Bifidobacterium animalis lactis and use thereof

Abstract

This invention relates to the field of microbial technology, and more particularly to a subsp. lactis of Bifidobacterium animalis and its applications. Specifically, the present invention provides a subsp. lactis of Bifidobacterium animalis BX-BC08, which helps restore the balance of the intestinal microecology and enhances the intestinal immune barrier function. Furthermore, experiments have demonstrated that the subsp. lactis of Bifidobacterium animalis BX-BC08 of this invention can regulate the immune system, reduce excessive immune responses, and for autoimmune-related diseases such as atopic dermatitis, can reduce the production of inflammatory factors and alleviate skin inflammation symptoms. Simultaneously, it exhibits relatively good human tolerance, greatly reducing adverse reactions in the human body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a subspecies of Bifidobacterium animalis and its applications. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease. It is characterized by dry, itchy skin, erythema, and scaly patches. In recent years, effective medications for treating atopic dermatitis include calcineurin inhibitors such as tacrolimus ointment and pimecrolimus cream, as well as antihistamines such as loratadine and cetirizine. Immunosuppressants such as cyclosporine and methotrexate can also be chosen depending on the patient's condition. However, caution is needed regarding potential side effects such as dizziness and nausea, as well as the risk of drug resistance.

[0003] For patients with mild to moderate Alzheimer's disease (AD) and those under 12 years old, finding safe, cost-effective, and long-term effective drugs while minimizing potential risks has become crucial. Summary of the Invention

[0004] The purpose of this invention is to address the technical problems existing in the background art by proposing a new subsp. lactis of Bifidobacterium animalis. The taxonomic name of this subsp. lactis of Bifidobacterium animalis is BX-BC08, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC, Beijing, China) with accession number CGMCC No. 30902 and deposit date of June 7, 2024.

[0005] Furthermore, the present invention also provides an article for treating atopic dermatitis, the article comprising Bifidobacterium animalis subsp. lactis of the present invention or comprising Bifidobacterium animalis subsp. lactis of the present invention and its metabolites.

[0006] Furthermore, the aforementioned metabolites include (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid, s7p sedoheptulose-7-phosphate, DL-2-(acetylamino)-3-phenylpropanoic acid, and alpha-ketoglutaric acid.

[0007] Furthermore, the aforementioned products include food or medicine.

[0008] Furthermore, the aforementioned drugs include pharmaceutical excipients, which may be at least one of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0009] Furthermore, the number of live Bifidobacterium animalis subsp. lactis in the above-mentioned products is not less than 1x10⁸ CFU / mL or 1x10⁸ CFU / g.

[0010] Furthermore, the present invention also provides the use of Bifidobacterium animalis subsp. lactis in the preparation of products for treating atopic dermatitis.

[0011] Furthermore, the aforementioned products include Bifidobacterium animalis subsp. lactis or include Bifidobacterium animalis subsp. lactis and its metabolites.

[0012] Furthermore, the number of live Bifidobacterium animalis subsp. lactis in the above-mentioned products is not less than 1x10⁸ CFU / mL or 1x10⁸ CFU / g.

[0013] Furthermore, the aforementioned metabolites include (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid, s7p sedoheptulose-7-phosphate, DL-2-(acetylamino)-3-phenylpropanoic acid, and alpha-ketoglutaric acid.

[0014] Furthermore, the present invention also provides a method for isolating Bifidobacterium animalis subsp. lactis, comprising the following steps: Samples of naturally fermented yogurt from households in Inner Mongolia were taken, diluted with sterile saline, and placed on a culture medium. Anaerobic culture at 37°C for 72 hours; Typical colonies were selected for Gram staining and microscopic examination, and Gram-positive strains were selected. Pure colonies were obtained by culturing on the culture medium.

[0015] Furthermore, the present invention also provides a method for culturing Bifidobacterium animalis subsp. lactis, comprising the following steps: Prepare BHI medium, which includes BHI agar medium and BHI liquid medium; Bifidobacterium animalis subsp. lactis was cultured on the above-mentioned BHI medium at 37 ℃ for 24 hours.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: 1. Bifidobacterium is chosen as a treatment product for atopic dermatitis because it is mild and highly effective. It can regulate the host's immune response, enhance the body's immunity, and affect atopic dermatitis by influencing the gut microbiota.

[0017] 2. Bifidobacteria are generally considered safe with no obvious side effects. This makes them an ideal treatment option.

[0018] 3. Bifidobacteria can be used in combination with other treatment methods (such as drugs, phototherapy, etc.) to improve the treatment effect.

[0019] 4. Since Bifidobacteria are naturally occurring microorganisms, they can exist in the body for a long time and be replenished when needed, making them sustainable and reproducible. Attached Figure Description

[0020] Figure 1 A graph showing the changes in the thickness of a mouse's ear; Figure 2 These are control photos of mouse ears taken at 1, 7, and 14 days after the modeling time. Figure 3 Scanned images of the Control group after staining; Figure 4 Scanned images of the stained slides from the Negative control group; Figure 5 Scanned images of the Target bacteria group after staining; Figure 6 Comparison images of single-image staining scans from the three groups; Figure 7 for Figure 6 A thickness analysis diagram of the epidermis layer; Figure 8 The graph shows the expression results of IL-4; Figure 9 The graph shows the expression results of IL-13; Figure 10 The graph shows the expression results of IL-25; Figure 11 The graph shows the expression results of TSLP; Figure 12 This is the POS graph analyzed by PLS-DA in Example 9; Figure 13 This is the NEG plot analyzed by PLS-DA in Example 9; Figure 14 This is the POS diagram of the volcano diagram in Example 9; Figure 15 This is the NEG plot of the volcano plot in Example 9; Figure 16 This is the POS diagram of the cluster analysis graph in Example 9; Figure 17 This is the NEG plot of the cluster analysis plot in Example 9; Figure 18 This is the POS graph analyzed by PLS-DA in Example 10; Figure 19 The NEG plot analyzed by PLS-DA in Example 10; Figure 20 This is the POS diagram of the volcano diagram in Example 10; Figure 21 This is the NEG plot of the volcano plot in Example 10; Figure 22 This is the POS diagram of the cluster analysis graph in Example 10; Figure 23 This is the NEG plot of the cluster analysis graph in Example 10; Figure 24 This is a phenotypic result diagram of the metabolite in animal experiment one of Example 11; Figure 25 This is a phenotypic result diagram of the metabolite in animal experiment 2 of Example 11; Figure 26 This is a phenotypic result diagram of the metabolite in animal experiment three of Example 11; Figure 27 This is a phenotypic result diagram of the metabolite in animal experiment four of Example 11. Detailed Implementation

[0021] The present invention will be further illustrated by the following embodiments. These embodiments are purely illustrative and are used only to specifically describe the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be purchased through legitimate channels.

[0022] Example 1: Screening and identification of Bifidobacterium animalis subsp. lactis BX-BC08 1. Screening Naturally fermented yogurt samples were collected from households in Inner Mongolia. 10g of the sample was diluted with 90g of sterile physiological saline, and 0.1 mL was spread onto selective RCM solid medium and anaerobically cultured at 37°C for 72 hours. Typical colonies were picked for Gram staining and microscopic examination. Gram-positive strains were then streaked onto RCM solid medium and cultured 2-3 times to obtain strain BX-BC08.

[0023] 2. Identification Under a microscope, this strain was observed to be Gram-positive, exhibiting short rod-shaped, slender rod-shaped, or spherical forms. It could form various branches or forks and exhibit multiple morphologies. It was not acid-resistant, did not spore, and was non-motile. The genome of BX-BC08 was extracted and screened. The 16S rDNA of BX-BC08 was amplified and sequenced. The sequence was compared with the nucleic acid sequence in NCBI. The results showed that the strain was Bifidobacterium animalis subsp. lactis, and it was named Bifidobacterium animalis subsp. lactis BX-BC08.

[0024] The aforementioned Bifidobacterium, taxonomically named Bifidobacterium animalis subsp. lactis BX-BC08, has been deposited at the China General Microbiological Culture Collection Center (CGMCC, Beijing, China) with accession number CGMCC No. 30902.

[0025] Example 2: Culture of Bifidobacterium animalis subsp. lactis BX-BC08 Preparation of BHI agar medium: Add 18.5g of OXOID Brain Heart Infusion Broth (BHI), model CM1135B, 3g of Sigma-Aldrich L3625 lactose, and 6g of Millipore agarose to a 1L glass bottle, and add ultrapure water to a final volume of 500ml. After autoclaving, pour the agar plates while still hot in a clean bench and allow them to solidify at room temperature for 20 minutes. Then, invert the plates, wrap them in packaging bags, and store them in a 4°C refrigerator.

[0026] This embodiment also provides a method for preparing BHI liquid culture medium: Add 4.37g of OXOID CM1135B type BHI and 5g of Sigma-Aldrich L3625 type lactose to a 1L glass bottle, and add ultrapure water to 1000ml. Affix the high-pressure sealing tape, tighten the cap, loosen it half a turn, cover with aluminum foil, and perform high-pressure sealing. Then store at room temperature or in a 4°C refrigerator.

[0027] BX-BC08, cryopreserved with glycerol, was cultured on BHI agar plates under standard anaerobic conditions (using a Mitsubishi MGCanaeroPack® anaerobic bag) at 37°C for 24 hours. The primary strain was sequenced using a single-colony cloning method to confirm it was Bifidobacterium and free from contamination before further experiments. The cultured sample was removed the following day and centrifuged at 4000 rpm for 15 minutes; the precipitate was used for animal studies.

[0028] Example 3: Effect of BX-BC08 on ear thickness in mice with atopic dermatitis Take overnight culture of Bifidobacterium BX-BC08 and BHI liquid culture medium from the anaerobic digester. Adjust the Thermo Fisher Scientific benchtop centrifuge (model ST16R) to 4000 rpm, 20°C, 15 minutes, with an increase of 9 and a decrease of 9. After removing the centrifuge, discard the supernatant from the culture tube and mix the remaining portion with a 3 ml Pasteur tube by pipetting. Adjust the final concentration to approximately 1 x 10⁻⁶. 8 CFU / mL, discard the same volume if the tube contains BHI liquid culture medium. Administer 0.2 mL of BX-BC08 to each experimental group mouse using a 10 mL disposable sterile syringe, and administer the same volume of BHI liquid culture medium to each control group mouse.

[0029] The obtained ear thickness image is as follows Figure 1 As shown, the Control group refers to the blank group that was administered BHI culture medium by gavage without applying the drug, the Negative control group refers to the group that was administered BHI culture medium by gavage and applied MC903, and the Target bacteria group refers to the group that was administered BX-BC08 by gavage and applied MC903.

[0030] Further, 10 mg of Sigma MC903 powder was dissolved in 1 mL of DMSO, then aliquoted and stored at -80°C. The stock solution was 240 times the volume of the working solution, diluted with anhydrous ethanol to a final working solution concentration of 100 μM. Quarantined, female, 7-week-old BALB / c mice were selected. The model group was colonized by gavage with Bifidobacterium lactis subsp. lactis combined with BHI. After anesthetizing the mice, 10 μL of MC903 was pipetted and applied to the inner ear of each mouse. The final total dose of MC903 in each ear was 1 nM. A one- to two-minute wait was allowed for ethanol evaporation to facilitate drug absorption. Application was performed once daily, and ear thickness was measured daily for 14 days. Photos were taken on day 1, day 7, and at the end of the modeling process.

[0031] The results are as follows Figure 2 As shown, the Control group refers to the blank group that was administered BHI culture medium by gavage without applying the drug, the Negative control group refers to the group that was administered BHI culture medium by gavage and applied MC903, and the Target bacteria group refers to the group that was administered BX-BC08 by gavage and applied MC903.

[0032] Observations showed that on day 7, mice in the negative control group exhibited significant ear congestion and thickening, along with some dandruff, while mice in the target bacteria group showed only slight ear congestion and thickening, without dandruff (dermatitis) symptoms. On day 14, mice in the negative control group developed extensive dandruff and dermatitis symptoms in their ears, with the ears becoming more curled, a significantly worsening condition compared to day 7. In contrast, mice in the target bacteria group showed only some dandruff, and the inflammation was significantly reduced compared to the negative control group. These analyses demonstrate that the Bifidobacterium BX-BC08 of this invention can significantly reduce ear thickness and alleviate ear swelling in mice with atopic dermatitis.

[0033] Example 4: Effects of BX-BC08 on pathological sections of mice with atopic dermatitis Tissue from the inner ear of a mouse in Example 3 was embedded in paraffin and stained with H&E. The results are as follows.

[0034] Control group: Description under scanning microscope: Scanning was performed at 20x, 100μm to obtain relatively normal cross-sections of skin tissue, such as... Figure 3 As shown, the epidermal cells are tightly arranged, exhibiting a regular multi-layered structure. Abundant blood vessels and fibrous connective tissue are visible in the dermis, and hair follicles can be observed in some sections. In the lowest section, a relatively clear distribution of adipocytes is visible. Morphology is normal.

[0035] Negative control group: Microscopic description of scans: Scans performed at 20x, 100μm revealed significant thickening of the epidermis in six images, with an increased number of cell layers, possibly accompanied by excessive stratum corneum proliferation. In the dermis, vasodilation and congestion were evident, with abundant inflammatory cell infiltration around the vessels, possibly including lymphocytes and eosinophils. Fibrous tissue proliferation was observed, and collagen fibers showed disordered arrangement. Partial deformation or destruction of hair follicle structures and abnormalities in the morphology and distribution of sebaceous glands were also observed. This indicates the presence of an atopic dermatitis model. Overall, the skin structure and layers, including the epidermis and dermis, were clearly visible. Specific results are as follows... Figure 4 As shown.

[0036] Target bacteria group: Microscopic description of slides: Slides were taken at 20x, 100μm. Compared to the control group, six images showed thickened epidermis and increased cell number, indicating a swollen atopic dermatitis model. In the dermis, vasodilation and congestion were evident, with less inflammatory cell infiltration around the vessels. Overall, the skin structure and layers, including the epidermis and dermis, were clearly visible. Specific results are as follows... Figure 5 As shown.

[0037] like Figure 6 As shown, comparing the stained scans of the three groups yielded more significant results. For each scan, three fields of view were taken, and five points were averaged in each field to analyze the epidermal thickness. The Control group consisted of 5 mice, the Negative Control group of 12 mice, and the Target bacteria group of 11 mice. The differences were statistically significant. Specific results are shown below. Figure 7 As shown.

[0038] Overall, compared with the Negative control group, the epidermis in the Target bacteria group was significantly thinner and the degree of swelling was reduced, indicating that BX-BC08 has an ameliorative effect on atopic dermatitis in mouse models.

[0039] Example 5: Effects of BX-BC08 on qPCR and interleukin-4 (IL-4) Mouse skin tissue was ground using a tissue homogenizer from Shanghai Jingxin. The homogenizing module was flash-frozen at -80°C for ten minutes. Each homogenizing EP tube contained pea-sized pieces of minced skin tissue, 300 μL of RNA extraction buffer (RNAsino Plus), and two 5 mm diameter white hyaluronic acid beads. The EP tube was then placed on the pre-chilled homogenizing module, and the homogenizing parameters were set to 60 Hz for 80 seconds, with two homogenization cycles performed at 1-minute intervals. After homogenization, the homogenization effect was observed; no visible tissue clumps were observed. Then, 700 μL of RNA extraction buffer (RNAsino Plus) was added to each EP tube and mixed thoroughly. The EP tubes were then placed at room temperature for further lysis for 5 minutes.

[0040] Then, extract RNA using chloroform and collect the aqueous phase containing RNA. Add 200 μL of chloroform to each EP tube, invert and mix for 15 seconds to ensure thorough mixing of the chloroform and RNA extract, and incubate at room temperature for 5 minutes. Pre-cool the centrifuge to 4°C, transfer the EP tubes to the centrifuge, and centrifuge at 12000g for 15 minutes. After centrifugation, the liquid in the EP tube will separate into three layers: a bottom layer of DNA, a middle layer of protein, and an upper layer of RNA (aqueous phase). Use a pipette to aspirate the upper aqueous phase in small, frequent amounts into new EP tubes, avoiding aspirating the middle or bottom layers to prevent cross-contamination.

[0041] RNA was precipitated using isopropanol and washed with 75% ethanol. Before aspirating the upper aqueous phase containing RNA, 500 μL of isopropanol was added to all new tubes to precipitate the RNA. The tubes were inverted and mixed for 15 seconds, then incubated at room temperature for 10 minutes. After centrifugation, RNA precipitate was observed on the sidewalls at the bottom of the EP tubes. The supernatant was discarded, and 1 mL of 75% ethanol solution prepared with DEPC-treated water was added to wash the RNA precipitate. The bottom of the EP tubes can be gently tapped to allow the RNA precipitate to float, ensuring thorough washing. The tubes were then centrifuged again at 12000 g for 5 minutes at 4°C, and the RNA precipitate was washed again with anhydrous ethanol solution using the same method.

[0042] After washing the RNA precipitate twice, the supernatant was discarded, and any remaining liquid in the EP tube was aspirated using a 10 μL pipette. The RNA precipitate was then dried at room temperature for 10 minutes. Approximately 20 μL of RNase-free DEPC-treated water was added to dissolve the RNA precipitate. The RNA concentration was then further determined using a nanodrop instrument. Reverse transcription and amplification were then performed using the Vazyme HiScrpt III AHn-one RT SuperMix Perfect for qPCR and Tag Pro Universal SYBR qPCR Master Mix kits. The final IL-4 expression results are shown below. Figure 8 As shown.

[0043] IL-4 (interleukin-4) is known to be one of the inflammatory markers associated with atopic dermatitis. In atopic dermatitis, IL-4 plays an important role in allergic diseases by promoting B cell differentiation and antibody class switching, especially promoting IgE production. Elevated serum IgE levels are usually associated with the onset of atopic dermatitis, and IL-4 is a key cytokine regulating IgE production. Therefore, IL-4 levels can serve as an indicator for assessing inflammatory activity and immune status in patients with atopic dermatitis.

[0044] Based on the analysis of the above figure, significant differences were observed between the Control group and the Negative control group, and between the Negative control group and the Target bacteria group, while no significant difference was found between the Control group and the Target bacteria group. This indicates that the Bifidobacterium of the present invention effectively reduces IL-4 levels, demonstrating good efficacy and thus helping to control Th2-type atopic dermatitis.

[0045] Example 6: Effect of BX-BC08 on interleukin-13 (IL-13) IL-13 (interleukin-13) is a key cytokine in atopic dermatitis. Similar to IL-4, it belongs to the Th2 cytokine family and plays an important role in allergic diseases and inflammatory responses.

[0046] Ground mouse ear tissue was extracted and subjected to qPCR and amplification as described in Example 5. The final IL-13 expression results were as follows: Figure 9 As shown.

[0047] Through the Figure 9 The analysis revealed significant differences between the Control group and the Negative control group, and between the Negative control group and the Target bacteria group, while the difference between the Control group and the Target bacteria group was not significant. This indicates that the Bifidobacterium of the present invention effectively reduces IL-13, demonstrating good efficacy and thus controlling atopic dermatitis.

[0048] Example 7: Effect of BX-BC08 on interleukin-25 (IL-25) IL-25 (interleukin-25) is a cytokine primarily produced by Th2 cells and is involved in the pathogenesis of atopic dermatitis (also known as eczema). IL-25 can promote the differentiation and activation of Th2 cells and enhance the production of Th2 cytokines, thus playing an important role in the pathogenesis of atopic dermatitis.

[0049] Ground mouse ear tissue was extracted and subjected to qPCR and amplification as described above. The final IL-25 expression results were as follows: Figure 10 As shown.

[0050] Through the Figure 10The analysis revealed an improvement between the Control group and the Target bacteria group compared to the Control group and the Negative control group, indicating that the Bifidobacterium of the present invention played a role in reducing IL-25.

[0051] Example 8: The effect of BX-BC08 on TSLP TSLP activates Th2 cells in the immune system by binding to specific receptors, thereby promoting the production of cytokines such as IL-4, IL-5, and IL-13. These cytokines play a key role in the pathogenesis of allergic diseases and atopic dermatitis (eczema), enhancing inflammatory responses, promoting IgE production, and affecting skin barrier function.

[0052] Ground mouse ear tissue was extracted and subjected to qPCR and amplification as described above. Significant differences were observed between the Control group and the Negative control group, and between the Negative control group and the Target bacteria group. The final TSLP expression results are as follows: Figure 11 As shown.

[0053] Through the Figure 11 The analysis revealed significant differences between the Negative control group and the Target bacteria group, and between the Control group and the Target bacteria group, while the difference between the Control group and the Negative control group was not significant. Therefore, it can be concluded that the Bifidobacterium of this invention has a very significant effect in reducing TSLP, thereby reducing atopic dermatitis.

[0054] Example 9: Effects of BX-BC08 on fecal metabolism in mice Fourteen fecal samples were collected from the Negative control group and the Target bacteria group, with seven samples from each group, designated as G1 and G2 groups, respectively. The metabolomics results were analyzed as follows: 1. Partial Least Squares Discriminant Analysis Partial Least Squares Discrimination Analysis (PLS-DA) is a supervised statistical method for discriminant analysis. This method uses partial least squares regression [7] to establish a model of the relationship between metabolite expression levels and sample categories in order to predict sample categories. PLS-DA models for each comparison group were established, and the model evaluation parameters (R2, Q2) were obtained through 7-fold cross-validation (seven-fold cross-validation, k-fold cross-validation when the number of biological replicates n <= 3, k = 2n). If R2 and Q2 are closer to 1, the model is more stable and reliable. PLS-DA analysis was performed through POS and NEG pathways, and the results are as follows, where Figure 12 For POS, Figure 13 For NEG: G1 is the feces of mice in the Negative Control group, and G2 is the feces of mice in the Target bacteria group. Significant differences were observed between the two groups at the group level. This indicates a significant difference in metabolomics between the two groups, and further, it allows us to identify the metabolites influencing these differences, providing valuable guidance for the search for differentially expressed metabolites.

[0055] 2. Volcano Map Volcano plots can visually display the overall distribution of differentially expressed metabolites. The horizontal axis represents the fold change of metabolites in different groups (log2(Fold Change)), and the vertical axis represents the significance level of the difference (-log10(P-value)). Each point in the volcano plot represents a metabolite. Metabolites that are significantly upregulated are represented by red points, and metabolites that are significantly downregulated are represented by blue points. The size of the circle represents the VIP value.

[0056] Further analysis of the volcano plot revealed that G2 exhibits both upregulation and downregulation of metabolites compared to G1. This indicates that the upregulated and downregulated metabolites are the focus of this study. Figure 14 For POS, Figure 15 It is NEG.

[0057] 3. Cluster analysis Cluster analysis is used to determine the metabolic patterns of metabolites under different experimental conditions. Metabolites with similar metabolic patterns may have similar functions or participate in the same metabolic process or cellular pathway. Therefore, by clustering metabolites with the same or similar metabolic patterns, the functions of certain metabolites can be inferred. The vertical axis represents sample clusters, and the horizontal axis represents metabolite clusters; shorter cluster branches indicate higher similarity. Horizontal comparisons reveal the relationship between metabolite content clusters in different groups. The cluster analysis diagram shows that G2 differs from G1 in the specific clustering characteristics of metabolites. Figure 16 For POS, Figure 17 For NEG, the specific screening of metabolites involves searching from differential clusters. This provides specific information and the degree of clustering for finding metabolites that affect the differences between G2 and G1, and thus determining the metabolites that affect atopic dermatitis.

[0058] Overall, fecal metabolomics revealed significant differences in non-targeted metabolites in mice with atopic dermatitis fed with different bacterial strains and those fed with standard culture media. Subsequent analysis of the bacterial supernatant further validated these potential differential metabolites.

[0059] Example 10: Metabolomics Analysis of Bacterial Fluid Supernatant Ten tubes of liquid were selected, taken from BHI medium supernatant and target bacterial supernatant, respectively, with five tubes from each group, named G1 and G2 groups. The metabolomics results were analyzed as follows: 1. Partial Least Squares Discriminant Analysis The following analysis was performed using PLS-DA. Figure 18 For POS, Figure 19 For NEG: contrast Figure 18 , 19 Significant differences were observed between groups G1 and G2. This indicates a clear difference in their metabolomics, and further, it allows us to identify the metabolites influencing these differences, providing valuable guidance for identifying differentially expressed metabolites.

[0060] 2. Volcano Map Volcano plot analysis can be used to determine the upregulation and downregulation of metabolites. Figure 20 For POS, Figure 21 For NEG. Metabolites that are significantly upregulated are indicated by red dots, and metabolites that are significantly downregulated are indicated by blue dots. The size of the dots represents the VIP value.

[0061] pass Figure 20 , 21It can be determined that G2 exhibits both upregulation and downregulation of metabolites compared to G1, meaning that the upregulated and downregulated metabolites are the focus of this study. Overall, G2 shows both upregulation and downregulation compared to G1; POS is on the left, and NEG is on the right.

[0062] 3. Cluster analysis Cluster analysis is used to determine the metabolic patterns of metabolites under different experimental conditions. Specifically, metabolite screening involves searching for metabolites within differential clusters. This provides specific information and the degree of clustering to identify metabolites affecting the differences between G2 and G1 metabolites, and thus to determine the metabolites influencing atopic dermatitis. Cluster analysis revealed specific clustering characteristics of metabolites in G2 compared to G1, allowing for the discovery of metabolites and mechanisms underlying the effects of Bifidobacteria. (About cluster analysis) Figure 22 For POS, Figure 23 For NEG: Metabolomics data from bacterial supernatants revealed significant differences in metabolites between BX-BC08 and BHI media. The aim is to identify common differential metabolites through combined analysis of both media, thereby uncovering the underlying mechanisms.

[0063] Example 11: Metabolomics Analysis of Mouse Feces and Bacterial Fluid Supernatant Gut microbiota has become a research hotspot in recent years, and metabolomics combined with other methods has provided new research tools and approaches in this field. High-throughput detection technologies can be used to qualitatively and quantitatively analyze all metabolites in an organism, revealing the full picture of metabolites. By performing non-targeted metabolomics analysis of the POS and NEG pathways in mouse feces and bacterial supernatant, metabolites were screened, Venn diagrams were used to identify upregulated common metabolites, and analysis of previous literature revealed the following four effective metabolites involved in this invention, BX-BC08: The purpose of untargeted metabolomics co-analysis is to identify target metabolites, and for target metabolites, it is necessary to confirm whether they originate from the host or are produced by target bacteria. By identifying target metabolites and their pathways of action, the source of differences between the Control group, the Negative control group, and the Target bacteria group can be determined.

[0064] Phenotypic experiments and tests involving inflammatory factors demonstrated that strain BX-BC08 alleviates atopic dermatitis in mice. Further metabolomics analysis revealed that the effective metabolites of BX-BC08 in this invention include the four mentioned above, and their effects can be verified through animal experiments using the following metabolites.

[0065] Metabolite animal experiments: Experiment 1: Metabolite (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid Twenty-four seven-week-old female BALB / c mice and the metabolite (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid were purchased. The mice were divided into a BHI group and a metabolite group, with 12 mice in each group. They were administered the metabolite via gavage for 21 days. Starting from day 8, MC903 modeling was performed. All mice were sacrificed on day 21. The phenotypic results are as follows: Figure 24 .

[0066] The phenotypic results shown in the figure revealed that, compared with the BHI group, the inflammation in the metabolite group was significantly reduced. The metabolite (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid had a relieving effect on atopic dermatitis in mice. Therefore, it can be inferred that the BX-BC08 strain has a relieving effect on atopic dermatitis, and the influencing factor is the metabolite (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid.

[0067] Experiment 2: Metabolite Sedum heptaose-7-phosphate (s7p) Twenty-four seven-week-old female BALB / c mice and the metabolite sedoheptulose-7-phosphate (s7p) were purchased. The mice were divided into a BHI group and a metabolite group, with 12 mice in each group. They were administered the treatment by gavage for 21 days. Starting from day 8, MC903 modeling was performed. All mice were sacrificed on day 21. The phenotypic results are as follows: Figure 25 .

[0068] The phenotypic results shown in the figure revealed that, compared with the BHI group, the inflammation in the metabolite group was significantly reduced. The metabolite sedoheptulose-7-phosphate (s7p) had a relieving effect on atopic dermatitis in mice. Therefore, it can be inferred that the BX-BC08 strain has a relieving effect on atopic dermatitis, and the metabolite sedoheptulose-7-phosphate (s7p) is an influencing factor.

[0069] Experiment 3: Metabolite DL-2-(acetylamino)-3-phenylpropanoic acid Twenty-four seven-week-old female BALB / c mice and the metabolite DL-2-(acetylamino)-3-phenylpropanoic acid were purchased. The mice were divided into a BHI group and a metabolite group, with 12 mice in each group. They were administered the treatment by gavage for 21 days. Starting from day 8, MC903 modeling was performed. All mice were sacrificed on day 21. The phenotypic results are as follows: Figure 26 .

[0070] The phenotypic results shown in the figure revealed that, compared with the BHI group, the inflammation in the metabolite group was significantly reduced. The metabolite DL-2-(acetylamino)-3-phenylpropanoic acid had a relieving effect on atopic dermatitis in mice. Therefore, it can be inferred that the BX-BC08 strain has a relieving effect on atopic dermatitis, and the influencing factor is the metabolite DL-2-(acetylamino)-3-phenylpropanoic acid.

[0071] Experiment 4: Metabolite α-ketoglutaric acid Twenty-four seven-week-old female BALB / c mice and their metabolite alpha-ketoglutaric acid were purchased. The mice were divided into a BHI group and a metabolite group, with 12 mice in each group. They were administered the treatment by gavage for 21 days. Starting from day 8, MC903 modeling was performed. All mice were sacrificed on day 21. The phenotypic results are as follows: Figure 27 .

[0072] The phenotypic results shown in the figure reveal that, compared to the BHI group, the inflammation in the metabolite group was significantly reduced. The metabolite alpha-ketoglutaric acid had a relieving effect on atopic dermatitis in mice. Therefore, it can be inferred that the BX-BC08 strain has a relieving effect on atopic dermatitis, and that the metabolite alpha-ketoglutaric acid is an influencing factor.

[0073] The above animal experiments demonstrate that the metabolites of strain BX-BC08 have a relieving effect on atopic dermatitis in mice.

[0074] In summary, this treatment plan utilizes Bifidobacterium, which helps restore the balance of the gut microbiota, potentially increasing the proportion of beneficial bacteria and enhancing the gut's immune barrier function. Furthermore, experiments have demonstrated that Bifidobacterium can regulate the immune system, reduce excessive immune responses, and, in autoimmune-related diseases such as atopic dermatitis, reduce the production of inflammatory factors and alleviate skin inflammation symptoms. Secondly, this strain, which generally originates from the human gut, is relatively well-tolerated by the body, with fewer serious adverse reactions.

[0075] The present invention has been illustrated with the above embodiments to explain the detailed method of the present invention. However, the present invention is not limited to the detailed method described above, that is, it does not mean that the present invention must rely on the detailed method described above to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A product for treating atopic dermatitis, characterized in that, The product includes Bifidobacterium lactis subsp. animalis (Bifidobacterium lactis) Bifidobacterium animalis subsp.lactis BX-BC08 and its metabolites, the animal Bifidobacterium lactis subsp. ( Bifidobacterium animalis subsp.lactis The accession number of BX-BC08 is CGMCC No. 30902, and the product is a pharmaceutical product. The metabolites include (3R,4S,5R)-(-)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid, s7p sedoheptulose-7-phosphate, DL-2-(acetylamino)-3-phenylpropanoic acid, and alpha-ketoglutaric acid.

2. An article for use in the treatment of atopic dermatitis according to claim 1, characterised in that, The product also includes pharmaceutical excipients, which include at least one of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

3. The product for treating atopic dermatitis according to claim 1, characterized in that, The animal Bifidobacterium lactis subspecies ( Bifidobacterium animalis subsp.lactis The number of live bacteria in BX-BC08 is no less than 1x10⁻⁶. 8 CFU / mL or 1x10 8 CFU / g.

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