Humanized bifidobacterium animalis probiotic strain and application thereof in preparation of medicine for preventing or treating atopic dermatitis

Abstract

The invention belongs to the field of probiotics and application thereof, and particularly relates to a humanized bifidobacterium animalis probiotic strain and application thereof in preparation of drugs for preventing or treating atopic dermatitis. The preservation number of the humanized bifidobacterium animalis is GDMCC No: 65044, the preservation unit is called GDMCC for short, and active metabolites secreted by the strain comprise acetate. The animal bifidobacterium disclosed by the invention has the advantages that the animal bifidobacterium is derived from the intestinal tract of a healthy individual, can normally survive in the intestinal tract of a human body, has no toxic or side effect and has relatively high safety. Besides, the strain can be used for building a microorganism-immune cell co-culture platform in a body, and the type 2 immune inflammation of an AD patient is effectively improved by regulating and controlling Th1 / Th2 immune balance. By means of targeted supplementation of deficient protective strains of patients, a new prevention and treatment means can be provided for AD patients.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, in particular to probiotics for preventing or treating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by an imbalance of T cell subsets. Its clinical symptoms are usually lifelong, mainly manifested as itching, polymorphic skin lesions accompanied by exudation, etc., which seriously interfere with the patient's quality of life and physical and mental health. The pathogenesis of AD is complex and involves multiple factors, including genetic predisposition, environmental factors, immune disorders and impaired skin barrier function. The immunological characteristics of AD are usually manifested as excessive activation of type 2 immune inflammation, especially the dominant role of Th2 immune cells in local inflammation. This immune response leads to the secretion of cytokines such as IL-4, IL-5 and IL-13, which in turn induces skin inflammatory reactions and allergic reactions. Type 2 immune inflammation not only plays a key role in the occurrence and development of AD, but is also closely related to the recurrence and chronicity of AD.

[0003] At present, there is no clinical cure for AD, and the recurrence of the disease in patients remains one of the difficulties in dermatology diagnosis and treatment. Therefore, the development of new and safe therapeutic targets or adjuvant drugs for the prevention and adjuvant treatment of AD has important research significance and clinical value. In recent years, fecal microbiota transplantation has made remarkable progress in the clinical treatment of AD. However, due to the complexity of fecal microbiota and the challenges of stool quality control, the widespread use of fecal microbiota transplantation still has great risks. In addition, some studies have attempted to use commercially available bacteria for the prevention and adjuvant treatment of AD, but the results are less reproducible. Therefore, the development of safer and more consistently effective treatment methods remains an urgent problem to be solved in this field. Studies have shown that after fecal microbiota from healthy people were transplanted into AD patients, the patients' skin itching, atopic dermatitis score (SCORAD), eczema area and severity index (EASI), pruritus numerical rating scale (NRS), and the number of Staphylococcus aureus on the skin surface were significantly improved. While filling the gap in the "missing protective flora" in the gut, the study also reshaped the patient's intestinal microbiome, significantly increasing the abundance of species such as Bifidobacterium. This suggests that the gut microbiome and skin immune homeostasis are linked through the "gut-skin axis," and that targeted transplantation (on-demand administration) of microorganisms (protective flora) that are abnormally missing from the gut of AD patients may be used for the prevention and treatment of AD. Summary of the Invention

[0004] The purpose of the present invention is to provide a humanized animal Bifidobacterium probiotic strain and its use in the preparation of a drug for preventing or treating atopic dermatitis, so as to solve the problems in the prior art of fecal microbiota transplantation in the clinical treatment of AD, which pose great risks and poor consistency in the treatment results of commercially available bacteria.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A humanized animal Bifidobacterium probiotic strain that can prevent or treat atopic dermatitis is animal Bifidobacterium (Bifidobacterium animalis), specifically named Bifidobacterium animalisSMUDHYang01, deposited on August 22, 2024, deposited with Guangdong Provincial Microbiological Culture Collection Center (GDMCC), with a deposit number of GDMCC No: 65044, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides a use of the humanized animal Bifidobacterium probiotic strain capable of preventing or treating atopic dermatitis in the preparation of a medicine capable of preventing or treating atopic dermatitis.

[0008] The present invention also provides an active metabolite of the humanized animal Bifidobacterium probiotic strain capable of preventing or treating atopic dermatitis.

[0009] The present invention also provides an active metabolite of the humanized animal Bifidobacterium probiotic strain capable of preventing or treating atopic dermatitis, wherein the active metabolite comprises acetate.

[0010] The present invention also provides a use of the active metabolite in preparing a drug for preventing or treating atopic dermatitis.

[0011] The present invention also provides a humanized probiotic composition, comprising the humanized animal Bifidobacterium probiotic strain Bifidobacterium animalis SMUDHYang01, with or without its active metabolites, and one or more physiologically acceptable excipients or carriers.

[0012] Furthermore, each gram of the humanized probiotic composition contains 1×10 8 to 1×10 12 CFU of live Bifidobacterium animalis SMUDHYang01 strain.

[0013] Furthermore, each gram of the humanized probiotic composition contains 180 μg of acetate, an active metabolite of the Bifidobacterium animalis SMUDHYang01 strain. This concentration, determined by targeted metabolomics quantitative detection, can significantly regulate immune responses and improve atopic dermatitis (AD) symptoms.

[0014] The present invention also provides an application of the humanized probiotic composition in the preparation of food or food additives.

[0015] The present invention also provides a use of the humanized probiotic composition in preparing a medicament for preventing or treating atopic dermatitis.

[0016] The advantages of this invention include the isolation of a protective bacterial strain, Bifidobacterium animalis SMUDHYang01, from the healthy human intestines, which is absent in AD patients. This Bifidobacterium animalis SMUDHYang01, originating from the healthy human intestine, can survive normally in the human intestine, is non-toxic, and demonstrates significant efficacy in subsequent immune regulation and improvement of type 2 immune inflammation in AD patients. By targeting and supplementing the missing protective strain, this approach provides a novel preventive and treatment option for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of this application, and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1-1 This is a diagram showing the results of the screening and identification experiments of different bifidobacteria;

[0019] Figure 1-2 The figure shows the experimental results of the mouse model of AD-like lesions induced by perfusion of MC903 and modeled by ear;

[0020] Figure 2 This is the statistical diagram of the proportion of Th2 immune cells in the AD-like dermatitis lesions and the immune analysis of mouse serum after Ba bacteria treatment;

[0021] Figure 3-1 This is one of the statistical graphs of the enrichment of characteristic metabolites of the Ba strain metabolomics and the relationship between metabolites and Th1 cell production;

[0022] Figure 3-2 This is the second statistical graph showing the enrichment statistics of characteristic metabolites of the Ba strain metabolomics and the relationship between metabolites and Th1 cell production;

[0023] Figure 3-3 This is the third statistical graph of the enrichment statistics of characteristic metabolites of the Ba strain metabolomics and the relationship between metabolites and Th1 cell production. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0026] The experimental materials involved in the following examples are as follows:

[0027] (1) Regarding the healthy volunteers mentioned in the instructions, the criteria are: no bad habits, no use of probiotics, antibiotics and other related products in the past two months, and no history of tumors or infectious diseases.

[0028] (2) Preparation of BSM agar medium (Bifidobacterium culture medium): Dissolve 10 g of glucose, 5 g of yeast extract, 10 g of peptone, and 5 g of NaCl in 1 L of deionized water, adjust the pH to 6.0-6.5, add 15 g of agar, and sterilize under high pressure at 121°C for 15-20 minutes to obtain BSM agar medium.

[0029] Example 1B. Isolation and identification of bacteria

[0030] The humanized animal Bifidobacterium probiotic strain Ba (Bifidobacterium animalis SMUDHYang01) was isolated from fecal samples of healthy volunteers. The specific isolation steps are as follows:

[0031] First, the abundance of target bacteria (Bifidobacterium) in fecal samples from 10 healthy volunteers was detected using quantitative PCR (qPCR) technology; then, based on the qPCR test results, the feces of healthy volunteers with the highest abundance of target bacteria in the intestine were selected for subsequent culture and isolation.

[0032] like Figure 1-1 As shown in A, fresh fecal samples were collected and immediately resuspended in 15 ml of PBS buffer and shaken for 15 minutes to evenly disperse the fecal samples. Subsequently, the remaining solid matter was removed by filtration through a 100 μm filter to obtain a clear fecal solution. The fecal solution was diluted in different concentration gradients and then spread on BSM agar medium. After incubation under anaerobic conditions at 37°C for 48 hours, several colonies formed on each agar medium. By visually observing the morphological characteristics of the colonies, such as size, color, and shape, four strains with potential protective effects were initially screened.

[0033] MALDI-TOF mass spectrometry can analyze the protein fingerprint of bacteria and compare it with the database of known strains to quickly and accurately determine the bacterial species. We used MALDI-TOF mass spectrometry (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) to preliminarily identify these colonies. The 16S rRNA gene is a commonly used landmark gene in bacterial taxonomy because its sequence is highly conserved among different bacteria and can effectively distinguish different bacterial genera and species. In order to further confirm the identity of the selected strain, we also used 16S rRNA gene amplification technology and used universal primers to amplify the 16S rRNA gene of the target strain. The amplified 16S rRNA gene product was sequenced by Sanger sequencing, and the isolated target bacterial species was finally confirmed by comparing the sequencing results with the known gene sequences in the public database. The Sanger sequencing comparison results are shown as follows. Figure 1-1 A. They are named Bifidobacterium Ba, B.br, B.bi, and Bl respectively.

[0034] The Ba bacteria were preserved and classified and named Bifidobacterium animalis (Bifidobacterium animalis), specifically named Bifidobacterium animalis SMUDHYang01, and the preservation unit is Guangdong Microbial Culture Collection Center (GDMCC). The preservation unit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The preservation date is August 22, 2024, and the preservation number is GDMCC No: 65044.

[0035] The rDNA sequence of Bifidobacterium animalis SMUDHYang0116S is 1394 bp in length, and its sequence is shown in SEQ ID NO.1:

[0036] TCGAACGGGATCCCTGGCAGCTTGCTGTCGGGGTGAGAGTGGCGAACGGG

[0037] TGAGTAATGCGTGACCAACCTGCCCTGTGCACCGGAATAGCTCCTGGAAA

[0038] CGGGTGGTAATACCGGATGCCTCCGCTCCATCGCATGGTGGGGTGGGAAATG

[0039] CTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTGGCGGGGTGA

[0040] TGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGACCGGCC

[0041] ACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG

[0042] AATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGAT

[0043] GGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTT

[0044] CGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAG

[0045] CAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTA

[0046] AAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCCTAA

[0047] CGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACT

[0048] GGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAAT

[0049] GGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGT

[0050] GGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGG

[0051] ATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTT

[0052] AAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATT

[0053] GACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACG

[0054] CGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTGGAGACACG

[0055] GTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGT

[0056] GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCAT

[0057] GTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACT

[0058] CGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGG

[0059] CTTCACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGT

[0060] GGGGCGGATCGCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTC

[0061] GACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGG

[0062] TGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTG

[0063] GGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGGGG

[0064] Example 2. Improvement of MC903-induced AD-like lesion progression by Bifidobacterium animalis SMUDHYang01<� <�

[0065] In order to explore the potential role of these strains in the prevention and treatment of AD-like dermatitis, Figure 1-2 B shows that one week before the experiment, mice were orally perfused with BHI medium (control group), E. coli, Ba bacteria, B. br bacteria, B. bi bacteria, and Bl bacteria for pretreatment, and then the MC903-induced AD-like lesion mouse model was established. In this model, mice were orally perfused with BHI medium (control group), E. coli, Ba bacteria, B. br bacteria, B. bi bacteria, and Bl bacteria every day until the end of the experiment. The experimental results are shown in Figure 2. Figure 1-2 CD showed that compared with the BHI culture medium control group and the E. coli bacterial control group, Ba bacteria and B. br bacteria can significantly reduce the AD-like dermatitis score of mice (scoring indicators include skin thickness, skin lesion severity, etc.), suggesting that these two strains have potential in alleviating the symptoms of AD-like dermatitis. Studies have shown that B. br bacteria can improve AD-like dermatitis in mice by regulating host tryptophan metabolism, and this finding has been successfully applied clinically, verifying its effectiveness in AD treatment. Our study also reached a similar conclusion. It was further found that Ba bacteria is particularly effective in improving AD-like dermatitis, especially in reducing skin inflammation and alleviating skin damage. This suggests that Ba bacteria may exert its anti-inflammatory effect by regulating the intestinal microbiota or through interactions with the host immune system.

[0066] In order to further verify the therapeutic effect of Ba bacteria, the results are as follows Figure 1-2 As shown in E, we conducted a dual-site modeling experiment, that is, MC903 was used to induce AD-like dermatitis in the mouse ears, and the treatments of each group were compared. The results are shown in Figure 1-2 FH showed that compared with the control group, the symptoms of AD-like dermatitis in the ears of mice treated with the bacterium Ba significantly improved, indicating that the bacterium Ba has a good therapeutic effect on AD-like dermatitis not only systemically but also in the localized inflammation area. The bacterium Ba was named Bifidobacterium animalis SMUDHYang01.

[0067] Example 3B.a bacteria (Bifidobacterium animalis SMUDHYang01) improves the Th2 immune response of MC903-induced AD-like lesions

[0068] AD is an immune-mediated disease dominated by Th2 immune cells. One of the characteristics of its pathogenesis is the imbalance between T cell subsets. In particular, in the immune response of AD, the overexpression of Th2 immune cytokines plays a vital role. In order to explore the role of Th2 immune response in the improvement of AD-like dermatitis by Ba bacteria (Bifidobacterium animalis SMUDHYang01). We designed an MC903-induced AD-like dermatitis mouse model and analyzed the proportion of Th2 immune cells in the lesions treated with BHI medium, E. coli bacteria, and Ba bacteria respectively. The experimental results are as follows. Figure 2 A shows that compared with the BHI culture medium control group and the E. coli bacterial control group, Ba bacteria significantly reduced the proportion of Th2 immune cells in the mouse skin lesions. This result shows that Ba bacteria can reshape the Th2 immune response in the MC903-induced AD-like lesion mouse model and reduce the local inflammatory response, suggesting that Ba bacteria plays an important role in regulating the distribution and function of immune cells. In order to further confirm the function of Ba bacteria in improving type 2 immune inflammation in mice, Figure 2 As shown in B, we performed immunoassays on 22 cytokines in mouse serum. Figure 2 C shows that compared with the BHI medium control group and the E. coli bacterial control group, Ba bacteria significantly reduced the levels of Th2 immune cytokines, especially IL-4 and IL-13. These two cytokines are hallmark Th2 immune cytokines of AD and have been confirmed as potential targets for the treatment of moderate to severe AD. These preliminary research results collectively suggest that Ba bacteria from healthy volunteers may become an innovative adjunctive therapy for alleviating AD-like symptoms by regulating Th2 immune responses.

[0069] Example 4B.a bacteria (Bifidobacterium animalis SMUDHYang01) releases acetate to regulate Th1 / Th2 immune balance

[0070] To identify immunomodulatory metabolites secreted by Bifidobacterium animalis SMUDHYang01, we performed metabolomic analysis of the low molecular weight fraction (less than 3 kDa) in the culture supernatant of Bifidobacterium animalis (Ba.CM) based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). Figure 3-1 As shown in A, through the score plot of principal component analysis (PCA), we found that the Ba strain exhibited a unique metabolomic profile compared with the BHI medium. Figure 3-1As shown in B, among the top 20 metabolites produced or metabolized by Ba bacteria, tryptophan derivatives indole lactic acid (ILA) and short-chain fatty acids (SCFAs) were significantly enriched (red arrows), including isobutyric acid and pyruvic acid. Therefore, we speculate that Ba bacteria may reshape the Th1 / Th2 immune balance by regulating the metabolic pathways of tryptophan and SCFAs production. In order to further confirm the specific role of immunomodulatory metabolites in the culture supernatant of Ba bacteria, we performed targeted metabolomics quantitative analysis, and the results are shown in Figure 2. Figure 3-2 As shown in C~3-2D, the quantitative analysis results showed that the enrichment concentrations of ILA and acetate in Ba.CM were 1200nM and 900μg / mL, respectively, which were significantly higher than those in the control group. Further T cell differentiation experiments found that Figure 3-3 As shown in Figures E-3-3F, ILA failed to induce Th1 cell differentiation, while acetate significantly promoted the generation of Th1 cells and significantly enhanced the balance of Th1 / Th2 immune responses. This indicates that acetate, rather than ILA, is the functionally active metabolite derived from the Ba strain.

[0071] In summary, Ba bacteria can establish a microbial-immune cell co-culture platform in the body, explaining the mechanism of action of Ba bacteria. By screening CD4+ T cell surface receptors, it can regulate the Th1 / Th2 immune balance, providing a new therapeutic target for improving type 2 immune inflammation in AD patients. In addition, acetate derived from the gut microbiota can improve skin barrier integrity and Th1 / Th2 immune balance in AD-like lesions, providing a new treatment option for AD.

[0072] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A humanized animal Bifidobacterium probiotic strain for preventing or treating atopic dermatitis, characterized by: It is Bifidobacterium animalis, specifically named Bifidobacterium animalis SMUDHYang01, the depository is abbreviated as GDMCC, the deposit number is GDMCC No: 65044, and its 16S rDNA sequence is shown in SEQ ID NO.

1.

2. Use of the humanized animal Bifidobacterium probiotic strain capable of preventing or treating atopic dermatitis as claimed in claim 1 in the preparation of a medicament capable of preventing or treating atopic dermatitis.

3. An active metabolite of the humanized animal Bifidobacterium probiotic strain capable of preventing or treating atopic dermatitis as claimed in claim 1.

4. The active metabolite of the humanized animal Bifidobacterium probiotic strain capable of preventing or treating atopic dermatitis according to claim 3, characterized in that: The active metabolites include acetate.

5. Use of the active metabolite according to claim 3 or 4 in the preparation of a medicament for preventing or treating atopic dermatitis.

6. A humanized probiotic composition, characterized in that: The invention comprises the humanized animal Bifidobacterium probiotic strain Bifidobacterium animalisSMUDHYang01 according to claim 1, with or without its active metabolites, and one or more physiologically acceptable excipients or carriers.

7. The humanized probiotic composition according to claim 6, characterized in that: Each gram of the humanized probiotic composition contains 1×10 8 to 1×10 12 CFU of live Bifidobacterium animalis SMUDHYang01 strain.

8. The humanized probiotic composition according to claim 6, characterized in that: Each gram of the humanized probiotic composition contains 180 μg of acetate, an active metabolite of the Bifidobacterium animalis SMUDHYang01 strain.

9. Use of the humanized probiotic composition according to claim 6 in preparing food or food additives.

10. Use of the humanized probiotic composition according to claim 6 in the preparation of a medicament for preventing or treating atopic dermatitis.

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