CN115537361A - Bifidobacterium adolescentis and application thereof - Google Patents
Bifidobacterium adolescentis and application thereof Download PDFInfo
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- CN115537361A CN115537361A CN202211390033.2A CN202211390033A CN115537361A CN 115537361 A CN115537361 A CN 115537361A CN 202211390033 A CN202211390033 A CN 202211390033A CN 115537361 A CN115537361 A CN 115537361A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
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- Mycology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Molecular Biology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Birds (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to bifidobacterium adolescentis and application thereof. The invention provides Bifidobacterium adolescentis (Bifidobacterium adolensis) with a preservation number of CCTCC NO: M20221223 and application thereof. Experiments show that the bifidobacterium adolescentis provided by the invention has excellent effects on promoting cell proliferation, repairing skin barriers, resisting aging and the like, and is suitable for preparing foods, medicines, cosmetics and the like for improving related functions of skin.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bifidobacterium adolescentis and application thereof.
Background
The epidermis acts as a line separating the body from the external environment and plays an important barrier role in resisting external stimuli and preserving moisture inside the skin. The epidermis not only prevents microorganisms, fungi, viruses, allergens, etc. from entering the skin, but also absorbs ultraviolet radiation and reduces mechanical damage. Briefly, the skin barrier functions to prevent the loss of water and electrolytes from the body and to maintain an intrinsic steady state in response to changes in the external environment. We can intuitively understand skin barriers as guarding city walls, damaged barriers like through-wind holes, and thus cause a series of skin problems when the skin barrier is damaged.
Meanwhile, the phenomenon of premature skin aging caused by exposure of the skin to UV radiation due to long-term exposure to sunlight is called a photo-aging phenomenon of the skin. The activity of keratinocyte of the epidermal layer of the photoaging skin is reduced, the renewal speed is reduced, the barrier function of the epidermis is weakened, and the skin is dry and peeled; the dermal layer has a reduced number of fibroblasts, and collagen and elastin synthesis is slowed down and breakdown is accelerated. Besides a reduction in the aesthetic appearance of aged skin, its function of protecting against damage is also significantly impaired: the integrity of the skin barrier is destroyed after excessive UV irradiation, the secretion function is obviously reduced, and the risk of skin inflammation and even skin malignant tumor is obviously increased.
Skin aging is associated with the huge micro-ecological flora system on the skin surface in addition to the above physiological changes. It has been shown that microorganisms living on the skin surface can increase peroxidase activity or scavenge ROS, reducing oxidative damage to the skin following UV irradiation. At the same time. The microorganism can also directly regulate the expression level of matrix metalloproteinase of skin cells by influencing a plurality of signal paths, and reduce the degradation of collagen and elastin after UV irradiation, thereby delaying skin aging.
Therefore, the skin microecological related product developed by utilizing the microecological technology is provided, and has wide application value in the aspect of improving the skin state.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide bifidobacterium adolescentis and its application in products for repairing skin barrier and delaying skin aging.
The invention provides Bifidobacterium adolescentis (Bifidobacterium adolescentis) with a preservation number of CCTCC NO: M20221223.
The bifidobacterium adolescentis is a gram-positive strain screened from feces of healthy girls in five years old, and experiments show that the bifidobacterium adolescentis has excellent effects on promoting cell proliferation, repairing skin barriers, resisting aging and the like.
Furthermore, the invention provides an application of the bifidobacterium adolescentis in preparing a product for promoting cell proliferation.
Still further, the cells include skin keratinocytes and/or fibroblasts.
In some specific embodiments, the invention uses HaCaT skin keratinocytes as a subject, and studies the cell proliferation promoting effect of the bifidobacterium adolescentis. The result shows that the bifidobacterium adolescentis has the function of promoting the proliferation of skin keratinocytes HaCaT, and the proliferation promoting rate is 9.02-44.80%.
In some specific embodiments, the HFF fibroblasts are used as the subject to study the cell proliferation promoting effect of the bifidobacterium adolescentis. The result shows that the bifidobacterium adolescentis has the function of promoting the proliferation of human fibroblast HFF, and the proliferation promoting rate is 19.88 to 30.63 percent.
Further, the invention provides an application of the bifidobacterium adolescentis in preparing a product for repairing a skin barrier.
Still further, the rejuvenating skin barrier includes at least one of:
1) Recovering the cell activity;
2) Promoting expression of a barrier repair-associated gene comprising at least one of OVOL1 and OCLN.
In some specific embodiments, the HaCaT keratinocytes are used as a subject, and the SDS damage repair capacity of the bifidobacterium adolescentis is studied, so that the bifidobacterium adolescentis can repair SDS-induced cell damage, increase the cell proliferation rate, and up-regulate the expression of non-damaged cell barrier repair-related genes.
Further, the invention provides an application of the bifidobacterium adolescentis in preparing an anti-aging product.
Still further, the anti-aging includes at least one of:
promoting expression of extracellular matrix-associated genes, the extracellular matrix-associated genes including at least one of MKX and COL13 A1;
ii, inhibiting expression of a gene associated with degrading extracellular matrix, said gene associated with degrading extracellular matrix comprising at least one member of the MMP family;
iii, promoting the expression of a cell antioxidant-related gene SIRT-1;
iv, promoting the expression of an immunomodulator related gene MOR;
v, promoting the expression of the gene BCL-2 related to the inhibition of the apoptosis;
vi, promoting the expression of the autophagy-related gene LC 3B;
vii, inhibiting expression of an apoptosis-related gene comprising at least one of BAX and Caspase-3;
viii, promoting the expression of a cell growth factor-related gene comprising at least one of FGF1, FGF2 and FGF 7.
In order to explore the anti-aging effect of the bifidobacterium adolescentis, indexes related to cell aging are measured, wherein the indexes comprise at least one of extracellular matrix synthesis, extracellular matrix degradation, cellular antioxidation, cellular immunoregulatory factors, apoptosis, autophagy, apoptosis inhibition and cell growth factors.
According to the invention, haCaT keratinocytes are taken as a subject, and the influence of the bifidobacterium adolescentis on HaCaT extracellular matrix degradation related genes is researched. The result shows that the bifidobacterium adolescentis can reduce and degrade the expression of extracellular matrix related genes MMP1 and inhibit the degradation of extracellular matrix.
The invention takes HaCaT keratinocytes as a subject, and researches the influence of the bifidobacterium adolescentis on HaCaT cell autophagy. The result shows that the bifidobacterium adolescentis can up-regulate the expression of the autophagy-related gene LC3B and promote autophagy of cells to eliminate aged cells.
The invention takes HFF fibroblasts as subjects, and researches the influence of the bifidobacterium adolescentis on synthesis related genes and degradation related genes of HFF extracellular matrix. The result shows that the bifidobacterium adolescentis can up-regulate the expression of extracellular matrix synthesis related genes COL13A1 and MKX, promote the synthesis of the extracellular matrix, down-regulate the expression of extracellular matrix related genes MMP3 and MMP10 and inhibit the degradation of the extracellular matrix.
The invention takes HFF fibroblasts as a subject and researches the influence of the bifidobacterium adolescentis on the apoptosis of the HFF cells. The result shows that the bifidobacterium adolescentis can reduce the expression of apoptosis related genes BAX and Caspase-3 and inhibit apoptosis.
The invention takes HFF fibroblasts as subjects, and researches the influence of the bifidobacterium adolescentis on the inhibition of apoptosis of the HFF cells. The result shows that the bifidobacterium adolescentis can up-regulate the expression of apoptosis-related gene BCL-2 and inhibit apoptosis.
The invention takes HFF fibroblasts as subjects and researches the influence of the bifidobacterium adolescentis on the expression of the antioxidant related genes of the HFF cells. The result shows that the bifidobacterium adolescentis can up-regulate the expression of an anti-oxidation related gene SIRT-1 and enhance the anti-oxidation capability of cells.
The invention takes HFF fibroblasts as subjects and researches the influence of the bifidobacterium adolescentis on the expression of genes related to the HFF cell immune regulatory factor. The result shows that the bifidobacterium adolescentis can up-regulate the expression of the gene MOR related to the immunoregulatory factor and enhance the immunoregulatory capacity of cells.
The invention takes HFF fibroblasts as subjects and researches the influence of the bifidobacterium adolescentis on the expression of genes related to the HFF cell growth factors. The result shows that the bifidobacterium adolescentis can up-regulate the expression of cell growth factor related genes FGF1, FGF2 and FGF7 and enhance the growth capacity of cells.
The invention also provides a product for improving skin conditions, and the raw materials of the product comprise the bifidobacterium adolescentis.
Further, the product comprises one or two of the following components I or II:
i, fermentation filtrate and/or fermentation lysate of the bifidobacterium adolescentis;
II, culture, exosome, lysate and/or extract of the bifidobacterium adolescentis.
Furthermore, the dosage form of the product of the present invention includes, but is not limited to, at least one of creams, emulsions, oils, aqueous solutions, gels, powders, and lyophilized powders, which is not limited in this respect.
The present invention also provides a method of improving skin condition comprising: the product of the invention is used. The application method of the product comprises oral administration or external application, the external application comprises smearing, external application, fumigation or injection, and the invention is not limited to the application.
The invention provides bifidobacterium adolescentis with a preservation number of CCTCC NO: M20221223. The bifidobacterium adolescentis provided by the invention has excellent effects on promoting cell proliferation, repairing skin barriers, resisting aging and the like, and is suitable for preparing foods, medicines, cosmetics and the like for improving related functions of skin.
Description of biological preservation
Bifidobacterium adolescentis ProfMIC-225 (Bifidobacterium adolensis ProfMIC-225) was deposited at the China center for type culture Collection at 8 months and 3 days in 2022 at the address of M20221223, CCTCC NO of Wuhan university in China.
Detailed Description
The invention provides bifidobacterium adolescentis and application thereof, and can be realized by appropriately improving process parameters by referring to the content in the text by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The Bifidobacterium adolescentis strain ProfMIC-225 is derived from feces of five-year-old healthy girls and is identified as Bifidobacterium adolescentis (Bifidobacterium adolescentis) through 16S rDNA. The strain is gram-positive and rod-shaped under a microscope; the bacterial colony grows on a BBL flat plate, can form a round bacterial colony with a smooth and opaque surface, is white and has a regular edge; the strain grows uniformly and turbulently in a BBL liquid culture medium, and the strain is white precipitate after being placed for a long time, and the optimal growth temperature is 37 ℃.
Bifidobacterium adolescentis ProfMIC-225 (Bifidobacterium adolensis ProfMIC-225), deposited in the collection: china center for type culture Collection, address: in the Wuhan university school of Wuhan 299 in the Wuchang area of Wuhan city, hubei province, the preservation date is as follows: 8 months and 3 days in 2022, the preservation number is CCTCC NO: M20221223.
Further, the bifidobacterium adolescentis ProfMIC-225 provided by the invention is present in the use or product according to the invention in a form that is live or dead or subjected to intermittent sterilization, or in the form of a lysate and/or extract, or in the form of a bacterial product or in the form of a fermentation filtrate or in the form of a derivative, preferably selected from: metabolites, metabolic biological products, exosomes, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation filtrate, fermentation lysate.
The test materials adopted by the invention are all common commercial products and can be purchased commercially, and the invention is further explained by combining the following embodiments:
EXAMPLE 1 isolation of ProfMIC-225
Sampling in feces of five-year-old healthy girls. Properly processing the sample, uniformly mixing the sample in normal saline by shaking, taking the supernatant, streaking the supernatant on a BBL solid plate, culturing the supernatant at the constant temperature of 37 ℃ for 48 hours, and selecting a white colony for repeated inoculation and screening until a uniform single colony is obtained, wherein the uniform single colony is named as ProfMIC-225.
Gram staining microscopy: the strain ProfMIC-225 is gram-positive and rod-shaped under a microscope; growing on a BBL flat plate to form white round microcolonies with smooth, mellow and non-transparent surfaces and regular edges; the bacteria grow uniformly and turbulently in BBL liquid culture medium, and the white bacteria precipitate after long-term placement.
Example 2 nucleic acid identification of ProfMIC-225
1. 16S rDNA gene sequence analysis:
picking single colony to be placed in BBL liquid culture medium, culturing overnight at 37 ℃, then rotating and centrifuging for 1min at 12000 ℃ to collect thalli, and operating according to the steps of a DNA extraction kit. The primers adopt bacterial universal primers 27F and 1492R, a PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 5min at 95 ℃;94 ℃ 15s,57 ℃ 15s,72 ℃ 40s,35 cycles; extension at 72 ℃ for 10min.
2. Results
The homology comparison (BLASTN) of the sequencing result of the PCR product and the standard sequence published in GenBank results in that the ProfMIC-225 strain is Bifidobacterium adolescentis (Bifidobacterium adolensis).
Example 3 ProfMIC-225 promotion of SDS-induced HaCaT Damage repair assay in human immortalized keratinocytes
1. ProfMIC-225 fermentation filtrate and fermentation lysate preparation:
selecting a single colony of the bifidobacterium adolescentis ProfMIC-225 to a BBL liquid culture medium, carrying out static culture in an anaerobic incubator at 37 ℃ for 16-18 h, and diluting with PBS to adjust OD 600 And (3) inactivating at 121 ℃ for 30min under high pressure, centrifuging at 12000 rpm for 2min, and filtering the supernatant with a 0.22 μm filter membrane to obtain fermentation filtrate. Washing the centrifugally precipitated thallus twice with PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial mud, re-suspending with PBS to the centrifugal volume, crushing with ultrasonic wave for 30min, and deactivating at 121 deg.c for 30min to obtain fermented lysate.
2. Experiment for promoting HaCaT cell repair
Inoculation of HaCaT cells (5X 10) 4 cells/well) to 96-well plates and cultured overnight until cells adhere. SDS was prepared at 50. Mu.g/ml, and 100. Mu.l of SDS was added to each well, and the mixture was incubated at 37 ℃ for 8 hours in a 5% carbon dioxide incubator. Each well was incubated for 24h with 5% fermentation filtrate and 10% fermentation lysate (control with equal volume of PBS instead of fermentation filtrate/fermentation lysate, respectively). Mu.l of CCK-8 solution was added to each well, incubated for 4h, and absorbance A at 450nm was measured. CellsThe formula for calculating the proliferation rate and the results are shown in Table 1.
TABLE 1 ProfMIC-225 promotes HaCaT cell proliferation
The results in the above table show that both the ProfMIC-225 fermentation filtrate and the fermentation lysate can promote and repair the damage of HaCaT keratinocytes caused by SDS, and increase the cell proliferation rate, which is 109.02% to 144.80%.
Example 4 ProfMIC-225 promotion of HaCaT Barrier repair-related Gene expression experiments
1. Preparation of ProfMIC-225 fermentation lysate:
the preparation method refers to example 3.
2. Experiment for promoting HaCaT barrier repair related gene expression
Inoculation of human immortalized keratinocytes HaCaT (2 ml/well, 5X 10 content) 5 Cells) to a 6-well plate, and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator until the cells adhere to the wall. Adding 10% (V/V) of fermentation lysate (the control group is replaced by PBS with the same volume), culturing for 24h, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, performing reverse transcription to obtain cDNA, and detecting the expression of OVOL1 and OCLN genes by real-time qPCR (quantitative polymerase chain reaction) by taking GAPDH as an internal reference gene. Control group treated with PBS added at equal volume (gene relative expression fold F = 1) using 2 -ΔΔCT The F value of each sample was calculated.
The formula: f =2 -ΔΔCT Wherein:
△CT experiment of ＝CT Experiment of -CT Internal reference (experiment) ；
△CT Control ＝CT Control -CT Internal reference (contrast) ；
△△CT＝△CT Experiment of -△CT Control 。
The results are shown in Table 2.
TABLE 2 expression of repair genes upregulated by ProfMIC-225 fermentation lysates
In vitro cell experiments show that the bifidobacterium adolescentis ProfMIC-225 fermentation lysate has the function of up-regulating the expression of a skin barrier repair related factor OVO-like transcription factor 1 gene OVOL1 and a tight junction protein gene OCLN, and the gene expression level is up-regulated by 1.27 to 1.89 times. The PROFMIC-225 is shown to have the effect of promoting skin barrier repair.
Example 5: profMIC-225 experiment for regulating photoaging HaCaT cell autophagy/degradation extracellular matrix related gene expression
1. Preparation of ProfMIC-225 fermentation filtrate and fermentation lysate:
the preparation method refers to example 3.
2. HaCaT cell preparation and ultraviolet ray damage
HaCaT cells were digested and then dispensed at 0.5 ml/well (2X 10 contents) 5 Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. Total dose of 2J/cm was applied to cells in the wells 2 Ultraviolet UVB radiation damage.
3. ProfMIC-225 addition
5% (V/V) of the fermentation filtrate and 10% (V/V) of the fermentation lysate were added to the stimulated HaCaT cells (control group replaced the volume of PBS for the fermentation filtrate/fermentation lysate, respectively). Each group 3 was incubated overnight at 37 ℃.
4. qPCR method for detecting relative expression multiple of relevant genes of autophagy/degradation extracellular matrix of cells
Removing the culture medium of the cells, adding a lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, taking GAPDH as an internal reference gene, and detecting the expression of the autophagy-related gene LC3B of the cells and the expression of the degradation extracellular matrix-related gene MMP1 by adopting real-time qPCR. Relative expression multiple F =1 of control group gene, using 2 -ΔΔCT The F value of each sample was calculated.
The fermentation filtrate can reduce and degrade extracellular matrix related gene MMP1. The results are shown in Table 3.
TABLE 3 ProfMIC-225 fermentation filtrates downregulating expression of extracellular matrix-associated genes
The fermentation lysate upregulates the autophagy gene LC3B. The results are shown in Table 4.
TABLE 4 expression of genes associated with autophagy in cells upregulated by ProfMIC-225 fermentation lysates
In vitro cell experiments show that the bifidobacterium adolescentis ProfMIC-225 has the function of up-regulating the expression of a microtubule-associated protein 1 light chain 3 beta gene LC3B related to HaCaT cell autophagy, and the relative expression multiple of the gene is 1.31-1.48; has the function of down-regulating the expression of matrix metalloproteinase family gene MMP1 related to degrading extracellular matrix, and the relative expression multiple of the gene is 0.36-0.70.
Example 6: profMIC-225 promotes the proliferation of HFF in human fibroblasts
1. Preparation of ProfMIC-225 fermentation filtrate and fermentation lysate:
the preparation method is referred to example 3.
2. HFF cell preparation and ProfMIC-225 addition
HFF cells cultured with DMEM were seeded at 100 ul/well (3X 10 cells per well) 4 Cells) were transferred to 96-well plates and cultured overnight until cells attached. Formulation 200. Mu. M H 2 O 2 Mu.l of the culture medium was added to each well, and the culture was carried out at 37 ℃ for 1 hour in a 5% carbon dioxide incubator. The original medium was discarded, PBS was washed twice, 100. Mu.l of fresh medium was added, 5% (V/V) of the fermentation filtrate and 10% (V/V) of the fermentation lysate were added to each well, respectively, (control group replaced the fermentation filtrate and the fermentation lysate with equal volume of PBS, respectively). And culturing for 24h. Adding 10 μ l CCK-8 solution into each well, culturing for 4 hr, and detectingThe absorbance at 450nm is A. The calculation formula and the results are shown in table 5.
TABLE 5 ProfMIC-225 promotes HFF cell proliferation
In vitro cell experiments show that the bifidobacterium adolescentis ProfMIC-225 fermentation filtrate and fermentation lysate both have the effect of promoting human fibroblast HFF proliferation, and the proliferation rate is 119.88-130.63%.
Example 7: profMIC-225 experiment for regulating oxidation damage to HFF extracellular matrix/antioxidation/immunoregulation factor/inhibition of apoptosis/cell growth factor/degradation of extracellular matrix/apoptosis related gene expression
1. Preparation of ProfMIC-225 fermentation filtrate and fermentation lysate:
the preparation method refers to example 3.
2. HFF cell preparation and H 2 O 2 Inducing oxidative damage
HFF cells cultured in DMEM were digested at a volume of 0.5 ml/well (2X 10 cells contained therein) 5 Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. H was added to each well to a final concentration of 200. Mu.M 2 O 2 Stimulating, and standing at 37 ℃ for 1h.
3. ProfMIC-225 addition
The fermentation filtrate 5% (V/V), fermentation lysate 10% (V/V) were added to the stimulated HFF cells (control groups replaced equal volumes of PBS for fermentation filtrate/fermentation lysate, respectively). Each group 3 was incubated overnight at 37 ℃.
4. qPCR method for detecting relative expression multiple of extracellular matrix/antioxidation/immunoregulation factor/inhibition apoptosis/cell growth factor/degradation extracellular matrix/apoptosis related gene
Removing the culture medium of the cells, adding lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, detecting extracellular matrix related genes MKX and COL13A1 and antioxidant related gene SIRT by using GAPDH as an internal reference gene and adopting real-time qPCR-1, an immunomodulatory factor MOR, an apoptosis-related gene BCL-2, cell growth factor-related genes FGF1, FGF2 and FGF7, degradation of extracellular matrix-related genes MMP3 and MMP10, and expression of apoptosis-related genes BAX and Caspase-3. Relative expression multiple F =1 of control group gene, using 2 -ΔΔCT F value was calculated for each sample.
The fermentation filtrate up-regulates extracellular matrix related genes MKX, antioxidant related genes SIRT-1, immunoregulation factor related genes MOR, apoptosis inhibiting genes BCL-2, cell growth factor related genes FGF1, FGF2 and FGF7; the apoptosis-related gene BAX was down-regulated, and the results are shown in Table 6.
TABLE 6 ProfMIC-225 fermentation filtrates Regulation of extracellular matrix/antioxidant/immunomodulatory factors/inhibition of apoptosis/cell growth factors/expression of apoptosis-related genes
The fermentation lysate up-regulates extracellular matrix related genes COL13A1 and MKX, an immune regulator related gene MOR, an apoptosis-inhibiting related gene BCL-2, and cell growth related genes FGF1, FGF2 and FGF7; down-regulating and degrading extracellular matrix related genes MMP3 and MMP10, and apoptosis related genes BAX and Caspase-3. The results are shown in Table 7.
TABLE 7 ProfMIC-225 fermentation lysates to modulate the expression of extracellular matrix/immunomodulatory factors/inhibit apoptosis/cell growth factors/degrade extracellular matrix/apoptosis-related genes
In vitro cell experiments show that the bifidobacterium adolescentis ProfMIC-225 has the effects of up-regulating the expression of collagen membrane protein 13 alpha chain gene COL13A1 and mohoke protein gene MKX related to HFF extracellular matrix, beta-endorphin receptor gene MOR related to immunoregulatory factors, sirtuins protein family gene SIRT-1 related to antioxidation, B-lymphocytoma-2 gene BCL-2 related to inhibition of apoptosis and fibroblast growth factor genes FGF1 and FGF2 related to cell growth factors and keratinocyte growth factor gene FGF7, and the relative expression multiple of the genes is 1.11-6.30 times; has the functions of down-regulating matrix metalloproteinase family genes MMP3 and MMP10 related to degrading extracellular matrix, and cell apoptosis-related BCL2-Associated X protein gene BAX and cysteine proteinase family gene Caspase-3, and has the relative expression multiple of 0.43-0.78.
The bacterial strain has the functions of promoting cell proliferation, repairing skin barrier, preserving moisture and resisting aging, and can be used for preparing related foods, medicines, cosmetics and the like.
Example 8 preparation of Bifidobacterium adolescentis ProfMIC-225 fermentation filtrate granule
Preparation of ProfMIC-225 fermentation filtrate granules, the formulation is shown in table 8.
TABLE 8 ProfMIC-225 ferment filtrate medicinal granules formulation table
The preparation process comprises the following steps: the ProfMIC-225 fermentation filtrate prepared in the example 3 is taken, spray-dried to prepare ProfMIC-225 fermentation powder, and then fish collagen, vitamin C, strawberry juice powder and silicon dioxide are added, and the ProfMIC-225 medicinal granules are obtained after even stirring and dispersion. The obtained instant granules of ProfMIC-225 is soaked in water, has sour and sweet taste, good palatability and convenient carrying, and can be used as oral beverage for caring skin.
Example 9 preparation of emulsion of Bifidobacterium adolescentis ProfMIC-225 fermentation lysate
Preparation of ProfMIC-225 emulsion, the formulation is shown in Table 9.
TABLE 9 ProfMIC-225 emulsion formulation Table
The preparation process comprises the following steps: mixing cyclopentasiloxane, vaseline, tween-80, vitamin E acetate, and glycerol, and heating to 75 deg.C to obtain phase A; then the ProfMIC-225 fermentation lysate prepared in example 3 is taken, added with xanthan gum, carbopol Ultrez 10, 1,3-butanediol and deionized water, mixed and dispersed evenly and heated to 75 ℃ as phase B; slowly adding the preheated phase A into the preheated phase B, homogenizing completely, adding triethanolamine to adjust the pH value to 6-7, stirring and cooling to 35 ℃. The emulsion of the ProfMIC-225 fermentation lysate is lubricated and comfortable.
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention.
Claims (10)
1. Bifidobacterium adolescentis (Bifidobacterium adolescentis) with a collection number of CCTCC NO: M20221223.
2. Use of a bifidobacterium adolescentis as claimed in claim 1 in the manufacture of a product for promoting cell proliferation.
3. The use of claim 2, wherein the cells comprise skin keratinocytes and/or fibroblasts.
4. Use of a bifidobacterium adolescentis as claimed in claim 1 for the preparation of a product for repairing skin barriers.
5. The use of claim 4, wherein the repair skin barrier comprises at least one of:
1) Recovering the cell activity;
2) Promoting expression of a barrier repair-associated gene comprising at least one of OVOL1 and OCLN.
6. Use of bifidobacterium adolescentis as claimed in claim 1 for the preparation of an anti-ageing product.
7. The use of claim 6, wherein the anti-aging comprises at least one of:
promoting expression of extracellular matrix-associated genes, the extracellular matrix-associated genes including at least one of MKX and COL13 A1;
ii, inhibiting expression of a gene associated with degradation of extracellular matrix, said gene associated with degradation of extracellular matrix comprising at least one member of the MMP family;
iii, promoting the expression of a cell antioxidant-related gene SIRT-1;
iv, promoting the expression of an immunomodulator related gene MOR;
v, promoting the expression of the gene BCL-2 related to the inhibition of the apoptosis;
vi, promoting the expression of cell autophagy related genes LC 3B;
vii, inhibiting expression of an apoptosis-related gene comprising at least one of BAX and Caspase-3;
viii, promoting the expression of a gene associated with a cellular growth factor comprising at least one of FGF1, FGF2 and FGF 7.
8. A product for improving skin conditions, the raw material of which comprises the bifidobacterium adolescentis of claim 1.
9. The product of claim 8, wherein the product comprises one or both of the following i or ii:
i, a fermentation filtrate and/or a fermentation lysate of Bifidobacterium adolescentis as claimed in claim 1;
II, a culture, exosome, lysate and/or extract of Bifidobacterium adolescentis according to claim 1.
10. The product of any of claims 8 or 9, wherein the product is in a dosage form comprising at least one of a cream, an emulsion, an oil, an aqueous, a gel, a powder, and a lyophilized.
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