CN119101614A - Bifidobacterium adolescentis YG3912, exosomes and extracts thereof and applications thereof in skin care - Google Patents
Bifidobacterium adolescentis YG3912, exosomes and extracts thereof and applications thereof in skin care Download PDFInfo
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- CN119101614A CN119101614A CN202411012565.1A CN202411012565A CN119101614A CN 119101614 A CN119101614 A CN 119101614A CN 202411012565 A CN202411012565 A CN 202411012565A CN 119101614 A CN119101614 A CN 119101614A
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- bifidobacterium adolescentis
- fermentation
- skin
- supernatant
- lysate
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Abstract
本发明涉及微生物领域，具体涉及一株青春双歧杆菌(Bifidobacterium adolescentis)YG3912，其灭活菌体、提取物、培养物、培养上清液、发酵物和/或发酵上清液，包含前述的组合物，以及其在制备用于促进皮肤修复，降低皮肤炎症或过敏，抗皮肤老化以及增强皮肤免疫功能的产品中的应用。本发明的青春双歧杆菌(Bifidobacterium adolescentis)YG3912及其相关产品具有较好的修复皮肤细胞的能力，减轻、抑制或消除皮肤炎症的能力以及显著的抗皮肤老化的能力，因此有望用于皮肤修复、缓解炎症以及对抗光老化等皮肤健康护理，具有显著的应用价值和市场潜力。The present invention relates to the field of microorganisms, and specifically to a strain of Bifidobacterium adolescentis YG3912, its inactivated bacterial body, extract, culture, culture supernatant, fermentation product and/or fermentation supernatant, comprising the aforementioned composition, and its use in the preparation of products for promoting skin repair, reducing skin inflammation or allergy, resisting skin aging and enhancing skin immune function. The Bifidobacterium adolescentis YG3912 and related products of the present invention have good ability to repair skin cells, reduce, inhibit or eliminate skin inflammation and significant ability to resist skin aging, and are therefore expected to be used for skin repair, inflammation relief and skin health care such as photoaging, and have significant application value and market potential.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912, an inactivated thallus, an extract, a culture supernatant, a ferment and/or a ferment supernatant thereof, a composition containing the composition, and application of bifidobacterium adolescentis (Bifidobacterium adolescentis) or the inactivated thallus, the extract, the culture and the composition containing the composition in preparation of products for repairing skin, resisting dermatitis, resisting skin aging and enhancing skin immunity.
Background
Conventional skin care products rely on chemical ingredients and may present a potential risk. With advances in biotechnology, particularly in microbiome research, important roles of microorganisms and their metabolites in skin health have been revealed. Exosomes are becoming research hotspots in the field of skin care and repair, as specific vesicles for intercellular communication, due to their unique biological properties. However, the exosomes currently on the market are mainly derived from human stem cells, which have limitations in ethics, acquisition costs and mass production.
Bifidobacterium adolescentis is a gram-positive, anaerobic bacterium belonging to the genus Bifidobacterium (bifidobacteria), and is an important representative of a class of probiotics. The prior art mainly focuses on researching the health effects of bifidobacterium adolescentis on hosts, including the effects of immunoregulation, diarrhea resistance, inflammation resistance, blood sugar reduction and other probiotics, and has no report on the application of bifidobacterium adolescentis in skin care.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
Object of the Invention
The invention aims to provide bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 with skin repairing, anti-dermatitis, anti-skin aging and skin immunity enhancing functions, inactivated bacteria, extract, culture supernatant, fermentation product and/or fermentation supernatant thereof, a composition containing the same, and application of bifidobacterium adolescentis (Bifidobacterium adolescentis) or inactivated bacteria, extract, culture thereof and the composition containing the same in preparation of products for repairing skin, anti-dermatitis, anti-skin aging and skin immunity enhancing functions.
Solution scheme
In order to achieve the purpose of the invention, the following technical scheme is adopted.
In a first aspect, the invention provides a bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 which is classified and named as bifidobacterium adolescentis (Bifidobacterium adolescentis) and is preserved in China general microbiological culture Collection center (China Committee for culture Collection) with a preservation unit address of North Star, national institute of China, national academy of sciences, no.3, korean area, beijing, china, with a preservation date of 2024, 6 months and 20 days, and a preservation number of CGMCC No.31020.
The bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 is isolated from a healthy adolescent stool sample, sequenced by 16S rRNA, and identified as bifidobacterium adolescentis (Bifidobacterium adolescentis) by combining morphological characteristics.
The 16S rRNA sequence of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 of the present invention is as follows:
GCGGGGCGGCGTGCTTACCATGCAGTCGAACGGGATCCCAGGAGCTTGCTCCTGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGGATGCATGTCCTTCTGGGAAAGATTCATCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTTGACTGGGAGCAAGCCCTTCGGGGTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGACCATTCCACGGTCTCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACAGCCCCAGAGATGGGGCCTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTGTGTTGCCAGCACGTCGTGGTGGGAACTCACGGGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACACCGTGAGGTGGAGCGGATCCCTTAAAACCGGTCTCAGTTCGGATTGGAGTCTGCAACCCGACTCCATGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCAACCTTTTGGGGGGAGCCGTCTAAGGTGAG(SEQ ID NO:1).
The bacterial colony features of the strain comprise that after the strain is cultured in an MRS agar culture medium for 24-48 h, the diameter is 0.5-2.0 mm, the edge of the bacterial colony is smooth, the surface is smooth, the bacterial colony is opaque and the bacterial colony is milky.
In vitro experiments prove that the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 HAS better repairing capability on human skin fibroblasts, and particularly, the inventor finds that the exosome of the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 can promote the generation of collagen such as COL1A2 and COL3A1, and the like, and fermentation liquor and lysate can enhance the generation of collagen such as COL1A1 and COL17A1 and hyaluronic acid (HAS 1 and HAS 3), thereby being beneficial to the deep repairing and regeneration of the skin fibroblasts.
The inventors have also found that exosomes, fermentation broths and lysates of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 of the present invention promote proliferation and survival of macrophages at suitable concentrations and stimulate macrophage cells, enhance their ability to produce cytokines such as TNF- α, IL-6, IL-1 β and iNOS, thereby promoting immunomodulation and enhancing the skin immune barrier function.
In addition, the inventor also proves that the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 also has obvious effects of inhibiting inflammation and resisting skin aging, and particularly, the inventor proves that the exosomes, fermentation liquor and lysate of the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 can obviously inhibit the expression of inflammatory factors (such as IL-1 beta, IL-6 and IL-8) induced by UVB and inhibit the generation of matrix metalloproteinases (MMP-2, MMP-9) and ROS under proper concentration, so that the effect of resisting photoaging is achieved.
In a second aspect, the present invention provides inactivated cells, extracts, cultures, culture supernatants, ferments and/or fermentation supernatants of bifidobacterium adolescentis (Bifidobacteriumadolescentis) YG3912 as described in the first aspect above.
Preferably, the extract of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 is lysate and/or exosome extracted from the extract.
In a possible embodiment, the method of extraction of the lysate comprises the following:
fermenting and culturing Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912, centrifuging the fermentation broth, collecting thallus, re-suspending the thallus with PBS and breaking cell wall by ultrasonic or enzymolysis treatment to obtain lysate, and preferably sterilizing the lysate.
In a possible embodiment, the extraction method of the exosomes comprises the following:
Fermenting and culturing Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912, centrifuging the fermentation liquid, collecting fermentation supernatant, centrifuging the obtained fermentation supernatant, filtering, and concentrating to obtain exosome;
preferably, the centrifugation procedure of the fermentation supernatant is that 200-400 g, preferably 300g, is centrifuged for 8-15min, preferably 10min, then 1500-3000 g, preferably 2000g, is centrifuged for 15-30min, preferably 20min, and finally 8000-12000 g, preferably 10000g, is centrifuged for 20-40min, preferably 30min;
preferably, the filtering of the fermentation supernatant is carried out by adopting a filter membrane with the pore diameter of 0.2-2.0 mu m, preferably, the filtering is carried out by adopting filter membranes with the pore diameters of 0.8 mu m and 0.45 mu m respectively in sequence;
Preferably, the concentration of the fermentation supernatant is to concentrate the fermentation supernatant by using 400-600 kd, preferably 500kd hollow fiber.
In a third aspect, the present invention provides a composition comprising as an active ingredient bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 as described in the first aspect above, or an inactivated thallus, extract, culture supernatant, fermented product and/or fermented supernatant of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 as described in the second aspect above.
In some possible embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier and/or excipient.
In other possible embodiments, the composition is a cosmetic composition further comprising a cosmetically acceptable carrier and/or excipient.
Preferably, the composition is in the form of an external dosage form;
further preferably, the external preparation is selected from the group consisting of aqueous solution, emulsion, ointment, cream, gel, powder and oil.
In a possible embodiment, the composition further comprises other active agents;
Preferably, the other active agents include any one or more of antioxidants, cellular active agents, moisturizing agents, anti-aging agents, and anti-dermatitis agents.
In a fourth aspect, the present invention provides the use of a bifidobacterium adolescentis (Bifidobacteriumadolescentis) YG3912 as described in the first aspect above, or an inactivated thallus, extract, culture supernatant, fermented product and/or fermented supernatant of a bifidobacterium adolescentis (Bifidobacteriumadolescentis) YG3912 as described in the second aspect above, or a composition comprising one or more of the foregoing, for the preparation of a product for use in:
1) Promoting skin repair;
2) Reducing, inhibiting or eliminating skin inflammation or allergy;
3) Anti-skin aging;
4) Enhancing skin immunity.
In a possible embodiment, the promoting skin repair is promoting sensitive muscle skin repair;
In a possible embodiment, the skin inflammation is aseptic skin inflammation, inflammation caused by an external stimulus;
in a possible embodiment, the skin allergy is a skin allergy symptom due to external factor irritation;
in a possible embodiment, the anti-skin aging is anti-skin photoaging.
In a fifth aspect, the present invention provides a method of repairing skin, reducing, inhibiting or eliminating skin inflammation or allergy, combating skin aging, and/or enhancing skin immune function, comprising administering to a subject in need thereof an effective amount of inactivated cells, extracts, cultures, culture supernatants, ferments and/or fermentation supernatants of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 as described in the first aspect above, or bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 as described in the second aspect above, or a composition comprising one or more of the foregoing.
In a possible embodiment, the promoting skin repair is promoting sensitive muscle skin repair;
In a possible embodiment, the skin inflammation is aseptic skin inflammation, inflammation caused by an external stimulus;
in a possible embodiment, the skin allergy is a skin allergy symptom due to external factor irritation;
in a possible embodiment, the anti-skin aging is anti-skin photoaging.
Advantageous effects
Compared with the prior art, the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 has the following obvious advantages:
(1) The exosomes can promote the generation of collagens such as COL1A2, COL3A1 and the like under proper concentration, and the fermentation liquor and lysate can enhance the generation of collagens such as COL1A1, COL17A1 and the like and hyaluronic acid (HAS 1, HAS 3), thereby being beneficial to the deep repair and regeneration of skin fibroblasts;
(2) The exosomes, fermentation liquor and lysate can promote proliferation and survival of macrophages under proper concentration, and can stimulate macrophage cells, enhance the capability of the macrophages to produce cytokines such as TNF-alpha, IL-6, IL-1 beta, iNOS and the like, thereby promoting immunoregulation and enhancing the skin immune barrier function;
(3) The exosomes, fermentation liquor and lysate can obviously inhibit the expression of inflammatory factors (such as IL-1 beta, IL-6 and IL-8) induced by UVB and inhibit the generation of matrix metalloproteinases (MMP-2, MMP-9) and ROS under proper concentration, thereby achieving the effect of resisting photoaging.
In conclusion, the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 and related products thereof have better capability of repairing skin cells, reducing, inhibiting or eliminating skin inflammation and resisting skin aging, so that the bifidobacterium adolescentis YG3912 is expected to be used for skin repair, inflammation relieving and skin health care such as photoaging resistance, and has remarkable application value and market potential.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a growth curve of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to the present invention.
FIG. 2 shows the particle concentration and particle size distribution of exosomes extracted from the fermentation broth of strain YG3912 of the invention, measured using NTA technique.
FIG. 3 shows HSF cell repair rate heatmaps of fermentation filtrate and lysates of different bifidobacterium adolescentis (Bifidobacterium adolescentis) strains.
Fig. 4 shows the effect of the exosomes of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 of the invention on HSF cell fusion rates, expressed as P <0.05.
Fig. 5 shows the effect of fermentation filtrate (panel a), lysate (panel B) and exosome (panel C) of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to the invention on expression of HSF cell repair related factors (including collagen COL1A1, COL1A2, COL3A1, COL17A and hyaluronic acid HAS1, HAS 3), wherein the abscissa indicates the type of repair related factor tested and the ordinate indicates the mRNA relative expression level of each repair related factor, P <0.05, P <0.01, P <0.001, P <0.0001.
FIG. 6 shows the effect of fermentation filtrate, exosomes, lysates of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 on RAW264.7 cell viability according to the invention.
Fig. 7 shows the effect of fermentation filtrate (a), lysate (B), exosome (C) of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to the invention on TNF- α secretion by RAW264.7 cells, wherein P <0.05 compared to the blank control group (i.e., con group), P <0.01 compared to the Con group, P <0.001 compared to the Con group, P <0.0001 compared to the Con group.
Fig. 8 shows the effect of fermentation filtrate (a-D), lysate (E-H), exosome (I-L) of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to the invention on secretion of inflammatory factors TNF- α, IL-6, IL-1 β and iNOS by RAW264.7 cells, wherein P <0.05 compared to the blank control group (i.e. Con group), P <0.01 compared to the Con group, P <0.001 compared to the Con group, P <0.0001 compared to the Con group.
FIG. 9 shows the effect of fermentation filtrate, lysate, exosomes of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 on the morphology and degree of fusion of UVB-damaged cells according to the invention.
FIG. 10 shows the effect of fermentation filtrate, lysate, exosomes of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 of the present invention on the expression levels of UVB-damaged cytokines and metalloproteinases, wherein panels A, B and C show the protein levels of IL-6, IL-8 and IL-1β, respectively, of each treatment group, and panel D shows the relative mRNA levels of IL-1β, MMP2 and MMP9 of each treatment group, respectively, representing 0.01< P <0.05 compared to the UVB group, 0.001< P <0.01 compared to the UVB group, representing P <0.001 compared to the UVB group, and ### representing P <0.001 compared to the blank control group (i.e., con group).
FIG. 11 shows the effect of fermentation filtrate, lysate, exosomes of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 of the invention on cell morphology, degree of fusion and cellular ROS levels of UVB lesions, wherein Panel A shows cell morphology and degree of fusion of each treatment group (upper panel) and fluorescent staining pictures of ROS (lower panel), panel B shows the relative fluorescence intensity (%) of ROS of each treatment group, wherein 0.01< P <0.05, represents 0.001< P <0.01, represents P <0.001, and ### represents P <0.001, compared to the UVB group, compared to the blank control group (i.e., con group).
The bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 is classified and named as bifidobacterium adolescentis (Bifidobacterium adolescentis), the preservation date is 2024, 6 and 20 days, the preservation unit is China general microbiological center of the culture Collection of microorganisms, the preservation address is North Star Xiyu No. 1, 3 national academy of sciences of China, the Korean area of Beijing, and the preservation number is CGMCC No.31020.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
In the examples described below, other reagents or materials were used, except as indicated otherwise, and were commercially available.
In addition, in the examples described below, the "concentration" of the strain fermentation filtrate, lysate, exosomes refers to the volume dilution factor by which the stock solution of the corresponding substance is diluted with the complete medium (i.e., serum-containing DMEM medium), and for example, the strain fermentation filtrate or lysate at the concentrations of 1/1000, 1/3000, 1/10000 and 1/30000 (V/V) refers to the dilution of the stock solution of the strain fermentation filtrate or lysate by 1000 times, 3000 times, 10000 times and 30000 times, respectively, by volume.
Example 1 isolation and identification of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912
(1) Isolation of strains
1G of healthy teenager faeces sample is put into 9mL of PBS buffer solution, ten-fold gradient dilution is carried out by using sterile physiological saline after vortex mixing, 10 -6、10-7、10-8 dilution gradients are selected and respectively coated on MRS agar culture medium, a solid flat plate is placed into a 37 ℃ anaerobic constant temperature incubator for culturing for 24-48 h, and colonies are picked and purified on the MRS agar culture medium for more than 3 times until the colony morphology on the flat plate is consistent. A plurality of clones were picked up and designated YGMCC2531、YGMCC3246、YGMCC3487、YGMCC3492、YGMCC3390、YGMCC3533、YGMCC3625、YGMCC0568、YGMCC0809、YGMCC0896、YGMCC0879、YGMCC1108、YGMCC1129、YGMCC1777、YGMCC3912, for strain identification by strain morphology and 16S rRNA sequencing results, and identified as bifidobacterium adolescentis (Bifidobacterium adolescentis), and, after efficacy screening, the bifidobacterium adolescentis (Bifidobacterium adolescentis) of the present invention, namely strain YGMCC3912, namely the bifidobacterium adolescentis strain YG3912 of the present invention (herein, strain YGMCC3912 is equivalent to strain YG3912, both of which are used interchangeably) was obtained.
(2) Identification of the strains of the invention
A. Colony morphology characterization
After the strain clone YG3912 is cultured in an MRS agar culture medium for 24-48 hours, the diameter is between 0.5-2.0 mm, the colony edge is smooth, the surface is smooth, and the opacity is milky.
B.16S rRNA sequencing
The strain YG3912 was sent to the stock of biological engineering (Shanghai) Co., ltd for 16S rRNA gene sequencing, the 16S rRNA sequence of which is shown as SEQ ID NO:1, and the 16S rRNA sequencing results were subjected to BLAST alignment (https:// BLAST. NCBI. Lm. Nih. Gov/BLAST. Cgi) on NCBI database, and the isolated strain YG3912 was determined to be bifidobacterium adolescentis (Bifidobacterium adolescentis) in combination with the morphological characteristics of the colonies.
Example 2 preparation of fermentation filtrate and lysate of Strain YG3912, extraction and characterization of exosomes
The culture medium involved in this example is as follows:
MRS culture medium (g/L) comprises 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, tri-ammonium citrate 2.0g/L, glucose 20.0g/L, manganese sulfate 0.05g/L, sodium acetate 5.0g/L, L-cysteine hydrochloride 0.5g/L, and water as solvent.
2.1 Culture of YG3912 Strain and preparation of fermentation broth thereof
The seed solution of strain YG3912 was inoculated into MRS liquid medium at an inoculum size of 2%, and subjected to anaerobic still culture at 37℃for 24 hours, and during the culture, OD600 values of the culture solution were measured by an enzyme-labeling instrument at intervals to obtain a growth curve of strain YG3912, as shown in FIG. 1. Fig. 1 shows that strain YG3912 reached growth plateau (od600=1.22) when cultured for 12 h.
The fermentation process parameters of the strain YG3912 are optimized to obtain the optimal fermentation parameters, wherein the optimal fermentation parameters comprise the following pH value of 6.25, inoculum size of 2.0% and culture time of 48 hours. The strain YG3912 was fermented according to the above optimized fermentation parameters to obtain a fermentation broth for later use.
2.2 Preparation of fermentation filtrate of YG3912 strain, lysate and exosome extraction and characterization
(1) And (3) preparing fermentation filtrate:
The fermentation broth of the strain YG3912 prepared above was centrifuged at 9000r/min×4 ℃ for 10min to obtain a fermentation supernatant, the fermentation supernatant was filtered with a 0.22 μm polyethersulfone filter membrane to obtain a cell-free fermentation filtrate of the strain YG3912, which was frozen at-80 ℃ for later use.
(2) Lysate preparation:
Harvesting the thalli after centrifugation in the step (1), adding pre-cooled sterile PBS for 2 times concentration and resuspension of the thalli, and adopting ultrasonic waves for cell wall breaking treatment, wherein the specific ultrasonic procedure is that the ultrasonic waves are carried out for 2s, the total ultrasonic time is 60min, and the whole ultrasonic procedure is carried out under the ice bath condition of-4~0 ℃. Then, the ultrasonic results were heat treated at 80℃for 30min using an autoclave to obtain sterilized lysates of the strain YG3912 broth, which were frozen at-80℃for later use.
(3) Exosome extraction and characterization:
The fermentation broth of strain YG3912 is centrifuged at 9000r/min×4 ℃ for 10min to obtain a fermentation supernatant, the fermentation supernatant is centrifuged at 300g×4 ℃ for 10min, then at 2000g×4 ℃ for 20min, and finally at 10000g×4 ℃ for 30min, and then the fermentation supernatant is obtained by filtering with a 0.8 μm and 0.45 μm polyethersulfone filter membrane in sequence. The resulting fermentation supernatant was concentrated to 50mL using 500kd hollow fiber and replaced with 200mL using PBS. And purifying the concentrated solution by using a protein purifier to obtain an exosome solution of the strain YG3912 fermentation broth. And measuring the particle number and the diameter distribution of exosomes in the obtained exosome solution by adopting NTA technology.
By the above procedure we obtained a fermentation filtrate, lysate and exosome solution of strain YG3912 with stable high yield. The number of exosomes and the diameter interval in the YG3912 exosome solution were measured based on NTA technique, see fig. 2, fig. 2 shows that the exosome diameter interval in the YG3912 exosome solution is between 100-500nm with an average value of 178.4nm (median diameter: 152.2 nm), which means that the major population has a broad size distribution despite its size of about 150nm, and the number of particles per milliliter of exosome solution is 1.8 x 10 10.
By adopting the above procedure, fermentation filtrate, lysate and exosome solution of strain YGMCC2531、YGMCC3246、YGMCC3487、YGMCC3492、YGMCC3390、YGMCC3533、YGMCC3625、YGMCC0568、YGMCC0809、YGMCC0896、YGMCC0879、YGMCC1108、YGMCC1129、YGMCC1777 were also prepared, respectively.
Example 3 detection of skin cell repair promoting ability of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 fermentation filtrate and lysate
Human skin fibroblasts, which are key cells of dermis, are vital to the maintenance of skin elasticity and tension, and can efficiently synthesize matrices such as elastin, collagen and the like, generate elastic fibers and collagen fibers, secrete repair factors, and endow skin with self-repair and renewal capacity.
In this example, human fibroblasts (hereinafter abbreviated as HSF) were used as the subjects, and the ability of the fermentation filtrate and lysate of various strains to repair them was examined, respectively.
The method for culturing HSF cells is as follows:
HSF cells were cultured in a 37 ℃ 5% co 2 cell incubator with high sugar medium (DMEM) containing 15% extra Fetal Bovine Serum (FBS) and 1%100 x green streptomycin mixed solution (S/P).
The procedure for measuring cell repair ability (cell repair rate) is as follows:
HSF cells which grow well and are within 10 generations of logarithmic growth phase are selected, the HSF cells are respectively inoculated to a 6-hole plate according to the inoculation density of 2X 10 5 cells/hole, after incubation is carried out for 24 hours in an incubator (37 ℃ and 5% CO 2), the fusion rate reaches 90%, a 200 mu L pipette tip is used for streaking on a cell culture plate, the streaked culture plate is washed for 2-3 times by using PBS, and floating cells are removed. Immediately after the scratch is finished, photographing is carried out for 0h, and 2mL of serum-free DMEM culture solution containing the sample is added after photographing is finished. Specifically, the control group was a DMEM-treated group, and the experimental group was a fermentation filtrate and lysate-treated group of different concentrations (including 1/1000, 1/3000, 1/10000, and 1/30000 (V/V) concentrations), of different strains, three replicates each. Photographs were taken at 0 and 48 hours, and scratch areas were calculated using Image J software, and cell repair rates were calculated. The cell repair rate was calculated as follows:
m- -mobility, expressed as percent (%), B 0h - -0h scratch area, B48 h - -48h scratch area.
The cell repair rates of each experimental group and control group were counted and a cell repair rate heat map was made, and the results are shown in FIG. 3. FIG. 3 shows that the repair ability of Bifidobacterium adolescentis showed strain selectivity, and the cell repair ability of the strain YG3912 of the present invention was significantly superior to that of the control group and other strains, regardless of the fermentation filtrate (32.3% -34.0%) or lysate (41.4% -46.9%). Therefore, YG3912 strain is an ideal candidate strain because of its excellent cell repair ability.
Further, selecting HSF cells which grow well and are within 10 generations of the logarithmic growth phase, inoculating the HSF cells to a 6-well plate according to the inoculation density of 2×10 5 cells/well, incubating for 24 hours in an incubator (37 ℃ and 5% CO 2), performing scratch 0h photographing, adding YG3912 exosomes with different concentrations, continuously culturing for 48h, photographing, calculating scratch areas by adopting Image J software, and calculating cell repair rate. The calculation formula of the cell repair rate is the same as the above.
The cell repair rate results for the respective concentrations of YG3912 exosomes and controls are shown in FIG. 4. FIG. 4 shows that YG3912 exosomes at concentrations 1/30000, 1/10000 and 1/3000 have the capacity to promote HSF cell repair, with a significant increase in YG3912 exosomes at concentrations 1/10000 (P < 0.05).
Effects on the expression of cell repair related factors
HSF cells which grow well and are within 10 generations of logarithmic growth phase are selected, the HSF cells are inoculated to a 6-hole plate according to the inoculation density of 2X 10 5 cells/hole respectively, after incubation for 24 hours in an incubator (37 ℃ and 5% CO 2), scratch 0h photographing is carried out, then test substances (YG 3912 fermentation filtrate with different concentrations, lysate and exosomes) are added, after continuous culture for 48 hours, the cells are collected, and TransZol (full gold, R61225) is used for cell lysis to extract RNA for measuring relative expression quantity of cell repair related factor mRNA.
The results are shown in FIG. 5.
Panel A of FIG. 5 shows the results of the detection of YG3912 fermentation filtrate treatment group, which shows that YGMCC03912 at 1/10000 and 1/3000 significantly increased mRNA expression of COL1A1 by HSF cells, wherein 1/10000 concentration increased most significantly (P < 0.001), and 1/10000 concentration increased mRNA expression of HAS3 by HSF cells (P < 0.05).
Panel B of FIG. 5 shows the results of the assay for YG3912 lysate treatment group, showing that YGMCC03912 at a lysate concentration of 1/10000 significantly increased mRNA expression of COL17A1 (P < 0.01) in HSF cells, 1/1000 significantly increased mRNA expression of HAS1 (P < 0.05) in HSF cells, and 1/30000 significantly increased mRNA expression of HAS3 (P < 0.05) in HSF cells.
Panel C of FIG. 5 shows the results of the detection of YG3912 exosome treatment, which showed that YGMCC03912 exosome concentration of 1/30000 significantly increased mRNA expression (P < 0.01) in HSF cells COL1A2 and COL3A 1.
In summary, the YG3912 fermentation filtrate, lysate and exosome can promote the production of collagen such as COL1A1, COL1A2, COL3A1, COL17A1 and hyaluronic acid such as HAS1, HAS3 at appropriate concentrations, which are proteins favorable for cell repair, and thus the YG3912 fermentation filtrate, lysate and exosome can promote repair of damaged cells by enhancing the expression of cell repair related proteins.
Example 4 detection of fermentation filtrate, lysate and exosomes of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 to enhance skin immune Barrier function
Macrophages are specialized phagocytes that are highly specialized in the removal of dying or dead cells and cell debris, and in addition, are the most important antigen presenting cells that play a critical role in initiating immune responses. Upon stimulation with antigen, macrophages present the antigen to the corresponding Th cells, and antigen presentation results in the production of antibodies, completing the immune modulatory response.
In this example, the effect of the YG3912 strain extract on the survival rate of RAW264.7 cells and on the secretion of cytokines by RAW264.7 cells was examined by taking macrophage RAW264.7 as a target.
(1) Effect of YG3912 fermentation filtrate, exosomes and lysates on survival of RAW264.7 cells
RAW264.7 cells (Prunocel, CL-0190) in the logarithmic growth phase were resuspended and diluted to 1X 10 5 cells/ml, 100. Mu.l of cell suspension was added to each well of a 96-well plate, 200. Mu.l of PBS was added as a moisturizing well to each well four weeks, and incubated in a 37℃5% CO 2 incubator for 24 hours. Afterwards, the cell culture solution was aspirated, YG3912 fermentation filtrate, exosomes and lysate dilutions of different concentrations were added, 100 μl of each well was filled with 6 duplicate wells, and the wells were placed in a 37℃5% CO 2 incubator for further incubation for 24h. Wherein the fermentation filtrate, lysate and exosome treatment groups respectively comprise Con group (complete medium group), LPS group (LPS working solution, 1 μg/ml), 1/100 (100 times lysate dilution), 1/300 group (300 times lysate dilution), 1/1000 group (1000 times lysate dilution), 1/3000 group (3000 times lysate dilution), 1/10000 (10000 times lysate dilution), 1/30000 (30000 times lysate dilution).
Then, 50. Mu.l of MTT solution (1 XMTT) was added to each well, and the mixture was incubated in a 37℃and 5% CO2 incubator for 4 hours, all under aseptic conditions. After the liquid in the culture plate was discarded, 150. Mu.l of DMSO was added to each well, and after mixing, absorbance was measured at 570nm using a microplate reader, and the measurement results were recorded. The experimental results were analyzed using GRAPHPAD PRISM. The concentration non-toxic to the cells was selected for subsequent testing.
The results are shown in FIG. 6. Fig. 6 shows:
1) The cell viability (76.15%) was lower for the LPS group than for the Con group, while the extract treatment groups for YG3912 were higher than for the LPS group;
2) For YG3912 fermentation filtrate treatment groups, there was no significant change in cell viability for the 1/30000, 1/10000, 1/3000 and 1/1000 concentration groups, and there was a decrease in the 1/100 and 1/300 concentration groups.
3) For YG3912 exosome treated groups, the cell viability of the other concentration groups was significantly improved, especially up to 121.74% for the 1/10000 concentration group, except for the 1/1000 concentration group which had a slightly reduced cell viability compared to the Con group.
4) For YG3912 lysate treated groups, the cell viability was improved for the 1/30000, 1/300 and 1/100 concentration groups compared to the Con group.
In summary, YG3912 exosomes and lysates promote macrophage proliferation and survival at appropriate concentrations.
(2) Effect of YG3912 fermentation filtrate, exosomes and lysates on the level of cytokine secretion by RAW264.7 cells
RAW264.7 cells in the logarithmic growth phase were taken, resuspended and diluted to 1X10 5 cells/ml, 2ml of cell suspension was added to each well in a 12-well plate, and incubated in a 37℃5% CO 2 incubator for 24 hours. Afterwards, the culture solution is sucked and removed, the complete culture medium, YG3912 fermentation filtrate with different concentrations, lysate and exosome diluent are added, 2ml of each well is provided with 3 compound holes, and the mixture is placed in a 37 ℃ and 5% CO 2 incubator for continuous culture for 24 hours. 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 (ELISA), IC 50325-1. The operating steps are carried out with reference to the product instructions provided by the kit company.
On the other hand, after collecting the cell fermentation filtrate, PBS was discarded after washing 2 times, and TransZol (full gold, R61225) was used to lyse the cells to extract RNA for the subsequent detection of the expression levels of the inflammatory factors TNF-. Alpha., IL-6, IL-1. Beta. And iNOS genes (calculated using the formula 2 -△△Ct after Ct values were obtained).
ELISA results of TNF- α detection of YG3912 fermentation filtrate, lysate and exosome treated groups are shown in FIG. 7, A, B, C respectively, and as can be seen from FIG. 7, A, B, C, the TNF- α content of LPS group is 11-15 times that of control group, demonstrating that LPS treatment stimulated macrophages to secrete cytokine TNF- α.
Panel A of FIG. 7 shows that the low concentration (1/300 and 1/100) YG3912 fermentation filtrate treated groups showed an increase in TNF- α secretion compared to the control group, with a significant difference (P < 0.05) in the 1/300 treated groups. Panel B of FIG. 7 shows that in YG3912 lysate treated groups, TNF- α secretion was significantly increased (P < 0.01) for the 1/10000 and 1/1000 concentration groups compared to the control group, and increased more (P < 0.0001) for the 1/300 and 1/100 concentration groups, by a factor of 13.03 and 20.24, respectively. Panel C of FIG. 7 shows that YG3912 exosome low concentration treatment group reduced TNF- α secretion, and only 1/100 concentration group showed a significant increase in TNF- α secretion (P < 0.001). These results indicate that the fermentation filtrate, lysate and exosomes of YG3912 at appropriate concentrations are capable of stimulating macrophages to secrete TNF- α, which can trigger cellular immunomodulation.
The results of gene expression measurements of TNF- α, IL-6, IL-1β and iNOS in YG3912 fermentation filtrate, lysate and exosome treated groups are shown in FIG. 8.
The A-D panels in FIG. 8 show the gene expression levels of TNF- α, IL-6, IL-1β and iNOS, respectively, in the YG3912 fermented filtrate treated group, showing no significant change in the TNF- α gene level in the YG3912 fermented filtrate treated group at each concentration compared to the control group, a significant increase in the IL-6 gene level in the 1/100 concentration group compared to the control group (P < 0.0001) by up to 17.89 times, a significant increase in the IL-1β gene level in the 1/100 and 1/300 concentration groups compared to the control group (P < 0.0001) by up to 8.21 and 36.81 times, respectively, and a significant increase in the iNOS gene level in all concentration groups compared to the control group.
The E-H diagram in FIG. 8 shows the gene expression levels of TNF- α, IL-6, IL-1β and iNOS, respectively, in YG3912 lysate treated groups, which shows that the levels of TNF- α gene in each lysate treated group were significantly increased by up to 3.75-fold, in particular, up to 176.20-fold, in 1/1000, 1/300 and 1/100 (P < 0.0001) concentration groups, in particular, up to 1003.59-fold, in 1/1000, 1/300 and 1/100 concentration groups, in particular up to 35-fold, in 1/1000, 1/300 and 1/100 concentration groups, in particular up to 1003.59-fold, in 1/1000 (P < 0.05), 1/300 (P < 0.0001) and 1/100 (P < 0.0001), in particular up to 41-fold, in 1/100 concentration groups, in comparison to control groups.
The I-L diagram in FIG. 8 shows the gene expression levels of TNF-. Alpha.IL-6, IL-1β and iNOS, respectively, in YG3912 exosome treated groups, showing a significant increase in the levels of TNF-. Alpha.gene in the 1/300 (P < 0.01) and 1/100 (P < 0.001) concentration groups compared to the control group, a significant decrease in the levels of IL-6 gene in all concentration groups compared to the control group, a significant increase in the levels of IL-1β gene in the 1/300 and 1/100 concentration groups compared to the control group (P < 0.0001), in particular, an increase in the 1/100 concentration group by up to 11.17 times, and a significant increase in the levels of iNOS gene in the 1/300 and 1/100 concentration groups compared to the control group (P < 0.0001), in particular, an increase in the 1/100 concentration group by up to 12.32 times.
EXAMPLE 5 evaluation of photo aging resistance of Bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912
(1) Establishing a HaCaT photoaging cell model of UVB radiation
Method for constructing cell photoaging model Protective Effect of Bifidobacteriumanimalis subs.lactis MG741 as Probiotics against UVB-Exposed Fibroblasts and HairlessMice(DOI:10.3390/microorganisms10122343).
Specifically, haCaT cells were cultured in a high sugar medium (DMEM) containing 10% extra Fetal Bovine Serum (FBS) and 1%100 XStreptomyces lividans (S/P) in a cell incubator at 37℃with 5% CO 2%, after the cell confluency reached about 80%, the medium was removed, and after washing the cells with sterile PBS, the cells were added with sterile PBS and irradiated with UVB under the conditions of 40S using a four-use ultraviolet analyzer (Linbell, ZF-2 type, UVB312nm, 30W) for constructing a cell photoaging model.
(2) YG3912 fermentation filtrate, lysate and exosomes alleviate UVB-induced HaCaT cell photoaging
HaCaT cells were cultured to log phase and cells were collected. A normal control group (Con), a model group (UVB) and a treatment group (S-10≡4, R-3X 10≡4 and W-3000) are arranged in the 12-well plate, the concentration of the cell suspension is adjusted to 3X 10 5/mL, and each well is inoculated with 1mL of the cell suspension. The cell culture plates were incubated in a 37℃5% CO 2 incubator for 24h.
After 24h incubation, the cells grew to a monolayer, the medium was removed, washed 2 times with sterile PBS, and 1mL of sterile PBS was added to each well. The control group was coated with tinfoil to avoid exposure to UVB. The model group and the treatment group were given UVB irradiation for 40 s. After irradiation, the control and model groups were each added with 1mL of complete medium, and the treatment group was added with 1mL of complete medium containing 10000-fold diluted YG3912 fermentation filtrate (i.e., the aforementioned "S-10≡4" group), 30000-fold diluted lysate (i.e., the aforementioned "R-3X 10≡4" group), and 3000-fold diluted exosome (i.e., the aforementioned "W-3000" group). After 24h of incubation, the change in cell morphology was observed with a microscope (20×), and cell supernatants were collected and protein levels of inflammatory factors IL-1. Beta., IL-6 and IL-8 in the cell supernatants were measured using the MQ60 PLUS fully automated chemiluminescence immunoassay system (heat Jing Shengwu), respectively. RNA was extracted from cells using TransZol (full gold, R61225) lysis for subsequent RT-PCR, and gene level content detection of inflammatory factors IL-1 beta and matrix metalloproteinases MMP-2 and MMP-9 in the cell supernatant (calculated using formula 2 -△△Ct after Ct values were obtained).
The results of observing the cell morphology with a microscope (20X) are shown in FIG. 9, and FIG. 9 shows that UVB causes large-area cells to fall off and the degree of fusion to deteriorate under the 20X microscope field of view, and the treatment of YG3912 fermentation filtrate, lysate and exosomes restores the cell state, demonstrating the function of repairing cell damage in YG3912 fermentation filtrate, lysate and exosomes.
The results of protein levels of inflammatory factors IL-1 beta, IL-6 and IL-8 are shown in FIG. 10, A, B, C respectively, and it can be seen from FIG. 10, A, B, C that the levels of IL-1 beta, IL-6 and IL-8 are significantly increased relative to the control group after UVB irradiation. However, YG3912 fermentation filtrate, lysate and exosome treatment significantly reduced the levels of these factors, with inhibition of IL-6 of 28%, 38% and 40%, respectively, and IL-8 of 43%, 39% and 26%, respectively, and IL-1. Beta. Of 43%, 44% and 32%, respectively.
The results of gene level measurements of inflammatory factors IL-1 beta and matrix metalloproteinases MMP2 and MMP9 are shown in FIG. 10, panel D. As can be seen from panel D of FIG. 10, the trend of the gene level of inflammatory factor IL-1β was consistent with the protein level. This further demonstrates that YG3912 fermentation filtrate, lysate and exosomes are able to inhibit UVB-induced increased expression of inflammatory factor IL-1 beta. Furthermore, as can be seen from the D plot in fig. 10, the gene expression levels of matrix metalloproteinases MMP2 and MMP9 were significantly increased after UVB irradiation, while YG3912 fermentation filtrate, lysate and exosome treatment significantly down-regulated the gene expression levels, indicating that YG3912 fermentation filtrate, lysate and exosome significantly inhibited UVB-induced increase in gene expression of MMP2 and MMP 9.
From the results, YG3912 fermentation filtrate, lysate and exosome can significantly inhibit the increase of the expression of inflammatory factors IL-6, IL-8 and IL-1 beta caused by UVB irradiation, and can relieve abnormal increase of the gene level of matrix metalloproteinase MMP2 and MMP9 caused by UVB, thereby playing a role in relieving the photo-aging of cells induced by UVB.
(3) YG3912 fermentation filtrate, lysate and exosomes alleviate UVB-induced HaCaT cell ROS content
HaCaT cells were cultured to log phase and cells were collected. A normal control group (Con), a model group (UVB) and a treatment group (S-10≡4, R-3 x 10≡4 and W-3000) are arranged in the 12-well plate, the concentration of the cell suspension is adjusted to 1.5X10 5 cells/mL, and each well is inoculated with 1mL of the cell suspension. The cell culture plates were incubated in a 5% CO 2, 37℃incubator for 24h.
After 24h incubation, the cells grew to a monolayer, the medium was removed, washed 2 times with sterile PBS, and 1mL of sterile PBS was added to each well. The control group was coated with tinfoil to avoid exposure to UVB. The model group and the treatment group were given UVB irradiation for 40s, respectively. After irradiation, 1mL of complete medium was added to each of the control and model groups, and 1mL of complete medium containing 10-4 times of YG3912 fermentation filtrate (i.e., the "S-10-4" group) or 3X 10-4 times of YG3912 lysate (i.e., the "R-3X 10-4" group) or 3000 times of YG3912 exosomes (i.e., the "W-3000" group) was added to the treatment group. After 24h incubation, changes in cell morphology were observed with a microscope (20×), after 2 washes with sterile PBS, intracellular ROS fluorescence levels were determined by DCFH-DA fluorescence using a reactive oxygen species detection kit (purchased from S0033S, bi-cloud days), and the relative fluorescence intensities of intracellular ROS were quantified using Image J to assess the effect of YG3912 fermentation filtrate, lysate, and exosomes on UVB-induced cellular ROS levels.
The results are shown in FIG. 11.
As can be seen from the a-graph in fig. 11:
1) Under the 20X microscope field of view, UVB causes large-area cells to fall off, the fusion degree is poor, and the treatment of YG3912 fermentation filtrate, lysate and exosomes causes the cell state to be recovered, which indicates that the YG3912 fermentation filtrate, lysate and exosomes have the function of repairing cell injury;
2) UVB can result in a significant increase in intracellular ROS levels under a 20 Xmicroscopic field of view, while YG3912 fermentation filtrate, lysate and exosome treatment can significantly inhibit UVB-induced increases in cellular ROS levels. This conclusion can also be drawn from the statistics of figure 11, panel B.
Photoaging is an important aspect of skin aging, and involves damage to skin cells by ultraviolet light, resulting in problems of skin pigmentation, reduced elasticity, increased wrinkles, and the like. Ultraviolet rays can destroy the natural barrier of skin, trigger the generation of free radicals, and further accelerate the skin aging process. And the extract of strain YG3912 (including fermentation filtrate, exosomes and lysates) can significantly improve cell morphology disorder, fusion degree deterioration and abnormal elevation of inflammatory factors and matrix metalloproteinases caused by UVB irradiation, thereby having an anti-photoaging effect. Thus, YG3912 strain or its extract can be used for developing natural anti-photoaging skin care product with economic benefit.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.
Claims (10)
1. A bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 is characterized in that the bifidobacterium adolescentis is preserved in the China general microbiological culture Collection center with the preservation date of 2024, 6 and 20 days and the preservation number of CGMCC No.31020.
2. The inactivated cells, extracts, cultures, culture supernatants, ferments, and/or fermentation supernatants of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 of claim 1.
3. The inactivated thallus, extract, culture supernatant, ferment and/or ferment supernatant of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to claim 2, characterized in that the extract of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 is lysate and/or exosome extracted therefrom.
4. The inactivated bacteria, extract, culture supernatant, fermentation product and/or fermentation supernatant of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to claim 3, wherein the extraction method of the lysate comprises the following steps:
Fermenting and culturing the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912, centrifuging fermentation liquor, collecting thalli, re-suspending the thalli by PBS and breaking cell walls by ultrasonic or enzymolysis treatment to obtain lysate;
And/or the extraction method of the exosome comprises the following steps:
Fermenting and culturing the bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912, centrifuging the fermentation liquid, collecting fermentation supernatant, centrifuging the obtained fermentation supernatant, filtering, and concentrating to obtain exosomes.
5. The inactivated bacteria, extract, culture supernatant, fermentation product and/or fermentation supernatant of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to claim 4, wherein the centrifugation procedure of the fermentation supernatant is that the fermentation supernatant is centrifuged for 8-15min at 200-400 g, then for 15-30min at 1500-3000 g and finally for 20-40min at 8000-12000 g;
and/or filtering the fermentation supernatant by adopting a filter membrane with the pore diameter of 0.2-2.0 mu m;
And/or concentrating the fermentation supernatant by adopting 400-600 kd hollow fiber.
6. A composition comprising as an active ingredient the inactivated cells, extracts, cultures, culture supernatants, ferments and/or fermentation supernatants of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to claim 1 and/or bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 according to any of claims 2 to 5.
7. The composition of claim 6, wherein the composition is a pharmaceutical or cosmetic composition, further comprising a pharmaceutically or cosmetically acceptable carrier and/or excipient.
8. The composition according to claim 6 or 7, wherein the composition is in a topical formulation selected from the group consisting of aqueous solutions, emulsions, ointments, creams, gels, powders, oils.
9. The composition of claim 6 or 7, further comprising other active agents including any one or more of antioxidants, cellular active agents, moisturizing agents, anti-aging agents, and anti-dermatitis agents.
10. Use of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 as defined in claim 1 or of an inactivated thallus, extract, culture supernatant, fermented product and/or fermented supernatant of bifidobacterium adolescentis (Bifidobacterium adolescentis) YG3912 as defined in any of claims 2-5, or of a composition comprising one or more of the foregoing, for the preparation of a product for use in:
1) Promoting skin repair;
2) Reducing, inhibiting or eliminating skin inflammation or allergy;
3) Anti-skin aging;
4) Enhancing skin immunity.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119530099A (en) * | 2025-01-20 | 2025-02-28 | 四川厌氧生物科技有限责任公司 | Bifidobacterium adolescentis beneficial to skin and derivative and application thereof |
| CN119530100A (en) * | 2025-01-20 | 2025-02-28 | 四川厌氧生物科技有限责任公司 | Lactobacillus polymorphus composition for improving skin and derivatives and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102296290B1 (en) * | 2020-12-31 | 2021-09-01 | 주식회사 바이오뱅크힐링 | Bifidobacterium adolescentis strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof |
| CN114292773A (en) * | 2021-12-07 | 2022-04-08 | 上海珈凯生物科技有限公司 | Bifidobacterium adolescentis as well as screening and culturing method and application thereof |
| CN115537361A (en) * | 2022-11-08 | 2022-12-30 | 廖梅香 | Bifidobacterium adolescentis and its application |
| CN116875516A (en) * | 2023-09-06 | 2023-10-13 | 上海上药信谊药厂有限公司 | Bifidobacterium adolescentis BA-3 and its application in resisting aging, oxidation and inflammation |
| CN117050907A (en) * | 2023-08-10 | 2023-11-14 | 深圳施诺华生物科技有限公司 | Combined fecal bacteria for improving skin aging, fecal bacteria transplanting capsule and application thereof |
-
2024
- 2024-07-26 CN CN202411012565.1A patent/CN119101614A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102296290B1 (en) * | 2020-12-31 | 2021-09-01 | 주식회사 바이오뱅크힐링 | Bifidobacterium adolescentis strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof |
| CN114292773A (en) * | 2021-12-07 | 2022-04-08 | 上海珈凯生物科技有限公司 | Bifidobacterium adolescentis as well as screening and culturing method and application thereof |
| CN115537361A (en) * | 2022-11-08 | 2022-12-30 | 廖梅香 | Bifidobacterium adolescentis and its application |
| CN117050907A (en) * | 2023-08-10 | 2023-11-14 | 深圳施诺华生物科技有限公司 | Combined fecal bacteria for improving skin aging, fecal bacteria transplanting capsule and application thereof |
| CN116875516A (en) * | 2023-09-06 | 2023-10-13 | 上海上药信谊药厂有限公司 | Bifidobacterium adolescentis BA-3 and its application in resisting aging, oxidation and inflammation |
Non-Patent Citations (1)
| Title |
|---|
| GAO, T.等: "The Role of Probiotics in Skin Health and Related Gut–Skin Axis: A Review", MDPI, vol. 15, 13 July 2023 (2023-07-13), pages 3123 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119530099A (en) * | 2025-01-20 | 2025-02-28 | 四川厌氧生物科技有限责任公司 | Bifidobacterium adolescentis beneficial to skin and derivative and application thereof |
| CN119530100A (en) * | 2025-01-20 | 2025-02-28 | 四川厌氧生物科技有限责任公司 | Lactobacillus polymorphus composition for improving skin and derivatives and application thereof |
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