Lactobacillus rhamnosus capable of stimulating CD103 + DC cells and application of lactobacillus rhamnosus

Abstract

The invention provides lactobacillus rhamnosus capable of stimulating CD103 + DC cells and application of the lactobacillus rhamnosus. The lactobacillus rhamnosus is lactobacillus rhamnosus B9-72, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31329. The lactobacillus rhamnosus B9-72 disclosed by the invention can be used for improving the symptoms of specific dermatitis (AD) of mice; after the allergen is removed, viable bacteria are applied in a smearing manner, so that the AD recovery can be obviously promoted, and the safety is extremely high.

Description

[0001] Cross-references to related applications

[0002] This invention claims priority to the earlier application filed on August 15, 2024, with patent application number 202411127070.3 and entitled "A strain of Lactobacillus rhamnosus that stimulates CD103+ DC cells and its application". The entire contents of that earlier application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of bacterial culture technology, and in particular to a type of Lactobacillus rhamnosus that stimulates CD103+DC cells and its applications. Background Technology

[0004] The direct causes of atopic dermatitis (AD) include genetic and environmental factors, skin barrier dysfunction, microbial imbalance, immune dysregulation, skin inflammation, and the interaction of various factors with the environment. According to Medscape, 85% of AD cases occur before the age of one, and 95% occur before the age of five. Three large-scale epidemiological surveys conducted in 1998, 2002, and 2014 showed that the prevalence of AD in Chinese children was 0.69%, 3.07%, and 12.94%, respectively. Among these, the majority of children with AD had mild symptoms (74.6%), followed by moderate symptoms (23.96%), with severe symptoms being relatively rare (1.44%).

[0005] It is generally believed that dysregulation of Th2 and Th22 cytokines leading to keratinocyte destruction is a key factor driving Alzheimer's disease (AD) and its development. Furthermore, damage to epidermal barrier proteins, resulting in impaired barrier function, can also increase cell and allergen penetration, playing a significant role in the occurrence and development of AD.

[0006] Childhood Alzheimer's disease (AD) is primarily characterized by Th2-type inflammation, and maintaining the Th1 / Th2 balance is crucial for alleviating AD symptoms. The body's immune response begins with antigen-presenting cells (APCs) capturing antigens, which are then processed and transmitted to lymphocytes, triggering an immune response. Dendritic cells (DCs) are the most potent APCs in the body. A significant characteristic of DCs is their regulatory role in T-cell immune responses, controlling the direction of the T-cell response. Population studies have found that DCs derived from allergic children produce more inflammatory factors compared to healthy children, promoting the transformation of CD4+ T cells into Th2-type cells in vitro.

[0007] The specific markers for dendritic cells (DCs) in humans and mice are CD11c+MHC-II+, while CD103+ serves as a marker for immunosuppressive DCs. CD103+ DCs are a type of regulatory DC that can be induced to differentiate from CD4+ T cells into Treg cells through cytokines such as TGF-β, playing a crucial role in regulating immune homeostasis and immune tolerance. Currently, there is a lack of effective methods for stimulating CD103+ DCs and for treating atopic dermatitis. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a novel method for treating atopic dermatitis by stimulating CD103+ DC cells with Lactobacillus rhamnosus and its application.

[0009] This invention uses CD103+CD11c+ as a marker for immunosuppressive dendritic cells (DCs) to screen for immunomodulatory bacterial strains. The resulting immunosuppressive strain, B9-72, showed superior efficacy compared to the commercial strain LGG. Animal experiments verified that this strain can improve AD symptoms during allergen exposure and significantly accelerate the recovery of AD symptoms after allergen removal.

[0010] In one aspect, the present invention provides a Lactobacillus rhamnosus, which is Lactobacillus rhamnosus B9-72, with accession number CGMCC No. 31329.

[0011] In one embodiment of the present invention, the Lactobacillus rhamnosus B9-72 does not contain antibiotic resistance genes or virulence factor genes.

[0012] In one embodiment of the present invention, the Lactobacillus rhamnosus B9-72 does not produce putrescine, cadaverine, histamine, or tyramine.

[0013] In one embodiment of the present invention, the Lactobacillus rhamnosus B9-72 can promote the increase of CD103+CD11c+DC cells.

[0014] In one embodiment of the present invention, Lactobacillus rhamnosus B9-72 can promote the increase of CD103+CD11c+ in bone marrow DC cells.

[0015] In one embodiment of the present invention, compared with Lactobacillus rhamnosus LGG, Lactobacillus rhamnosus B9-72 can promote the increase of more CD103+CD11c+DC cells.

[0016] In one embodiment of the present invention, the Lactobacillus rhamnosus B9-72 has one or more characteristics selected from the following:

[0017] 1) Promotes the increase of CD103+CD11c+ DC cells;

[0018] 2) Regulates DC cell differentiation;

[0019] 3) Promotes the differentiation of CD4+ T cells into Treg cells;

[0020] 4) Regulates the body's immune balance and immune tolerance;

[0021] 5) Prevention or treatment of allergic reactions or inflammatory skin diseases;

[0022] 6) Improve the speed of recovery of allergic reaction symptoms after allergen removal.

[0023] In one embodiment of the present invention, the allergic reaction or inflammatory skin disease includes atopic dermatitis and allergic dermatitis.

[0024] In a second aspect, the present invention provides a microbial agent comprising the aforementioned Lactobacillus rhamnosus B9-72.

[0025] In one embodiment of the present invention, the dosage of Lactobacillus rhamnosus B9-72 in the bacterial agent is approximately 1 × 10⁻⁶. 4 ~1×10 12 / dose.

[0026] In another embodiment, the therapeutically effective amount of Lactobacillus rhamnosus B9-72 in the bacterial agent is approximately 1 × 10⁻⁶. 6 ~1×10 9 / dose.

[0027] In one embodiment of the present invention, the bacterial agent includes live or inactivated Lactobacillus rhamnosus B9-72.

[0028] In one embodiment of the present invention, the microbial agent further includes pharmaceutically acceptable excipients. Preferably, the excipients include sweeteners or flavoring agents, colorants, stabilizers, flow aids, fillers, humectants, thickeners, emulsifiers, preservatives, film-forming agents, antioxidants, and any combination thereof.

[0029] In one embodiment of the present invention, the sweeteners include, but are not limited to, sucrose, fructose, glucose, honey, maple syrup, stevia, monk fruit extract, aspartame, sucralose (Splenda), saccharin, and acesulfame K. The flavoring agents include, but are not limited to, vanilla extract, citric acid, malic acid, peppermint oil, vanillin, cinnamon, turmeric, chili powder, and rosemary extract. The coloring substances include, but are not limited to, natural pigments such as carotene, beetroot red, safflower yellow, chlorophyll, curcumin, and carmine; and artificial pigments such as Tartzine, Sunset Yellow FCF, Allura Red AC, and Brilliant Blue FCF. The stabilizers include thickeners such as gelatin, gum arabic, guar gum, xanthan gum, and carrageenan. The anti-caking agents include silica, aluminum trichloride, and tricalcium phosphate. The flow aids include, but are not limited to, silica gel, magnesium stearate, talc, microcrystalline cellulose, and calcium hydrogen phosphate. The fillers include, but are not limited to, lactose, microcrystalline cellulose, starch, hydroxypropyl methylcellulose, and calcium phosphate. The humectants include, but are not limited to, glycerin, propylene glycol, sorbitol, sodium lactate, and polyethylene glycol. The thickeners include, but are not limited to, xanthan gum, guar gum, pectin, carrageenan, gelatin, and hydroxypropyl methylcellulose. The emulsifiers include, but are not limited to, lecithin, polysorbate 80 (Tween 80), monoglycerides, sodium stearate, phospholipids, and emulsifying waxes. The preservatives include, but are not limited to, sodium benzoate, potassium sorbate, parabens, sodium sulfate, sodium sulfite, and benzoic acid. The film-forming agents include, but are not limited to, polyvinyl alcohol, hydroxypropyl methylcellulose, ethyl cellulose, shellac, and cellulose acetate. The antioxidants include, but are not limited to, ascorbic acid (vitamin C), tocopherol (vitamin E), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and citric acid.

[0030] In one embodiment of the present invention, the microbial agent can be any dosage form, including emulsions, pills, tablets, capsules, powders, liquids, or gels. In a specific embodiment of the present invention, the microbial agent is an emulsion or powder.

[0031] In one embodiment of the present invention, the bacterial agent can be administered in any form, including oral, transdermal, injection, inhalation, and topical administration. In a specific embodiment of the present invention, the administration method is transdermal administration.

[0032] A third aspect of the present invention provides a culture comprising the above-mentioned *Lactobacillus rhamnosus* and bacterial agent.

[0033] In one embodiment of the present invention, the culture is a bacterial suspension of Lactobacillus rhamnosus.

[0034] In one embodiment of the present invention, the culture further comprises nutrients (e.g., solid or liquid culture medium, feeder cell layer).

[0035] In one embodiment of the present invention, the nutrients are selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof.

[0036] In one embodiment of the present invention, the culture further comprises a cell-free culture filtrate of Lactobacillus rhamnosus.

[0037] In one embodiment of the present invention, the culture further comprises a derivative of Lactobacillus rhamnosus.

[0038] In one embodiment of the present invention, the derivative is selected from metabolites, enzymes, cellular structural components (e.g., cell walls or components thereof), extracellular polysaccharides, bacteriocins, or any combination thereof.

[0039] In a fourth aspect, the present invention provides a composition comprising the above-mentioned Lactobacillus rhamnosus, bacterial agent, and culture.

[0040] In one embodiment of the present invention, the composition further comprises lipids, carbohydrates, proteins, vitamins, and / or minerals. The proteins include, but are not limited to, skim milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and any combination thereof. The carbohydrates include, but are not limited to, lactose, glucose, corn syrup solids, maltodextrin, sucrose, starch, rice syrup solids, and any combination thereof. The lipids include, but are not limited to, palm oil, soybean oil, palm olein, coconut oil, medium-chain triglyceride oil, high-oleic sunflower oil, high-oleic safflower oil, long-chain polyunsaturated fatty acids (LCPUFA), and any combination thereof. LCPUFA includes, but is not limited to, α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), ARA, and DHA. The minerals include, but are not limited to, iron, zinc, potassium, sodium, calcium, magnesium, and any combination thereof. The vitamins include, but are not limited to, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, and any combination thereof.

[0041] In one embodiment of the present invention, the composition further includes one or more other combinations of probiotics. In some embodiments, any probiotics known in the art are acceptable. In one embodiment of the present invention, the other probiotics are selected from lactic acid bacteria.

[0042] In one embodiment of the present invention, the composition further includes one or more prebiotic combinations. In some embodiments, any prebiotic known in the art is acceptable. In one embodiment of the present invention, the prebiotic may include lactulose, galactooligosaccharides, fructooligosaccharides, isomalto-oligosaccharides, soybean oligosaccharides, lactosucrose, xylo-oligosaccharides, and gentio-oligosaccharides.

[0043] In a fifth aspect, the present invention provides the use of the above-mentioned Lactobacillus rhamnosus, bacterial agent, culture, and composition in the preparation of pharmaceuticals for the following purposes.

[0044] 1) Promotes the increase of CD103+CD11c+ DC cells;

[0045] 2) Regulates DC cell differentiation or DC cell secretion of the cytokine TGF-β;

[0046] 3) Promotes the differentiation of CD4+ T cells into Treg cells;

[0047] 4) Regulates the body's immune balance and immune tolerance;

[0048] 5) Prevention or treatment of allergic reactions or inflammatory skin diseases;

[0049] 6) Improve the speed of recovery of allergic reaction symptoms after allergen removal.

[0050] In one embodiment of the present invention, the allergic reaction or inflammatory skin disease includes atopic dermatitis and allergic dermatitis.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The genome of Lactobacillus rhamnosus B9-72 of the present invention does not contain antibiotic resistance genes or virulence factor genes, and has extremely high safety at the gene level; the strain does not produce putrescine, cadaverine, histamine and tyramine, and has high safety at the metabolic level.

[0053] (2) The strain of the present invention can promote the proportion of CD103+ cells in bone marrow DC cells (mBMDC) and has the potential to be used for the prevention and treatment of diseases such as atopic dermatitis and allergic dermatitis.

[0054] (3) Animal experiments of the present invention have shown that during continuous exposure to allergens, the administration of live or dead bacteria by application can improve the symptoms of atopic dermatitis (AD) in mice; and the administration of live bacteria by application after the removal of allergens can significantly promote the recovery of AD.

[0055] (4) The strains or agents of the present invention can be applied directly to the skin and exert their effects directly on the dermatitis lesions, which can quickly and effectively treat or relieve dermatitis symptoms. Attached Figure Description

[0056] Figure 1 The results are from flow cytometry analysis of CD103+CD11c+ cells;

[0057] Figure 2 A graph showing the proportion of CD103+ cells in CD11c cells;

[0058] Figure 3 The back score of mice during allergen stimulation;

[0059] Figure 4 The thickness of the mouse's back during allergen stimulation;

[0060] Figure 5 The comprehensive back score of mice after removal of allergen stimulation;

[0061] Figure 6 The thickness of the back skin of mice after the removal of allergen stimulation. Detailed Implementation

[0062] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0063] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0064] The term "naked 1640" or "naked 1640" refers to basic RPMI-1640 medium without any of the above-mentioned additives.

[0065] Example 1: Isolation and Identification of Lactobacillus rhamnosus B9-72

[0066] 1. Isolation of Lactobacillus rhamnosus B9-72

[0067] (1) Stool sample collection: Take about 5g of the middle part of the stool sample (the center of the strip-shaped stool) from the stool sample of a healthy infant and put it into a 50mL centrifuge tube. Transport it to the laboratory at low temperature using an anaerobic box.

[0068] (2) Sample dilution: After weighing the sample, transfer it to the anaerobic chamber and dilute the fecal sample 10 times with physiological saline, denoted as 10. -1 Take 1 mL of 10 -1 Add the diluent to 9 mL of physiological saline to make a 10⁻⁶ solution. -2 ), and so on, diluting stepwise to 10. -9 During dilution, the centrifuge tube should be shaken up and down, and the mixture should be blown around several times with a pipette until it is thoroughly mixed.

[0069] (3) Plate coating: Take 200 μl and coat it with dilutions of -7 and -8 on MRS plates; the incubation conditions are 37℃ and 70% relative humidity;

[0070] (4) Enrichment culture: Select plates with a moderate number of bacteria (-7, -8, -9), observe the colony morphology, and observe the colony color, size, surface moisture or dryness. Use an inoculation loop to select single clones with different morphologies and add them to 3ml of the corresponding liquid culture medium for enrichment culture.

[0071] (5) When visible turbidity appears, take 200 μl for 16S DNA full-length PCR amplification and sequencing. Use a pipette to transfer 700 μl of the remaining bacterial culture into a 2 ml preservation tube (prepared with 300 μl of glycerol preservation solution). Preserve two copies of each bacterial strain. Store at -80℃.

[0072] 2. Identification of Lactobacillus rhamnosus B9-72

[0073] (1) Morphological identification

[0074] Strawberry strain B9-72 was streaked on MRS medium and incubated at 37°C for 48 hours. Colony morphology was observed. The colonies were approximately 0.5-1 mm in size, uniform in texture, white, moist, with uniform color, neat edges, and a smooth, moist surface. They were short rod-shaped.

[0075] (2) 16S rDNA molecular assay (SEQ ID NO:1)

[0076]

[0077] After comparison using NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), strain B9-72 was identified as *Lactaseibacillus rhamnosus*, and its accession number was assigned as *Lactaseibacillus rhamnosus* B9-72, or simply *Lactaseibacillus rhamnosus* B9-72 or B9-72. *Lactaseibacillus rhamnosus* B9-72 was deposited on July 17, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31329.

[0078] Lactobacillus rhamnosus B-9, Lactobacillus rhamnosus 277, and Lactobacillus rhamnosus LAC were isolated using the same method and identified as Lactobacillus rhamnosus.

[0079] Example 2: Physicochemical property testing of Lactobacillus rhamnosus B9-72

[0080] 1. Determination of biogenic amines

[0081] B9-72 strain was cultured anaerobically in MRS liquid medium at 37℃ for 48 h, centrifuged, and the wet bacterial cells were weighed. 10% trichloroacetic acid was added, and the mixture was extracted in a boiling water bath for 1 h. The supernatant was collected, and the contents of putrescine, cadaverine, histamine, and tyramine were determined by chromatography. The results were based on the percentage of wet bacterial cells. The limits of detection were (putrescine, histamine, and tyramine: 1.25 mg / kg; cadaverine: 0.75 mg / kg) (National Standard GB5009.208-2016 Food Safety Standard - Determination of Biogenic Amines in Food, Method I). The results are shown in Table 1.

[0082] Table 1. Results of Biogenic Amine Detection in B9-72

[0083] Biogenic amines Test results Detection limit putrescine Not detected 1.25 mg / L histamine Not detected 1.25 mg / L Tyramine Not detected 1.25 mg / L Cadaniamine Not detected 0.75 mg / L

[0084] 2. Quantitative Detection Method for L / D Lactic Acid Produced by Lactic Acid Bacteria

[0085] The B9-72 strain was inoculated into MRS liquid medium and cultured at 37°C for 24 h, followed by three generations of activation. The supernatant from the three generations after 24 h of activation was collected, and the lactic acid content was determined using high-performance liquid chromatography (HPLC). The results are shown in Table 2. The results indicate that the strain mainly produces L-lactic acid, with only trace amounts of D-lactic acid.

[0086] Table 2B9-72 Lactic Acid Production

[0087] Types of lactic acid concentration L-lactic acid 9.261 mg / mL D-lactic acid 0.320 mg / mL

[0088] 3. Antibiotic resistance gene testing

[0089] With the discovery and use of antibiotics, an increasing number of drug-resistant bacterial strains have emerged. The development of drug-resistant strains increases the difficulty and cost of disease treatment. The ARDB database was the first database to integrate drug-resistant genes from various microorganisms. The CARD (Comprehensive Antibiotic Research Database) contains all resistance information from the ARDB database and has established a data-sharing platform based on volunteer contributions, ensuring real-time updates and data validity. The core of the CARD database is the ARO (Antibiotic Resistance Ontology), which contains information on resistance genes, resistance mechanisms, and antibiotics. Bacterial genomes with a similarity (identify) >80% to known antibiotic resistance genes are considered highly probable and are identified as positive.

[0090] According to the identification and comparison results, there are no genes with a similarity greater than 80% in the CARD database for B9-72, so it is confirmed that there are no resistance genes in the genome.

[0091] 4. Virulence factor detection

[0092] Virulence factors (VFs) are molecules produced by the metabolism of bacteria, viruses, fungi, etc., containing invasive and toxin-like virulence components. They are primarily used by microorganisms to enter and exit host cells by inhibiting or evading the host's immune response, and to obtain nutrients and reproduce from the host. Virulence factors can be encoded on mobile genetic elements (such as plasmids, gene islands, bacteriophages, etc.) and undergo horizontal gene transfer, transforming harmless bacteria into dangerous pathogens. Therefore, gene introduction and secreted proteins are generally considered when identifying virulence factors. The Virulence Factor Database (VFDB), developed by the Chinese Academy of Medical Sciences, collects and organizes the composition, structure, function, pathogenic mechanism, virulence islands, sequences, and genomic information of known virulence factors from various important medical pathogens. It is widely used for the identification of virulence factor genes. A bacterial genome with a similarity (identify) >80% to a known virulence factor is considered highly probable and is identified as positive.

[0093] According to the identification and comparison results, there are no genes with a similarity greater than 80% in the VFDB database for B9-72, so it is confirmed that there are no virulence factors in the genome.

[0094] Example 3: Lactobacillus rhamnosus B9-72 cell assay

[0095] 1. BMDC cell collection

[0096] Mice were euthanized by cervical dislocation and immersed in 75% alcohol. The femur and tibia were removed with scissors and placed in naked 1640 containing 2% penicillin and antibiotics. In a clean bench, the mice were first washed twice with pre-cooled naked 1640, then the leg bones were held with gauze, ankles facing upwards, and muscles and cartilage were removed before being placed in a new culture dish containing naked 1640. The bone marrow cavity was rinsed with naked 1640 using a 2mL syringe to obtain bone marrow. Then, using a syringe, disperse the bone marrow cells by agitation. Transfer the cell suspension through a 200-mesh nylon mesh to a 15 mL or 50 mL centrifuge tube and collect the cells by centrifugation at 400 g for 5 min. Discard the supernatant, resuspend the pellet in erythrocyte lysis buffer (2 mL / mouse), mix well, and incubate at 4°C for 8 min. Add PBS to stop lysis, centrifuge at 400 g for 5 min to collect the cells, and resuspend them in a medium containing 1640 + 10% FBS + 1% PS + 20 ng / ml GM-CSF + 10 ng / ml IL-4 for counting and seeding. Follow the 2.5 × 10⁻⁶ mcg / mL method. 7 Cells were seeded in 6 T75cm culture flasks; this is Day 0 of culture.

[0097] 2. BMDC cell induction

[0098] On day 3 of culture, take 6 culture flasks, aspirate all the culture medium from the flasks into 50mL centrifuge tubes, centrifuge at 400g for 5min, and then aspirate half of the supernatant and mix it thoroughly with fresh culture medium (1640 + 10% FBS + 1% PS + 40ng / ml GM-CSF + 20ng / ml IL-4). Slowly add this mixture into the culture flasks. Incubate at 37℃ in a CO2 incubator until day 5. Gently pipette the adherent cells in the flasks, aspirate all the culture medium, and centrifuge at 400g for 5min. Discard the supernatant, resuspend the cells in 80mL of fresh culture medium (1640 + 10% FBS + 1% PS + 20ng / ml GM-CSF + 10ng / ml IL-4), add the cells to new culture flasks, and incubate at 37℃ in a CO2 incubator until day 6. Collect the cells, shake the culture flasks, resuspend the collected BMDC cells in culture medium, count them, and plate them into plates at 1×10⁶ cells per well. 6 cells.

[0099] 3. Co-incubation of BMDC cells with Lactobacillus rhamnosus B9-72

[0100] Add 2×10 to each of the above cells in each well 8The test bacteria (including *Lactobacillus rhamnosus* B9-72 obtained in Example 1, commercially available *Lactobacillus rhamnosus* LGG, *Lactobacillus rhamnosus* B-9, *Lactobacillus rhamnosus* 277, and *Lactobacillus rhamnosus* LAC; the dosage for each group is shown in Table 3) were incubated with the cells and bacterial strains for 24 hours. Cells and supernatant were collected separately and centrifuged at 400g for 5 minutes. Cell collection involved repeatedly shaking the 24-well plate or gently pipetting to detach the semi-adherent dendritic cells from the cell culture dish. The collected cells were washed once with DPBS and centrifuged at 400g for 5 minutes. The cells were resuspended in 1 mL of DPBS, with each 1 mL of cell suspension containing 1 x 10⁻⁶ cells. 6 / mL~1x10 7 Add 1 μL of FVS780 solution to the 1 / mL container, vortex immediately, and then incubate at 4°C in the dark for 20 minutes; wash the cells twice with an appropriate amount of Stain Buffer, and centrifuge at 400g for 5 minutes. Carefully aspirate or discard the supernatant; resuspend the cells in 100 μL of Stain Buffer. Before staining, add 2 μL (i.e., 2 μg) of purified CD16 / CD32 antibody to each tube (mixed with 100 μL Stain Buffer + 2 μL of purified CD16 / CD32 antibody), and block at 4°C in the dark for 10 min. Except for blank, add each antibody to the sample tubes according to the recommended dosage (antibody dosage is shown in Table 4), and incubate at 4°C in the dark for 30 min (for samples other than single-positive tubes, prepare a mix by adding 2 to the sample quantity). Add 1 mL of Stain Buffer to each tube, vortex, centrifuge at 400g, 4°C for 5 min, and discard the supernatant (repeat this step once). Resuspend the cells in 0.5 mL of Stain Buffer to each tube. If you are not able to use the flow cytometer on the same day, please proceed to the next step. Add 0.5 mL of Stain Buffer to each tube to resuspend the cells, and add 80 μL of... The cell suspension was fixed with 4% paraformaldehyde to a final concentration of 0.55% at 4°C for 30 min, and washed twice with 1 mL of Stain Buffer. The cell suspension was then passed through a 100-mesh sieve for analysis. Experimental results are as follows: Figure 1 , Figure 2 As shown.

[0101] Table 3. Co-incubation of bacterial strains with BMDC cells

[0102] Group deal with Dosage source Crtl PBS 20μl B9-72 Lactobacillus rhamnosus B9-72 <![CDATA[2×10 8 / 20μlPBS]]> Healthy infant gut LGG Rhamnose LGG <![CDATA[2×10 8 / 20μlPBS]]> Commercially available products B-9 Lactobacillus rhamnosus B-9 <![CDATA[2×10 8 / 20μlPBS]]> Healthy infant gut 277 Lactobacillus rhamnosus 277 <![CDATA[2×10 8 / 20μlPBS]]> Healthy infant gut LAC Lactobacillus rhamnosus LAC <![CDATA[2×10 8 / 20μlPBS]]> Healthy infant gut

[0103] Table 4. List of Antibody Dosages for Cell Experiments

[0104]

[0105] The results showed that B9-72 stimulation significantly increased the number of CD103+CD11c+ double-positive cells, with better results than LGG, B-9, 277, and LAC. Figure 1The proportion of CD103+CD11c+ cells was 7.08%, higher than that of the commercial strain *Lactobacillus rhamnosus* LGG and other simultaneously isolated *Lactobacillus rhamnosus* B-9, 277, and LAC; compared with the PBS control group, the percentage of CD103+CD11c+ double-positive cells increased by 2.5 times under B9-72 stimulation. Figure 2 ).

[0106] Example 4: Strain B9-72 improves AD symptoms in mice

[0107] 1. Animal Information: 36 BALB / c female mice, untagged, provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0108] 2. Disease model: AD model induced by OXA (acetone: olive oil = 4:1).

[0109] 3. Grouping: After all mice entered the animal facility, they were acclimatized for at least one week, and hair was removed on day 9. The mice were marked by their tails. On day 0, they were divided into 5 groups according to their weight, right ear, back skin thickness, and back score.

[0110] 4. Model Establishment: A 2cm × 2cm area in the middle of the neck and back of mice was sensitized with 5% OXA (20 μl volume). After grouping, starting on Day 1, the back area was stimulated with 0.2% OXA (50 μl volume), and the right ear was stimulated with 0.3% OXA (20 μl volume, 10 μl for the inner and outer sides of the right ear). Stimulation of the ear and back was performed every 2 days for 3 weeks (D1, D3, D5, D7, D9, D11, D13, D15).

[0111] 5. Administer the medication as shown in Table 5.

[0112] Table 5 Drug administration process

[0113]

[0114] 6. Data Collection

[0115] Weight checks were conducted every two days; back skin thickness was measured every two days; and back disease scores were assessed every two days using a model.

[0116] 7. The scoring criteria for skin lesions on the neck and back are shown in Table 6.

[0117] Table 6 Scoring Criteria for Skin Damage on the Neck and Back

[0118]

[0119] 9. Abnormal handling: If the mice are in abnormal condition (emaciation, decreased mobility, weight loss of more than 15%), feed them nutritional gel and house them in individual cages.

[0120] 10. Experimental Results

[0121] Animal studies have shown that during sustained exposure to allergens, applying live or dead bacteria topically can improve Alzheimer's disease (AD) symptoms in mice, including the back score. Figure 3 ), back skin thickness ( Figure 4 As shown in the figure, both the live bacteria group and the dead bacteria group improved OXA-induced AD symptoms in mice, reduced the overall back score, and reduced back thickness during the intervention.

[0122] Applying live bacteria topically after removing the allergen can significantly promote AD recovery, including the overall back score. Figure 5 ), back skin thickness ( Figure 6 Experimental results showed that this strain could promote the recovery process of AD after the removal of allergens, significantly reducing the back comprehensive score and improving the recovery of back edema at 19 days and 21 days.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A type of Lactobacillus rhamnosus, characterized in that: It is Lactobacillus rhamnosus B9-72, with accession number CGMCC No. 31329.

2. The *Lactobacillus rhamnosus* as described in claim 1, characterized in that: The Lactobacillus rhamnosus B9-72 does not contain antibiotic resistance genes or virulence factor genes; Preferably, the Lactobacillus rhamnosus B9-72 does not produce putrescine, cadaverine, histamine, or tyramine; Preferably, the Lactobacillus rhamnosus B9-72 can promote the increase of CD103+CD11c+ DC cells; more preferably, the Lactobacillus rhamnosus B9-72 can promote the increase of CD103+CD11c+ in bone marrow DC cells. Preferably, compared to Lactobacillus rhamnosus LGG, Lactobacillus rhamnosus B9-72 can promote the increase of more CD103+CD11c+ DC cells; Preferably, the Lactobacillus rhamnosus B9-72 has one or more characteristics selected from the following: 1) Promotes the increase of CD103+CD11c+ DC cells; 2) Regulates DC cell differentiation; 3) Promotes the differentiation of CD4+ T cells into Treg cells; 4) Regulates the body's immune balance and immune tolerance; 5) Prevention or treatment of allergic reactions or inflammatory skin diseases; 6) Improve the recovery speed of allergic reaction symptoms after allergen removal; Preferably, the allergic reaction or inflammatory skin disease includes atopic dermatitis and allergic dermatitis.

3. A microbial agent, characterized in that: Includes Lactobacillus rhamnosus as described in claim 1 or 2.

4. The microbial agent as described in claim 3, characterized in that: The dosage of Lactobacillus rhamnosus B9-72 in the bacterial agent is 1×10⁻⁶. 4 ~1×10 12 / Dosage; Preferably, the therapeutically effective dose of Lactobacillus rhamnosus B9-72 in the bacterial agent is 1×10 6 ~1×10 9 / dose; Preferably, the bacterial agent comprises live or inactivated Lactobacillus rhamnosus B9-72; Preferably, the microbial agent further includes pharmaceutically acceptable excipients; more preferably, the excipients include sweeteners or flavoring agents, colorants, stabilizers, flow aids, fillers, humectants, thickeners, emulsifiers, preservatives, film-forming agents, antioxidants, and any combination thereof.

5. The microbial agent as described in claim 3, characterized in that: The microbial agent includes emulsions, pills, tablets, capsules, powders, liquids, or gels; preferably, the microbial agent is an emulsion or powder. Preferably, the administration method of the bacterial agent includes oral, transdermal, injection, inhalation, and local administration; more preferably, the administration method is transdermal administration.

6. A culture, characterized in that: It contains the Lactobacillus rhamnosus as described in claim 1 or 2, and the bacterial agent as described in any one of claims 3-5; Preferably, the culture is a bacterial suspension of Lactobacillus rhamnosus; Preferably, the culture further contains nutrients; Preferably, the nutrients are selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof. Preferably, the culture further comprises a cell-free culture filtrate of Lactobacillus rhamnosus; Preferably, the culture further comprises a derivative of Lactobacillus rhamnosus; Preferably, the derivative is selected from metabolites, enzymes, cellular structural components, extracellular polysaccharides, bacteriocins, or any combination thereof.

7. A composition, characterized in that: Includes the Lactobacillus rhamnosus as described in claim 1 or 2, the bacterial agent as described in any one of claims 3-5, and the culture as described in claim 6.

8. The composition according to claim 7, characterized in that: The composition also contains lipids, carbohydrates, proteins, vitamins and / or minerals; Preferably, the composition further includes one or more other probiotic combinations; Preferably, the composition further includes one or more prebiotic combinations.

9. The use of *Lactobacillus rhamnosus* according to claim 1 or 2, the bacterial agent according to any one of claims 3-5, the culture according to claim 6, or the composition according to claim 7 or 8 in the preparation of a pharmaceutical product for the following purposes. 1) Promotes the increase of CD103+CD11c+ DC cells; 2) Regulates DC cell differentiation or DC cell secretion of the cytokine TGF-β; 3) Promotes the differentiation of CD4+ T cells into Treg cells; 4) Regulates the body's immune balance and immune tolerance; 5) Prevention or treatment of allergic reactions or inflammatory skin diseases; 6) Improve the speed of recovery of allergic reaction symptoms after allergen removal.

10. The application as described in claim 9, characterized in that: The allergic reactions or inflammatory skin diseases mentioned include atopic dermatitis and allergic dermatitis.

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