Akkermansia muciniphila YG2602 with repair, soothing and anti-aging effects and its applications

Abstract

The present invention relates to the field of microorganisms, and particularly relates to an Akkermansia muciniphila YG2602 with the effects of repair, soothing and anti-aging, its inactivated cells, extracts, cultures, culture supernatants, fermentation products and / or fermentation supernatants, compositions containing the foregoing, and their applications in the preparation of products for anti-dermatitis, anti-skin aging, repairing and soothing the skin. The Akkermansia muciniphila YG2602 and its related products of the present invention have good ability to repair skin cells, reduce, inhibit or eliminate skin inflammation, and significant anti-skin aging ability. Therefore, they are expected to be used in skin health care such as skin repair, inflammation relief and anti-photoaging, and have significant application value and market potential.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to an Akkermansia muciniphila YG2602 with repair, soothing and anti-aging effects, its inactivated cells, extracts, cultures, culture supernatants, fermented products and / or fermented supernatants, compositions containing the foregoing, and the application of Akkermansia muciniphila or its inactivated cells, extracts, cultures and compositions containing the foregoing in the preparation of products for anti-dermatitis, anti-skin aging, repairing and soothing the skin. Background Art

[0002] People's pursuit of beauty is endless, and the causes of skin problems such as pigmentation, wrinkles, aging and dehydration are complex. Researchers are committed to developing safe and effective solutions, but chemicals in cosmetics such as titanium dioxide may pose health risks. Although herbal ingredients have potential, their complexity affects the stability of the effects. With the increasing demand for green skincare, microbial fermentation products, such as bacterial lysates, fermented broths and filtrates, are leading the trend of microecological skincare and have broad market prospects.

[0003] Probiotics, due to their low toxicity, are used in skin health and treatment. They can reduce inflammation and improve skin conditions, such as resisting allergic dermatitis, enhancing the skin barrier and delaying aging. Probiotic exosomes are rich in active compounds, such as lipopolysaccharides and lipoteichoic acids of Gram-negative and positive bacteria, which are beneficial for skin anti-aging. Research shows that the exosomes of Lactobacillus.plantarum can inhibit MMP-1 and elastase and increase the expression of filaggrin mRNA, contributing to anti-aging and skin barrier function.

[0004] Akkermansia muciniphila is an intestinal probiotic that plays a key role in human intestinal health, especially related to the maintenance of the mucus layer and the regulation of the immune system. Currently, there are research reports on the application of Akkermansia muciniphila in various disease fields such as cancer, diabetes, obesity, progeria, amyotrophic lateral sclerosis, epilepsy, IBD, hypertension, autism, etc., but there is no report showing its role in the field of skin care.

[0005] The information disclosed in this background art section is only intended to increase the understanding of the general background of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0006] Object of the Invention

[0007] The object of the present invention is to provide a strain of Akkermansia muciniphila YG2602 with repair, soothing and anti-aging effects, its inactivated cells, extracts, cultures, culture supernatants, fermented products and / or fermented supernatants, compositions containing the foregoing, and the use of Akkermansia muciniphila or its inactivated cells, extracts, cultures and compositions containing the foregoing in the preparation of products for anti-dermatitis, anti-skin aging, repairing and soothing the skin.

[0008] Solution

[0009] To achieve the object of the present invention, the present invention adopts the following technical solutions.

[0010] In a first aspect, the present invention provides a strain of Akkermansia muciniphila YG2602. The taxonomic name of this strain is Akkermansia muciniphila, and it is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The address of the depositary institution is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The deposit date is April 22, 2024, and the deposit number is CGMCC No. 30411.

[0011] The Akkermansia muciniphila YG2602 of the present invention is isolated from a fecal sample of a healthy adult. Through 16S rRNA sequencing, it is identified as Akkermansia muciniphila.

[0012] The colony characteristics of this strain include: after culturing on BHI agar medium for 24 - 48 h, the diameter is between 0.01 - 1.0 mm, the colony edge is smooth, milky white, and viscous.

[0013] In vitro experiments have confirmed that the fermentation broth, lysate, and exosomes of Akkermansia muciniphila YG2602 of the present invention all have good ability to repair skin cells. Specifically, the inventors found that the fermentation broth, lysate, and exosomes of Akkermansia muciniphila YG2602 of the present invention can significantly enhance the expression of type XVII collagen (COL17A1), and at the same time promote the production of COL1A1, COL3A1, and hyaluronic acid (HAS1, HAS3), and inhibit the activity of matrix metalloproteinase (MMP1), thereby achieving deep repair of skin cells.

[0014] In addition, the inventors found and confirmed through in vitro experiments that the lysate of Akkermansia muciniphila YG2602 of the present invention also has the ability to inhibit the expression of inflammatory factors. Specifically, the inventors found that the lysate of the Akkermansia muciniphila strain of the present invention can significantly reduce the levels of inflammatory factors such as TNF-α, IL-6, IL-1β, and COX-2 caused by skin irritation (such as LPS stimulation).

[0015] In addition, it has also been confirmed that the lysate and exosomes of Akkermansia muciniphila YG2602 of the present invention have obvious anti-skin aging effects. Specifically, it may achieve the effect of anti-photoaging by inhibiting the expression of UVB-induced inflammatory factors (such as IL-1β, IL-6, and IL-8) and restoring the expression of skin epidermal physical barrier proteins (IVL, LOR, and FLG) damaged by UVB.

[0016] In a second aspect, the present invention provides the inactivated cells, extracts, cultures, culture supernatants, fermentates, and / or fermentation supernatants of Akkermansia muciniphila YG2602 as described in the first aspect above.

[0017] Preferably, the extract of Akkermansia muciniphila YG2602 is the lysate and / or exosomes extracted therefrom.

[0018] In a feasible embodiment, the extraction method of the lysate includes the following:

[0019] Ferment and culture Akkermansia muciniphila YG2602, centrifuge the fermentation broth, collect the bacterial cells, resuspend the bacterial cells with PBS and break the cell wall by ultrasonic treatment or enzymatic hydrolysis to obtain a lysate; preferably, sterilize the obtained lysate.

[0020] In feasible embodiments, the method for extracting the exosomes includes the following:

[0021] Ferment and culture Akkermansia muciniphila YG2602, centrifuge the fermentation broth, and collect the fermentation supernatant; centrifuge, filter, and concentrate the obtained fermentation supernatant to obtain exosomes;

[0022] Preferably, the centrifugation procedure for the fermentation supernatant is as follows: first centrifuge at 200 - 400 g, preferably 300 g for 8 - 15 min, preferably 10 min, then centrifuge at 1500 - 3000 g, preferably 2000 g for 15 - 30 min, preferably 20 min, and finally centrifuge at 8000 - 12000 g, preferably 10000 g for 20 - 40 min, preferably 30 min;

[0023] Preferably, the filtration of the fermentation supernatant is as follows: filter with a filter membrane having a pore size of 0.2 - 2.0 μm, preferably filter successively with filter membranes having pore sizes of 0.8 μm and 0.45 μm;

[0024] Preferably, the concentration of the fermentation supernatant is as follows: concentrate the clarified fermentation broth with a hollow fiber having a molecular weight cut-off of 400 - 600 kD, preferably 500 kD.

[0025] In a third aspect, the present invention provides a composition, which comprises Akkermansia muciniphila YG2602 as described in the first aspect above, or inactivated bacterial cells, extracts, cultures, culture supernatants, fermentation products, and / or fermentation supernatants of Akkermansia muciniphila YG2602 as described in the second aspect above as active ingredients.

[0026] In some feasible embodiments, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier and / or excipient.

[0027] In other feasible embodiments, the composition is a cosmetic composition, which further comprises a cosmetically acceptable carrier and / or excipient.

[0028] Preferably, the composition is in an external preparation form;

[0029] Further preferably, the topical dosage form is a dosage form selected from the following: aqueous solution, emulsion, ointment, cream, gel, powder, and oil.

[0030] In a feasible embodiment, the composition further comprises other active agents;

[0031] Preferably, the other active agents include any one or more of antioxidants, cell activators, moisturizing agents, anti-aging agents, and anti-dermatitis agents.

[0032] In a fourth aspect, the present invention provides the use of Akkermansia muciniphila, or inactivated cells, extracts, cultures, culture supernatants, fermentates, and / or fermentation supernatants of Akkermansia muciniphila, or a composition comprising one or more of the foregoing substances, in the preparation of a product for the following uses:

[0033] 1) Repairing and / or soothing the skin;

[0034] 2) Alleviating, inhibiting, or eliminating skin inflammation or allergy;

[0035] 3) Anti-skin aging or skin senescence.

[0036] In a preferred embodiment, the Akkermansia muciniphila is the Akkermansia muciniphila YG2602 as described in the first aspect above, the inactivated cells, extracts, cultures, culture supernatants, fermentates, and / or fermentation supernatants of Akkermansia muciniphila are the inactivated cells, extracts, cultures, culture supernatants, fermentates, and / or fermentation supernatants of Akkermansia muciniphila YG2602 as described in the second aspect above, and the composition is a composition comprising one or more of the foregoing substances;

[0037] In a feasible embodiment, the repairing and soothing of the skin is for repairing and soothing sensitive skin;

[0038] In a feasible embodiment, the skin inflammation is aseptic skin inflammation or inflammation caused by external stimuli;

[0039] In a feasible embodiment, the skin allergy is skin allergy symptoms caused by external factor stimuli;

[0040] In a feasible embodiment, the anti-skin aging is anti-skin photoaging.

[0041] In a fifth aspect, the present invention provides a method for repairing and / or soothing the skin, and / or reducing, inhibiting or eliminating skin inflammation or allergy, and / or anti-skin aging or anti-skin senescence, which comprises: administering an effective amount of Akkermansia muciniphila to a subject in need, or inactivated cells, extracts, cultures, culture supernatants, fermentates and / or fermentation supernatants of Akkermansia muciniphila, or a composition comprising one or more of the foregoing substances.

[0042] In a preferred embodiment, the Akkermansia muciniphila is Akkermansia muciniphila YG2602 as described in the first aspect above, the inactivated cells, extracts, cultures, culture supernatants, fermentates and / or fermentation supernatants of Akkermansia muciniphila are the inactivated cells, extracts, cultures, culture supernatants, fermentates and / or fermentation supernatants of Akkermansia muciniphila YG2602 as described in the second aspect above, and the composition is a composition comprising one or more of the foregoing substances;

[0043] In a feasible embodiment, the repairing and soothing of the skin is for repairing and soothing sensitive skin;

[0044] In a feasible embodiment, the skin inflammation is aseptic skin inflammation, inflammation caused by external stimuli;

[0045] In a feasible embodiment, the skin allergy is skin allergy symptoms caused by external factor stimuli;

[0046] In a feasible embodiment, the anti-skin aging is anti-skin photoaging.

[0047] Beneficial Effects

[0048] Compared with the prior art, Akkermansia muciniphila YG2602 of the present invention has the following obvious advantages:

[0049] (1) Its fermentation broth, lysate and exosomes can significantly enhance the expression of type XVII collagen (COL17A1), promote the production of collagens such as COL1A1 and COL3A1, and hyaluronic acid (HAS1, HAS3), and inhibit the activity of matrix metalloproteinase (MMP1), thus being beneficial to skin regeneration and achieving deep repair of the skin.

[0050] (2) Its lysate can effectively reduce the expression of intracellular inflammatory factors (such as TNF-α, IL-6, IL-1β and COX-2, etc.) caused by external stimuli (such as LPS), thereby providing excellent soothing protection for sensitive skin and significantly improving the inflammatory response.

[0051] (3) Its lysate and exosomes have obvious anti-skin photoaging effects. Specifically, they may achieve the anti-photoaging effect by inhibiting the expression of UVB-induced inflammatory factors (such as IL-1β, IL-6 and IL-8) and restoring the expression of skin epidermal physical barrier proteins (IVL, LOR and FLG) damaged by UVB.

[0052] In summary, Akkermansia muciniphila YG2602 of the present invention and its related products have good abilities to repair skin cells, reduce, inhibit or eliminate skin inflammation, and have significant anti-skin aging abilities. Therefore, they are expected to be used in skin repair, inflammation alleviation, and anti-photoaging and other skin health care, and have significant application value and market potential.

[0053] In addition, the inventors also first discovered that other Akkermansia muciniphila and their fermentation broth, lysate and exosomes in addition to Akkermansia muciniphila YG2602 of the present invention also have the effects of repairing and soothing the skin and anti-skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] One or more embodiments are illustrated by way of example in the accompanying drawings, and these exemplary illustrations do not limit the embodiments. The special word "exemplary" here means "serving as an example, embodiment or illustrative". Any embodiment illustrated as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0055] Figure 1 is the growth curve of Akkermansia muciniphila YG2602 of the present invention.

[0056] Figure 2Shows the particle concentration and particle size distribution of exosomes extracted from the fermentation broth of the strain YG2602 of the present invention measured by NTA technology.

[0057] Figure 3 Shows the heat map of the HSF cell repair rate of the fermentation filtrate and lysate of different Akkermansia muciniphila strains.

[0058] Figure 4 Shows the effects of the fermentation filtrate, lysate, and exosomes of Akkermansia muciniphila YG2602 of the present invention at different concentrations on the survival rate of HSF cells.

[0059] Figure 5 Shows the effect of the fermentation filtrate of Akkermansia muciniphila YG2602 of the present invention on HSF cell repair; wherein, Figure A is a schematic diagram of HSF cell fusion; Figure B is a statistical chart of the HSF cell fusion rate, where * indicates P<0.05 and ** indicates P<0.01; Figure C is a statistical chart of the relative expression levels of mRNAs of HSF cell repair-related factors, where * indicates P<0.05 and **** indicates P<0.0001.

[0060] Figure 6 Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on HSF cell repair; wherein, Figure A is a schematic diagram of HSF cell fusion; Figure B is a statistical chart of the HSF cell fusion rate, where *** indicates P<0.001; Figure C is a statistical chart of the relative expression levels of mRNAs of HSF cell repair-related factors, where * indicates P<0.05 and **** indicates P<0.0001.

[0061] Figure 7 Shows the effect of the exosomes of Akkermansia muciniphila YG2602 of the present invention on HSF cell repair; wherein, Figure A is a schematic diagram of HSF cell fusion; Figure B is a statistical chart of the HSF cell fusion rate, where ** indicates P<0.01 and **** indicates P<0.0001; Figure C is a statistical chart of the relative expression levels of mRNAs of HSF cell repair-related factors, where **** indicates P<0.0001.

[0062] Figure 8Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the viability of RAW264.7 cells in the evaluation of soothing efficacy; wherein, # indicates: compared with the blank control group (i.e., Con group), p < 0.05; ## indicates: compared with the blank control group (i.e., Con group), p < 0.01.

[0063] Figure 9 Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the cell morphology of the LPS-induced RAW264.7 cell soothing model; the pictures show the cell morphology of different treatment groups observed under a 20× microscope.

[0064] Figure 10 Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the TNF-α secretion of the LPS-induced RAW264.7 cell soothing model; wherein, ## indicates: compared with the blank control group (i.e., Con group), p < 0.01; * indicates: compared with the LPS group, p < 0.05.

[0065] Figure 11 Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the gene expression levels of related inflammatory factors TNF-α (Figure A), COX-2 (Figure B), IL-6 (Figure C) and IL-1β (Figure D) in the LPS-induced RAW264.7 cell soothing model; wherein, # indicates: compared with the blank control group (i.e., Con group), p < 0.0001; * indicates: compared with the LPS group, p < 0.05, ** indicates: compared with the LPS group, p < 0.01, *** indicates: compared with the LPS group, p < 0.001, **** indicates: compared with the LPS group, p < 0.0001.

[0066] Figure 12 Shows the effect of different UVB treatments on cell viability; wherein, ### indicates: compared with the blank control group (i.e., Con group), p < 0.001.

[0067] Figure 13 Shows the effect of the exosomes of Akkermansia muciniphila YG2602 of the present invention on the cell morphology and fusion degree of UVB damage.

[0068] Figure 14Shows the effect of exosomes of Akkermansia muciniphila YG2602 of the present invention on the inflammatory factors of cells damaged by UVB; wherein, Figure A, Figure B, and Figure C respectively show the protein levels of IL-1β, IL-6, and IL-8 in each treatment group, and Figure D shows the mRNA levels of IL-6, IL-8, IL-1β, and COX-2 in each treatment group; wherein, * indicates: compared with the UVB group, 0.01 < p < 0.05, ** indicates: compared with the UVB group, 0.001 < p < 0.01, *** indicates: compared with the UVB group, p < 0.001; ### Indicates: compared with the blank control group (i.e., Con group), p < 0.001.

[0069] Figure 15 Shows the effect of exosomes of Akkermansia muciniphila YG2602 of the present invention on the cell skin barrier-related factors damaged by UVB; wherein, Figure A shows the mRNA levels of the antimicrobial peptides hBD-2 and CAMP in each treatment group, Figure B shows the mRNA levels of TGF-β and MMP-9 in each treatment group, and Figure C shows the FLG level in each treatment group; wherein, * indicates: compared with the UVB group, 0.01 < p < 0.05, ** indicates: compared with the UVB group, 0.001 < p < 0.01, *** indicates: compared with the UVB group, p < 0.001; # Indicates: compared with the blank control group (i.e., Con group), 0.01 < p < 0.05, ## Indicates: compared with the blank control group (i.e., Con group), 0.001 < p < 0.01, ### Indicates: compared with the blank control group (i.e., Con group), p < 0.001.

[0070] Figure 16 Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the cell morphology and fusion degree damaged by UVB.

[0071] Figure 17Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the inflammatory factors of cells damaged by UVB; among them, Figure A, Figure B and Figure C respectively show the protein levels of IL-1β, IL-6 and IL-8 in each treatment group, and Figure D shows the mRNA levels of IL-6, IL-8 and IL-1β in each treatment group; where, * indicates: compared with the UVB group, 0.01 < p < 0.05, ** indicates: compared with the UVB group, 0.001 < p < 0.01, *** indicates: compared with the UVB group, p < 0.001; ## Indicates: compared with the blank control group (i.e., Con group), 0.001 < p < 0.01, ### Indicates: compared with the blank control group (i.e., Con group), p < 0.001.

[0072] Figure 18 Shows the effect of the exosomes of Akkermansia muciniphila YG2602 of the present invention on the cell skin barrier proteins IVL (Figure A), LOR (Figure B) and FLG (Figure C) damaged by UVB; among them, * indicates: compared with the UVB group, 0.01 < p < 0.05, *** indicates: compared with the UVB group, p < 0.001; # Indicates: compared with the blank control group (i.e., Con group), 0.01 < p < 0.05, ## Indicates: compared with the blank control group (i.e., Con group), 0.001 < p < 0.01, ### Indicates: compared with the blank control group (i.e., Con group), p < 0.001.

[0073] Figure 19 Shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the viability of RAW264.7 cells in the evaluation of immunomodulatory efficacy; among them, * indicates: compared with the blank control group (i.e., Con group), p < 0.05, **** indicates: compared with the blank control group (i.e., Con group), p < 0.0001.

[0074] Figure 20It shows the effect of the lysate of Akkermansia muciniphila YG2602 of the present invention on the secretion of TNF-α in the RAW264.7 cell immunomodulation model (Figure A), and the effect on the gene expression levels of related inflammatory factors TNF-α (Figure B), IL-6 (Figure C), IL-1β (Figure D), and iNOS (Figure E); wherein, * indicates: compared with the blank control group (i.e., Con group), p < 0.05, ** indicates: compared with the blank control group (i.e., Con group), p < 0.01, **** indicates: compared with the blank control group (i.e., Con group), p < 0.0001.

[0075] Akkermansia muciniphila YG2602 of the present invention is taxonomically named Akkermansia muciniphila, deposit date: April 22, 2024; depository: General Microbiology Center of China Committee for Culture Collection of Microorganisms; depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; deposit number is CGMCC NO. 30411. Detailed implementation manners

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention. Unless otherwise clearly stated, throughout the specification and claims, the term "comprise" or its variations such as "comprises" or "including" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0077] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0078] In the following embodiments, unless otherwise specified, other reagents or raw materials used can be obtained commercially.

[0079] In addition, in the following examples, the "concentration" of the strain fermentation filtrate, lysate, and exosome refers to the volume dilution multiple obtained by diluting the stock solution of the corresponding substance with a complete medium (i.e., DMEM medium containing serum); for example, strain fermentation filtrates or lysates at concentrations of 1 / 100, 1 / 1000, and 1 / 10000 (V / V) mean that the stock solutions of the strain fermentation filtrates or lysates are diluted 100-fold, 1000-fold, and 10000-fold by volume, respectively.

[0080] Example 1: Isolation and identification of Akkermansia muciniphila

[0081] (1) Strain isolation

[0082] Ninety-four fecal samples were collected from healthy adults in Beijing, China. One gram of fecal specimens was collected, and fecal genomic DNA was extracted using a fecal genomic DNA extraction kit (Qiagen, Germany) according to the operation manual. The concentration and purity were detected using a microplate reader. The fecal DNA concentration was adjusted to 10 ng / μL as the qPCR template for detection, and samples with positive detection results were subjected to strain isolation.

[0083] One gram of positive fecal sample was placed into 9 mL of PBS buffer, vortexed and mixed well, and then serially diluted 10-fold with sterile saline. Three dilution gradients of 10-4, 10-5, and 10-6 were selected. For each gradient, 0.5 mL of the diluted solution was added to 4.5 mL of enrichment tubes containing mucin culture medium and anaerobically cultured at 37 °C for 72 h. Serial gradient dilution was performed using PBS, and the diluted liquids with dilution factors of 10-3 to 10-6 were respectively spread on BHI agar medium. Two plates were spread for each dilution factor. The solid plates were placed in an anaerobic constant temperature incubator at 37 °C for 48 - 72 h, and the colonies were picked and purified on BHI agar medium more than 3 times until the colony morphology on the plate was consistent.

[0084] (2) Strain identification

[0085] A. Colony morphological characteristics

[0086] After culturing the 5 isolated strains on BHI agar medium for 24 - 48 h, the diameter was between 0.01 - 1.0 mm, the colony edges were smooth, milky white, and viscous.

[0087] B. 16S rRNA sequencing

[0088] The five isolated strains were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA gene sequencing. The 16S rRNA sequencing results were BLAST aligned on the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Combining their colony morphological characteristics, it was preliminarily determined that the five isolated strains were all Akkermansia muciniphila, named YGMCC02592, YGMCC02618, YGMCC02594, YGMCC02586, and YGMCC02602 respectively.

[0089] Through the efficacy screening of the following examples, it was identified that YGMCC02602 had the ability to repair skin cells well, reduce, inhibit or eliminate skin inflammation, and significantly resist skin aging. That is, Akkermansia muciniphila YG2602 of the present invention (hereinafter, "YGMCC02602" and "YG2602" can be used interchangeably, both referring to the strain of the present invention), and its 16S rRNA sequence is shown in SEQ ID NO:1.

[0090] Example 2: Preparation of fermentation filtrate and lysate of strain YGMCC02602, and extraction and characterization of exosomes

[0091] The culture media involved in this example are as follows:

[0092] Modified BHI medium (g / L): Brain heart infusion 12.5 g / L, bovine heart infusion 5.0 g / L, proteose peptone 10.0 g / L, glucose 2.0 g / L, sodium chloride 5.0 g / L, disodium hydrogen phosphate 2.5 g / L, L-cysteine hydrochloride 0.5 g / L, L-threonine 5 g / L, N-acetylglucosamine 5 g / L, and the solvent is water; its preparation method is: Mix the components except L-threonine and N-acetylglucosamine evenly and autoclave at 121 °C for 15 min. After the medium cools down, add L-threonine and N-acetylglucosamine filtered and sterilized through a 0.22 μm polyethersulfone membrane to obtain it.

[0093] 2.1 Cultivation of YGMCC02602 and preparation of its fermentation broth

[0094] The seed solution of strain YGMCC02602 was transferred to the modified BHI liquid medium at an inoculation amount of 5%, and anaerobically statically cultured at 37 °C for 24 h. During the culture process, the OD600 value of the culture broth was measured by a microplate reader at regular intervals to obtain the growth curve of strain YGMCC02602, as Figure 1 shown.Figure 1 It was shown that when the strain YGMCC02602 was cultured for 16 h, it could reach the growth stationary phase (OD600 = 0.92).

[0095] The fermentation process parameters of the strain YGMCC02602 were optimized to obtain the optimal fermentation parameters as follows: pH value 6.85, inoculum size 4.5%, and culture time 48 h.

[0096] Based on the above optimized fermentation process parameters, the seed liquid of the strain YGMCC02602 was transferred to the improved BHI liquid medium (pH value 6.85) at an inoculum size of 4.5%, and anaerobically statically cultured at 37 °C for 48 h to obtain the fermentation broth.

[0097] 2.2 Preparation of fermentation filtrate, lysate and extraction and characterization of exosomes of YGMCC02602

[0098] (1) Preparation of fermentation filtrate:

[0099] The fermentation broth of the strain YGMCC02602 prepared above was centrifuged at 9000 r / min at 4 °C for 10 min to obtain the fermentation supernatant. The fermentation supernatant was filtered through a 0.22 μm polyethersulfone membrane to obtain the cell-free fermentation filtrate of the strain YGMCC02602, which was frozen at -80 °C for later use.

[0100] (2) Preparation of lysate:

[0101] The cells after centrifugation in the above (1) were harvested, added with pre-cooled sterile PBS for 2-fold concentration and resuspended, and the cell wall was broken by ultrasonic treatment. The specific ultrasonic program was as follows: ultrasonic for 2 s, stop for 2 s, and the total ultrasonic time was 60 min, and the whole ultrasonic program was carried out under the ice bath condition of -4 to 0 °C. Then, the ultrasonic product was heat-treated at 80 °C for 30 min by a high-pressure sterilization device to obtain the sterilized lysate of the YGMCC02602 fermentation broth, which was frozen at -80 °C for later use.

[0102] (3) Extraction and characterization of exosomes:

[0103] The fermentation broth of strain YGMCC02602 was centrifuged at 9000 r / min at 4 °C for 10 min to obtain the fermentation supernatant. The fermentation supernatant was first centrifuged at 300 g at 4 °C for 10 min, then at 2000 g at 4 °C for 20 min, and finally at 10000 g at 4 °C for 30 min. Then, it was filtered successively through 0.8-μm and 0.45-μm polyethersulfone membranes to obtain the clarified fermentation broth. The obtained clarified fermentation broth was concentrated to 50 mL using a 500-kd hollow fiber, and the buffer was exchanged with 200 mL of PBS. The concentrated solution was purified using a protein purification instrument to obtain the exosome solution of the YGMCC02602 fermentation broth. The particle number and diameter distribution of exosomes in the obtained exosome solution were measured using NTA technology.

[0104] After the above procedures, we obtained the fermentation filtrate, lysate, and exosome solution of YGMCC02602 with stable high yields. Based on the NTA technology, the number of exosome particles and the diameter range in the YGMCC02602 exosome solution were measured, and the results are shown in Figure 2 ; Figure 2 It shows that the diameter range of exosomes in the YGMCC02602 exosome solution is between 100 - 1000 nm, with an average value of 280.2 nm (median diameter: 249.5 nm). This means that although the size of the main population is about 250 nm, it has a wide size distribution; and the number of particles per milliliter of the exosome solution is 1.4*10 10 particles.

[0105] Using the above procedures, the fermentation filtrate, lysate, and exosome solution of the other four strains, YGMCC02592, YGMCC02618, YGMCC02594, and YGMCC02586, were also prepared respectively.

[0106] Example 3: Comparison of the ability of fermentation filtrates and lysates of different Akkermansia muciniphila strains to repair cells

[0107] Human skin fibroblasts, as key cells in the dermis layer, are crucial for maintaining skin elasticity and tension. They can efficiently synthesize matrix such as elastin and collagen, generate elastic fibers and collagen fibers, secrete repair factors, and endow the skin with the ability of self-repair and renewal.

[0108] In this example, human fibroblast cells (hereinafter referred to as HSF) were used as the research object, and the repair abilities of the fermentation filtrates and lysates of the five strains on them were detected respectively.

[0109] The culture method of HSF cells is as follows:

[0110] HSF cells were cultured in a cell culture incubator at 37°C and 5% CO2 in a high glucose medium (DMEM) containing 15% special fetal bovine serum (FBS) and 1% 100× penicillin-streptomycin mixture (S / P).

[0111] The detection procedure of cell repair ability is as follows:

[0112] HSF cells were cultured at 2 × 10 5 The inoculation density of cells / well was inoculated into 6-well plates, and after incubation in an incubator (37°C, 5% CO2) for 24 hours, the fusion rate reached 90%; streaked the cell culture plate with a 200μL pipette tip, and washed the streaked culture plate 2 to 3 times with PBS to remove floating cells. Take a photo immediately after the scratching at 0h, and add 2mL of serum-free DMEM culture medium containing the sample after the photo is taken. Specifically, the control group was a 1×PBS treatment group, and the experimental group was a strain fermentation filtrate and lysate treatment group with different concentrations (including 1 / 100, 1 / 1000 and 1 / 10000 (V / V) concentrations), with three replicates in each group. Take pictures at 0 hours and 48 hours, and use Image J software to calculate the scratch area and cell repair rate. The calculation formula is as follows:

[0113]

[0114] M--Migration, expressed as a percentage (%), B 0h --0h scratch area, B48 h --48h scratch area.

[0115] The cell repair rates of each experimental group and control group were counted and a cell repair rate heat map was made. The results are as follows Figure 3 As shown; Figure 3 The results showed that the fermentation filtrate or lysate of each Akkermansia muciniphila bacterium showed obvious cell repair ability at appropriate concentrations, among which the cell repair ability of the strain YGMCC2602 of the present invention was significantly better than that of the control group and other strains, whether its fermentation filtrate (33.3%-35.4%) or lysate (25.7%-30.0%). Therefore, the strain YGMCC2602 has become an ideal candidate strain due to its excellent cell repair ability.

[0116] Example 4: Determination of the cell repair-promoting ability of the fermentation filtrate, lysate and exosomes of strain YGMCC02602

[0117] Effect on the proliferation of HSF cells

[0118] Select HSF cells that are growing well and in the logarithmic growth phase within 10 generations and culture them at a rate of 1×10 4Cells were inoculated into 96-well plates at an inoculation density of

[0119] cells / well. After incubation in an incubator (37 °C, 5% CO2) for 24 h, a zeroing group, a blank control group, a positive control group, and a sample group (including fermentation filtrate, lysate, and exosome treatment groups) were set up for treatment. In the sample group, the fermentation filtrate, lysate, and exosomes were serially diluted at a final concentration of 1 / 30000 - 1 / 300 (V / V). The treated cells were further cultured in the incubator for 24 h, then CCK-8 working solution was added, and the cells were incubated at 37 °C in the dark for 1 - 1.5 h. After incubation, the OD value was read at 450 nm, and the cell viability (%) was calculated according to the formula: cell viability (%) = (OD of sample group - OD of zeroing group) / (OD of blank control group - OD of zeroing group) * 100%, and the cytotoxicity was determined. Figure 4 The results are shown as Figure 4 follows; it was shown that the fermentation filtrate, lysate, and exosomes of different concentrations of YGMCC02602 had no obvious toxicity to HSF cells (the survival rate was higher than 80.5%). Notably, the fermentation filtrate (1 / 30000 and 1 / 10000), lysate (except 1 / 300), and exosomes (except 1 / 300) at specific concentrations also showed a persistent promoting effect on the growth of HSF cells, indicating the potential to repair cells.

[0120] Effect on the repair of HSF cells

[0121] HSF cells within 10 passages in good growth and in the logarithmic growth phase were selected. The HSF cells were inoculated into 6-well plates at an inoculation density of 2×10 5 cells / well. After incubation in an incubator (37 °C, 5% CO2) for 24 h, a scratch was made and photographed at 0 h, then after adding the test substances (fermentation filtrate, lysate, and exosomes of different concentrations of YGMCC02602) and culturing for 48 h, photographs were taken again, and the scratch area was calculated using Image J software.

[0122] After culturing with the test substances for 48 h, the cells were harvested, and TransZol (TransGen Biotech, R61225) was used to lyse the cells to extract RNA for the determination of the relative expression levels of mRNAs of cell repair-related factors.

[0123] The results of the fermentation filtrate of YGMCC02602 are shown as Figure 5 follows; Figure 5 in which, Figure A is a schematic diagram of HSF cell fusion, Figure B is a statistical chart of the HSF cell fusion rate, and Figure C is a statistical chart of the relative expression levels of mRNAs of HSF cell repair-related factors. Figure 5Figures A and B show that, compared with the control group, the fermentation filtrate of YGMCC02602 at a concentration of 1 / 10000 had the most significant promoting effect on cell migration (33.76% ± 4.67%, P < 0.01), and was superior to the 1 / 3000 concentration group (32.42% ± 4.53%, P < 0.05); Figure C indicates that the fermentation filtrates of YGMCC02602 at concentrations of 1 / 10000 and 1 / 3000 could enhance the mRNA expression of COL1A2 and HAS1 in HSF cells, among which the enhancing effect at a concentration of 1 / 10000 was the most obvious (P < 0.0001), while the 1 / 3000 concentration mainly enhanced the expression of COL1A2 (P < 0.05) and COL17A1 (P < 0.05).

[0124] The results of the YGMCC02602 lysate are as Figure 6 shown; Figure 6 In it, Figure A is a schematic diagram of HSF cell fusion, Figure B is a statistical chart of HSF cell fusion rate, and Figure C is a statistical chart of the relative mRNA expression levels of HSF cell repair-related factors. Figure 6 Figures A and B show that, compared with the control group, the YGMCC02602 lysate (at concentrations of 1 / 10000, 1 / 3000, and 1 / 1000) promoted the migration of HSF cells, among which the effect at a concentration of 1 / 3000 was the best, and there was a significant difference from the control group (36.11% ± 5.71%, P < 0.001); Figure C shows that the YGMCC02602 lysate at concentrations of 1 / 3000 and 1 / 1000 promoted the mRNA expression of collagen (COL1A1, COL17A1, P < 0.001; COL3A1, P < 0.05) and hyaluronic acid (HAS1, P < 0.0001) in HSF cells, and at the same time, had an inhibitory effect on MMP expression.

[0125] The results of the YGMCC02602 exosomes are as Figure 7 shown; Figure 7 In it, Figure A is a schematic diagram of HSF cell fusion, Figure B is a statistical chart of HSF cell fusion rate, and Figure C is a statistical chart of the relative mRNA expression levels of HSF cell repair-related factors. Figure 7 Figures A and B show that the YGMCC02602 exosomes (at concentrations of 1 / 10000, 1 / 3000, and 1 / 1000) all showed a significant promoting effect on migration (P < 0.01), among which the effect at a concentration of 1 / 3000 was the best (42.90% ± 4.29%); and Figure C shows that the YGMCC02602 exosomes at concentrations of 1 / 10000 and 1 / 3000 could significantly promote the mRNA expression of collagen (COL17A1 and HAS1, P < 0.0001) and hyaluronic acid in HSF cells, and at the same time, inhibit MMP.

[0126] Based on the above results, the fermentation filtrate, lysate, and exosomes of strain YGMCC02602 all significantly promoted the repair of HSF cells, with the most significant effect at a concentration of 1 / 3000; these findings provide a scientific basis for the potential application of strain YGMCC02602 in the field of skin regeneration and repair.

[0127] Example 5: Evaluation of the soothing effect of the lysate of strain YGMCC02602

[0128] 1. Toxic effect of YGMCC2602 lysate on RAW264.7 cells

[0129] 1.1 Preparation of LPS working solution

[0130] LPS stock solution: Dissolve LPS in sterile PBS at a concentration of 100 μg / ml, filter through a 0.22 μm filter membrane, aliquot, and store at -80 °C.

[0131] LPS working solution: Dilute the LPS stock solution to 1 μg / ml with complete medium for later use.

[0132] 1.2 Cell seeding

[0133] Resuspend RAW264.7 cells in the logarithmic growth phase and dilute to 1×10 5 cells / ml. Add 100 μl of the cell suspension to each well of a 96-well plate, and add 200 μl of PBS to each well around the perimeter as a humidifying well. Incubate in a 37 °C, 5% CO2 incubator for 24 h, then add the test substance (YGMCC02602 lysate at different concentrations), and culture in the incubator for another 24 h. Observe the morphology, density, and even distribution of RAW264.7 cells to determine whether they meet the requirements for subsequent experiments.

[0134] 1.3 Treatment of cells with YGMCC2602 lysate

[0135] After aspirating the culture medium from the cells cultured for 24 h, add 100 μl of the dilution of YGMCC2602 lysate at different concentrations to each well, set 6 replicates, and continue to culture in a 37 °C, 5% CO2 incubator for 24 h. This includes a Con group (complete medium group), an LPS group (LPS working solution), a 1 / 100 group (100-fold lysate dilution + LPS), a 1 / 300 group (300-fold lysate dilution + LPS), a 1 / 1000 group (1000-fold lysate dilution + LPS), and a 1 / 3000 group (3000-fold lysate dilution + LPS).

[0136] 1.4 Detection of cell toxicity by MTT method

[0137] Add 50 μl of MTT solution (1×MTT) to each well and incubate in a 37°C, 5% CO2 incubator for 4 h. The above operations are carried out under sterile conditions. After discarding the liquid in the culture plate, add 150 μl of DMSO to each well, mix well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Record the measurement results. Select the concentration that is non-toxic to cells for subsequent experiments.

[0138] 1.5 Data processing and analysis

[0139] The experimental results were analyzed using Graphpad prism.

[0140] The results are as Figure 8 shown; Figure 8 showed that the cell survival rate in the LPS group (70.1%) was significantly lower than that in the control group, while the cell survival rates in the 1 / 100 YGMCC2602 lysate group (52.9%) and the 1 / 300 YGMCC2602 lysate group (53.4%) were slightly lower than that in the LPS group. There was no significant difference between the 1 / 1000 YGMCC2602 lysate group and LPS, while the cell survival rate in the 1 / 3000 YGMCC2602 lysate group was as high as 84.3%, significantly higher than that in the LPS group. This indicates that: the YGMCC2602 lysate group can significantly increase the cell survival rate at appropriate concentrations, showing a cytoprotective effect. Based on this result, the subsequent study will select the two concentrations of 1 / 1000 and 1 / 3000 to explore the soothing effect of the YGMCC2602 lysate.

[0141] 2. Effect of YGMCC2602 lysate on the relaxation model of LPS-induced RAW264.7 cells

[0142] Skin irritation is mainly regulated by barrier, neurovascular, and immune-inflammatory processes. Soothing inflammation can improve irritation. When cells are stimulated, they will deform and release inflammatory factors such as TNF-α, IL-6, IL-1β, and COX-2, which trigger irritation. In this experiment, the RAW264.7 macrophage model was stimulated with LPS, and the soothing effect of the YGMCC2602 lysate was evaluated by detecting the effect of the YGMCC2602 lysate on the secretion of inflammatory factors. The specific procedure is as follows.

[0143] 2.1 Cell seeding

[0144] Take RAW264.7 cells in the logarithmic growth phase, resuspend them, and dilute them to 1×10 5 cells / ml. Add 2 ml of the cell suspension to each well of a 12-well plate and place it in a 37°C, 5% CO2 incubator for 24 h.

[0145] 2.2 Treatment of cells with the YGMCC2602 lysate

[0146] After discarding the culture medium of the cells cultured for 24 h, complete medium, lysate dilutions of different concentrations of YGMCC2602, and dexamethasone working solution (DEX, 100 μg / ml) were added, 2 ml per well, with 3 replicate wells set, and then placed in an incubator at 37 °C and 5% CO2 for continued culture for 24 h. This included the Con group, LPS group, DEX group (DEX + LPS), 1 / 1000 group, and 1 / 3000 group. After 24 h, the cell culture fermentation filtrate was collected, and the level of the cytokine TNF-α was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Enzyme-linked Biology, IC50325-1). The operation steps were carried out according to the product instructions provided by the kit company.

[0147] After collecting the cell fermentation filtrate, it was washed twice with PBS, 1 ml of PBS was added to each well, and it was observed under an inverted microscope. The PBS was discarded, and TransZol (TransGen Biotech, R61225) was used to lyse the cells to extract RNA for subsequent detection of the gene expression levels of the inflammatory factors TNF-α, IL-6, IL-1β, and COX-2 (calculated using the 2-△△Ct formula after obtaining the Ct value).

[0148] 2.3 Data processing and analysis

[0149] The results of the microscopic observation were as Figure 9 shown; Figure 9 shown that compared with the Con group, the cells in the LPS group were slightly enlarged, and some cells showed spindle-shaped deformation; while the cell size in the DEX group and the YGMCC2602 lysate group changed insignificantly, and the number and degree of cells with spindle-shaped deformation showed a decreasing trend compared with the LPS group, indicating that DEX and YGMCC2602 lysate had varying degrees of soothing effects on LPS-induced cell deformation.

[0150] The ELISA detection results of the inflammatory factor TNF-α were as Figure 10 shown; Figure 10 shown that for the inflammatory factor TNF-α, the TNF-α content in the LPS group was 15.1 times that of the control group; compared with the LPS group, the TNF-α secretion in the DEX group and the 1 / 1000 YGMCC2602 lysate group decreased, but there was no significant difference; while the TNF-α content in the 1 / 3000 YGMCC2602 lysate group decreased significantly, a 41.1% decrease compared with the LPS group. The above results indicate that YGMCC2602 lysate and DEX can inhibit the excessive production of TNF-α, especially the 1 / 3000 YGMCC2602 lysate treatment group can effectively and significantly reduce the production of TNF-α.

[0151] The detection results of the gene expression levels of TNF-α, IL-6, IL-1β, and COX-2 were as Figure 11 shown;Figure 11 The results showed that the gene expressions of TNF-α, IL-6, IL-1β and COX-2 in the LPS group were significantly higher than those in the control group, while the expression of these genes was reduced in the DEX and YGMCC2602 lysate treatment groups. This shows that YGMCC2602 lysate may have a significant soothing effect by inhibiting the gene expression of a series of inflammatory factors.

[0152] Example 6: Evaluation of the anti-photoaging efficacy of lysates and exosomes of strain YGMCC02602

[0153] (1) Establishment of UVB-irradiated HaCaT photoaging cell model

[0154] HaCaT cells were cultured at 5 × 10 4 Cells were inoculated at a density of 100 / well in a 96-well plate, and the 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours. When the cell proliferation covered 80% of the plate area, the culture medium was removed, washed twice with sterile PBS, 100 μL of sterile PBS was added to each well, and UVB irradiation was started. The normal control group was wrapped with tin foil to avoid UVB irradiation. The conditions for UVB irradiation were: using a four-use ultraviolet analyzer (Qilin Bell, ZF-2, UVB 312nm, 30W), and ultraviolet irradiation was given for 0s, 30s, 40s, 45s, 50s, 60s and 75s respectively. The CCK-8 kit (Biyuntian, C0038) was used to detect and calculate the cell survival rate.

[0155] The cell survival rate results of different UVB treatment groups are as follows Figure 12 As shown; Figure 12 The results showed that the survival rate of HaCaT cells decreased with the increase of UVB irradiation dose. When the UVB irradiation time was 40s, the survival rate of HaCaT cells reached 56.2%, and this dose was selected as the dose of UVB damage HaCaT cell model.

[0156] (2) YGMCC02602 exosomes alleviate UVB-induced photoaging of HaCaT cells

[0157] HaCaT cells were cultured to the logarithmic phase and the cells were collected. A normal control group (Con), a model group (UVB), and a treatment group (W-3000) were set up in a 12-well plate. The concentration of the cell suspension was adjusted to 3×10 5 Each well was inoculated with 1 mL of cell suspension and the cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 h.

[0158] After 24 h of culture, the cells grew to confluence. The culture medium was removed, and the cells were washed twice with sterile PBS. 1 mL of sterile PBS was added to each well. The control group was wrapped with tin foil to avoid UVB irradiation. The model and treatment groups were given 40 s of UVB irradiation. After irradiation, 1 mL of complete medium was added to the control and model groups, and 1 mL of complete medium containing YGMCC02602 exosomes diluted 3000-fold was added to the treatment group. After 24 h of culture, the morphological changes of the cells were observed under a microscope (20×). The cell supernatant was collected, and the protein levels of inflammatory factors IL-1β, IL-6, and IL-8 in the cell supernatant were measured using an MQ60 PLUS fully automatic chemiluminescence immunoassay system (Hotgen Biotech). TransZol (TransGen Biotech, R61225) was used to lyse the cells to extract RNA for subsequent RT-PCR, and the gene levels of inflammatory factors IL-1β, IL-6, and IL-8, inflammation pathway-related genes COX-2, skin barrier protein FLG, MMPs (TGF-β, MMP-9), and antimicrobial peptides (hBD-2, CAMP) in the cell supernatant were detected (after obtaining the Ct value, the 2 -△△Ct formula was used for calculation).

[0159] The results of observing the cell morphology under a microscope (20×) were as Figure 13 shown; Figure 13 It was shown that under a 20× microscope field of view, UVB caused large-scale cell detachment and poor confluence, and the treatment with YGMCC02602 exosomes restored the cell state, demonstrating the function of YGMCC02602 exosomes in repairing cell damage.

[0160] The results of measuring the protein levels of inflammatory factors IL-1β, IL-6, and IL-8 were respectively as Figure 14 shown in Figures A, B, and C of Figure 14 ; it could be seen from Figures A, B, and C of Figure 14 that after UVB irradiation, the contents of IL-1β, IL-6, and IL-8 factors increased significantly compared with the control group. However, the treatment with YGMCC02602 exosomes significantly reduced the contents of these factors, and the inhibition rates of IL-1β, IL-6, and IL-8 were 51.85%, 40.4%, and 34.18%, respectively. Further, through RT-PCR detection, we found that the changes in the gene expression levels of inflammatory factors (IL-1β, IL-6, and IL-8) (as

[0161] shown in D) were consistent with the changes in their protein levels, and the gene expression level of the inflammation pathway-related gene COX-2 also showed a similar change trend, which further confirmed the significant effect of YGMCC02602 exosomes on inhibiting the expression of IL-1β, IL-6, IL-8, and COX-2 induced by UVB.

[0161] In addition, as Figure 15As shown, after UVB irradiation, MMPs (TGF-β, MMP-9) and antimicrobial peptides (hBD-2, CAMP) were significantly increased at the gene level, and treatment with exosomes of YGMCC02602 significantly decreased their contents; moreover, treatment with exosomes of YGMCC02602 also significantly restored the physical barrier protein (FLG) of the skin epidermis damaged by UVB.

[0162] From the above results, exosomes of YGMCC2602 significantly inhibited the expression of IL-6, IL-8, and IL-1β, repaired the physical barrier protein (FLG) of the epidermis, and alleviated the abnormal increase of MMPs (TGF-β, MMP-9) and antimicrobial peptides (hBD-2, CAMP), thus playing a role in alleviating UVB-induced cellular photoaging.

[0163] (3) YGMCC02602 lysate alleviates UVB-induced photoaging of HaCaT cells

[0164] HaCaT cells were cultured to the logarithmic phase and the cells were collected. In a 12-well plate, there were a normal control group (Con), a model group (UVB), and a treatment group (R-600, R-1200). The concentration of the cell suspension was adjusted to 3×10 5 cells / ml, and 1 mL of the cell suspension was inoculated into each well. The cell culture plate was placed in a 5% CO2, 37 °C incubator and cultured for 24 h.

[0165] After culturing for 24 h, the cells grew to a monolayer. The culture medium was removed, and the cells were washed twice with sterile PBS. 1 mL of sterile PBS was added to each well. The control group was wrapped with tin foil to avoid UVB irradiation. The model and treatment groups were given 40 s of UVB irradiation. After irradiation, 1 mL of complete medium was added to the control group and the model group, and 1 mL of complete medium containing YGMCC02602 lysate diluted 600-fold or 1200-fold was added to the treatment group. After culturing for 24 h, the changes in cell morphology were observed under a microscope (20×). The cell supernatants were collected, and the protein levels of the inflammatory factors IL-1β, IL-6, and IL-8 in the cell supernatants were measured using an MQ60PLUS fully automatic chemiluminescence immunoassay system (Hotgen Biotech). TransZol (TransGen Biotech, R61225) was used to lyse the cells to extract RNA for subsequent RT-PCR, and the gene levels of the inflammatory factors IL-1β, IL-6, and IL-8 and the skin barrier proteins IVL, LOR, and FLG in the cell supernatants were detected (after obtaining the Ct values, the calculation was performed using the 2 -△△Ct formula).

[0166] The results of observing cell morphology under a microscope (20×) are as Figure 16 shown; Figure 16It was shown that under a 20× microscope field of view, UVB caused large-scale cell detachment and poor confluence, and the treatment with the lysate of YGMCC02602 restored the cell state to some extent, indicating that the lysate of YGMCC02602 has the function of repairing cell damage.

[0167] The measurement results of the protein levels of inflammatory factors IL-1β, IL-6 and IL-8 are shown in Figures A, B, and C of Figure 17 respectively; as can be seen from Figures A, B, and C of Figure 17 after UVB irradiation, the contents of IL-1β, IL-6 and IL-8 factors in the cell supernatant increased significantly compared with the control group. However, the treatment with the lysate of YGMCC02602 (1 / 600, 1 / 1200) significantly reduced the contents of these factors. Specifically, the inhibition rates of the lysate of YGMCC02602 (1 / 600) on IL-1β, IL-6 and IL-8 were 66.87%, 88.56% and 95.48% respectively, and the inhibition rates of the lysate of YGMCC02602 (1 / 1200) on IL-1β, IL-6 and IL-8 were 70.83%, 92.73% and 96.39% respectively.

[0168] Furthermore, through RT-PCR detection, we found that the changes in the gene expression levels of inflammatory factors (IL-1β, IL-6 and IL-8) (as shown in Figure 17 Figure D) were consistent with the changes in their protein levels, which further confirmed the significant effect of the lysate of YGMCC02602 on inhibiting the expression of IL-1β, IL-6 and IL-8 induced by UVB.

[0169] In addition, as shown in Figure 18 after UVB irradiation, the physical barrier proteins of the skin epidermis (IVL, LOR and FLG) were significantly reduced at the gene level, and the treatment with the lysate of YGMCC02602 significantly restored the physical barrier proteins of the skin epidermis (IVL, LOR and FLG) damaged by UVB. It shows that the lysate of YGMCC02602 can significantly restore the expression of the barrier proteins IVL, LOR and FLG damaged by UVB.

[0170] In summary, the lysate of YGMCC2602 can not only significantly inhibit the expression of IL-6, IL-8 and IL-1β, but also repair the physical barrier proteins of the epidermis (IVL, LOR and FLG), thus playing a role in alleviating the photoaging of cells induced by UVB.

[0171] Anti-photoaging is an important aspect of skin aging. It involves the damage of ultraviolet rays to skin cells, leading to problems such as skin pigmentation, weakened elasticity, and increased wrinkles. Ultraviolet rays can damage the natural barrier of the skin, trigger the generation of free radicals, and further accelerate the skin aging process. The YGMCC2602 extract has potential research and application value in anti-photoaging research. Its exosomes and lysates both possess anti-photoaging ability, and are more remarkable in improving cell morphology and fusion degree, anti-inflammation and repairing the epidermal physical barrier. Thus, the YGMCC2602 strain or its extract can be applied to develop natural anti-photoaging skin care products with economic benefits.

[0172] Example 7 Evaluation of the immunomodulatory effect of the YGMCC02602 lysate

[0173] (1) Effect of the YGMCC2602 lysate on the cell viability of RAW264.7 cells

[0174] After resuspending RAW264.7 cells in the logarithmic growth phase and diluting them to 1×10 5 cells / ml, add 100 μl of the cell suspension to each well of a 96-well plate, and add 200 μl of PBS to each well around as a moisturizing well. Place it in an incubator at 37 °C and 5% CO2 for 24 h. After aspirating the culture medium of the cells cultured for 24 h, add different concentrations of the YGMCC2602 lysate dilution, 100 μl per well, set 6 replicates, and place it in an incubator at 37 °C and 5% CO2 for continued culture for 24 h. It includes the Con group (complete medium group), LPS group (LPS working solution, 1 μg / ml), 1 / 100 (100-fold lysate dilution), 1 / 300 group (300-fold lysate dilution), 1 / 1000 group (1000-fold lysate dilution), 1 / 3000 group (3000-fold lysate dilution), 1 / 10000 (10000-fold lysate dilution), 1 / 30000 (30000-fold lysate dilution).

[0175] Add 50 μl of MTT solution (1×MTT) to each well, place it in an incubator at 37 °C and 5% CO2 for 4 h. The above operations are carried out under sterile conditions. After discarding the liquid in the culture plate, add 150 μl of DMSO to each well, mix well, and measure the absorbance at a wavelength of 570 nm with an enzyme-labeled instrument, and record the measurement results. Select the concentration that is non-toxic to cells for subsequent experiments.

[0176] The experimental results were analyzed using Graphpad prism.

[0177] The effect of the YGMCC2602 lysate on the cell viability of RAW264.7 cells is as Figure 19 shown; Figure 19The results showed that the cell survival rate of the LPS group (73.49%) was significantly lower than that of the control group. The survival rates of the groups treated with the lysates of YGMCC2602 were all higher than those of the LPS group. Among them, the 1 / 100 YGMCC2602 lysate group (75.79%) and the 1 / 1000 YGMCC2602 lysate group (83.47%) were also significantly lower than the control group. The 1 / 300 YGMCC2602 lysate group (91.94%), the 1 / 3000 YGMCC2602 lysate group (99.44%) and the 1 / 10000 YGMCC2602 lysate group (93.18%) had no significant effect on the cell survival rate. However, the cell survival rate of the 1 / 30000 YGMCC2602 lysate group was as high as 104.16%, which was higher than that of the control group. This indicated that the lysates of YGMCC2602 could promote cell proliferation and improve the cell survival rate at appropriate concentrations. According to this result, subsequent studies will explore the immunomodulatory effects of the lysates of YGMCC2602 at concentrations between 1 / 300 and 1 / 30000.

[0178] (2) Effects of the lysates of YGMCC02602 on cytokine secretion and gene expression

[0179] RAW264.7 cells in the logarithmic growth phase were resuspended and diluted to 1×10 5 cells / ml. 2 ml of the cell suspension was added to each well of a 12-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 h. After discarding the culture medium of the cells cultured for 24 h, complete medium and diluted lysates of YGMCC2602 at different concentrations (2 ml per well) were added. Three replicate wells were set up and cultured in an incubator at 37°C and 5% CO2 for another 24 h. These included the Con group, the LPS group, the 1 / 300 group, the 1 / 1000 group, the 1 / 3000 group, the 1 / 10000 group, and the 1 / 30000 group. After 24 h, the cell culture fermentation filtrate was collected, and the level of the cytokine TNF-α was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Enzyme-linked Biotechnology, IC50325-1). The operation steps were carried out according to the product instructions provided by the kit company.

[0180] After collecting the cell fermentation filtrate, the cells were washed twice with PBS and then the PBS was discarded. TransZol (TransGen Biotech, R61225) was used to lyse the cells to extract RNA for subsequent detection of the gene expression levels of the inflammatory factors TNF-α, IL-6, IL-1β and iNOS (the calculation was carried out using the 2 -△△Ct formula after obtaining the Ct value).

[0181] Macrophages are professional phagocytes that can highly specifically remove dying or dead cells and cell debris. In addition, macrophages are also the most important antigen-presenting cells and play a crucial role in initiating immune responses. After receiving antigen stimulation, macrophages will present the antigen to the corresponding Th cells, and antigen presentation leads to the production of antibodies, completing the immune regulation reaction.

[0182] The ELISA detection results of the inflammatory factor TNF-α are as Figure 20 shown in Figure A of Figure 20 Figure A shows that for the inflammatory factor TNF-α, the TNF-α content in the LPS group is 11.1 times that of the control group; compared with the control group, the TNF-α secretion in the 1 / 30000 YGMCC2602 lysate group decreased, and the TNF-α secretion in the 1 / 10000 YGMCC2602 lysate group increased, but there were no significant differences; while the TNF-α content in the 1 / 3000 YGMCC2602 lysate group, 1 / 1000 YGMCC2602 lysate group, and 1 / 300 YGMCC2602 lysate group increased significantly, and with the increase in the concentration of YGMCC2602 lysate, the increase in TNF-α content showed a dose-dependent manner. Since the TNF-α secretion in the 1 / 30000 YGMCC2602 lysate group decreased compared with the control group and there was no significant difference in the detection of the inflammatory factor TNF-α, this concentration will no longer be used in the determination of gene expression levels in subsequent experiments.

[0183] The detection results of the gene expression levels of TNF-α, IL-6, IL-1β, and iNOS are as Figure 20 shown in Figures B, C, D, and E of Figure 20 Figure B shows that the TNF-α gene expression in the 1 / 1000 YGMCC2602 lysate group and 1 / 300 YGMCC2602 lysate group was significantly higher than that in the control group, while the TNF-α gene expression in the 1 / 10000 YGMCC2602 lysate group, 1 / 3000 YGMCC2602 lysate group, and LPS group was slightly lower than that in the control group. This may be due to the differences in the time and intensity of cytokine protein secretion and gene expression, and the expression of cytokine mRNA precedes protein secretion. Figure 20 Figures C, D, and E show that the gene expressions of IL-6, IL-1β, and iNOS in the LPS group were much higher than those in the control group, indicating that the YGMCC2602 lysate group could stimulate cells to increase the expression of these factors and showed a dose-dependent manner. The above results indicate that the YGMCC2602 lysate can stimulate cells to produce an immune regulation reaction and enhance skin immune function.

[0184] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Akkermansia muciniphila ( Akkermansia muciniphila ) YG2602, characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit date being April 22, 2024 and the deposit number being CGMCC No.30411.

2. The Akkermansia muciniphila according to claim 1 ( Akkermansia muciniphila ) Culture, lysate, fermentation supernatant and / or exosomes extracted therefrom of YG2602.

3. The Akkermansia muciniphila according to claim 2 ( Akkermansia muciniphila ) A culture, a lysate, a fermentation supernatant and / or exosomes extracted therefrom of YG2602, characterized in that: The method for extracting the lysate comprises the following: The Akkermansia muciniphila ( Akkermansia muciniphila ) YG2602 was fermented and cultured, the fermentation broth was centrifuged, the cells were collected, the cells were resuspended in PBS and the cell walls were broken by ultrasound or enzymatic hydrolysis to obtain lysate; And / or, the method for extracting exosomes comprises the following: The Akkermansia muciniphila ( Akkermansia muciniphila ) YG2602 is fermented and cultured, the fermentation broth is centrifuged, and the fermentation supernatant is collected; the obtained fermentation supernatant is centrifuged, filtered, and concentrated to obtain exosomes.

4. The Akkermansia muciniphila according to claim 3 ( Akkermansia muciniphila ) A culture, a lysate, a fermentation supernatant and / or exosomes extracted therefrom of YG2602, characterized in that: The method for extracting the lysate further comprises: sterilizing the obtained lysate; In the method for extracting exosomes: The centrifugation procedure of the fermentation supernatant is: first centrifuge at 200-400 g for 8-15 min, then centrifuge at 1500-3000 g for 15-30 min, and finally centrifuge at 8000-12000 g for 20-40 min; And / or, the filtration of the fermentation supernatant is: filtering with a filter membrane having a pore size of 0.2-2.0 μm; And / or, the fermentation supernatant is concentrated by using a 400-600 kd hollow fiber to concentrate the fermentation supernatant.

5. The Akkermansia muciniphila according to claim 4 ( Akkermansia muciniphila ) A culture, a lysate, a fermentation supernatant and / or exosomes extracted therefrom of YG2602, characterized in that: In the method for extracting exosomes: The centrifugation procedure of the fermentation supernatant was as follows: first centrifugation at 300 g for 10 min, then at 2000 g for 20 min, and finally at 10000 g for 30 min; And / or, the filtration of the fermentation supernatant is: filtering using filter membranes with pore sizes of 0.8 μm and 0.45 μm respectively in sequence; And / or, the fermentation supernatant is concentrated by using a 500 kd hollow fiber to concentrate the fermentation supernatant.

6. A composition, characterized in that The composition comprises the Akkermansia muciniphila according to claim 1 ( Akkermansia muciniphila ) YG2602 and / or Akkermansia muciniphila as described in any one of claims 2 to 5 ( Akkermansia muciniphila ) The culture, lysate, fermentation supernatant and / or exosomes extracted therefrom of YG2602 are used as active ingredients.

7. The composition according to claim 6, characterized in that The composition is a pharmaceutical composition or a cosmetic composition, which further comprises a pharmaceutically or cosmetically acceptable carrier and / or excipient.

8. The composition according to claim 6, characterized in that The composition is in the form of an external dosage form.

9. The composition according to claim 8, characterized in that The external dosage form is selected from the following dosage forms: aqueous solution, emulsion, ointment, cream, gel, powder, oil.

10. The composition according to any one of claims 6 to 9, characterized in that The composition may also include other active agents; The other active agents include any one or more of antioxidants, cell active agents, moisturizing agents, anti-aging agents and anti-dermatitis agents.

11. The Akkermansia muciniphila according to claim 1 ( Akkermansia muciniphila ), or the muciniphilic Akkermansia according to any one of claims 2 to 5 ( Akkermansia muciniphila ) and / or exosomes extracted therefrom, or a composition comprising one or more of the foregoing substances in the preparation of a product for the following purposes: 1) Repair and / or soothe the skin; 2) Reduce, inhibit or eliminate skin inflammation; 3) Anti-skin photoaging or skin aging caused by photoaging.

12. The use according to claim 11, characterized in that: The repairing and soothing of the skin is to repair and soothe sensitive skin; And / or, the skin inflammation is sterile skin inflammation or inflammation caused by external stimulation.

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