Bifidobacterium adolescentis beneficial to skin as well as derivative and application of bifidobacterium adolescentis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium adolescentis beneficial to skin as well as a derivative and application of the bifidobacterium adolescentis. The derivative of bifidobacterium adolescentis has remarkable antioxidant and anti-inflammatory effects, can inhibit acne and other common pathogenic bacteria infected by skin, such as propionibacterium acnes, staphylococcus aureus and malassezia furfur, can eliminate induced pathogenic bacteria, can also inhibit skin lipid accumulation, avoids hair follicle blockage, and has a good anti-inflammatory effect. The skin problem, especially the acne skin problem, is improved in multiple aspects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bifidobacterium adolescentis beneficial to the skin and derivatives and applications thereof. Background Art

[0002] The skin can be divided into the epidermis, dermis and subcutaneous tissue, which wraps the surface of the human body and serves as the first line of defense against the invasion of adverse external factors. The skin is in direct contact with the external environment and microorganisms. Various skin microbial communities, skin tissues, cells and various secretion microenvironments constitute the skin microecological system, which together maintain the balance of the epidermal microecological system. As a component of the microecological system, the skin flora will not cause disease under normal circumstances. However, once pathogenic microorganisms reproduce or normal skin microorganisms overproliferate, or abnormal immune responses occur in the skin, it will lead to an imbalance in the skin microecology, which will cause skin diseases or aggravate existing diseases.

[0003] Acne is a chronic inflammatory skin disease of the pilosebaceous gland unit, which is clinically manifested as comedones, papules, pustules, cysts or nodules. Acne patients generally have vigorous sebaceous gland secretion, excessive keratinization of hair follicles, and Propionibacterium acnes ( Propionibacterium acnes, P. acnes ) dominates, and the severity of acne is positively correlated with its content. Pathogenic Staphylococcus aureus is also highly correlated with the incidence of acne. These bacteria produce a large amount of reactive oxygen species (ROS) during metabolism, leading to increased levels of local oxidative stress. Oxidative stress can damage cell membranes, proteins and DNA, destroy the integrity of hair follicle walls, and activate a series of pro-inflammatory signaling pathways. In addition, excessive sebum itself also contains a high concentration of free fatty acids, which can be converted into more toxic compounds under oxidative conditions, further exacerbating cell damage and inflammatory responses. Activation of the immune system caused by Propionibacterium acnes and other factors is a key driver of acne inflammation. When the number of microorganisms in the hair follicles increases, the body activates the immune defense mechanism and releases chemokines to attract white blood cells to the site of infection. White blood cells remove pathogens through phagocytosis, but also release various enzymes and free radicals, causing damage to surrounding tissues. This local inflammation not only manifests as symptoms such as redness, swelling, and pain, but may also cause the blockage of the hair follicle mouth to worsen, forming different forms of acne lesions such as acne, papules, pustules, and even cysts. Long-standing chronic inflammation can also induce collagen degradation, which may lead to scarring. Oxidative stress and inflammation reinforce each other, forming a vicious cycle that drives acne from initial subtle changes to overt clinical symptoms.

[0004] Acne is common in adolescent boys and girls, with the incidence rate among teenagers as high as 93%. It has become the eighth largest chronic disease in the world. According to statistics, 31.8% of common acne may cause secondary sensitive skin, and 3% to 7% may leave scars, seriously affecting the patient's appearance and physical and mental health.

[0005] Current methods for treating acne include the use of retinoic acid drugs, benzoyl peroxide, antibiotics, hormones, and photodynamic therapy, which target different pathological mechanisms. However, each of these methods has its limitations: retinoic acid and benzoyl peroxide may cause skin irritation such as erythema, dryness, and peeling, and retinoic acid has a risk of teratogenicity; antibiotics not only destroy the normal microbial balance of the skin, but long-term use may also lead to the development of drug resistance; hormones can only be used for specific populations and have a limited scope of application; photodynamic therapy may aggravate the imbalance of the skin microbial community and bring potential skin problems.

[0006] As a new method for treating acne, microbial preparations have gradually attracted widespread attention in recent years. Compared with existing therapies, they are milder and more sustainable, and have shown some potential effects. KR1020110127342A discloses a Bifidobacterium adolescentis SPM0308 with antibacterial activity against Propionibacterium acnes and an antibacterial composition for improving acne with the bacterium or its culture as an effective ingredient. CN115537361A provides a derivative of the Bifidobacterium adolescentis strain ProfMIC-225 that can improve skin conditions, including promoting the expression of barrier repair-related genes, repairing damage, promoting the proliferation of human fibroblasts HFF, regulating the expression of genes related to oxidative damage / cell apoptosis, etc. However, these existing methods are not targeted enough, and it is necessary to explore more potential microecological therapies. Summary of the invention

[0007] The purpose of the present invention is to provide a more potential improvement plan for skin problems, in particular to provide an improvement plan for acne symptoms.

[0008] The present invention first provides a strain of Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacteria, the deposit number of the Badol-1 strain is CCTCC NO:M 20242593.

[0009] Furthermore, the present invention provides a composition, wherein the active ingredient of the composition is Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacteria, the deposit number of the Badol-1 strain is CCTCC NO:M 20242593.

[0010] In some embodiments, the composition is a pharmaceutical composition, a food composition, or a cosmetic composition.

[0011] The present invention also provides Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) A method for culturing the Badol-1 strain or a pure culture thereof, the method comprising the step of inoculating the Bifidobacterium adolescentis into a culture medium for proliferation and cultivation.

[0012] The present invention also provides a Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or killed bacterial derivatives.

[0013] In some specific embodiments, the derivative is a lysate, an extract, a fermentation product lysate or a combination thereof of the Badol-1 strain or a pure culture or inactivated bacterial cell thereof.

[0014] Furthermore, the present invention provides a cosmetic composition or a pharmaceutical composition containing Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or killed bacterial derivatives.

[0015] In some embodiments, the cosmetic or pharmaceutical composition contains Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) lysate, extract, fermentation product lysate or combination thereof of Badol-1 strain or its pure culture or inactivated bacteria. The content of the multi-lactobacillus derivative in the cosmetic composition or pharmaceutical composition is an amount that can play a pharmacological reaction, which is easy to determine for those skilled in the art. The preferred content is 1-100%, more preferably 1-50%, further preferably 5-15%, and further 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% can be selected.

[0016] The present invention also provides an application of a probiotic preparation for non-disease treatment purposes, wherein the active ingredient of the probiotic preparation contains Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacterial derivatives, wherein the non-disease treatment use is selected from one or more of anti-oxidation, antibacterial, anti-inflammatory, reduction of skin redness and swelling, and inhibition of skin lipid accumulation.

[0017] In some specific embodiments, the antibacterial activity refers to the inhibition of Propionibacterium acnes ( Propionibacterium acnes ), Staphylococcus aureus ( Staphylococcus aureus ) and Malassezia ( Malassezia ) one, two or three of the following.

[0018] Furthermore, the present invention provides an application of a probiotic preparation in the preparation of a drug for treating acne, wherein the active ingredient of the probiotic preparation contains Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or killed bacterial derivatives.

[0019] The present invention also provides a method for preparing the aforementioned derivative, the method comprising: Bifidobacterium adolescentis ) The Badol-1 strain or its pure culture is inactivated, and the bacteria are crushed and filtered.

[0020] The Bifidobacterium adolescentis derivative of the present invention has significant antioxidant and anti-inflammatory effects, can be effective for skin problems, especially the clinical symptoms of acne, and can inhibit the pathogenic bacteria of acne and other common skin infections, such as Propionibacterium acnes, Staphylococcus aureus and Malassezia, can eliminate inducing pathogens, and can also inhibit skin lipid accumulation, avoid hair follicle blockage, and improve skin problems, especially acne skin problems, in all aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the results of total skin lesion score detection in the mouse acne model.

[0022] Figure 2 shows the skin thickness test results of the mouse acne model.

[0023] Figure 3 shows the results of inflammatory factor detection in the mouse acne model.

[0024] Figure 4 is a representative picture of the endpoint of the mouse acne model experiment. DETAILED DESCRIPTION

[0025] The meaning of the "strain" with the specific deposit number claimed in the present invention includes but is not limited to: (1) A strain with a specific accession number deposited in the above-mentioned depository; (2) Strains with the same genome as the strain described in (1), including clonal strains; (3) A non-mutated strain based on the above (1) or (2), including progeny strains; (4) A bacterial strain based on (1), (2) or (3) above that accumulates minor mutations during subculture but has no substantial changes in toxicity, immunogenicity or biological activity.

[0026] As is known in the art, minor mutations are inevitably introduced into strains through passage and application. When mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect the toxicity, immunogenicity and biological activity of the strain (for example, it may be a connecting amino acid residue between two domains, or a minor mutation residue located inside the higher-level structure of a protein that does not contact immune cells and does not affect toxicity, immunogenicity and biological activity), it can be reasonably expected that when these minor changes do not significantly affect the toxicity, immunogenicity and biological activity of the offspring strains, the purpose of the present invention can still be achieved, and they are derived from the strains contributed by the present invention, and therefore are still within the scope of the substantial technical contribution of the present invention. These minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity and biological activity.

[0027] There is no substantial change in toxicity, immunogenicity and biological activity, including but not limited to, within the limits of detection technology such as detection sensitivity and detection limit and acceptable or unavoidable errors, the toxicity, immunogenicity and biological activity are considered to be the same. When the toxicity, immunogenicity and biological activity of the offspring of the strain are measured using cells and animals, there is no substantial change due to differences in cell strains, animal species, age, sex, health status, culture conditions, etc., as well as foreseeable or unavoidable systematic errors.

[0028] "Progeny strain" refers to a descendant strain produced by an original strain or parent strain, including those strains that have undergone multiple generations but still retain the key characteristics and functional properties described in this patent application.

[0029] "Clone strain" means a genetically identical offspring strain that is directly derived from a single parent strain or its progeny through asexual reproduction (such as cell division or gene duplication).

[0030] A "pure culture" is a culture that consists of a single species of microorganism. A pure culture means that all the microorganisms growing on the culture medium are descendants of the same microorganism and have the same genetic characteristics.

[0031] "Composition" refers to a product or a collection of substances composed of a mixture of two or more different ingredients. These ingredients can be chemical substances, biological materials (such as cells, proteins, nucleic acids, etc.), active pharmaceutical ingredients, excipients or other additives.

[0032] "Pharmaceutical composition" refers to a preparation consisting of one or more active ingredients with therapeutic, preventive or diagnostic effects and pharmaceutically acceptable carriers, excipients or other additives. These compositions are intended to enter the body through appropriate administration routes (such as oral, injection, inhalation, topical application, etc.) to achieve the desired medical effect.

[0033] "Food composition" refers to a product intended for human consumption, which is composed of various ingredients, nutritional enhancers, flavoring substances, pigments, preservatives, etc., and is processed to form a specific form, taste and nutritional value. Such a composition not only provides basic energy and nutritional needs, but also may have active ingredients with specific health benefits.

[0034] "Cosmetic composition" refers to a product designed for personal care purposes such as beauty and skin care, usually composed of active ingredients, matrix materials, auxiliary ingredients and other additives, and is intended to clean, beautify, attract or change the appearance of the human body, while maintaining the health of the skin, hair and other parts.

[0035] "Inactivated bacteria" refers to microbial cells or their structurally intact cell walls and cell membranes that have lost their ability to reproduce after physical, chemical or other treatment methods. Although these cells are no longer viable (i.e., they cannot carry out metabolic activities or divide and proliferate), they still maintain a certain degree of structural integrity and can retain certain biologically active components.

[0036] "Derivative" in this patent refers to the product obtained by one or more steps of inactivation, crushing, separation, extraction, filtration, etc. of a strain, pure culture or inactivated bacteria through physical and chemical methods. The product contains the structural components, fermentation products, metabolites, etc. of the bacteria.

[0037] "Lysate" refers to a mixture formed by the destruction of the cell walls and cell membranes of microbial cells by physical, chemical or enzymatic methods, resulting in the release of cell contents.

[0038] "Extract" refers to a product obtained by physical, chemical or enzymatic treatment, in which the cell walls and cell membranes of microbial cells are destroyed, resulting in the release of cell contents, and then specific ingredients or components are selectively separated and concentrated by physical, chemical or biological methods.

[0039] "Fermentation product lysate" refers to a mixture of cell contents and metabolites obtained from a microbial culture after a fermentation process by physical, chemical or enzymatic methods. During this process, the cell wall and cell membrane of the microorganism are destroyed, resulting in the release of intracellular substances into the external environment. The lysis process not only exposes and separates intracellular components such as proteins, nucleic acids, enzymes, and secondary metabolites, but also retains the extracellular metabolites produced during the fermentation process.

[0040] Reagent / medium preparation Preparation of anaerobic MRS liquid culture medium: weigh 54.0 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312) and 0.5 g of cysteine ​​hydrochloride monohydrate, dissolve in 1 L of distilled water, deoxygenate with N2 replacement, and sterilize at 121°C for 15 min.

[0041] Preparation of anaerobic tri-mix liquid culture medium: weigh 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3), dissolve in 1 L of distilled water, deoxygenate by N2 replacement and package, sterilize at 121°C for 30 min, and store in a cool and dry place.

[0042] Preparation of anaerobic and resazurin-free three-mixed liquid culture medium (BHI+MRS+modified GAM): weigh 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3), dissolve in 1 L of distilled water, heat to boil, cool to room temperature, add 0.5 g of cysteine ​​hydrochloride, stir to dissolve, adjust the pH to 6.5-7.0, install a quantitative dispenser and pass N2, heat to boiling, keep at a slight boiling state for 30 min, cool and package, sterilize at 121°C for 15 min, and store in a cool and dry place.

[0043] TSB (Tryptone Soy Broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114) medium was prepared according to the instructions, weighed and dissolved, sterilized at 121°C for 30 min, and stored in a cool and dry place.

[0044] Example 1 Isolation and identification of strains Fresh feces from healthy volunteers were collected, shaken, resuspended and filtered in an appropriate amount of anaerobic PBS. Under N2 protection, the samples were gradient diluted and anaerobically cultured at 37°C in anaerobic three-mixed liquid medium for 9 days. The culture was deduplicated by MALDI-TOF mass spectrometry and then amplified and sequenced by 16S rDNA gene (Beijing Qingke Biotechnology Co., Ltd. Chengdu Branch) for identification. The sequencing results were compared by BLAST, and it was preliminarily confirmed that 1 strain of Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), formerly known by the Latin name: Bifidobacterium stercoris , formerly known as fecal bifidobacterium, was named Badol-1.

[0045] Example 2 Whole genome analysis and identification Badol-1 was inoculated into 5 mL of anaerobic three-mixed liquid medium at a 2% (v / v) inoculation volume and cultured until the late logarithmic growth period. The strain DNA was extracted for whole genome sequencing. After quality control of the offline data, it was assembled and annotated using conventional methods. The obtained protein sequence was compared with the VFDB (Virulence Factor Databases) and CARD (The ComprehensiveAntibiotic Resistance Database) databases to analyze virulence factors and drug resistance genes. The results showed that the strain was safe at the genetic level.

[0046] The novelty of the strains was analyzed using Average Nucleotide Identity (ANI) and Single Nucleotide Polymorphism (SNP)-based Variant analysis. By searching Genbank, 29 published Bifidobacterium_adolescentis The related whole genomes were compared by fastANI (v1.33) and snippy (v4.6.0), and it was found that the strain with the closest ANI to Badol-1 was GCA_003030905.1 (ANI=98.27%), so Badol-1 can be considered a new strain.

[0047] The microbial preservation information of Bifidobacterium adolescentis Badol-1 is as follows: Strain name: Bifidobacterium adolescentis ( Bifidobacterium adolescentis )Badol-1 Deposit date: November 18, 2024 Depository: China Center for Type Culture Collection (CCTCC), Address: Wuhan University, Wuhan, Hubei, Postal Code: 430072, Tel: 027-68754052 Deposit number: CCTCC NO:M 20242593.

[0048] Example 3 Inhibition of pathogenic bacteria Bacterial inhibition assay Two common pathogenic bacteria that cause acne were selected for antibacterial ability testing. The source information of the pathogenic strains is as follows: Table 1 Information on the source of pathogenic bacteria beads Strain name Strain accession number Strain collection unit Staphylococcus aureus CMCC(B)26003 China Food and Drug Inspection Institute Propionibacterium acnes BNCC330605 (ATCC6919 second generation) Henan Industrial Microbial Engineering Technology Research Center Preparation of the sample solution to be tested: After the preserved Bifidobacterium adolescentis Badol-1 is activated, it is inoculated into 15 mL of anaerobic MRS liquid medium at a 4% (v / v) inoculum. Badol-1 is cultured in anaerobic MRS liquid medium at 37°C for 48 h, then pasteurized at 65°C for 30 min, and then treated with an ultrasonic disruptor (working power of 120 W, ultrasound on for 3s, off for 4s, and ultrasound in an ice bath for a total of 10 min). After ultrasound, the fermentation product lysate is filtered with a 0.22μm filter membrane to obtain the sample solution to be tested.

[0049] Bifidobacterium saccharomyces cerevisiae (Shanghai Yanfu Industrial Co., Ltd.) was used as the positive control stock solution.

[0050] Preparation of test samples: Dilute the test sample stock solution and the bifid yeast stock solution to 10% with anaerobic resazurin-free three-mixed liquid culture medium as test samples.

[0051] Preparation of pathogenic bacteria: Staphylococcus aureus was activated with TSB medium and diluted in TSB medium to OD 600 acnes was activated by anaerobic three-mixed liquid medium and diluted to OD 600 It is 0.16-0.24.

[0052] Co-culture with pathogenic bacteria: The experiment was divided into a test sample group of the fermentation product lysate of Bifidobacterium adolescentis Badol-1 (Badol-1), a positive control test sample group of difidobacterium bifidum (difidobacterium bifidum) and a blank control group. The Badol-1 group and difidobacterium bifidum group were respectively placed in a 96-well plate with 0.1 mL of two pathogenic bacteria and 0.1 mL of the test sample, and each group had 3 parallel groups; the blank control group was respectively placed in a 96-well plate with 0.1 mL of two pathogenic bacteria and 0.1 mL of the corresponding culture medium, and each group had 3 parallel groups; Staphylococcus aureus was cultured aerobically for 24 h, and Propionibacterium acnes was cultured anaerobically for 24 h, and the OD at 0 h and 24 h of culture was detected 600 .

[0053] Data processing: Calculate the inhibition rate (R) using the formula R=(1−ΔOD1 / ΔOD0) × 100%, where ΔOD1 is the OD value of the sample group before and after 24 h of culture. 600 ΔOD0 is the OD value of the blank control group of the same pathogen before and after 24 h of culture. 600 changes.

[0054] The results were expressed as Mean ± SEM of the inhibition rate, and statistical analysis was performed using T-test. The antibacterial ability of Bifidobacterium adolescentis Badol-1 was compared with that of Saccharomyces cerevisiae; "***" indicates that compared with Saccharomyces cerevisiae, p<0.001, "**" indicates comparison with Bifida Saccharomyces cerevisiae, p <0.01, "*" indicates comparison with Bifida Saccharomyces cerevisiae, p <0.05.

[0055] Table 2 Results of pathogenic bacteria inhibition test Group Propionibacterium acnes inhibition rate Staphylococcus aureus inhibition rate Bifidobacterium 31.21±1.78 27.14±1.67 Badol-1 77.73±0.17 *** 60.70±12.67 * The experimental results are shown in Table 2. Badol-1 has antibacterial ability against both Staphylococcus aureus and Propionibacterium acnes (inhibition rate>50%), and its antibacterial ability against Propionibacterium acnes and Staphylococcus aureus is significantly stronger than that of Bifidobacterium.

[0056] Malassezia furfur inhibition test Select the fungus Malassezia furfur ( Malassezia furfur , Henan Industrial Microbial Strain Engineering Technology Research Center, BNCC324536) were used as pathogenic bacteria to detect the antibacterial ability of the samples.

[0057] Sample to be tested: stock solution of lysate of fermentation product of Bifidobacterium adolescentis Badol-1.

[0058] Determination method: Refer to WS / T 650-2019 "Antibacterial and Antibacterial Effect Evaluation Method" 5.1. Suspension quantitative antibacterial test, in which the positive control is anaerobic and resazurin-free three-mixed liquid culture medium, the experimental group is the culture stock solution of Bifidobacterium adolescentis Badol-1 strain, and the sample action time is 24h.

[0059] Experimental results: The antibacterial rate of Badol-1 stock solution against Malassezia is 53%, which shows antibacterial ability.

[0060] Example 4 Detection of total antioxidant capacity of Bifidobacterium adolescentis Badol-1 Sample to be tested: Bifidobacterium adolescentis Badol-1 was cultured for 24 hours according to the method of Example 3 to obtain a stock solution. Bifidobacterium bifida stock solution (Shanghai Yanfu Industrial Co., Ltd.) was used as a control.

[0061] A BCA protein concentration assay kit (purchased from Beijing Solebow Technology Co., Ltd., PC0020) was used to draw a standard curve and detect the sample protein concentration according to the instructions of the kit.

[0062] The total antioxidant capacity detection kit of strains (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC1315) was used to determine the antioxidant capacity of the sample (stock solution) according to the instructions of the kit and the standard curve. The total antioxidant capacity unit is μmol / mg prot.

[0063] Data processing: Three parallel groups were set in this experiment, and the results were expressed as Mean ± SEM. t-test was used for comparative analysis. "*" indicates comparison with bifid yeast. p <0.05.

[0064] Table 3 Total antioxidant test results Group Total Antioxidant Test Results Bifidobacterium 7.81±0.55 Badol-1 10.33±0.26 * Experimental results: As shown in Table 3, the total antioxidant capacity of Bifidobacterium adolescentis Badol-1 reached 10.33 ± 0.26 μmol / mg prot, which was significantly better than that of Bifidobacterium bifidum ( p < 0.05), indicating that Badol-1 has a strong antioxidant capacity in vitro.

[0065] Example 5 Detection of the DPPH free radical scavenging ability of Bifidobacterium adolescentis Badol-1 Preparation of stock solution: Bifidobacterium adolescentis Badol-1 and Bifidobacterium adolescentis standard strain (CCAM85, China Anaerobic Microbial Culture Collection Center) were treated according to the method of Example 3 to obtain stock solution.

[0066] Samples to be tested: Each stock solution and bifid yeast (Shanghai Yanfu Industrial Co., Ltd.) were diluted 5 times with anaerobic and resazurin-free liquid culture medium for detection. Bifid yeast and Bifidobacterium adolescentis standard strain CCAM85 were used as positive controls.

[0067] The DPPH free radical scavenging ability of the samples was determined using a strain DPPH free radical scavenging ability detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC4755) according to the instructions of the kit.

[0068] Data processing: Three parallel groups were set in this experiment, and the results were expressed as Mean ± SEM. One-way ANOVA was used for multiple comparison analysis. "***" indicates comparison with bifid yeast. p <0.001, "##" indicates comparison with standard strains, p <0.01.

[0069] Table 4 DPPH free radical scavenging test results Group DPPH free radical scavenging test results Bifidobacterium -3.00±2.34 Badol-1 36.79±4.21 ***## CCAM85 13.16±3.41 Experimental results: As shown in Table 4, the DPPH free radical scavenging ability of Bifidobacterium adolescentis Badol-1 reached 36.79% ± 4.21%, which was significantly better than the standard strain of Bifidobacterium adolescentis CCAM85 ( p < 0.01), significantly better than Bifida Saccharomyces cerevisiae ( p <0.001), indicating that Badol-1 has a strong DPPH free radical scavenging ability in vitro.

[0070] Example 6 Detection of ABTS free radical scavenging ability of Bifidobacterium adolescentis Badol-1 Preparation of stock solution: Bifidobacterium adolescentis Badol-1 and Bifidobacterium adolescentis standard strain (CCAM85, China Anaerobic Microbial Culture Collection Center) were treated according to the method of Example 3 to obtain stock solution.

[0071] Samples to be tested: Each stock solution and bifid yeast (Shanghai Yanfu Industrial Co., Ltd.) were diluted 5 times for testing, and bifid yeast and Bifidobacterium adolescentis standard strain CCAM85 were used as positive controls.

[0072] The strain ABTS free radical scavenging ability detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC4775) was used to determine the ABTS free radical scavenging ability of the sample according to the instructions of the kit.

[0073] Data processing: Three parallel groups were set up in this experiment, and the results were expressed as Mean ± SEM. One-way ANOVA was used for multiple comparison analysis. "***" indicates comparison with bifid yeast. p <0.001, "##" indicates comparison with standard strains, p <0.01.

[0074] Table 5 ABTS free radical scavenging test results Group ABTS free radical scavenging test results Bifidobacterium 9.57±1.35 Badol-1 85.67±1.01 ***## CCAM85 64.15±2.80 Experimental results: As shown in Table 5, the ABTS free radical scavenging ability of Bifidobacterium adolescentis Badol-1 reached 85.67% ± 1.01%, which was significantly better than the standard strain of Bifidobacterium adolescentis CCAM85 (p < 0.01) and significantly better than Saccharomyces cerevisiae (p < 0.001), indicating that Badol-1 has a strong ABTS free radical scavenging ability in vitro.

[0075] Example 7 The ability of Bifidobacterium adolescentis to clear lipid accumulation Sample processing: Bifidobacterium adolescentis Badol-1 and Bifidobacterium adolescentis standard strain (CCAM85, China Anaerobic Microbial Culture Collection Center) were processed according to the method of Example 3 to obtain stock solutions.

[0076] Linoleic acid (Sigma-Aldrich (Shanghai) Trading Co., Ltd., L1376) was used to induce lipid secretion in immortalized human sebaceous gland cell line SZ95 cells (Qingqi (Shanghai) Biotechnology Development Co., Ltd., BFN60807569) to induce cellular lipid accumulation. The ability of Bado-1 to clear lipid accumulation was evaluated by the amount of cellular lipid accumulation.

[0077] Linoleic acid was dissolved in a mixture of DMSO and ethanol (1:1, v / v) to prepare a 1 M linoleic acid stock solution, which was then diluted 1000 times using serum-free DMEM culture medium (Thermo Fisher Scientific (China) Co., Ltd., C11065500BT) to prepare a linoleic acid working solution.

[0078] Isotretinoin (Sigma-Aldrich (Shanghai) Trading Co., Ltd., I0800000) was dissolved in DMSO to prepare a 100 mM isotretinoin stock solution, which was then diluted with linoleic acid working solution to prepare a 100 μM isotretinoin working solution.

[0079] 5% (v / v) bifid yeast linoleic acid working solution was prepared by adding bifid yeast stock solution (Shanghai Yanfu Industrial Co., Ltd.) to linoleic acid working solution.

[0080] 5% (v / v) Badol-1 linoleic acid working solution and 5% (v / v) CCAM85 linoleic acid working solution were prepared by adding the corresponding Badol-1 or CCAM85 stock solution to the linoleic acid working solution.

[0081] SZ95 cells were collected at 3×10 4 The cells / well were inoculated in a 96-well plate and cultured at 37°C, 5% CO2 for 24 hours. The supernatant was removed and the experiment was divided into 6 groups. The blank control group was added with 200 μL of serum-free DMEM medium, the model control group was added with 200 μL of linoleic acid working solution, the isotretinoin group was added with 200 μL of isotretinoin working solution, the bifid yeast group, the Badol-1 group and the CCAM85 group were added with 200 μL of bifid yeast linoleic acid working solution, Badol-1 linoleic acid working solution or CCAM85 linoleic acid working solution, respectively.

[0082] After adding the sample, the cells were cultured at 37°C and 5% CO2 for 24 hours. According to the requirements of the lipid droplet red fluorescence detection kit (Biyuntian Biotechnology Co., Ltd., Nile Red C2051M), 100 μL of fluorescent dye was added for staining, and the cells were incubated at 37°C in the dark for 10 minutes before detection. The excitation light was 485 nm and the emission light was 535 nm. The detection results are shown in Table 6.

[0083] Data processing: This experiment set 6 parallel groups in each group, and the results were expressed as Mean ± SEM. One-way ANOVA was used for multiple comparison analysis. "***" indicates comparison with the model control. p <0.001, "**" indicates comparison with model control, p <0.01, "#" indicates comparison with standard strains, p <0.05.

[0084] Table 6 Lipid accumulation removal ability test results Group Lipid accumulation clearance ability test results Blank control 100.00±6.07 *** Model comparison 176.53±3.72 Isotretinoin 119.34±3.04 *** Bifidobacterium 162.09±7.17 CCAM85 145.55±5.45 ** Badol-1 128.08±4.90 ***# The results showed that after linoleic acid induction, cellular lipid secretion increased and obvious lipid accumulation occurred. The lipid accumulation in the model control was significantly higher than that in the blank control. After isotretinoin treatment, the lipid accumulation was significantly decreased compared with the model control. However, no obvious drug effect was observed in bifid yeast, and there was no significant difference in lipid accumulation compared with the model control.

[0085] After treatment with Badol-1 and CCAM85, the amount of lipid accumulation was significantly decreased compared with the model control, and Bado-1 was superior to the standard strain CCAM85, and the amount of lipid accumulation of Bado-1 was significantly reduced compared with CCAM85.

[0086] Example 8 Detection of the anti-inflammatory effect of Bifidobacterium adolescentis Badol-1 Sample processing: Bifidobacterium adolescentis Badol-1 was processed according to the method of Example 3 to obtain a stock solution.

[0087] Human keratinocytes Hacat cells (Chengdu Gumeng Biotechnology Co., Ltd., BNCC339817) were stimulated with Propionibacterium acnes (Henan Industrial Microbial Strain Engineering Technology Research Center, BNCC330605 (ATCC6919 second generation)) to induce Hacat cells to produce cellular inflammatory factors, and the anti-inflammatory effect of Badol-1 was evaluated by the level of inflammatory factors.

[0088] Preparation of heat-killed Propionibacterium acnes working solution: Select Propionibacterium acnes in the logarithmic growth phase, heat at 80°C for 30 min to inactivate, centrifuge at 12000 rpm / min for 5 min after inactivation, remove the supernatant, reconstitute with serum-free DMEM medium, and adjust the final concentration of Propionibacterium acnes to 5×10 8 CFU / ml.

[0089] Preparation of salicylic acid working solution: Use heat-killed Propionibacterium acnes working solution to prepare 200 μg / ml salicylic acid working solution.

[0090] 5% (v / v) bifid yeast Propionibacterium acnes working solution was prepared by adding heat-inactivated Propionibacterium acnes working solution to bifid yeast stock solution (Shanghai Yanfu Industrial Co., Ltd.).

[0091] 5% (v / v) Badol-1 Propionibacterium acnes working solution is prepared by adding heat-killed Propionibacterium acnes working solution to Badol-1 stock solution.

[0092] Hacat cells were collected at 3 × 10 4The cells were inoculated in a 96-well plate at a concentration of 1000 μl / well and cultured at 37°C, 5% CO2 for 24 hours. The supernatant was removed and the cells were divided into 5 groups. 200 ul serum-free DMEM medium was added to the blank control, 200 ul heat-killed Propionibacterium acnes working solution was added to the model control, 200 ul salicylic acid working solution was added to the salicylic acid group, 200 ul bifid yeast Propionibacterium acnes working solution and 200 ul Badol-1 Propionibacterium acnes working solution were added to the bifid yeast and Badol-1, respectively.

[0093] After adding the sample, the cells were cultured at 37°C and 5% CO2 for 24 h, and the cell culture supernatant was collected. Inflammatory factors were detected according to the instructions of the human interleukin 8 (IL-8) enzyme-linked immunosorbent assay kit (Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H6008).

[0094] Data processing: Three parallel groups were set up in each group of this experiment. The results were expressed as Mean±SEM. One-way ANOVA was used for multiple comparison analysis. "****" means that compared with the model control, p<0.0001; "*" means that compared with the model control, p<0.05.

[0095] Table 7 Inflammatory factor detection results Group Anti-inflammatory test results Blank control 11.13±0.12 *** Model comparison 36.69±3.45 Salicylic acid 15.72±0.80 *** Bifidobacterium 28.05±2.02 * Badol-1 15.52±1.41 *** The results are shown in Table 7. After induction by Propionibacterium acnes, the IL-8 content in the model control group was significantly higher than that in the blank control group. The salicylic acid group and the bifid yeast group had obvious anti-inflammatory effects, and the IL-8 content after treatment was significantly decreased compared with the model control group. The Badol-1 group had obvious anti-inflammatory effects, and the IL-8 content after treatment was significantly decreased compared with the model control group.

[0096] Example 9 Bifidobacterium adolescentis Badol-1 improves acne in mice Sample processing: Bifidobacterium adolescentis Badol-1 and standard strain CCAM85 were processed according to the method of Example 3 to obtain stock solutions.

[0097] Preparation of test samples: Take 20 ml of the above-prepared Badol-1 and CCAM85 stock solutions and the purchased positive control bifid yeast stock solution (Repair Complex CLR™ PF, purchased from Chemisches Laboratorium Dr. Kurt Richter GmbH), add 0.4 g of hydroxyethyl cellulose (thickener, purchased from Shandong Heda Co., Ltd.), mix well, and then divide them into portions with 1 ml sterile syringes for storage at -20°C. Take one vial daily for application.

[0098] Experimental grouping and administration: 30 SPF female ICR mice aged 8-9 weeks and weighing 23-26 g were purchased from Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd. and kept in an SPF animal room. After 7 days of adaptive feeding, the mice were randomly divided into 6 groups according to their body weight, namely blank control group, model control group, adapalene group, bifida yeast group, Badol-1 group and standard strain CCAM85 group, with 5 mice in each group. Before the experiment, all mice were depilated with depilatory cream on the back. Except for the blank control group, the other groups were injected with Propionibacterium acnes modeling for two consecutive days between 9:00 and 10:00 am on the first and second days of the experiment. The injection site was the left and right sides of the back of the mouse, and 50 μl was injected on each side, with an injection volume of 1×10 8 CFU, after the injection on the first day, 0.1g of artificial sebum (purchased from Beijing Solebow Technology Co., Ltd.) was immediately applied to the injection site for 7 consecutive days, and the sebum application was performed in the morning. The blank control group was injected with the same amount of saline and no artificial sebum was applied. The positive drug adapalene group was applied with adapalene (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.) at 0.1g / mouse, and the bifid yeast group, Badol-1 group and CCAM85 group were all applied with the above-prepared bifid yeast, Badol-1 and CCAM85 test products at 0.2ml / mouse, and the drugs were administered once a day for 7 consecutive days, and the drugs were administered in the afternoon. All mice were dissected on the 8th day, and the skin tissues at the modeling site were collected and stored for later use.

[0099] Skin lesion score: The diameter of the papule / pustule / erythema formed at the modeling site was measured with a vernier caliper, and the skin lesion status of all mice was scored according to the scoring criteria in the table below. The total score of skin lesions was obtained by adding the scores of each indicator in the table below. The test results were expressed as Mean ± SEM, and the differences between the groups were tested using non-parametric tests, such as the Kruskal-Wallis test.

[0100] Table 8 Skin lesion scoring table Lesion type Rating content Diameter of papule / pustule / erythema 0~1.9mm 0 points 2~3.9mm 1 point 4~5.9mm 2 points 6~8mm 3 points Abscess (degree of swelling) None 0 points Small 1 point Medium 2 points Large 3 points Black scab None 0 points Mild 1 point Moderate 2 points Severe 3 points Dry skin None 0 points Mild 1 point Moderate 2 points Severe 3 points Desquamation None 0 points Mild 1 point Moderate 2 points Severe 3 points Skin thickness: After the experiment, all mice were dissected, the back skin was cut open, and the skin thickness of the modeling sites on both sides was measured with a vernier caliper.

[0101] Inflammatory factor detection: According to the instructions, the TNF-α content in mouse skin tissue was detected using a mouse ELISA detection kit.

[0102] The experimental results are expressed as Mean±SEM, and One-way ANOVA was used for multiple comparison analysis. Compared with the model group, "*" p<0.05; "***" p<0.001; "****" p<0.0001, "##" indicates a significant difference compared with the standard strain CCAM85 group, p<0.01, and "#" indicates a significant difference compared with the standard strain CCAM85 group, p<0.05.

[0103] Table 9 Results of mouse acne model testing Group Skin lesion scoring results Skin thickness test results TNF-α test results Blank control 0.00±0.00 *** 0.56±0.03 *** 11.06±0.81 ** Model comparison 7.60±0.40 1.05±0.07 20.32±2.39 Adapalene 3.60±0.40 ** 0.79±0.02 * 13.44±1.24 * Bifidobacterium 4.00±0.95 * 0.87±0.07 16.29±1.23 Badol-1 1.00±0.32 *** 0.64±0.04 *** 12.83±1.00 ** CCAM85 5.00±0.84 ## 0.93±0.06 # 17.24±1.94 Test results: Figure 1 is the total score of skin lesions at the end of the experiment. Compared with the blank control group, the total score of skin lesions in the model control group was significantly increased. Compared with the model control group, the skin lesion scores in the adapalene group, the bifid yeast group and the Badol-1 group were significantly decreased. The improvement effect of the Badol-1 group was better than that of the adapalene group and the bifid yeast group, and significantly better than that of the standard strain CCAM85 group.

[0104] Figure 2 As for the skin thickness results, compared with the blank control group, the skin thickness of the model control group at the modeling site was significantly increased. Compared with the model control group, the adapalene group and the Badol-1 group could significantly reduce the skin thickness of the modeling site. The improvement effect of the Badol-1 group was better than that of the adapalene group and the bifida yeast group, and was significantly better than that of the standard strain CCAM85 group.

[0105] Figure 3 The results of TNF-α detection showed that compared with the blank control group, the TNF-α content in the skin tissue of the model control group was significantly increased. Compared with the model control group, the adapalene group and the Badol-1 group could significantly reduce the TNF-α content in the skin tissue. The improvement effect of the Badol-1 group was better than that of the bifida yeast group and the standard strain CCAM85 group.

[0106] Figure 4 The following are representative pictures of the back skin of mice in each group at the end point of the experiment. The back skin of mice in the normal control group was normal, while the back skin of mice in the model control group was rough, hardened, and had increased dandruff. The modeling site was raised, and there were black scabs formed by the transformation of pustules. The other groups all had different degrees of improvement. Adapalene had a certain improvement effect, but increased skin dandruff was observed, and there were certain side effects. Among the other groups, the improvement effect of the Badol-1 group was the most obvious. At the end of the drug administration, its skin was smooth and the modeling site was flat, and the effect was better than that of the bifid yeast and the standard strain CCAM85.

Claims

1. A Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacteria, the deposit number of the Badol-1 strain is CCTCC NO:M 20242593.

2. A composition, characterized in that The active ingredient of the composition is Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacteria, the deposit number of the Badol-1 strain is CCTCC NO:M 20242593.

3. A method for culturing the Bifidobacterium adolescentis described in claim 1 ( Bifidobacterium adolescentis ) A method for producing the Badol-1 strain or a pure culture thereof, characterized in that The method comprises the step of inoculating the bifidobacterium adolescentis into a culture medium for proliferation culture.

4. A Bifidobacterium adolescentis according to claim 1 ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or killed bacterial derivatives.

5. The derivative according to claim 4, characterized in that The derivative is a lysate, an extract, a fermentation product lysate or a combination thereof of the Badol-1 strain or its pure culture or inactivated bacteria.

6. A cosmetic composition or a pharmaceutical composition, characterized in that: The active ingredient contains the derivative according to claim 4 or 5.

7. A use of a probiotic preparation for non-disease treatment purposes, characterized in that: The active ingredient of the microecological preparation contains the derivative according to claim 4 or 5, and the non-disease treatment purpose is selected from one or more of anti-oxidation, antibacterial, anti-inflammatory, reduction of skin redness and swelling, and inhibition of skin lipid accumulation.

8. The use according to claim 7, characterized in that: The antibacterial activity refers to the inhibition of one, two or three of Propionibacterium acnes, Staphylococcus aureus and Malassezia furfur.

9. Use of a probiotic preparation in the preparation of a drug for treating acne, wherein the active ingredient of the probiotic preparation contains the derivative according to claim 4 or 5.

10. A method for preparing the derivative according to claim 4 or 5, the method comprising the steps of: Bifidobacterium adolescentis ) The Badol-1 strain or its pure culture is inactivated, and the bacteria are crushed and filtered.

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