD600 = 0.1 for standby.

[0056] 3. Culture of brain microvascular endothelial cells The culture method of brain microvascular endothelial cell BEND3 was referred to Example 7.

[0057] 4. Experiment on NHNK-622 improving the survival rate of brain microvascular endothelial cells damaged by Klebsiella pneumoniae BEND3 cells were seeded into 96-well cell culture plates at 1×10^4 cells / well and cultured for 24 h until the cells adhered. After removing the medium, 100 μL of DMEM medium containing 1% (v / v) live NHNK-622 or fermentation product was added to the experimental group, and an equal volume of DMEM medium was added to the control group. After incubation for 3 h, 1 μL of Klebsiella pneumoniae suspension was added to each well, and the cells were incubated at 37 °C and 5% CO2 for 24 h. Then, 10 μL of cck-8 (GLPbio, USA) reagent was added to each well, and the absorbance at 450 nm was measured after culturing at 37 °C for 3 h to quantify the number of live cells. The calculation formula and results are as follows: Table 7 NHNK-622 improving the survival rate of brain microvascular endothelial cells damaged by Klebsiella pneumoniae

[0058] The results showed that the fermentation product of NHNK-622 could increase the relative survival rate of brain microvascular endothelial cell BEND3 under the damage induced by Klebsiella pneumoniae.

[0059] Example 9 NHNK-622 regulating the expression of genes related to the barrier and inflammation of brain microvascular endothelial cells damaged by Klebsiella pneumoniae 1. Preparation of live NHNK-622 and fermentation product The preparation method was referred to Example 3.

[0060] 2. Preparation of Klebsiella pneumoniae suspension The preparation method of Klebsiella pneumoniae suspension was referred to Example 8.

[0061] 3. Culture of brain microvascular endothelial cells The culture method of brain microvascular endothelial cell BEND3 was referred to Example 7.

[0062] 4. NHNK-622 regulating the expression of genes related to the barrier and inflammation of brain microvascular endothelial cells damaged by Klebsiella pneumoniae Inoculate BEND3 cells at a density of 1×10^6 cells per well in a 6-well cell culture plate and incubate for 12 h until the cells adhere to the plate. Remove the cell culture medium, wash twice with sterile PBS, then add 1.9 mL of DMEM medium and 100 μL of live bacteria / fermentation products of NHNK-609. Add an equal volume of DMEM medium to the control group. After 3 h of treatment, add 20 μL of Klebsiella pneumoniae bacterial suspension to induce injury and culture at 37 °C under 5% CO2 for 24 h. After the culture is completed, discard the supernatant, wash twice with sterile PBS, then add 1 mL of cell RNA extraction reagent to each well, and extract total RNA according to the reagent instructions and measure the concentration and purity. After extraction, reverse transcribe it into cDNA, and use qPCR to measure the expression levels of ZO-1, OCLD, CLDN5, TNF-α, IL-6, and IL-1β genes. The relative expression fold of the genes in the control group is F = 1, and the F value of each sample is calculated using the 2-ΔΔCT method. The results are shown in the following table: Table 8 Regulation of the expression of genes related to the barrier and inflammation of BEND3 cells induced by Klebsiella pneumoniae by live bacteria of NHNK-622

[0063] Table 9 Regulation of the expression of genes related to the barrier and inflammation of BEND3 cells induced by Klebsiella pneumoniae by fermentation products of NHNK-622

[0064] The results showed that NHNK-622 upregulated the expression of the vascular barrier-related genes ZO-1, CLDN5, and OCLD, downregulated the expression of the inflammation-related genes TNF-α, IL-6, and IL-1β, and could reduce the barrier injury of brain microvascular endothelial cells induced by Klebsiella pneumoniae and reduce inflammation.

[0065] Example 10 Regulation of the expression of genes related to the barrier and inflammation of intestinal epithelial cells induced by Klebsiella pneumoniae by NHNK-622 1. Preparation of live bacteria and inactivated bacteria of NHNK-622 The preparation method refers to Example 6.

[0066] 2. Preparation of Klebsiella pneumoniae bacterial suspension The preparation of Klebsiella pneumoniae bacterial suspension refers to Example 9.

[0067] 3. Culture of human intestinal epithelial cells Caco-2 After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, Caco-2 cells are cultured at 37 °C under 5% CO2. When the cell confluence reaches 80-90%, subculture or plating operations are performed.

[0068] 4. NHNK-622 Regulates the Expression of Genes Related to Intestinal Epithelial Cell Barrier and Inflammation Induced by Klebsiella pneumoniae Caco-2 cells were seeded into 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. Subsequently, 1.9 mL of DMEM medium and 100 μL of live / inactivated NHNK-622 bacteria were added. In the control group, an equal volume of DMEM medium was added. After 3 h of treatment, 20 μL of Klebsiella pneumoniae suspension was added to induce injury, and 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. Then, 1 mL of cell RNA extraction reagent was added to each well, and total RNA was extracted according to the reagent instructions, and the concentration and purity were measured. After extraction, it was reverse transcribed into cDNA, and the expression levels of ZO-1, CLDN4, TNF-α, IL-6, and IL-1β genes were measured by qPCR. The relative expression fold of the genes in the control group was F = 1, and the F values of each sample were calculated using the 2-ΔΔCT method. The results are shown in the following table: Table 10 Regulation of the Expression of Genes Related to Intestinal Epithelial Cell Barrier and Inflammation Induced by Klebsiella pneumoniae by Live NHNK-622

[0069] Table 11 Regulation of the Expression of Genes Related to Intestinal Epithelial Cell Barrier and Inflammation Induced by Klebsiella pneumoniae by Inactivated NHNK-622

[0070] The results showed that NHNK-622 could upregulate the expression of intestinal barrier-related genes ZO-1 and CLDN4 and downregulate the expression of inflammatory factor-related genes TNF-α, IL-6, and IL-1β. NHNK-622 could reduce intestinal permeability and inflammation caused by Klebsiella pneumoniae, thereby reducing the possibility of Klebsiella pneumoniae entering the circulatory system through the intestine.

[0071] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), and this strain is Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) NHNK-622, which was deposited at the China Center for Type Culture Collection on December 05, 2024, and its deposit number is CCTCC NO: M 20242734.

2. The product prepared from the Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), characterized in that The product is live bacteria, inactivated bacteria or fermentation products of Bifidobacterium adolescentis ( Bifidobacterium adolescentis ).

3. The product prepared from the Bifidobacterium adolescentis as claimed in claim 1, characterized in that, Bifidobacterium adolescentis The product further comprises excipients and / or adjuvants.

4. The application of the Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) or the product prepared from the Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) as claimed in claim 2 in the preparation of a product for improving urticaria and Alzheimer's disease.

5. The application according to claim 4, wherein The improvement of urticaria and Alzheimer's disease includes at least one of the following a)-h): a), inhibiting the growth and / or alcohol production of Klebsiella pneumoniae ( Klebsiella pneumoniae ); b), agglutinating Klebsiella pneumoniae; c), growing tolerantly to 10% alcohol; d), reduce alcohol-induced damage to the skin keratin barrier and inflammation, up-regulate barrier-related genes ZO-1, CLDN4 and OCLD , down-regulate the expression of at least one of the inflammation-related genes TNF-α , IL-6 and IL-1β ; e), reducing alcohol-induced damage to the cerebral microvascular barrier and inflammation, and upregulating barrier-related genes ZO-1, CLDN5 and OCLD , and downregulating the expression of at least one of the inflammation-related genes TNF-α , IL-6 and IL-1β ; f), increasing the survival rate of Klebsiella pneumoniae-induced injury in brain microvascular endothelial cells; g), reducing Klebsiella pneumoniae-induced damage to the brain microvascular barrier and inflammation, and upregulating the expression of barrier-related genes ZO-1 , CLDN5 and OCLD , and downregulating the expression of at least one of the inflammation factor-related genes TNF-α , IL-6 and IL-1β ; h), reducing Klebsiella pneumoniae-induced intestinal barrier damage and inflammation, upregulating the expression of barrier-related genes ZO-1 , CLDN4 , downregulating the expression of at least one of the inflammation factor-related genes TNF-α , IL-6 and IL-1β .

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