Bifidobacterium animalis subsp. Lactis and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a bifidobacterium animalis subsp. Lactis and application thereof, and particularly provides a bifidobacterium animalis subsp. Lactis BX-BC08 which is beneficial for restoring the microecological balance of the intestinal tract and enhancing the immune barrier function of the intestinal tract. Experiments prove that the bifidobacterium animalis subsp. Lactis BX-BC08 disclosed by the invention can regulate an immune system and relieve excessive immune response, can reduce generation of inflammatory factors and relieve skin inflammation symptoms for autoimmune related diseases such as atopic dermatitis, is relatively good in tolerance to a human body, and greatly reduces adverse reaction of the human body.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an animal Bifidobacterium lactis subspecies and an application thereof. Background Art

[0002] Atopic dermatitis (AD) is a chronic, recurrent, inflammatory skin disease. Atopic dermatitis is characterized by dry, itchy, erythematous, and scaly skin. In recent years, effective drugs for the treatment of atopic dermatitis include calcineurin inhibitors such as tacrolimus ointment and pimecrolimus cream, antihistamines such as loratadine and cetirizine, and immunosuppressants such as cyclosporine and methotrexate can also be selected according to the patient's condition. However, we need to be vigilant about the side effects of drugs such as dizziness and nausea, as well as drug resistance.

[0003] For patients with mild to moderate AD and patients under the age of 12, how to find safe, cost-effective, and long-term effective drugs while minimizing potential risks has become the key at present. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problems existing in the background technology and propose an animal bifidobacterium subsp. lactis. The animal bifidobacterium subsp. lactis of the present invention is taxonomically named as animal bifidobacterium subsp. lactis BX-BC08, which has been deposited in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), with a deposit number of CGMCC No. 30902 and a deposit date of June 7, 2024.

[0005] Furthermore, the present invention also provides a product for treating atopic dermatitis, wherein the product comprises the Bifidobacterium animalis subsp. lactis of the present invention or comprises the Bifidobacterium animalis subsp. lactis of the present invention and a metabolite thereof.

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

[0007] Furthermore, the above-mentioned products include food or medicine.

[0008] Furthermore, the above-mentioned medicine includes pharmaceutical excipients, and the pharmaceutical excipients may be at least one of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

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

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

[0011] Furthermore, the above product comprises Bifidobacterium animalis subsp. lactis or comprises Bifidobacterium animalis subsp. lactis and its metabolites.

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

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

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

[0015] Furthermore, the present invention also provides a method for culturing the Bifidobacterium animalis subsp. lactis of the present invention, comprising the following steps: preparing a BHI medium, wherein the BHI medium includes a BHI agar medium and a BHI liquid medium; Bifidobacterium animalis subsp. lactis was cultured on the above BHI medium at 37°C for 24 hours.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: 1. Bifidobacterium is selected as a therapeutic product for atopic dermatitis. It is mild and highly effective. It can regulate the host's immune response, enhance the body's immunity, and affect atopic dermatitis by affecting the intestinal flora.

[0017] 2. Bifidobacterium is generally considered safe and has no significant side effects. This makes it an ideal treatment option.

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

[0019] 4. Since bifidobacteria are naturally occurring microorganisms, they can exist in the body for a long time and be replenished when needed, which is sustainable and reproducible. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the thickness changes of mouse ears; Figure 2 These are the control photos of the mouse ears at 1, 7, and 14 days of modeling; Figure 3 This is the stained scan of the Control group; Figure 4 This is the staining scan of the Negative control group; Figure 5 This is the staining scan of the Target bacteria group; Figure 6 This is a comparison of single-image stained scans of the three groups; Figure 7 for Figure 6 Thickness analysis of the epidermis; Figure 8 The expression result diagram of IL-4; Fig. 9 The expression result diagram of IL-13; Fig.10 The expression result diagram of IL-25; Fig.11 The expression result diagram of TSLP; Fig.12 is the POS diagram analyzed by PLS-DA in Example 9; Fig.13 This is the NEG diagram analyzed by PLS-DA in Example 9; Fig.14 It is the POS map of the volcano map in Example 9; Fig.15 It is the NEG diagram of the volcano diagram in Example 9; Fig.16 It is the POS diagram of the cluster analysis diagram in Example 9; Fig.17 It is the NEG diagram of the cluster analysis diagram in Example 9; Fig.18 is the POS diagram analyzed by PLS-DA in Example 10; Fig.19 is the NEG diagram analyzed by PLS-DA in Example 10; Fig. 20 It is the POS map of the volcano map in Example 10; Fig.21 It is the NEG diagram of the volcano diagram in Example 10; Fig. 22 It is the POS diagram of the cluster analysis diagram in Example 10; Fig.23 It is the NEG diagram of the cluster analysis diagram in Example 10; Fig.24 This is a diagram showing the phenotypic results of the metabolite animal experiment 1 in Example 11; Fig.25 This is a diagram showing the phenotypic results of the metabolite animal experiment 2 in Example 11; Fig.26 This is a diagram showing the phenotypic results of the metabolite animal experiment 3 in Example 11; Fig. 27 This is a diagram showing the phenotypic results of the metabolite animal experiment 4 in Example 11. DETAILED DESCRIPTION

[0021] The present invention is further described by the following examples. These examples are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

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

[0023] 2. Identification The strain was Gram-positive under microscope, and was short rod-shaped, slender rod-shaped or spherical. It could form various branches or bifurcations, was not acid-resistant, had no spores, and was unmotile. The genome of BX-BC08 was extracted and screened, and the 16S rDNA of BX-BC08 was amplified and sequenced. The sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Bifidobacterium animalis subsp. lactis, and it was named Bifidobacterium animalis subsp. lactis BX-BC08.

[0024] The obtained bifidobacterium is taxonomically named Bifidobacterium animalis subsp. lactis BX-BC08, and has been deposited in China General Microbiological Culture Collection Center (CGMCC, Beijing, China), and its deposit number is CGMCC No.30902.

[0025] Example 2: Cultivation of Bifidobacterium animalis subsp. lactis BX-BC08 Preparation of BHI agar medium: Add 18.5g OXOID CM1135B model Brain Heart Infusion Broth (BHI), 3g Sigma-Aldrich L3625 model lactose, and 6g Millipore agarose to a 1L glass bottle, and add ultrapure water to 500ml. After high pressure, pour the plate in the clean bench while it is still hot, solidify at room temperature for 20 minutes, then invert the plate, wrap the bag and store it in a 4 degree refrigerator.

[0026] This embodiment also provides a preparation of a BHI liquid culture medium: add 4.37g OXOID brand CM1135B model BHI and 5g Sigma-Aldrich brand L3625 model lactose to a glass bottle with a volume of 1L, add ultrapure water to 1000ml, apply a high pressure seal, tighten the bottle cap and loosen it half a turn, put on tin foil, and perform high pressure, and then store it at room temperature or in a refrigerator at 4 degrees.

[0027] Take the glycerol frozen preservation BX-BC08 and carry out routine culture on the above-mentioned BHI agar medium plate. The culture conditions are anaerobic environment (Japan Mitsubishi MGC AnaeroPack® anaerobic bag is placed in the anaerobic tank), the temperature is 37°C, and the culture is 24 hours. BX-BC08 was cultured in BHI medium at 37 degrees Celsius for 24 hours. The primary strain was sequenced by the single colony cloning method to confirm that the strain was bifidobacterium and was not contaminated, and then the subsequent experiments were carried out. Take it out every other day after culture, centrifuge it at 4000rpm for 15min, and the precipitate is used for animal experimental research.

[0028] Example 3: Effect of BX-BC08 on ear thickness in mice with atopic dermatitis Take the overnight culture of Bifidobacterium BX-BC08 and BHI liquid medium from the anaerobic tank, adjust the Thermo Fisher desktop centrifuge (model ST16R) to 4000 rpm, 20°C, 15 minutes, 9 up and 9 down, discard the supernatant of the bacterial tube after taking it out, and mix the remaining part with a 3ml Pasteur pipette and adjust the final concentration to about 1x10 8 CFU / mL, and the same volume of BHI liquid culture medium tube was poured out. Use a 10mL disposable sterile syringe to inject 0.2mL of BX-BC08 into each experimental group mouse, and inject the same volume of BHI liquid culture medium into each control group mouse.

[0029] The ear thickness map obtained is as follows Figure 1 As shown, the Control group refers to the blank group that was gavaged with BHI medium and not applied with medicine, the Negative control group refers to the group that was gavaged with BHI medium and applied with medicine MC903, and the Target bacteria refers to the group that was gavaged with BX-BC08 and applied with medicine MC903.

[0030] Further, 10 mg of MC903 powder from Sigma was dissolved in 1 mL of DMSO, then packaged and stored in a -80 °C refrigerator. The storage solution is 240 times the working solution, diluted to the working solution with anhydrous ethanol, and the final concentration of the working solution is 100 μM. Select BALB / C mice that have passed the quarantine, female, 7 weeks old, and the modeling group is given Bifidobacterium lactis subspecies combined with BHI gavage modeling for colonization. After the mice are anesthetized, use a pipette to draw 10ul of MC903, and apply 10uL of working solution to the inside of the ears of the mice on both sides. The final total dose of MC903 in each ear is 1nM, and wait for one or two minutes for the alcohol to evaporate, which is conducive to the absorption of the drug. The frequency of application is once a day, the ear thickness is measured once a day, and the modeling time is 14 days. Take pictures on the first day, the seventh day, and at the end of the modeling.

[0031] The result is as follows Figure 2 As shown, the Control group refers to the blank group that was gavaged with BHI medium and not applied with medicine, the Negative control group refers to the group that was gavaged with BHI medium and applied with medicine MC903, and the Target bacteria refers to the group that was gavaged with BX-BC08 and applied with medicine MC903.

[0032] Through observation, on the 7th day, the ears of the mice in the Negative control group showed obvious congestion and thickening, and a small amount of dandruff, while the ears of the mice in the Target bacteria group showed a little congestion and thickening, and no dandruff (dermatitis) symptoms. On the 14th day, the ears of the mice in the Negative control group showed a large number of dandruff dermatitis symptoms, and the ears were curled, and the dermatitis condition was significantly aggravated compared with the 7th day, while the ears of the mice in the Target bacteria group showed a small amount of dandruff, and the inflammation was significantly relieved compared with the Negative control group. From the above analysis, it can be concluded that the Bifidobacterium BX-BC08 of the present invention can significantly reduce the thickness of the ears of mice with atopic dermatitis and alleviate the swelling of the ears of mice.

[0033] Example 4: Effect of BX-BC08 on pathological sections of mice with atopic dermatitis The internal tissue of the mouse ear in Example 3 was taken, embedded in paraffin, and stained with H&E, and the results were as follows.

[0034] Control group: Scanning microscope description: Scanning at 20x, 100μm to obtain a relatively normal cross-section of skin tissue, such as Figure 3 As shown, the epidermal cells are closely arranged, presenting a regular multi-layer structure. Abundant blood vessels and fibrous connective tissue can be seen in the dermis, and hair follicles can be observed in some sections. In the bottom section, more obvious distribution of fat cells can be seen. The morphology is normal.

[0035] Negative control group: Description under the scanning microscope: Scanning was performed at 20x, 100μm, and it was found that the epidermis was significantly thickened in the six pictures, and the number of cell layers increased, which may be accompanied by excessive proliferation of the stratum corneum. In the dermis, the vascular dilation and congestion are more obvious, and there are more inflammatory cell infiltrations around the blood vessels, which may include lymphocytes, eosinophils, etc. The fibrous tissue may proliferate, and the collagen fibers are disordered. The hair follicle structure is partially deformed or destroyed, and the morphology and distribution of the sebaceous glands are also partially abnormal. This indicates that an atopic dermatitis model has appeared. Overall, the structure and layers of the skin are clearly visible, including the epidermis, dermis and other parts. The specific results are as follows Figure 4 shown.

[0036] Target bacteria group: Scanning microscope description: Scanning at 20x, 100μm, found that in the six images, compared with the control group, the epidermis was thickened and the number of cell layers increased, indicating that a swollen atopic dermatitis model appeared. In the dermis, the vascular dilation and congestion were obvious, and there was less inflammatory cell infiltration around the blood vessels. Overall, the structure and layers of the skin were clearly visible, including the epidermis, dermis and other parts. The specific results are as follows Figure 5 shown.

[0037] like Figure 6 As shown, the single images of the three groups were taken for staining and scanning comparison, and the results were more obvious. For the scanning results, three fields of view were taken for each field of view, and 5 points were taken for each field of view for average, so as to analyze the thickness of the epidermis. A total of 5 mice were taken for the Control group, 12 mice were taken for the Negative Control group, and 11 mice were taken for the Target bacteria group. It can be seen that the significance is obvious. The specific results are as follows Figure 7 shown.

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

[0039] Example 5: Effect of BX-BC08 on qPCR and interleukin 4 (IL-4) The tissue grinder of Shanghai Jingxin was used to grind the mouse skin tissue, and the grinding module was placed in a -80 refrigerator for quick freezing for ten minutes. Soybean-sized shredded skin tissue, 300μL RNA extract RNAsino Plus and two 5mm diameter white beads were added to each grinding EP tube. Then the grinding EP tube was placed on the pre-cooled grinding metal module, and the machine was set to 60Hz, 80 seconds, and the grinding parameters were set to 60Hz, 80 seconds, and the grinding was performed twice with an interval of 1 minute. After the grinding is completed, the grinding effect can be checked, and there are no tissue clumps visible to the naked eye. Then 700μL RNA extract RNAsinoPlus was added to each grinding EP tube to mix well, and the grinding EP tube was placed at room temperature and continued to be fully lysed for 5 minutes.

[0040] Then use chloroform to extract RNA and take the aqueous phase containing RNA. Add 200μL of chloroform to each EP tube, mix it upside down for 15 seconds to fully mix the chloroform and RNA extract, and place it at room temperature for 5 minutes. Precool the centrifuge to 4°C in advance, transfer the EP tube to the centrifuge, and centrifuge it at 12000g for 15 minutes. After the centrifugation, you can see that the liquid in the EP tube can be divided into 3 layers, namely the DNA component in the bottom layer, the protein component in the middle layer, and the RNA component in the upper aqueous phase. Use a pipette to aspirate the upper aqueous phase into a new EP tube in small amounts and multiple times to avoid aspirating the liquid in the middle or bottom layer, which may cause cross contamination.

[0041] Use isopropanol to precipitate RNA and 75% ethanol to wash the RNA precipitate. Before aspirating the upper aqueous phase containing RNA, add 500μL of isopropanol to all new tubes to precipitate RNA, mix by inverting for 15 seconds, and leave at room temperature for 10 minutes. Centrifuge at 12000g for 10 minutes in a 4℃ centrifuge. After the centrifugation, you can see RNA precipitate on the side wall at the bottom of the EP tube. Then discard the supernatant and add 1mL of 75% ethanol solution prepared with DEPC-treated water to wash the RNA precipitate. You can flick the bottom of the EP tube to make the RNA precipitate float, so that the RNA precipitate can be fully washed. Then centrifuge at 12000g for 5 minutes in a 4℃ centrifuge, and wash the RNA precipitate again with anhydrous ethanol solution in the same way.

[0042] After washing the RNA precipitate twice, discard the supernatant, and use a 10μL range pipette to remove the residual liquid in the EP tube, and dry the RNA precipitate at room temperature for 10 minutes. According to the amount of RNA precipitate, add about 20μL of RNase-free DEPC-treated water to dissolve the RNA precipitate. Finally, use the nanodrop machine to further determine the RNA concentration, and then perform reverse transcription and amplification operations according to Vazyme's HiScrpt ⅢAHn-one RT SuperMix Perfed forqPCR and Tag Pro Universal SYBRqPCR Master Mix kits. Finally, the expression results of IL-4 are as follows Figure 8 shown.

[0043] It is known that IL-4 (interleukin 4) is one of the inflammatory indicators associated with atopic dermatitis. In atopic dermatitis, IL-4 plays an important role in allergic diseases by promoting the differentiation of B cells and antibody class switching, especially promoting the production of IgE. Increased levels of IgE in serum are usually associated with the onset of atopic dermatitis, and IL-4 is the key cytokine that regulates the production of IgE. Therefore, the level of IL-4 can be used as an indicator to evaluate the inflammatory activity and immune status in patients with atopic dermatitis.

[0044] By judging from the above figure, it can be found that there are obvious differences between the Control group and the Negative control group, and between the Negative control group and the Target bacteria group, while there is no significant difference between the Control group and the Target bacteria group. This means that the bifidobacterium of the present invention plays a good role in reducing IL-4, and the effect is relatively good, so as 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 is a Th2 cytokine that plays an important role in allergic diseases and inflammatory responses.

[0046] The ground mouse ear tissue was taken and extracted, qPCR and amplified as described above (Example 5). Finally, the expression results of IL-13 were obtained as shown in the following figure. Fig. 9 shown.

[0047] Through Fig. 9 It can be found that there are obvious differences between the Control group and the Negative control group, and between the Negative control group and the Target bacteria group, while there is no significant difference between the Control group and the Target bacteria group. This means that the bifidobacteria of the present invention play a good role in reducing IL-13, and the effect is relatively good, thereby controlling atopic dermatitis.

[0048] Example 7: Effect of BX-BC08 on interleukin 25 (IL-25) IL-25 (interleukin 25) is a cytokine mainly produced by Th2 cells and is associated with 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] The ground mouse ear tissue was taken and extracted, qPCR and amplified as above. Finally, the expression results of IL-25 were obtained as shown in the following figure. Fig.10 shown.

[0050] Through Fig.10It can be found that there is a certain improvement between the Control group and the Target bacteria group compared with the Control group and the Negative control group, indicating that the bifidobacterium of the present invention plays a certain role in the reduction of IL-25.

[0051] Example 8: 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), and they can enhance inflammatory responses, promote the production of IgE, and affect the skin barrier function.

[0052] The ground mouse ear tissue was extracted and qPCR and amplified as above. There were obvious differences between the control group and the negative control group, and between the negative control group and the target bacteria group. The expression results of TSLP were as follows: Fig.11 shown.

[0053] Through Fig.11 It can be judged that there are obvious differences between the Negative control group and the Target bacteria group, and between the Control group and the Target bacteria group, while there is no significant difference between the Control group and the Negative control group. Therefore, it can be judged that the bifidobacterium of the present invention has a very significant effect on reducing TSLP, thereby reducing atopic dermatitis.

[0054] Example 9: Effect of BX-BC08 on Fecal Metabolism in Mice 14 tubes of feces were selected, from the Negative control and Target bacteria groups, 7 tubes in each group, named G1 and G2 groups respectively. According to the metabolomics results, the analysis is as follows: 1. Partial least squares discriminant analysis Partial Least Squares Discrimination Analysis (PLS-DA) is a supervised discriminant analysis statistical method. This method uses partial least squares regression [7] to establish a relationship model between metabolite expression and sample category to predict the sample category. The PLS-DA model of each comparison group was established, and the model evaluation parameters (R2, Q2) were obtained through 7-fold cross-validation (seven cycles of cross-validation, when the number of biological replicates of the sample n<=3, it is k cycles of cross-validation, k=2n). If R2 and Q2 are closer to 1, the model is more stable and reliable. PLS-DA analysis was performed on the POS and NEG pathways, and the results were as follows: Fig.12 For POS, Fig.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. It can be found that there are obvious differences between the two groups at the group level. Therefore, it can be judged that the two are significantly different at the metabolomics level, and further it can be judged what metabolites affect the difference between the two, which plays a good guiding role in finding differential metabolites.

[0055] 2. Volcano map The volcano plot can intuitively display the overall distribution of differential metabolites. The horizontal axis represents the fold change (log2 (Fold Change)) of metabolites in different groups, and the vertical axis represents the significance level of the difference (-log10 (P-value)). Each point in the volcano plot represents a metabolite, significantly up-regulated metabolites are represented by red dots, and significantly down-regulated metabolites are represented by blue dots. The size of the dot represents the VIP value.

[0056] Further through the volcano plot, it can be determined that compared with G1, G2 has up-regulated and down-regulated metabolites, which means that the up-regulated and down-regulated metabolites are the focus of this study. Fig.14 For POS, Fig.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 direction is the clustering of samples, and the horizontal direction is the clustering of metabolites. The shorter the cluster branch, the higher the similarity. The relationship between the clustering of metabolite content between groups can be seen through horizontal comparison. The cluster analysis diagram shows that G2 has specific clustering characteristics of differential metabolites compared to G1. Fig.16 For POS, Fig.17 For NEG, the specific screening of metabolites is to search from the differential clustering, which provides specific information and the degree of clustering for finding metabolites that affect the differences between G2 and G1, and then determining the metabolites that affect atopic dermatitis.

[0058] In general, fecal metabolomics revealed that mice with atopic dermatitis fed with strains and ordinary culture media had very different non-targeted metabolites in their bodies. The metabolites in the subsequent bacterial supernatant can be used to jointly verify the potential differential metabolites.

[0059] Example 10: Metabolomics analysis of bacterial supernatant Ten tubes of liquid were selected, which were taken from the supernatant of BHI culture medium and the supernatant of target bacteria, with 5 tubes in each group, named G1 and G2 groups respectively. According to the metabolomics results, the analysis is as follows: 1. Partial least squares discriminant analysis The PLS-DA analysis is as follows: Fig.18 For POS, Fig.19 For NEG: contrast Fig.18 , 19 It can be found that there are obvious differences between the G1 and G2 groups. Therefore, it can be judged that the two groups are significantly different at the metabolomics level, and further it can be judged what metabolites affect the difference between the two groups, which plays a good guiding role in finding differential metabolites.

[0060] 2. Volcano map The up-regulation and down-regulation of metabolites were determined by volcano plot analysis. Fig. 20 For POS, Fig.21 The significantly up-regulated metabolites are indicated by red dots, the significantly down-regulated metabolites are indicated by blue dots, and the size of the dots represents the VIP value.

[0061] pass Fig. 20 , 21, it can be determined that compared with G1, G2 has up-regulated and down-regulated metabolites, which means that the up-regulated and down-regulated metabolites are the focus of this study. Overall, G2 has up-regulated and down-regulated metabolites compared with G1, with POS on the left and NEG on the right.

[0062] 3. Cluster analysis Cluster analysis is used to determine the metabolic patterns of metabolites under different experimental conditions. Specific screening of metabolites means searching from differential clusters, which provides specific information and the degree of clustering for finding metabolites that affect the differences between G2 and G1, and then determining the metabolites that affect atopic dermatitis. The cluster analysis diagram shows that G2 has specific clustering characteristics of differential metabolites compared to G1, which can help discover the metabolites and mechanisms that cause bifidobacteria to work. About cluster analysis Fig. 22 For POS, Fig.23 For NEG: The metabolomics data of the bacterial supernatant showed that the metabolites in the BX-BC08 and BHI culture media were significantly different. We wanted to find common and differential metabolites through a joint analysis of the two, so as to explore the mechanism.

[0063] Example 11: Combined metabolomics analysis of mouse feces and bacterial supernatant Intestinal microecology has been a research hotspot in recent years, and metabolomics combined analysis has provided new research means and methods for this field. Qualitative and quantitative analysis of all metabolites in an organism using high-throughput detection technology can reveal the full picture of metabolites in the organism. Non-targeted metabolomics analysis was performed through the POS and NEG pathways of mouse feces and bacterial supernatant to screen metabolites, and the upregulated common metabolites were found using the Venn diagram method. Through the analysis of past literature, the following four effective metabolites involved in BX-BC08 of the present invention can be obtained: The purpose of non-targeted metabolomics co-analysis is to identify the target metabolites. For the target metabolites, it is necessary to confirm whether they are derived from the host or produced by the target bacteria. By identifying the target metabolites and their action pathways, the source of the differences between the control group, the negative control group, and the target bacteria group can be determined.

[0064] Through phenotypic experiments, inflammatory factors and other related experiments, it can be determined that the BX-BC08 strain has a relieving effect on atopic dermatitis in mice. Through further metabolomics analysis, it can be concluded that there are the above 4 effective metabolites of BX-BC08 of the present invention, and the effects of these metabolites can be verified by the following metabolite animal experiments.

[0065] Metabolite Animal Experiments: Experiment 1: Metabolite (3R, 4S, 5R)-(-)-3, 4, 5-trihydroxycyclohex-1-ene-1-carboxylic acid Twenty-four 7-week-old BALB / c female 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, 12 mice in each group, and were treated with gavage for 21 days. MC903 modeling was performed from the 8th day, and all mice were killed on the 21st day. The phenotypic results are shown in the figure. Fig.24 .

[0066] The phenotypic results shown in the figure show that compared with the BHI group given by oral administration, the inflammation in the metabolite group was significantly reduced, and the metabolite (3R, 4S, 5R)-(-)-3, 4, 5-trihydroxycyclohex-1-ene-1-carboxylic acid alleviated the atopic dermatitis in mice. It can be inferred that the BX-BC08 strain has an alleviating effect on atopic dermatitis, and its influencing factors include the metabolite (3R, 4S, 5R)-(-)-3, 4, 5-trihydroxycyclohex-1-ene-1-carboxylic acid.

[0067] Experiment 2: Metabolite Sedoheptulose-7-phosphate (S7P) Twenty-four seven-week-old BALB / c female 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, and were treated by gavage for 21 days. MC903 modeling was performed from the 8th day, and all mice were killed on the 21st day. The phenotypic results are shown in the figure. Fig.25 .

[0068] From the graphical phenotypic results, it can be found that compared with the BHI group given by gavage, the inflammation in the metabolite group was significantly reduced, and the metabolite sedoheptulose-7-phosphate (s7p) had an alleviating effect on the atopic dermatitis in mice. It can be inferred that the BX-BC08 strain has an alleviating effect on atopic dermatitis, and its influencing factors include the metabolite sedoheptulose-7-phosphate (s7p).

[0069] Experiment 3: Metabolite DL-2-(acetylamino)-3-phenylpropanoic acid Twenty-four 7-week-old BALB / c female mice and the metabolite DL-2-(acetylamino)-3-phenylpropanoic acid were purchased. The mice were divided into a BHI group and a metabolite group, 12 mice in each group, and were treated by gavage for 21 days. MC903 modeling was performed from the 8th day, and all mice were killed on the 21st day. The phenotypic results are shown in the following table. Fig.26 .

[0070] The phenotypic results shown in the figure show that compared with the BHI group given by oral gavage, the inflammation in the metabolite group was significantly reduced, and the metabolite DL-2-(acetylamino)-3-phenylpropanoic acid had a relieving effect on the atopic dermatitis in mice. It can be inferred that the BX-BC08 strain has a relieving effect on atopic dermatitis, and its influencing factors include the metabolite DL-2-(acetylamino)-3-phenylpropanoic acid.

[0071] Experiment 4: Metabolite alpha-ketoglutaric acid Twenty-four 7-week-old BALB / c female mice and the metabolite alpha-ketoglutaric acid were purchased. The mice were divided into a BHI group and a metabolite group, with 12 mice in each group, and were treated with gavage for 21 days. MC903 modeling was performed from the 8th day, and all mice were killed on the 21st day. The phenotypic results are as follows Fig. 27 .

[0072] The phenotypic results shown in the figure show that compared with the BHI group given by oral administration, the inflammation in the metabolite group was significantly reduced, and the metabolite alpha-ketoglutaric acid had a relieving effect on the atopic dermatitis in mice. It can be inferred that the BX-BC08 strain has a relieving effect on atopic dermatitis, and its influencing factors include the metabolite alpha-ketoglutaric acid.

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

[0074] In summary, this scheme selects bifidobacteria, which helps to restore the balance of intestinal microecology, for example, it may increase the proportion of beneficial bacteria in the intestine and enhance the immune barrier function of the intestine. And it has been proved through experiments that bifidobacteria can regulate the immune system and reduce excessive immune response. For autoimmune diseases such as atopic dermatitis, it can reduce the production of inflammatory factors and relieve skin inflammation symptoms. Secondly, for this strain that generally comes from the human intestine, the human body has relatively good tolerance to it and rarely has serious adverse reactions.

[0075] The present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various 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 Bifidobacterium animalis subsp. lactis, characterized in that: The deposit number of the Bifidobacterium animalis subsp. lactis is CGMCC No.30902.

2. A product for treating atopic dermatitis, characterized in that: The product comprises the Bifidobacterium animalis subsp. lactis according to claim 1 or comprises the Bifidobacterium animalis subsp. lactis according to claim 1 and a metabolite thereof.

3. A product for treating atopic dermatitis according to claim 2, characterized in that: The metabolites include (3R, 4S, 5R)-(-)-3, 4, 5-trihydroxycyclohex-1-ene-1-carboxylic acid, sedoheptulose-7-phosphate (s7p), DL-2-(acetylamino)-3-phenylpropanoic acid and alpha-ketoglutaric acid.

4. A product for treating atopic dermatitis according to claim 2, characterized in that: The product includes food or medicine.

5. A product for treating atopic dermatitis according to claim 4, characterized in that: The medicine also includes pharmaceutical excipients, which include at least one of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

6. A product for treating atopic dermatitis according to claim 2, characterized in that: The number of live bacteria of the Bifidobacterium animalis subsp. lactis is not less than 1x10 8 CFU / mL or 1x10 8 CFU / g.

7. Use of the Bifidobacterium animalis subsp. lactis according to claim 1 in the preparation of a product for treating atopic dermatitis.

8. The use of Bifidobacterium animalis subsp. lactis in the preparation of a product for treating atopic dermatitis according to claim 7, characterized in that: The preparation comprises the Bifidobacterium animalis subsp. lactis or comprises the Bifidobacterium animalis subsp. lactis and a metabolite thereof.

9. The use of Bifidobacterium animalis subsp. lactis in the preparation of a product for treating atopic dermatitis according to claim 8, characterized in that: The number of live bacteria of the Bifidobacterium animalis subsp. lactis is not less than 1x10 8 CFU / mL or 1x10 8 CFU / g.

10. The use of Bifidobacterium animalis subsp. lactis in the preparation of a product for treating atopic dermatitis according to claim 8, characterized in that: The metabolites include (3R, 4S, 5R)-(-)-3, 4, 5-trihydroxycyclohex-1-ene-1-carboxylic acid, sedoheptulose-7-phosphate (s7p), DL-2-(acetylamino)-3-phenylpropanoic acid, and alpha-ketoglutaric acid.

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