Lactobacillus reuteri YG3708, exosome and extract of lactobacillus reuteri YG3708 and application of lactobacillus reuteri YG3708 in skin care

Abstract

The invention relates to the field of microorganisms, in particular to lactobacillus reuteri YG3708, inactivated thalli, an extract, a culture, a culture supernatant, a fermentation product and / or a fermentation supernatant of the lactobacillus reuteri YG3708 and application of the lactobacillus reuteri YG3708 to preparation of a composition for promoting skin repair, reducing skin inflammation or allergy, improving skin quality and improving skin quality. The invention also relates to application of the composition in products for resisting skin aging and enhancing skin immune function. The lactobacillus reuteri YG3708 and related products of the lactobacillus reuteri YG3708 disclosed by the invention have relatively good skin cell repairing capacity, skin inflammation relieving, inhibiting or eliminating capacity and remarkable skin aging resisting capacity, so that the lactobacillus reuteri YG3708 is expected to be used for skin health care such as skin repairing, inflammation relieving and light aging resisting; and the method has obvious application value and market potential.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus reuteri YG3708, its inactivated bacterial bodies, extracts, cultures, culture supernatants, fermented products and / or fermented supernatants, a composition comprising the aforementioned, and use of Lactobacillus reuteri YG3708 or its inactivated bacterial bodies, extracts, cultures and the composition comprising the aforementioned in preparing products for repairing skin, resisting dermatitis, resisting skin aging and enhancing skin immune function. Background Art

[0002] Traditional skin care products rely on chemical ingredients and may have potential risks. With the advancement of biotechnology, especially the in-depth study of microbiome, the important role of microorganisms and their metabolites in skin health has been revealed. Exosomes, as specific membrane vesicles for intercellular communication, are becoming a research hotspot in the field of skin care and repair due to their unique biological properties. However, the exosomes currently on the market are mainly derived from human stem cells, which has limitations in terms of ethics, acquisition costs and large-scale production.

[0003] Lactobacillus reuteri is a dominant lactic acid bacteria naturally present in the intestines of humans and animals. It has many excellent probiotic functions such as inhibiting pathogens, improving the intestinal environment, and regulating intestinal flora. It is an important microorganism widely used in food, medicine and feed additives. However, there is no report in the prior art on its use in skin care.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] Purpose of the Invention

[0006] The present invention aims to provide a strain of Lactobacillus reuteri YG3708 having the functions of repairing skin, resisting dermatitis, resisting skin aging and enhancing skin immunity, its inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant, a composition comprising the aforementioned, and use of Lactobacillus reuteri YG3708 or its inactivated bacteria, extract, culture and the composition comprising the aforementioned in the preparation of products for repairing skin, resisting dermatitis, resisting skin aging and enhancing skin immunity.

[0007] Solution

[0008] To achieve the purpose of the present invention, the present invention adopts the following technical scheme.

[0009] In the first aspect, the present invention provides a strain of Limosilactobacillus reuteri YG3708, which is classified and named as Limosilactobacillus reuteri and is deposited in the General Microbiology Center of China Culture Collection Administration. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The deposit date is July 9, 2024, and the deposit number is CGMCC No.31243.

[0010] The Lactobacillus reuteri YG3708 of the present invention is separated from a healthy breast milk sample and is identified as Lactobacillus reuteri through 16S rRNA sequencing and combined with its morphological characteristics.

[0011] The 16S rRNA sequence of Limosilactobacillus reuteri YG3708 of the present invention is as follows:

[0012]

[0013] The colony characteristics of this strain include: after culturing on MRS agar medium for 24 to 48 hours, the diameter is between 1 and 2 mm, the colony edges are neat, the surface is smooth, opaque, milky white, and the texture is moist.

[0014] In vitro experiments have confirmed that the Limosilactobacillus reuteri YG3708 of the present invention has good repair ability on human skin fibroblasts; specifically, the inventors found that the fermentation filtrate of the Limosilactobacillus reuteri YG3708 of the present invention is rich in reuterin, which is a key effector molecule that plays a role in cell repair.

[0015] In addition, the exosomes and lysates of Limosilactobacillus reuteri YG3708 of the present invention also have the ability to resist cell aging (especially UVB-induced photoaging); specifically, it may alleviate UVB-induced cell photoaging by inhibiting the abnormally high expression of inflammatory factors (IL-6, IL-1β, IL-8 and COX-2), MMPs (MMP-2, MMP-9), barrier proteins (FLG and IVL) and antimicrobial peptides (CAMP and hBD-2) caused by UVB damage, inhibiting the increase of ROS caused by UVB, etc.

[0016] The inventors also found that the exosomes, fermentation broth and lysate of Limosilactobacillus reuteri YG3708 of the present invention can promote the proliferation and survival of macrophages at appropriate concentrations, and can stimulate macrophages and enhance their ability to produce cytokines such as TNF-α, IL-6, IL-1β and iNOS, thereby promoting immune regulation and enhancing the skin immune barrier function.

[0017] In addition, the inventors have also confirmed that the fermentation filtrate and exosomes of Limosilactobacillus reuteri YG3708 of the present invention have the effect of soothing the skin. Specifically, it may achieve the effect of soothing the skin by regulating the abnormally high expression of cytokines such as TNF-α, IL-6, IL-1β and iNOS caused by skin irritation; and, it has been identified that reuterin in the fermentation broth of the strain may be the key active substance that exerts the skin soothing effect.

[0018] In a second aspect, the present invention provides the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 as described in the first aspect above.

[0019] Preferably, the extract of Limosilactobacillus reuteri YG3708 is a lysate and / or exosomes extracted therefrom.

[0020] In a feasible embodiment, the method for preparing the fermentation supernatant comprises the following:

[0021] The Lactobacillus reuteri YG3708 is fermented and cultured in the presence of glycerol, the fermentation liquid is centrifuged, and the supernatant is collected and filtered to obtain the fermentation supernatant.

[0022] Preferably, in the fermentation system, the final concentration of glycerol is 200-300, preferably 250 mmol / L.

[0023] Preferably, the centrifugation treatment refers to centrifugation at 8000-10000 r / min and 3-8°C for 8-15 min.

[0024] Preferably, the filtration is performed using a filter membrane with a pore size of 0.2-2.0 μm, preferably 0.22 μm.

[0025] In a feasible embodiment, the method for extracting the lysate comprises the following:

[0026] Limosilactobacillus reuteri YG3708 is fermented and cultured, the fermentation broth is centrifuged, the bacteria are collected, the bacteria are resuspended in PBS and the cell walls are broken by ultrasound or enzymatic hydrolysis to obtain a lysate; preferably, the obtained lysate is sterilized.

[0027] In a feasible embodiment, the method for extracting exosomes comprises the following:

[0028] Fermenting Lactobacillus reuteri YG3708, centrifuging the fermentation liquid, and collecting the fermentation supernatant; centrifuging, filtering, and concentrating the obtained fermentation supernatant to obtain exosomes;

[0029] Preferably, the centrifugation procedure of the fermentation supernatant is: first centrifuge at 200-400g, preferably 300g for 8-15min, preferably 10min, then centrifuge at 1500-3000g, preferably 2000g for 15-30min, preferably 20min, and finally centrifuge at 8000-12000g, preferably 10000g for 20-40min, preferably 30min;

[0030] Preferably, the filtration of the fermentation supernatant is: filtering with a filter membrane having a pore size of 0.2-2.0 μm, preferably, filtering with filter membranes having pore sizes of 0.8 μm and 0.45 μm respectively;

[0031] Preferably, the fermentation supernatant is concentrated by using a hollow fiber of 400-600 kd, preferably 500 kd, to concentrate the fermentation clarified liquid.

[0032] In a third aspect, the present invention provides a composition comprising Limosilactobacillus reuteri YG3708 as described in the first aspect above, or the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 as described in the second aspect above, as an active ingredient.

[0033] In some feasible embodiments, the composition is a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier and / or excipient.

[0034] In other possible embodiments, the composition is a cosmetic composition, which further comprises a cosmetically acceptable carrier and / or excipient.

[0035] Preferably, the composition is in the form of an external dosage form;

[0036] More preferably, the external dosage form is selected from the following dosage forms: aqueous solution, emulsion, ointment, cream, gel, powder, oil.

[0037] In a feasible embodiment, the composition further comprises other active agents;

[0038] Preferably, the other active agents include any one or more of antioxidants, cell active agents, moisturizing agents, anti-aging agents and anti-dermatitis agents.

[0039] In a fourth aspect, the present invention provides the use of the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 as described in the first aspect, or the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 as described in the second aspect, or a composition comprising one or more of the foregoing substances in the preparation of a product for the following purposes:

[0040] 1) Promote skin repair;

[0041] 2) Reduce, inhibit or eliminate skin inflammation or allergies;

[0042] 3) Anti-skin aging;

[0043] 4) Enhance skin immune function.

[0044] In a feasible embodiment, the promoting skin repair is promoting the repair of sensitive skin;

[0045] In a feasible embodiment, the skin inflammation is sterile skin inflammation or inflammation caused by external stimulation;

[0046] In a feasible embodiment, the skin allergy is a skin allergy symptom caused by stimulation from external factors;

[0047] In a feasible embodiment, the anti-skin aging is anti-skin photoaging.

[0048] In a fifth aspect, the present invention provides a method for repairing skin, reducing, inhibiting or eliminating skin inflammation or allergy, resisting skin aging, and / or enhancing skin immune function, comprising: administering to a subject in need an effective amount of Limosilactobacillus reuteri YG3708 as described in the first aspect above, or the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 as described in the second aspect above, or a composition comprising one or more of the foregoing substances.

[0049] In a feasible embodiment, the promoting skin repair is promoting the repair of sensitive skin;

[0050] In a feasible embodiment, the skin inflammation is sterile skin inflammation or inflammation caused by external stimulation;

[0051] In a feasible embodiment, the skin allergy is a skin allergy symptom caused by stimulation from external factors;

[0052] In a feasible embodiment, the anti-skin aging is anti-skin photoaging.

[0053] Beneficial Effects

[0054] Compared with the prior art, the Limosilactobacillus reuteri YG3708 of the present invention has the following obvious advantages:

[0055] (1) It has good repair ability on human skin fibroblasts;

[0056] (2) Its exosomes and lysates have the ability to resist cell aging (especially UVB-induced photoaging); its possible mechanism is that it achieves the anti-photoaging effect by inhibiting the abnormally high expression of UVB-induced inflammatory factors (IL-6, IL-1β, IL-8 and COX-2), MMPs (MMP-2, MMP-9), barrier proteins (FLG and IVL) and antimicrobial peptides (CAMP and hBD-2) and increased ROS production;

[0057] (3) Its exosomes, fermentation broth and lysate can promote the proliferation and survival of macrophages at appropriate concentrations, and can stimulate macrophages and enhance their ability to produce cytokines such as TNF-α, IL-6, IL-1β and iNOS, thereby promoting immune regulation and enhancing the skin immune barrier function;

[0058] (4) It has a good effect of soothing the skin.

[0059] In summary, the Limosilactobacillus reuteri YG3708 and related products of the present invention have good ability to repair skin cells, resist skin aging, reduce, inhibit or eliminate skin inflammation, enhance skin immune barrier function and soothe the skin. Therefore, they are expected to be used for skin health care such as skin repair, inflammation relief and anti-photoaging, and have significant application value and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0061] Figure 1It is the growth curve of Limosilactobacillus reuteri YG3708 of the present invention.

[0062] Figure 2 It shows the particle concentration and particle size distribution of exosomes extracted from the fermentation broth of strain YG3708 of the present invention measured by NTA technology.

[0063] Figure 3 It shows a heatmap of the HSF cell repair rate of the fermentation filtrates and lysates of different Limosilactobacillus reuteri strains.

[0064] Figure 4 It shows the determination results of the content of reuterin in the fermentation filtrate and lysate of Limosilactobacillus reuteri YG3708 of the present invention; among them, Figure A shows the standard curve of tryptophan-acrolein, and Figure B shows the determination results of the reuterin concentration in the fermentation filtrate and lysate of the strain.

[0065] Figure 5 It shows the effect of the fermentation product of strain YG3708 rich in reuterin on the repair of HSF cells; among them, the abscissa is the type of fermentation product, and the ordinate is the cell fusion rate (%); * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.

[0066] Figure 6 It shows the effect of 3-HPA and the fermentation broth of strain YG3708 on the repair of HSF cells; among them, the abscissa is the experimental group, and the ordinate is the cell fusion rate (%); ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001.

[0067] Figure 7 It shows the effect of the lysate (A), fermentation filtrate (B), and exosomes (C) of Limosilactobacillus reuteri YG3708 of the present invention on the survival rate of HaCaT cells after UVB irradiation; among them, * indicates: compared with the model group (i.e., UVB), 0.01<P<0.05; ** indicates: compared with the model group, 0.001<P<0.01; *** indicates: compared with the model group, P<0.001; indicates: compared with the control group (i.e., Con), P<0.001.

[0068] Figure 8Shows the effects of the lysate, fermentation filtrate, and exosomes of Limosilactobacillus reuteri YG3708 of the present invention on the morphology and fusion degree of HaCaT cells after UVB irradiation.

[0069] Fig. 9 Shows the effects of the lysate, fermentation filtrate, and exosomes of Limosilactobacillus reuteri YG3708 of the present invention on the secretion of inflammatory factors IL-6, IL-1β, IL-8, and COX-2 by HaCaT cells after UVB irradiation; among them, Figure A shows the protein content of IL-6 in the cell culture supernatant of each treatment group, Figure B shows the protein content of IL-1β in the cell culture supernatant of each treatment group, Figure C shows the protein content of IL-8 in the cell culture supernatant of each treatment group, and Figure D shows the relative mRNA levels of IL-6, IL-1β, IL-8, and COX-2 in the cells of each treatment group; and, among them, * indicates: compared with the model group (i.e., UVB), 0.01 < P < 0.05; ** indicates: compared with the model group, 0.001 < P < 0.01; *** indicates: compared with the model group, P < 0.001; indicates: compared with the control group (i.e., Con), P < 0.001.

[0070] Fig.10 Shows the effects of the lysate, fermentation filtrate, and exosomes of Limosilactobacillus reuteri YG3708 of the present invention on the secretion of MMPs (MMP-2, MMP-3, MMP-9), TGF-β, barrier proteins (FLG and IVL), and antimicrobial peptides (CAMP and hBD-2) by HaCaT cells after UVB irradiation; among them, Figure A shows the relative mRNA levels of MMP-2, MMP-3, MMP-9, and TGF-β in the cells of each treatment group, Figure B shows the relative mRNA levels of the barrier proteins FLG and IVL in the cells of each treatment group, and Figure C shows the relative mRNA levels of the antimicrobial peptides CAMP and hBD-2 in the cells of each treatment group; and, among them, * indicates: compared with the model group (i.e., UVB), 0.01 < P < 0.05; ** indicates: compared with the model group, 0.001 < P < 0.01; *** indicates: compared with the model group, P < 0.001; # indicates: compared with the control group (i.e., Con), 0.01 < P < 0.05; ## indicates: compared with the control group, 0.001 < P < 0.01; indicates: compared with the control group, P < 0.001.

[0071] Fig.11Shows the effects of the lysate, fermentation filtrate, and exosomes of Limosilactobacillus reuteri YG3708 of the present invention on the production of ROS in HaCaT cells after UVB irradiation; wherein, Figure A shows the cell morphology and confluence of each treatment group (upper figure) and the fluorescence staining pictures of ROS (lower figure), and Figure B shows the relative fluorescence intensity (%) of ROS in each treatment group; and, wherein, * indicates: compared with the model group (i.e., UVB), 0.01 < P < 0.05, ** indicates: compared with the model group, 0.001 < P < 0.01, *** indicates: compared with the model group, P < 0.001; ### Indicates: compared with the blank control group (i.e., Con group), P < 0.001.

[0072] Fig.12 Shows the effects of the fermentation filtrate and lysate of Limosilactobacillus reuteri YG3708 of the present invention on the viability of RAW264.7 cells; wherein, * indicates: compared with the blank control group (i.e., Con group), P < 0.05; **** indicates: compared with the blank control group, P < 0.0001.

[0073] Fig.13 Shows the effects of the fermentation filtrate (A) and lysate (B) of Limosilactobacillus reuteri YG3708 of the present invention on the secretion of TNF-α by RAW264.7 cells; wherein, * indicates: compared with the blank control group (i.e., Con group), P < 0.05; ** indicates: compared with the blank control group, P < 0.01.

[0074] Fig.14 Shows the effects of the fermentation filtrate (Figures A - D) and lysate (Figures E - H) of Limosilactobacillus reuteri YG3708 of the present invention on the secretion of inflammatory factors TNF-α, IL-6, IL-1β, and iNOS by RAW264.7 cells; wherein, * indicates: compared with the blank control group (i.e., Con group), P < 0.05; ** indicates: compared with the blank control group, P < 0.01; *** indicates: compared with the blank control group, P < 0.001; **** indicates: compared with the blank control group, P < 0.0001.

[0075] Fig.15 Shows the effects of the fermentation filtrate (A) and exosomes (B) of Limosilactobacillus reuteri YG3708 of the present invention on the viability of RAW264.7 cells treated with LPS.

[0076] Fig.16 The effects of the fermentation filtrate (A) and exosomes (B) of Limosilactobacillus reuteri YG3708 of the present invention on the secretion of TNF-α in the LPS-induced RAW264.7 cell relief model are shown; wherein, * 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; #### Means: Compared with the blank control group (ie, Con group), P < 0.0001.

[0077] Fig.17 The results show the effects of the fermentation filtrate (AC) and exosomes (DF) of Limosilactobacillus reuteri YG3708 of the present invention on the gene expression levels of inflammatory factors IL-6, IL-1β and iNOS in the LPS-induced RAW264.7 cell relief model; where * 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<0.0001; #### Means: Compared with the blank control group (ie, Con group), P < 0.0001.

[0078] Fig.18 It shows that the key active substance with soothing effect in the fermentation filtrate of Limosilactobacillus reuteri YG3708 of the present invention is 3-HPA; wherein, Figure A shows the effect of different fermentation filtrate groups on TNF-α secretion in the LPS-induced RAW264.7 cell soothing model, and Figure B shows the determination of reuterin concentration in the fermentation filtrate with soothing effect; Figure C shows the effect of 3-HPA on TNF-α secretion in the LPS-induced RAW264.7 cell soothing model.

[0079] The Limosilactobacillus reuteri YG3708 of the present invention is classified and named as Limosilactobacillus reuteri, with a preservation date of July 9, 2024; the preservation unit is the General Microbiology Center of the China Culture Collection Administration; the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the preservation number is CGMCC NO.31243. DETAILED DESCRIPTION

[0080] In order to make the purpose, 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0081] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0082] In the following examples, unless otherwise specified, other reagents or raw materials used are commercially available.

[0083] In addition, in the following examples, the "concentration" of the fermentation filtrate, lysate, and exosomes of the strain refers to the volume dilution multiple of the original solution of the corresponding substance diluted with a complete medium (i.e., DMEM medium containing serum); for example, the fermentation filtrate or lysate of the strain at a concentration of 1 / 1000, 1 / 3000, 1 / 10000, and 1 / 30000 (V / V) refers to the original solution of the fermentation filtrate or lysate of the strain diluted 1000 times, 3000 times, 10000 times, and 30000 times by volume, respectively. Further, in the result figures of the following examples, the fermentation filtrate, lysate, and exosome treatment groups of the strain can be abbreviated as S+dilution multiple, R+dilution multiple, and W+dilution multiple, respectively; for example, R-90W represents a strain lysate diluted 90W times, S-60W represents a strain fermentation filtrate diluted 60W times, and W-120W represents a strain exosome diluted 120W times.

[0084] Example 1: Isolation and identification of Limosilactobacillus reuteri YG3708

[0085] (1) Strain isolation

[0086] Take 1 mL of healthy breast milk sample and put it into 9 mL of PBS buffer. Vortex and mix well. Then dilute it tenfold with sterile saline. -2 , 10 -3 , 10 -4Three dilution gradients were spread on MRS agar medium respectively, and the solid plates were placed in an anaerobic constant temperature incubator at 37°C for 24 to 48 hours. Colonies were picked and purified on MRS agar medium for more than 3 times until the colony morphology on the plate was consistent.

[0087] A plurality of clones were picked and named YGMCC03708, YGMCC03709, YGMCC03920, YGMCC03921 and YGMCC04335, respectively. The strains were identified by strain morphology and 16S rRNA sequencing results, and they were all identified as Limosilactobacillus reuteri. Moreover, based on the cell repair ability of human skin fibroblasts (HSF), the Limosilactobacillus reuteri of the present invention, i.e. strain YGMCC03708 (also known as YG3708), was screened and obtained.

[0088] (2) Identification of the strains of the present invention

[0089] A. Colony morphology

[0090] After the strain clone YG3708 of the present invention is cultured in MRS agar medium for 24 to 48 hours, the diameter is between 1 and 2 mm, the colony edge is neat, the surface is smooth, opaque and milky white, and the texture is moist.

[0091] B. 16S rRNA sequencing

[0092] The strain YG3708 was sent to Shanghai Bioengineering Co., Ltd. for 16S rRNA gene sequencing, and its 16S rRNA sequence is shown in SEQ ID NO: 1. The 16S rRNA sequencing results were compared with the NCBI database by BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and combined with its colony morphology, the isolated strain YG3708 was determined to be Limosilactobacillus reuteri.

[0093] Example 2: Preparation of fermentation filtrate and lysate of strain YG3708, and extraction and characterization of exosomes

[0094] The culture medium involved in this embodiment is as follows:

[0095] MRS medium (g / L): peptone 10.0g / L, beef powder 5.0g / L, Tween-80 1mL / L, dipotassium hydrogen phosphate 2.0g / L, yeast powder 4.0g / L, magnesium sulfate 0.2g / L, triammonium citrate 2.0g / L, glucose 20.0g / L, manganese sulfate 0.05g / L, sodium acetate 5.0g / L, L-cysteine ​​hydrochloride 0.5g / L, solvent is water; the preparation method is: mix all the ingredients evenly and sterilize them under high pressure at 121℃ for 15min.

[0096] 2.1 Cultivation of strain YG3708 and preparation of fermentation broth in the presence of glycerol

[0097] The seed solution of strain YG3708 was transferred to MRS liquid medium at a 1% inoculation rate and cultured anaerobically at 37°C for 24 h. During the culture process, the OD600 value of the culture solution was measured by an ELISA instrument at regular intervals to obtain the growth curve of strain YG3708. Figure 1 shown. Figure 1 The results showed that strain YG3708 reached a stable growth phase (OD600=1.30) after 10 hours of cultivation.

[0098] The seed liquid of strain YG3708 was transferred to MRS liquid culture medium at a 1% inoculation rate, and cultured anaerobically at 37°C for 24 hours. Sterile glycerol was then added to make the final concentration of glycerol 250 mmol / L. After anaerobic incubation at 37°C for 2 hours, the fermentation was completed to obtain the reuteri-enriched fermentation liquid for later use.

[0099] 2.2 Preparation of fermentation filtrate and lysate of strain YG3708 and extraction and characterization of exosomes

[0100] (1) Preparation of fermentation filtrate:

[0101] The fermentation broth of strain YG3708 prepared above was centrifuged at 9000 r / min×4°C for 10 min to obtain the fermentation supernatant, which was filtered using a 0.22 μm polyethersulfone filter membrane to obtain the fermentation filtrate of strain YG3708, which was frozen at -80°C for later use.

[0102] (2) Lysate preparation:

[0103] The cells after centrifugation in (1) were harvested, and pre-cooled sterile PBS was added to resuspend the cells and supplement to 25% of the original fermentation broth mass. The cells were enzymatically hydrolyzed in a 37°C constant temperature water bath for 24 h using a composite enzyme (the composite enzyme consisted of lysozyme (4000 U / mg), neutral protease (50 U / mg) and α-chymotrypsin (800 U / mg), and the mass ratio of the three enzymes was 1:1:1, and the amount of the composite enzyme added was 0.6 wt %). Ultrasound was used to break the cell wall. The specific ultrasonic procedure was as follows: ultrasonic for 2 seconds, stop for 2 seconds, and the total ultrasonic time was 60 minutes. The entire ultrasonic procedure was carried out under ice bath conditions at -4 to 0°C. After the ultrasonic was completed, pre-cooled sterile water was added to 50% of the original fermentation broth mass and mixed. Then, the fermentation broth was heat treated at 80°C for 30 minutes using an autoclave to obtain a sterilized lysate of the fermentation broth of strain YG3708, which was frozen at -80°C for later use.

[0104] (3) Exosome extraction and characterization:

[0105] The fermentation broth of strain YG3708 was centrifuged at 9000r / min×4℃ for 10min to obtain the fermentation supernatant. The fermentation supernatant was first centrifuged at 300g×4℃ for 10min, then at 2000g×4℃ for 20min, and finally at 10000g×4℃ for 30min, and then filtered with 0.8μm and 0.45μm polyethersulfone filter membranes in sequence to obtain the fermentation clarification liquid. The obtained fermentation clarification liquid was concentrated to 50mL using a 500kd hollow fiber, and 200mL of PBS was used to replace the liquid. The concentrate was purified using a protein purifier to obtain the exosome solution of the fermentation broth of strain YG3708. The particle number and diameter distribution of exosomes in the obtained exosome solution were measured using NTA technology.

[0106] Through the above procedures, we obtained the fermentation filtrate, lysate and exosome solution of the stable and high-yield strain YG3708. The number and diameter range of exosome particles in the YG3708 exosome solution were measured based on NTA technology. The results are shown in Figure 2 ; Figure 2 The results showed that the diameter of exosomes in the YG3708 exosome solution ranged from 10 to 1000 nm, with an average of 195.5 nm (median diameter: 169.0 nm), which means that although the size of the main population was about 200 nm, it had a wide size distribution; and the number of particles per milliliter of exosome solution was 8.4*10 10 indivual.

[0107] Using the above procedure, lysates of strains YGMCC03709, YGMCC03920, YGMCC03921, YGMCC04129, and YGMCC04335 were also prepared.

[0108] Example 3: Detection of the ability of strain YG3708 to promote skin cell repair

[0109] Human skin fibroblasts are key cells in the dermis, and they play an indispensable role in maintaining skin elasticity and tension. These cells can efficiently synthesize matrices such as elastin and collagen, generate elastic fibers and collagen fibers, and secrete repair factors, giving the skin the ability to repair and renew itself.

[0110] In this example, human fibroblasts (hereinafter referred to as HSF) were used as the research object, and the repair abilities of lysates and / or fermentation filtrates of various strains were tested.

[0111] The culture method of HSF cells is as follows:

[0112] 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).

[0113] The detection procedure of cell repair ability (cell repair rate) is as follows:

[0114] Select HSF cells that are growing well and in the logarithmic growth phase within 10 generations and culture them at a rate of 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. After taking the photo, aspirate the PBS and add 2mL of serum-free DMEM culture medium containing the sample. Specifically, the control group was the DMEM treatment group, and the experimental group was the fermentation filtrate and lysate treatment group of different concentrations (including 1 / 1000, 1 / 3000 and 1 / 10000 (V / V) concentrations) and different strains, 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 the cell repair rate. The calculation formula for the cell repair rate is as follows:

[0115]

[0116] M--Migration, expressed as a percentage (%), B 0h --0h scratch area, B48 h --48h scratch area.

[0117] 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 3It was shown that the repair ability of Lactobacillus reuteri showed strain selectivity; the lysate of the strain YG3708 of the present invention showed a strong cell repair ability (31.2%) at a concentration of 1 / 10000 (m / V), and at this concentration, its repair ability was significantly better than that of the control group and other strains; more importantly, the fermentation filtrate of the strain YG3708 of the present invention showed good cell repair ability (30.5% to 31.6%) in the concentration range of 1 / 10000 to 1 / 1000 (m / V). Therefore, strain YG3708 has become an ideal candidate strain due to its excellent cell repair ability.

[0118] Example 4: Determination of the key active substances in the skin cell repair-promoting ability of strain YG3708

[0119] Lactobacillus reuteri has the ability to synthesize reuterin, a process catalyzed by coenzyme B12-dependent glycerol dehydratase. Reuterin is produced by metabolizing glycerol, and reuterin can be stably accumulated in cells.

[0120] In order to analyze the effect of glycerol on the content of reuterin in the fermentation product of YG3708, two experimental groups were set up: Experimental Group 1 (MRS medium, ordinary fermentation) and Experimental Group 2 (MRS medium, glycerol fermentation). The specific preparation method of the fermentation broth of Experimental Group 1 is as follows: the seed liquid of strain YG3708 is transferred to MRS liquid medium at a 1% inoculation rate, and the fermentation is completed after anaerobic static culture at 37°C for 48 hours; the preparation method of the fermentation broth of Experimental Group 2 is the same as that described in Example 2; the above two fermentation broths are subjected to the fermentation filtrate preparation method described in Section 2.2 (1) of Example 2 to obtain two fermentation filtrates.

[0121] Acrolein was used as the standard substance to establish a standard curve, and the standard curve equation was: y = 0.0958x + 0.0445 (e.g. Figure 4 The results of determination of reuterin concentration in the fermentation filtrate and lysate of the two experimental groups are shown in Figure A. Figure 4 As shown in Figure B, it shows that the content of reuterin in the fermentation filtrate of experimental group 2 is significantly higher than that in experimental group 1, while there is no significant difference in the content of reuterin in the lysates of experimental group 1 and experimental group 2; in addition, no reuterin was detected in the exosomes of experimental group 1 and experimental group 2. These results indicate that the YG3708 fermentation filtrate obtained by glycerol fermentation is rich in reuterin, and therefore, this YG3708 fermentation filtrate is also referred to as "fermentation filtrate rich in reuterin" or "fermentation filtrate enriched in reuterin" hereinafter.

[0122] The repair ability of YG3708 fermentation filtrate rich in reuteri and other fermentation products of YG3708 on HSF cells was further studied. The experiment was divided into a control group (DMEM) and an experimental group (samples of different concentrations), with three replicates in each group. Pictures were taken at 0 hours and 48 hours, and the scratch area was calculated using Image J software. The results are shown in Figure 2. Figure 5 shown. Figure 5 The results showed that the repair capacity of the fermentation filtrate and lysate of YG3708 rich in reuteri was significantly better than that of the control group at the concentration of 1 / 100000-1 / 10000 (m / V) (fermentation filtrate: 32.31%-33.18%, lysate: 31.24%-32.63%, P<0.01), and the repair capacity of exosomes was significantly better than that of the control group at the concentration of 1 / 30000 (30.12%, P<0.05).

[0123] In order to explore whether reuterin is the key effector molecule for YG3708 fermentation filtrate to play a role in cell repair, we conducted the following experiment. Since 3-HPA is the main representative substance of reuterin, we set up multiple experimental groups: control group (DMEM), experimental group 1 (ordinary YG3708 fermentation filtrate), experimental group 2 (YG3708 fermentation filtrate rich in reuterin), experimental group 3 (ordinary YG3708 fermentation filtrate + 3-HPA), and 3-HPA alone treatment group. The concentrations of YG3708 and YG3708 fermentation filtrate rich in reuterin are both 3 ten thousandths by mass (1 / 30000, m / V). The experimental results are as follows. Figure 6 shown. Figure 6 The results showed that compared with the control group, there was no significant difference in the cell repair ability of experimental group 1, while the cell repair ability of experimental group 2 was significantly improved, which was significantly different from the control group and experimental group 1 (P<0.01), indicating that the YG3708 fermentation filtrate rich in reuterin has better cell repair ability; after adding a certain amount of 3-HPA to the YG3708 ordinary fermentation filtrate, the cell repair ability of experimental group 3 was also significantly improved, which was significantly different from the control group and experimental group 1 (P<0.001), and the difference with experimental group 2 was not significant, which showed that the addition of 3-HPA can enhance the cell repair ability of YG3708 ordinary fermentation filtrate; and the 3-HPA alone treatment group also showed significant cell repair ability (P<0.001), and there was no significant difference with experimental groups 2 and 3. In summary, we speculate that reuterin may be the main active ingredient of YG3708 fermentation filtrate to exert HSF cell repair effect.

[0124] Example 5: Evaluation of the anti-photoaging efficacy of the fermentation product of strain YG3708

[0125] (1) Establishment of UVB-irradiated HaCaT photoaging cell model

[0126] For the construction method of the cell photoaging model, please refer to the article Protective Effect of Bifidobacteriumanimalis subs.lactis MG741 as Probiotics against UVB-Exposed Fibroblasts and Hairless Mice (DOI:10.3390 / microorganisms10122343).

[0127] Specifically, HaCaT cells were cultured in a high glucose medium (DMEM) containing 10% special grade fetal bovine serum (FBS) and 1% 100× penicillin-streptomycin mixture (S / P) in a cell culture incubator at 37°C and 5% CO2; when the cell confluence reached about 80%, the culture medium was removed, the cells were washed with sterile PBS, sterile PBS was added to the cells, and UVB irradiation was started; the UVB irradiation conditions were: using a four-purpose ultraviolet analyzer (Qilin Bell, ZF-2, UVB 312nm, 30W) for 40s of irradiation to construct a cell photoaging model.

[0128] (2) Effects of YGMCC3912 fermentation filtrate, lysate, and exosomes on the survival rate of UVB-irradiated HaCaT cells

[0129] 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 were set up in a 96-well plate. The concentration of the cell suspension was adjusted to 1×10 4 Each well was inoculated with 0.1 mL of cell suspension and the cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 h.

[0130] After 24 hours of culture, the cells grew to a monolayer, the culture medium was removed, and the cells were washed twice with sterile PBS, and 1 mL of sterile PBS was added to each well. The control group was wrapped in tin foil to avoid UVB irradiation. The model group and the treatment group were irradiated with UVB for 40 seconds. After irradiation, 1 mL of complete culture medium was added to the control group and the model group, and 1 mL of complete culture medium containing lysate, fermentation filtrate and exosomes of strain YG3708 at different dilutions was added to the treatment group. After 24 hours of culture, the cell survival rate was detected and calculated using the CCK-8 kit (Biyuntian, C0037) and according to the instructions of the kit.

[0131] The cell survival rates of the lysate, fermentation filtrate and exosome treatment groups of strain YG3708 were shown in Figure 2. Figure 7 As shown in Figures A, B, and C; Figure 7The results showed that the YG3708 lysate diluted 90w times, the fermentation filtrate diluted 60w times, and the exosomes diluted 120w times significantly increased the survival rate of UVB-induced light-damaged cells; specifically, compared with the UVB group, the cell survival rate of the YG3708 lysate diluted 90w times group increased by 27%, the cell survival rate of the fermentation filtrate diluted 60w times group increased by 51.9%, and the cell survival rate of the exosomes diluted 120w times group increased by 66.8%. Therefore, these three dilution schemes were selected for subsequent efficacy experiments.

[0132] (3) Fermentation filtrate, lysate and exosomes of YG3708 alleviate UVB-induced photoaging of HaCaT cells

[0133] HaCaT cells were cultured to the logarithmic phase and the cells were collected. A normal control group (Con), a model group (UVB), and treatment groups (S-60W, R-90W, and W-120W) 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.

[0134] After 24 hours of culture, the cells grew to a monolayer, the culture medium was removed, and the cells were washed twice with sterile PBS, and 1 mL of sterile PBS was added to each well. The control group was wrapped with tin foil to avoid UVB irradiation. The model group and the treatment group were irradiated with UVB for 40 seconds. After irradiation, 1 mL of complete culture medium was added to the control group and the model group, and 1 mL of complete culture medium containing 90w-fold diluted YG3708 lysate (i.e., R-90W group), 60w-fold diluted fermentation filtrate (i.e., S-60W group), and 120w-fold diluted exosomes (i.e., W-120W group) was added to the treatment group. After 24 hours of culture, the changes in cell morphology were observed under a microscope (20×), and the cell supernatant was collected. The protein levels of inflammatory factors IL-1β, IL-6, and IL-8 in the cell supernatant were determined using the MQ60 PLUS fully automatic chemiluminescence immunoassay system (Thermogene Bio). TransZol (Full Gold, R61225) was used to lyse cells and extract RNA for subsequent RT-PCR detection of gene expression levels of inflammatory factors (IL-6, IL-1β, IL-8, and COX-2), matrix metalloproteinases (MMP-2, MMP-3, and MMP-9), TGF-β, barrier proteins (FLG and IVL), and antimicrobial peptides (CAMP and hBD-2) (after obtaining Ct values, 2 -△△Ct formula for calculation).

[0135] The results of observing cell morphology under a microscope (20×) are as follows Figure 8 As shown; Figure 8The results showed that under a 20× microscope field of view, UVB caused large-area cell detachment and poor fusion. Treatment with the fermentation filtrate, lysate and exosomes of strain YG3708 restored the cell state, proving that the fermentation filtrate, lysate and exosomes of strain YG3708 have the function of repairing cell damage.

[0136] The protein levels of inflammatory factors IL-6, IL-1β and IL-8 were measured as follows: Fig. 9 As shown in Figures A, B, and C; Fig. 9 As can be seen from Figures A, B, and C, after UVB irradiation, the protein content of inflammatory factors IL-6, IL-1β, and IL-8 increased significantly compared with the control group; however, the fermentation filtrate, lysate, and exosome treatment of YG3708 significantly reduced the content of these factors, with the inhibition rates of IL-6 being 48.22%, 32.56%, and 57.91%, respectively, for IL-8 being 57.26%, 27.77%, and 49.08%, respectively, and for IL-1β being 61.08%, 34.96%, and 44.03%, respectively.

[0137] The results of gene level determination of inflammatory factors IL-6, IL-8, IL-1β and COX-2 are as follows Fig. 9 As shown in Figure D. Fig. 9 As can be seen from Figure D, the trend of changes in the gene level of inflammatory factors is consistent with their protein level. This further confirms that the fermentation filtrate, lysate and exosomes of YG3708 can inhibit the increased expression of inflammatory factors induced by UVB.

[0138] The results of gene level determination of matrix metalloproteinases (MMP-2, MMP-3 and MMP-9) and TGF-β, barrier proteins (FLG and IVL) and antimicrobial peptides (CAMP and hBD-2) were shown as follows: Fig.10 As shown in Figures A, B, and C; Fig.10 As can be seen from Figures A, B, and C, at appropriate concentrations, fermentation filtrate, lysate, and exosome treatment of YG3708 can significantly inhibit the increase in gene expression of these factors induced by UVB.

[0139] Judging from the above results, the fermentation filtrate, lysate and exosomes of YG3708 can significantly inhibit the increased expression of inflammatory factors, matrix metalloproteinases, barrier proteins and antimicrobial peptides caused by UVB irradiation, thereby alleviating UVB-induced cellular photoaging.

[0140] (4) YG3708 fermentation filtrate, lysate and exosomes alleviate UVB-induced ROS content in HaCaT cells

[0141] HaCaT cells were cultured to the logarithmic phase and the cells were collected. A normal control group (Con), a model group (UVB), and treatment groups (S-60W, R-90W, and W-120W) were set up in a 12-well plate. The concentration of the cell suspension was adjusted to 1.5×10 5 Each well was inoculated with 1 mL of cell suspension and the cell culture plate was placed in a 5% CO2, 37°C incubator for 24 h.

[0142] After 24 h of culture, the cells grew to a monolayer, the culture medium was removed, and the cells were washed twice with sterile PBS, and 1 mL of sterile PBS was added to each well. The control group was wrapped in tin foil to avoid UVB irradiation. The model group and the treatment group were irradiated with UVB for 40 seconds. After irradiation, 1 mL of complete culture medium was added to the control group and the model group, and 1 mL of complete culture medium containing 90w-fold diluted YG3708 lysate (ie, R-90W group), 60w-fold diluted fermentation filtrate (ie, S-60W group), and 120w-fold diluted exosomes (ie, W-120W group) was added to the treatment group. After culturing for 24 h, the changes in cell morphology were observed under a microscope (20×). After washing twice with sterile PBS, the intracellular ROS fluorescence level was determined by DCFH-DA fluorescence using a reactive oxygen species detection kit (purchased from S0033S, Bio-Tech), and the relative fluorescence intensity of intracellular ROS was quantified using Image J to evaluate the effects of fermentation filtrate, lysate and exosomes of YG3708 on UVB-induced cellular ROS levels.

[0143] The results are as follows Fig.11 shown.

[0144] Depend on Fig.11 As can be seen from Figure A:

[0145] 1) Under a 20× microscope, UVB caused a large area of ​​cells to fall off and the degree of fusion to deteriorate, while the fermentation filtrate, lysate and exosomes of YG3708 restored the cell state, indicating that the fermentation filtrate, lysate and exosomes of YG3708 have the function of repairing cell damage;

[0146] 2) Under a 20× microscope field of view, UVB can cause a significant increase in the level of ROS in cells, while the fermentation filtrate, lysate and exosome treatment of YG3708 can significantly inhibit the increase in cellular ROS levels induced by UVB. Fig.11 The statistical results of Figure B are obtained.

[0147] In summary, the fermentation filtrate, lysate and exosomes of YG3708 can significantly inhibit the increase of intracellular ROS levels caused by UVB, thereby alleviating UVB-induced cellular photoaging.

[0148] Anti-photoaging is an important aspect of skin aging, which involves the damage of ultraviolet rays to skin cells, leading to skin pigmentation, reduced elasticity, increased wrinkles and other problems. Ultraviolet rays can destroy the natural barrier of the skin, trigger the production of free radicals, and further accelerate the skin aging process. The extract of strain YG3708 (including fermentation filtrate, exosomes and lysates) can significantly improve the cell morphological disorder, poor fusion, and abnormal increase of inflammatory factors, matrix metalloproteinases, antimicrobial peptides and intracellular ROS caused by UVB irradiation, thereby having an anti-photoaging effect. It can be seen that strain YG3708 or its extract can be used to develop economically effective natural anti-photoaging skin care products.

[0149] Example 6: Detection of the fermentation filtrate and lysate of strain YG3708 to enhance the skin immune barrier function

[0150] Macrophages are professional phagocytes that can remove dying or dead cells and cell fragments in a highly specialized manner. In addition, macrophages are the most important antigen-presenting cells and play a vital role in initiating immune responses. After receiving antigen stimulation, macrophages will present the antigen to the corresponding Th cells. Antigen presentation leads to the production of antibodies and completes the immune regulatory response.

[0151] In this example, macrophages RAW264.7 were used as the subjects to explore the effect of the strain YG3708 extract on the survival rate of RAW264.7 cells and the secretion of cytokines by RAW264.7 cells to explore its effect on the skin immune barrier function.

[0152] (1) Effects of YG3708 fermentation filtrate and lysate on the survival rate of RAW264.7 cells

[0153] RAW264.7 cells (Pnosai, CL-0190) in the logarithmic growth phase were resuspended and diluted to 1×10 5 Cells / ml, 100μl cell suspension was added to each well of the 96-well plate, 200μl PBS was added to each well around the plate as a moisturizing well, and the plate was placed in a 37°C, 5% CO2 incubator for 24h. Afterwards, the cell culture medium was discarded, and different concentrations of YG3708 fermentation filtrate and lysate dilutions were added, 100μl per well, 6 replicates were set, and the plate was placed in a 37°C, 5% CO2 incubator for 24h. Among them, the fermentation filtrate and lysate treatment groups included the Con group (complete culture medium group) and 1 / 100, 1 / 300, 1 / 1000, 1 / 3000, 1 / 10000, 1 / 30000, 1 / 100000, 1 / 300000 times dilution groups, respectively.

[0154] Then, add 50 μl of MTT solution (1×MTT) to each well and place in a 37°C, 5% CO2 incubator for 4 hours. All the above operations are performed under sterile conditions. After discarding the liquid in the culture plate, add 150 μl of DMSO to each well, mix well, measure the absorbance at a wavelength of 570 nm with an enzyme marker, and record the measurement results. The experimental results are analyzed using Graphpad prism. Select a concentration that is non-toxic to cells to continue the subsequent experiments.

[0155] The results are as follows Fig.12 shown. Fig.12 show:

[0156] 1) For the YG3708 fermentation filtrate treatment group, there was no significant change in the cell survival rates of the 1 / 300000, 1 / 30000, 1 / 10000 and 1 / 1000 dilution groups, but the cell survival rates of the 1 / 100 and 1 / 300 dilution groups decreased significantly, among which the cell survival rate of the 1 / 100 group was as low as 18.13%.

[0157] 2) For the YG3708 lysate treatment group, except for a slight decrease in cell survival rate in the 1 / 100,000 dilution group, other concentrations of lysate had no significant effect on cell survival rate.

[0158] In summary, YG3708 fermentation filtrate and lysate had no significant effect on the proliferation and survival of macrophages at appropriate concentrations, that is, no obvious toxicity; these dilution multiples can be selected for subsequent experiments.

[0159] (2) Effects of YG3708 fermentation filtrate and lysate on the levels of cytokine secretion by RAW264.7 cells

[0160] Take RAW264.7 cells in the logarithmic growth phase, resuspend them and dilute them to 1×10 5 Cells / ml, 2mL of cell suspension was added to each well of a 12-well plate, and the plate was placed in a 37°C, 5% CO2 incubator for 24h. Afterwards, the culture medium was discarded, and the treatment solution was added (depending on the group, the treatment solution added was different, namely: Con group (complete culture medium), LPS group (1μg / mL LPS working solution, prepared in DMEM), fermentation filtrate or lysate treatment group (different concentrations of YG3708 fermentation filtrate or lysate dilution)), 2mL per well, 3 replicates were set, and the plate was placed in a 37°C, 5% CO2 incubator for 24h. After 24h, the cell culture fermentation filtrate was collected, and the level of cytokine TNF-α was detected by enzyme-linked immunosorbent assay (ELISA) detection kit (Enzyme-linked Biology, IC50325-1). The operation steps were carried out according to the product instructions provided by the kit company.

[0161] On the other hand, after collecting the cell fermentation filtrate, the cells were washed twice with PBS and then the PBS was discarded. The cells were lysed with TransZol (Full Formula Gold, R61225) to extract RNA for subsequent detection of the gene expression levels of inflammatory factors TNF-α, IL-6, IL-1β and iNOS (after obtaining the Ct value, 2 -△△Ct formula for calculation).

[0162] The ELISA test results of TNF-α in the YG3708 fermentation filtrate and lysate treatment groups were as follows: Fig.13 As shown in Figures A and B.

[0163] Fig.13 Figure A shows that the TNF-α secretion of the YG3708 fermentation filtrate treatment group at the concentration of 1 / 3000 (p < 0.05) and 1 / 10000 (p < 0.01) was significantly higher than that of the control group, which was 3.67 times and 3.79 times that of the control group respectively; Fig.13 Figure B shows that the TNF-α secretion of the YG3708 lysate-treated group at concentrations of 1 / 100 (p<0.01) and 1 / 300 (p<0.05) was significantly higher than that of the control group, which was 5.66 times and 4.50 times that of the control group, respectively.

[0164] These results indicate that the fermentation filtrate and lysate of strain YG3708 can stimulate macrophages to secrete TNF-α at appropriate concentrations, thereby inducing cellular immune regulation.

[0165] The gene expression results of TNF-α, IL-6, IL-1β and iNOS in the YG3708 fermentation filtrate and lysate treatment groups are shown in Fig.14 shown.

[0166] Fig.14 Figures AD in the figure respectively show the gene expression levels of TNF-α, IL-6, IL-1β and iNOS in the YG3708 fermentation filtrate treatment group, which show that in the LPS group, the gene expression levels of TNF-α, IL-6, IL-1β and iNOS were significantly higher than those in the control group, indicating that LPS induced abnormally high expression of these inflammatory factors; while in the fermentation filtrate treatment groups of various concentrations, the expression levels of these genes showed an upward trend compared with the control group, but were significantly lower than those in the LPS group.

[0167] Fig.14Figures EH in the figure show the gene expression levels of TNF-α, IL-6, IL-1β and iNOS in the YG3708 lysate treatment group, respectively, which show that in the LPS group, the gene expression levels of TNF-α, IL-6, IL-1β and iNOS were significantly higher than those in the control group, indicating that LPS induced abnormally high expression of these inflammatory factors; and in the lysate treatment groups of each concentration, the expression levels of these genes showed an upward trend compared with the control group, but the expression levels of TNF-α and IL-1β genes induced by low concentration lysate treatment and IL-6 and iNOS genes induced by higher concentration lysate treatment were significantly lower than those in the LPS group.

[0168] These results indicate that the fermentation filtrate and lysate of strain YG3708 can stimulate cells to enhance the expression of inflammatory factors, promote immune regulation, and thus enhance the skin immune barrier function.

[0169] Example 7: Evaluation of the soothing efficacy of the fermentation filtrate and exosomes of strain YG3708

[0170] 1. Construction of LPS-induced cell relaxation model

[0171] Take RAW264.7 cells in the logarithmic growth phase, resuspend them and dilute them to 1×10 5 cells / ml, added 2 mL of cell suspension to each well of a 12-well plate, and cultured in a 37°C, 5% CO2 incubator for 24 h; then, aspirated the culture medium, added 1 μg / ml of LPS working solution, and continued to culture in a 37°C, 5% CO2 incubator for 24 h.

[0172] The preparation method of 1μg / ml LPS working solution is as follows:

[0173] 1) Prepare LPS stock solution: Dissolve LPS in sterile DMEM to a concentration of 100 μg / ml, filter through a 0.22 μm filter membrane, and store at -80°C after aliquoting;

[0174] 2) Prepare LPS working solution: Before use, dilute the LPS stock solution to 1 μg / ml with complete culture medium.

[0175] 2. Effects of YG3708 fermentation filtrate and exosomes on the survival rate of RAW264.7 cells

[0176] The experimental groups are as follows:

[0177] Con group: prepared according to the method described in Example 6;

[0178] LPS group (i.e., model group): prepared as described in the above section (I);

[0179] Fermentation filtrate or lysate treatment group: Add different concentrations of YG3708 fermentation filtrate or exosome dilution to the above-mentioned LPS-treated cells (i.e., LPS cell relief model), 2 mL per well, set up 3 replicates, and place in a 37°C, 5% CO2 incubator for continued culture for 24 hours.

[0180] Then, the cell survival rate curve was measured according to the method described in Example 6. The results are as follows: Fig.15 shown.

[0181] Fig.15 Figure A shows the effect of YG3708 fermentation filtrate on the survival rate of RAW264.7 cells treated with LPS; specifically, compared with the control group, the cell survival rate of the LPS group was reduced, but the cell survival rate of each fermentation filtrate treatment group compared with the LPS group was not reduced, especially, the cell survival rate of the 1 / 1200000 group was even higher than that of the LPS group, showing a cell protective effect.

[0182] Fig.15 Figure B shows the effect of YG3708 exosomes on the survival rate of RAW264.7 cells treated with LPS; specifically, compared with the control group, the cell survival rate of the LPS group was reduced; among the exosome-treated groups, except for the 1 / 300000 and 1 / 100000 groups, the cell survival rates of the other treatment groups were not significantly reduced compared with the LPS group.

[0183] 3. Effects of YG3708 fermentation filtrate and exosomes on LPS-induced RAW264.7 cell relief model

[0184] Skin irritation is mainly regulated by barrier, neurovascular and immune inflammatory processes, and relieving inflammation can improve irritation. When cells are stimulated, they deform and release inflammatory factors such as TNF-α, IL-6, IL-1β and COX-2 to cause irritation. This experiment used LPS to stimulate the RAW264.7 macrophage model, and evaluated its soothing effect by detecting the effects of YG3708 fermentation filtrate and exosomes on the secretion of inflammatory factors. The specific procedures are as follows.

[0185] The Con group and LPS group were prepared by the method described in the above part (ii), and the cells treated with different concentrations of YG3708 fermentation filtrate or exosomes were diluted with LPS working solution; in addition, this study also set up a dexamethasone (DEX) treatment group as a positive control, and its specific treatment method was: the treatment solution was changed to 100 μg / ml dexamethasone working solution.

[0186] After 24 hours of treatment, the cell culture supernatant was collected and the level of cytokine TNF-α was detected by enzyme-linked immunosorbent assay (ELISA) kit (ELISA Biotechnology, IC50325-1). The operation steps were carried out according to the product instructions provided by the kit company.

[0187] In addition, after collecting the cell culture supernatant, wash it twice with PBS, add 1 ml of PBS to each empty space, and observe it under an inverted microscope. Discard the PBS, use TransZol (Quanshijin, R61225) to lyse the cells and extract RNA for subsequent detection of inflammatory factors IL-6, IL-1β and COX-2 gene expression levels (after obtaining the Ct value, use the 2-△△Ct formula for calculation).

[0188] The results of ELISA test for TNF-α are as follows Fig.16 shown.

[0189] Fig.16 A shows that compared with the blank control group (i.e., Con group), the TNF-α content in the LPS group was significantly increased (P<0.0001), which indicates that the cell soothing model was successfully constructed; compared with the LPS group, the TNF-α content in the DEX group and the YG3708 fermentation filtrate groups at various concentrations was significantly decreased; this shows that even at very low concentrations, the YG3708 fermentation filtrate can significantly inhibit the LPS-induced TNF-α secretion, thereby reducing the LPS-induced inflammatory response and playing a certain protective role.

[0190] Fig.16 B shows that compared with the blank control group (i.e., Con group), the TNF-α content in the LPS group was significantly increased (P<0.0001), which indicates that the cell soothing model was successfully constructed; compared with the LPS group, the TNF-α content in the DEX group and the 1 / 30000 concentration of YG3708 exosome group was significantly decreased, and the TNF-α content in the 1 / 10000 concentration of YG3708 exosome group was also decreased; this indicates that at the appropriate concentration, YG3708 exosomes can significantly inhibit LPS-induced TNF-α secretion, thereby alleviating the LPS-induced inflammatory response to a certain extent and playing a certain protective role.

[0191] The results of gene expression level detection of IL-6, IL-1β and COX-2 are as follows Fig.17 shown.

[0192] Fig.17Figure AC in the figure shows the gene expression levels of IL-6, IL-1β and COX-2 in the YG3708 fermentation filtrate treatment group. It shows that compared with the control group, the gene expression levels of IL-6, IL-1β and COX-2 in the LPS group were significantly increased, while those in the DEX group were significantly downregulated compared with the LPS group. For the fermentation filtrate treatment group, at appropriate concentrations, the gene expression levels of IL-6, IL-1β and COX-2 were significantly decreased compared with the LPS group. This shows that at appropriate concentrations, the YG3708 fermentation filtrate can significantly inhibit the abnormally high expression of inflammatory factors induced by LPS, thereby having a soothing effect.

[0193] Fig.17 The DF graph in the figure shows the gene expression levels of IL-6, IL-1β and COX-2 in the YG3708 exosome-treated group. It shows that compared with the control group, the gene expression levels of IL-6, IL-1β and COX-2 in the LPS group were significantly increased, while those in the DEX group were significantly downregulated compared with the LPS group. For the exosome-treated group, at appropriate concentrations, the gene expression levels of IL-6, IL-1β and COX-2 were significantly decreased compared with the LPS group. This shows that at appropriate concentrations, YG3708 exosomes can significantly inhibit the abnormally high expression of inflammatory factors induced by LPS, thereby having a soothing effect.

[0194] Example 8: Determination of active substances with soothing effects in the fermentation filtrate of strain YG3708

[0195] In this example, the method described in Example 7 was used to determine the effects of different fermentation filtrate groups (including a common fermentation filtrate group and a reuteri-enriched fermentation filtrate group at dilution multiples of 1 / 90W and 1 / 120W) and different concentrations of 3-HPA on the production of TNF-α by LPS-induced macrophages; and the reuteri-enriched fermentation filtrate content was also determined.

[0196] The results are as follows Fig.18 shown.

[0197] Fig.18 Figure A in the figure shows the effects of different fermentation filtrate groups on the production of TNF-α by LPS-induced macrophages; specifically, it shows that the ordinary fermentation filtrate group failed to inhibit the secretion of inflammatory factor TNF-α by LPS-induced macrophages, while the reuteri-enriched fermentation filtrate group significantly reduced the secretion of TNF-α at the same dilution concentration, thereby reducing the inflammatory response.

[0198] Fig.18Figure B in the figure shows the results of the determination of the content of reuterin in the fermentation filtrate enriched with reuterin; specifically, in the fermentation filtrate with soothing effect, the content of reuterin was between 0.001 μg / mL and 0.003 μg / mL. In contrast, the content reading of reuterin in the ordinary fermentation filtrate group was close to zero and had no statistical significance.

[0199] Fig.18 Figure C in the figure shows the effect of different concentrations of 3-HPA on the production of TNF-α by LPS-induced macrophages; specifically, it shows that when the concentration of 3-HPA is 0.001μg / mL and 0.005μg / mL, it can significantly reduce the secretion of TNF-α by LPS-induced macrophages, which indicates that 3-HPA can alleviate the LPS-induced inflammatory response within a certain concentration range.

[0200] Since 3-HPA is the main representative substance of reuterin, it can be inferred based on the above results that the key substance that exerts a soothing effect in the reuterin-enriched fermentation filtrate may be 3-HPA.

[0201] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Lactobacillus reuteri YG3708, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit date being July 9, 2024 and the deposit number being CGMCC No.31243.

2. The inactivated bacterial cell, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 according to claim 1.

3. The inactivated bacterial body, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 according to claim 2, characterized in that: The extract of Limosilactobacillus reuteri YG3708 is a lysate and / or exosome extracted therefrom.

4. The inactivated bacterial body, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 according to claim 3, characterized in that: The method for preparing the fermentation supernatant comprises: the following: Fermenting the Lactobacillus reuteri YG3708 in the presence of glycerol, centrifuging the fermentation liquid, collecting the supernatant and filtering it to obtain the fermentation supernatant; And / or, the method for extracting the lysate includes the following: The Lactobacillus reuteri YG3708 is fermented and cultured in the presence of glycerol, the fermentation broth is centrifuged, the cells are collected, the cells are resuspended in PBS, and the cell walls are broken by ultrasound or enzymatic hydrolysis to obtain a lysate; preferably, the obtained lysate is sterilized; And / or, the method for extracting exosomes comprises the following: The Lactobacillus reuteri YG3708 is fermented and cultured in the presence of glycerol, the fermentation liquid is centrifuged, and the fermentation supernatant is collected; the obtained fermentation supernatant is centrifuged, filtered, and concentrated to obtain exosomes.

5. The inactivated bacterial body, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 according to claim 4, characterized in that: In the method for preparing the fermentation supernatant: In the fermentation system, the final concentration of glycerol is 200-300, preferably 250 mmol / L; And / or, the centrifugation treatment refers to centrifugation at 8000-10000 r / min and 3-8° C. for 8-15 min; And / or, the filtration is performed using a filter membrane with a pore size of 0.2-2.0 μm, preferably 0.22 μm.

6. The inactivated bacterial body, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 according to claim 4, characterized in that: In the method for extracting exosomes, the centrifugation procedure of the fermentation supernatant is: first centrifugation at 200-400g for 8-15min, then centrifugation at 1500-3000g for 15-30min, and finally centrifugation at 8000-12000g for 20-40min; And / or, the filtration of the fermentation supernatant is: filtering with a filter membrane having a pore size of 0.2-2.0 μm, preferably 0.22 μm; And / or, the fermentation supernatant is concentrated by using a hollow fiber of 400-600 kd, preferably 500 kd, to concentrate the fermentation clarified liquid.

7. A composition, characterized in that The composition comprises the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Limosilactobacillus reuteri YG3708 as described in claim 1 and / or Limosilactobacillus reuteri YG3708 as described in any one of claims 2 to 6 as active ingredients.

8. The composition according to claim 7, 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; Preferably, the composition is an external dosage form; the external dosage form is a dosage form selected from the following: aqueous solution, emulsion, ointment, cream, gel, powder, oil.

9. The composition according to claim 7 or 8, characterized in that The composition further comprises other active agents; the other active agents comprise any one or more of antioxidants, cell active agents, moisturizing agents, anti-aging agents and anti-dermatitis agents.

10. Use of the Lactobacillus reuteri YG3708 according to claim 1, or the inactivated bacteria, extract, culture, culture supernatant, fermentation product and / or fermentation supernatant of Lactobacillus reuteri YG3708 according to any one of claims 2 to 6, or a composition comprising one or more of the foregoing substances in the preparation of a product for the following purposes: 1) Promote skin repair; 2) Reduce, inhibit or eliminate skin inflammation or allergies; 3) Anti-skin aging; 4) Enhance skin immune function.

Images