CN116355770B - Debaryomyces hansenii and application thereof - Google Patents
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- 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
- A23L33/14—Yeasts or derivatives thereof
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- A—HUMAN NECESSITIES
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- 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
- A23L33/14—Yeasts or derivatives thereof
- A23L33/145—Extracts
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A61K36/064—Saccharomycetales, e.g. baker's yeast
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- 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
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
The invention discloses Debaryomyces hansenii and application thereof, and relates to the technical field of microorganisms. The invention discloses a Debaryomyces hansenii, which is Debaryomyces hansenii SEUNEU-124 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221433 in 2022, 9 months and 14 days. Experiments show that SEUNEU-124 has effects of promoting cell proliferation, repairing skin barrier, moistening skin, resisting aging, whitening skin, removing black skin, and refreshing aged cutin, and can be used for preparing food, medicine, and cosmetics.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Debaryomyces hansenii and application thereof.
Background
The skin barrier is a structural barrier formed by the epidermis-forming cells of the stratum corneum and the lipids between the horns. The skin barrier prevents excessive moisture release from the human body and prevents harmful substances such as chemicals or microorganisms from entering our body. The keratinocyte cortex, which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. However, as the age increases, the moisture content of the stratum corneum gradually decreases, and when the moisture content is less than 10%, various problems of the skin are caused.
Skin aging is 80% of photoaging caused by ultraviolet light, while natural aging is only 20%. At the same time, the proliferation of cells is reduced, the apoptosis of cells is increased, and the extracellular matrix components are obviously reduced along with the aging of skin. In the research of aging mechanism, it is found that autophagy can delay organ function degradation, and can repair DNA by removing damaged proteins and lipids in cells irradiated by UV, help cells recover metabolic homeostasis, and provide protection for photoaged damaged skin. In addition, it has been demonstrated that reducing apoptosis, reducing cell inflammation, increasing extracellular matrix synthesis and reducing degradation thereof, improving antioxidant capacity of cells, etc. can slow down skin cell aging.
Skin microorganisms are important members of the skin micro-ecosystem, and the flora on the skin surface can be generally divided into resident bacteria, which are a group of microorganisms colonizing healthy skin, and transient bacteria, which are a class of microorganisms obtained by contact with the external environment. Most microorganisms living on the skin appear to be symbiotic or reciprocal symbiotic under steady state conditions, and skin microorganisms play an important role in the maturation and homeostasis of skin immunity. Recent studies have demonstrated that the skin microbiota regulates the expression of various innate factors, skin resident bacteria are not just passive resident bacteria; they actively participate in host immunity through the intact skin barrier. The innate immune system of the skin in combination with the skin microbiota is a barrier of the human body against pathogenic microorganisms and opportunistic pathogens. The skin has self-renewing ability. Therefore, the probiotics related product developed by utilizing the microecology technology has important practical significance.
Disclosure of Invention
In view of the above, the invention provides Debaryomyces hansenii and application thereof.
The invention provides the Debaryomyces hanseniiDebaryomyces hansenii) The strain is Debaryomyces hansenii SEUNEU-124, and has been preserved in China center for type culture collection (CCTCC for short, address: eight paths 299 in Wuchang district of Wuhan, university of Wuhan, post code 430072) at 9 months and 14 days 2022, and has a preservation number of Debaryomyces hansenii, of which the CCTCC NO is M20221433.
It is a further object of the present invention to provide the use of the above-mentioned debaryomyces hansenii for the preparation of a product for improving skin conditions.
Further, the improving skin condition is at least one of promoting cell proliferation, repairing skin barrier, moisturizing, anti-aging, whitening, blacking, and refreshing aged keratinous materials.
In some embodiments, the pro-cell proliferation is an effect that promotes skin keratinocyte proliferation;
the repairing the skin barrier comprises restoring cell viability and/or up-regulating expression of a barrier repair related gene; the barrier repair related gene isOVOL1AndOCLNat least one of (a) and (b);
the moisturizing is up-regulating moisturizing related genesGBAIs expressed by (a);
the whitening and blacking effects are inhibition effect of cytomelanin generation.
In some embodiments, the anti-aging is at least one of the following a) -g):
a) Up-regulating the expression of genes involved in extracellular matrix synthesis, includingTIMP1、SPTSSA、LN、MKXAndCOL13A1at least one of (a) and (b);
b) Down-regulating the expression of genes related to degradation of extracellular matrix; the genes related to the degradation of the extracellular matrix compriseMMPAt least one of the family genes;
c) Down-regulating expression of apoptosis-related genes; the apoptosis-related genes includeBAXAnd/orCaspaseAny one or more of the families;
d) Up-regulating apoptosis-inhibiting related genesBCL-2Is expressed by (a);
e) Up-regulating cell antioxidant related geneNRF2、PTENAnd/orSIRT-1Is expressed by (a);
f) Up-regulating cell immunoregulatory factor related geneMORExpression of (2)
g) Up-regulating cell growth factor related geneFGF1、FGF2、FGF7、FGF21、HGFIs expressed by (a).
In some embodiments, the whitening and blacking comprises at least one of the following a) to f):
a) Down-regulating melanin synthesis-related genesMITF、AKT、GSK3βAnd/orP13KIs expressed by (a);
b) Down-regulating melanogenesis-related genesASIPIs expressed by (a);
c) Down-regulating melanin secretion-related genesEDNIs expressed by (a);
d) Down-regulating melanin transport-related genesRAB27A、SLAC2A、VaAnd/orHMGB1Is expressed by (a);
e) Down-regulating melanosome maturation-related geneOA1、MATPIs expressed by (a);
f) Down-regulating melanocyte migration and apoptosis-related genesE-cadherinIs expressed by (a).
In some embodiments, the up-regulation of the aging keratinocyte-associated gene promotes the renewal of the keratinocyteCDSNDown-regulating desmin-related genesDSG1、DSG3、DSC1Is expressed by (a).
In some embodiments, the product is a food product, a pharmaceutical product, or a cosmetic product.
It is another object of the present invention to provide a product for improving skin conditions, made from raw materials comprising debaryomyces hansenii as described above.
Further, debaryomyces hansenii in the above product comprises:
the flora and/or microbial inoculum of debaryomyces hansenii.
The invention discloses SEUNEU-124, which has a preservation number of CCTCC NO: M20221433. Experiments show that the Debaryomyces hansenii provided by the invention has the functions of promoting cell proliferation, repairing skin barrier, moisturizing, resisting aging, whitening, removing black and updating aged cutin, and can be used for preparing foods, medicines, cosmetics and the like.
Description of biological preservation
Hansenula debaryomyces hanseniiDebaryomyces hansenii) SEUNEU-124, deposited at 2022, 9, 14The China center for type culture collection (CCTCC, address is No. 299 of Wuchang district of Wuhan, university of Wuhan), and the preservation number is CCTCC NO: M20221433.
Detailed Description
The invention provides Debaryomyces hansenii and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The Debaryomyces hansenii strain SEUNEU-124 is derived from sugar cane molasses of the guest city of the Guangxi Zhang autonomous region, and is identified as Debaryomyces hansenii through fungus ITS gene sequence analysisDebaryomyces hansenii). The strain is single-cell, oval or spherical under a microscope; the growth on the wort agar plate can form a round colony with smooth and opaque surface, milky white and neat edge; the growth is uniform and turbid in malt juice liquid medium, and the optimal growth temperature is 24 ℃.
Hansenula debaryomyces hanseniiDebaryomyces hansenii) SEUNEU-124, accession number: china center for type culture Collection, address: in the Jiuqiu No. 299 university of Wuhan, hubei province, the date of preservation: and 2022, 9 and 14 days, wherein the preservation number is CCTCC NO: M20221433.
Further, the invention provides debaryomyces hansenii SEUNEU-124 in the use or product according to the invention, in the form of a live or dead or batch sterilized, or in the form of a lysate and/or extract, or in the form of a bacterial product, or in the form of a supernatant or derivative, preferably selected from: metabolites, metabolic biological products, exosomes, probiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from the group consisting of: supernatant and thallus lysate.
The test materials adopted in the invention are all common commercial products and can be purchased commercially, and the invention is further described below by combining examples:
example 1 isolation of SEUNEU-124
Sampling in cane molasses. After the sample is properly treated, shaking and uniformly mixing the sample in normal saline, streaking the supernatant on a wort agar plate, culturing the wort agar plate at the constant temperature of 24 ℃ for 16-24 hours, and then picking and repeatedly inoculating and screening milky white colonies until uniform single colonies are obtained, and the colony is named as SEUNEU-124.
Gram staining microscopy: the strain SEUNEU-124 is in a single cell, oval or spherical shape under a microscope; growing on the wort flat plate to form round colony with smooth and opaque surface, milky white and regular edge; the growth is uniform and turbid in malt juice liquid medium, and the optimal growth temperature is 24 ℃.
EXAMPLE 2 nucleic acid identification of SEUNEU-124
1. Fungal ITS gene sequence analysis:
single colony is selected and placed in malt juice liquid culture medium, after the culture is carried out at 24 ℃ overnight, the culture medium is centrifuged for 1min at 12000 revolutions to collect thalli, and the operation is carried out according to the step of extracting the yeast genome DNA kit. The primers adopt saccharomycete universal primers ITS1 and ITS4, and the PCR amplification system is a 15 mu L system, and the primers are pre-denatured for 5min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 60s,35 cycles; extending at 72℃for 7min.
2. Results
The PCR product sequencing result is compared with the published standard sequence in GenBank (BLASTN) to obtain SEUNEU-124 strain which is debaryomyces hanseniiDebaryomyces hansenii）。
Example 3 SEUNEU-124 promoting SDS-induced repair of HaCaT injury in human immortalized keratinocytes
1. Preparation of SEUNEU-124 supernatant:
selecting a single colony of Debaryomyces hansenii SEUNEU-124 in a malt juice liquid culture medium, shake culturing at 24 ℃ for 16-24 hours, detecting by an enzyme-labeling instrument, and diluting by PBS to adjust OD 600 The cells were separated and precipitated by centrifugation at 12000 rpm for 2min, the supernatant was removed, inactivated at 121 ℃ for 30min under high pressure, and filtered through a 0.22 μm filter membrane to obtain an inactivated supernatant.
2. Promoting HaCaT cell repair experiments
Inoculation of HaCaT cells (5×10) 4 cells/well) to 96-well plates, cultured overnight to cell attachment. 50. Mu.g/ml SDS was prepared, 100. Mu.l of each well was added thereto, and the mixture was incubated in a 5% carbon dioxide incubator at 37℃for 8 hours. Each well was incubated with 5% supernatant (V/V) (control was replaced with equal volume of PBS) for 24h. To each well, 10. Mu.l of CCK-8 solution was added, and the mixture was incubated for 4 hours, and absorbance A at 450nm was measured.
Cell proliferation rate calculation formula and results are shown in the following table:
as shown in the above table, both the SEUNEU-124 supernatant and the bacterial lysate can promote repairable HaCaT keratinocyte injury caused by SDS, increase the cell proliferation rate, and promote the proliferation rate to be 12.96-16.87%
Example 4SEUNEU-124 promoting HaCaT Barrier maintenance related Gene expression experiments
1. Preparation of SEUNEU-124 supernatant and bacterial lysate:
selecting a single colony of Debaryomyces hansenii SEUNEU-124 in a malt juice liquid culture medium, shake culturing at 24 ℃ for 16-24 hours, detecting by an enzyme-labeling instrument, and diluting by PBS to adjust OD 600 The cells were separated and precipitated by centrifugation at 12000 rpm for 2min, the supernatant was removed, inactivated at 121 ℃ for 30min under high pressure, and filtered through a 0.22 μm filter membrane to obtain an inactivated supernatant. The method comprises the steps of washing the centrifugally precipitated thalli twice by PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial mud, re-suspending the crushed bacterial mud to the volume of the centrifugally precipitated thalli by PBS, crushing the crushed thalli by ultrasonic waves for 20 minutes, and inactivating the crushed thalli at 121 ℃ for 30 minutes under high pressure to obtain a thalli lysate.
2. Promote HaCaT barrier repair related gene expression experiment
Inoculated human immortalized keratinocyte HaCaT (2 ml/well, 5X 10 contained therein) 5 Cells) to 6-well plate, 5% carbon dioxide incubator at 37 ℃ overnight until cells adhere to the wall. Respectively adding 5% (V/V) supernatant and 10% (V/V) thallus lysate (the control group respectively replaces supernatant and thallus lysate with equal volume PBS), culturing for 24 hr, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNA to obtain final productGAPDHAs reference gene, real-time qPCR detection was usedOVOL1AndOCLNexpression of the genes. The control group (relative gene expression fold f=1) was treated with PBS added in equal volume, using 2 -ΔΔCT The F value of each sample was calculated by the method.
The formula: f=2 -ΔΔCT Wherein:
△CT experiment =CT Experiment -CT Internal reference (experiment);
△CT control =CT Control -CT Internal control (control);
△△CT=△CT experiment -△CT And (3) controlling.
The results are shown in the following table:
SEUNEU-124 supernatant up-regulates the expression of repair genes
SEUNEU-124 thallus lysate up-regulates expression of repair gene
In vitro cell experiments show that the supernatant and the thallus lysate of the Debaryomyces hansenii SEUNEU-124 have OVO-like transcription factor 1 genes related to up-regulation skin barrier repairOVOL1And a claudin geneOCLNThe expression effect is that the gene expression quantity is up-regulated by 1.29-2.64 times. SEUNEU-124 has been shown to have an effect of promoting skin barrier repair.
Example 5SEUNEU-124 up-regulation of HaCaT moisture-retention-related Gene expression experiments
1. Preparation of SEUNEU-124 thallus lysate:
the preparation is described in example 4.
2. Up-regulating HaCaT moisture-preserving related gene expression experiment
Inoculated human immortalized keratinocyte HaCaT (2 ml/well, 5X 10 contained therein) 5 Cells) to 6-well plates, 5% carbon dioxide incubator at 37 ℃ overnight until cells adhere. Adding 10% (V/V) of cell lysate (the control group is replaced by equal volume PBS), culturing for 24 hr, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNA to obtain the final productGAPDHAs reference gene, real-time qPCR detection was usedGBAExpression of the genes. The control (relative gene expression fold f=1) was equal volume PBS treated group, using 2 -ΔΔCT The F value of each sample was calculated by the method.
The results are shown in the following table:
in vitro cell experiments show that the Debaryomyces hansenii SEUNEU-124 has glucose cerebrosidase gene related to up-regulation and moisture preservationGBAThe expression effect is that the gene expression quantity is up-regulated by 1.13-1.43 times. SEUNEU-124 has been shown to have skin moisturizing effects.
Example 6: SEUNEU-124 regulated light aging HaCaT extracellular matrix/antioxidant related Gene expression experiments
1. Preparation of SEUNEU-124 thallus lysate:
the preparation is described in example 4.
2. HaCaT cell preparation and ultraviolet injury
HaCaT cells were digested and then subjected to a reaction at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. The total dose of cells in the well was 2J/cm 2 Is damaged by ultraviolet UVB irradiation.
3. SEUNEU-124 addition
Cell lysates 10% (V/V) were added to each of the stimulated HaCaT cells (control group replaced cell lysates with equal volume of PBS). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of extracellular matrix/antioxidant related genes
Removing culture medium from above cells, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHAs reference genes, real-time qPCR was used to detect extracellular matrix-related genesTIMP1Antioxidant related genesNRF2Genes associated with degradation of extracellular matrixMMP1Is expressed by (a). Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
Up-regulating extracellular matrix gene by thallus lysateTIMP1，Antioxidant related geneNRF2The method comprises the steps of carrying out a first treatment on the surface of the Down-regulating degradation of extracellular matrix related genesMMP1. The results are shown in the following table:
in vitro cell experiments show that the Debaryomyces hansenii SEUNEU-124 has the function of up-regulating the tissue metalloproteinase inhibitor 1 gene related to the extracellular matrix of HaCaTTIMP1Antioxidation-related nuclear factor E2-related factor2NRF2The gene expression function, the relative expression times of the genes are 1.15-1.36; family of matrix metalloproteinases with downregulation of extracellular matrix degradationMMP1The gene expression function, and the relative expression multiple of the gene is 0.52-0.75.
Example 7: SEUNEU-124 regulated oxidative damage HFF extracellular matrix/apoptosis/antioxidant/immunomodulatory factor/cytokine expression experiments
1. Preparation of SEUNEU-124 supernatant and thallus lysate:
the preparation is described in example 4.
2. HFF cell preparation and H 2 O 2 Induced oxidative damage
After digestion of HFF cells in DMEM culture, the cells were subjected to digestion at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. H was added to each well at a final concentration of 200. Mu.M 2 O 2 Stimulation was performed and the mixture was allowed to stand at 37℃for 1 hour.
3. SEUNEU-124 addition
Supernatant 5% (V/V) and cell lysate 10% (V/V) were added to stimulated HFF cells, respectively (control group replaced supernatant/cell lysate with equal volume of PBS, respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of extracellular matrix/apoptosis/antioxidation/immunoregulation factor/cell growth factor related genes
Removing culture medium from above cells, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHAs reference genes, real-time qPCR was used to detect extracellular matrix-related genesSPTSSA、LN、MKXAndCOL13A1inhibiting apoptosis-related genesBCL-2Antioxidant related genesPTENAndSIRT-1immunomodulatory factor-related genesMOR，Cell growth factor related genesFGF1、FGF2、FGF7、FGF21AndHGFthe method comprises the steps of carrying out a first treatment on the surface of the Genes associated with degradation of extracellular matrixMMPFamily, apoptosis-related genesCaspaseFamily(s)BAX. Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
Supernatant up-regulates extracellular matrix-related genesSPTSSAAndCOL13A1，gene inhibiting apoptosisBCL-2Antioxidant related genesPTENAndSIRT-1immunomodulatory factor-related genesMOR，Cell growth factorFGF2The method comprises the steps of carrying out a first treatment on the surface of the Down-regulating degradation of extracellular matrix related genesMMPFamily, apoptosis-related genesCaspaseFamily(s)BAXThe results are shown in the following table:
up-regulating and inhibiting extracellular matrix related gene by thallus lysateLN、MKX、SPTSSAAndCOL13A1inhibiting apoptosis-related genesBCL-2Antioxidant related genesPTENAndSIRT-1immunomodulatory factor-related genesMORCell growth factor related genesFGF1、FGF2、FGF7、FGF21AndHGFthe method comprises the steps of carrying out a first treatment on the surface of the Down-regulating apoptosisRelated genesCaspase-3AndBAX. The results are shown in the following table:
in vitro cell experiments show that the Debaryomyces hansenii SEUNEU-124 has serine palmitoyltransferase genes related with up-regulation of HFF extracellular matrixSPTSSALaminin geneLNMo Huoke protein geneMKXAnd type XIII collagen alpha 1 chain geneCOL13A1Inhibiting apoptosis-related B lymphocytoma-2 geneBCL-2，Antioxidant related No. 10 chromosome deleted phosphatase and tensin homolog genePTENSirtuins protein family genesSIRT-1，Immunomodulatory factor-related beta-endorphin receptor genesMORAnd a fibroblast growth factor related to the cell growth factorFGF1Fibroblast growth factorFGF2Keratinocyte growth factorFGF7Fibroblast growth factorFGF21And hepatocyte growth factorHGFThe expression function, the relative expression times of genes are 1.11-2.99 times; matrix metalloproteinase family genes associated with downregulation degradation of extracellular matrixMMPApoptosis-related BCL2-Associated X protein genesBAXAnd cysteine protease family genesCaspaseThe expression function, the relative expression multiple of genes is 0.53-0.91.
Example 8 SEUNEU-124 Regulation of expression experiments of A875 human melanoma cell melanin synthesis/pheomelanin production/melanin secretion/melanin transport/melanosome maturation/melanocyte migration-related genes
1. Preparation of SEUNEU-124 supernatant:
the preparation is described in example 3.
2. Preparation of A875 human melanoma cells and ultraviolet injury
A875 human melanoma cells were digested and then subjected to 0.5 mL/well (containing 8.5X10 s) 4 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. The total dose of cells in the well was 1J/cm 2 Is irradiated by ultraviolet UVBInjury.
3. SEUNEU-206 addition
Supernatant 5% (V/V) was added to stimulated a875 human melanoma cells (control group replaced supernatant with equal volume of PBS) separately. Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of gene related to pheomelanin generation/melanin secretion/melanosome maturation/melanin transport/melanocyte migration
Removing culture medium from above cells, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHAs reference genes, real-time qPCR was used to detect melanin synthesis-related genesMITF、AKT、GSK3βAnd/orP13KGene involved in melanogenesisASIPMelanin secretion-related geneEDNMelanin transport-related genesRAB27A、SLAC2A、VaAnd/orHMGB1Gene related to melanosome maturationOA1、MATPMelanocyte migration-associated geneE-cadherinExpression of the genes. Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
The formula: f=2 -ΔΔCT Wherein:
△CT experiment =CT Experiment -CT Internal reference (experiment);
△CT control =CT Control -CT Internal control (control);
△△CT=△CT experiment -△CT And (3) controlling.
Supernatant down-regulating melanin synthesis-related genesMITF、AKT、GSK3βAnd/orP13KGene involved in melanogenesisASIPMelanin secretion-related geneEDNMelanin transport-related genesRAB27A、SLAC2A、VaAnd/orHMGB1Gene related to melanosome maturationOA1、MATPMelanocyte migration-associated geneE-cadherinExpression of the genes. The results are shown in the following table:
in vitro cell experiments show that the Debaryomyces hansenii SEUNEU-124 has the related transcription factor genes for down regulating melanin synthesis related microoculopathyMITF、Protein kinase B geneAKTGlycogen synthase kinase 3 beta geneGSK3βAnd phosphatidylinositol 3-kinase geneP13K、Pheomelanin generation related spiny mouse signal protein geneASIPEndothelin gene related to melanin secretionEDNMelanin transport-related GTPase family protein genesRAB27AMelanin avidin geneSLAC2A、Myosin geneVaAnd high mobility group protein B1 genesHMGB1Melanosome formation-related melanosome membrana protein geneOA1Gene membrane-associated transporter gene related to melanosome maturation-associated geneMATPCadherin gene related to melanocyte migrationE-cadherinAction of gene expression. The relative expression multiple of the genes is 0.31-0.76, which shows that SEUNEU-124 has the function of inhibiting melanin generation.
Example 9: experiment for regulating HaCaT cell to update and age cutin related gene expression by SEUNEU-124
1. Preparation of SEUNEU-124 supernatant and thallus lysate:
the preparation is described in example 4.
2. HaCaT cell preparation and ultraviolet photoaging
HaCaT cells were digested and then subjected to 0.5 mL/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. The total dose of cells in the well was 2J/cm 2 Uv UVB photoaging of (c).
3. SEUNEU-124 addition
The supernatant 5% (V/V) and the cell lysate 10% (V/V) were added to stimulated HaCaT respectively (control group replaced supernatant/cell lysate with equal volume of PBS respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of updated aging cutin related gene
Removing culture medium from above cells, adding lysate, and extractingDetecting the concentration and purity of total RNA in cells, and reverse transcribing the total RNA into cDNA to obtainGAPDHAs reference genes, real-time qPCR detection is adopted to promote updating of keratinocyte related genesCDSNThe method comprises the steps of carrying out a first treatment on the surface of the Desmoplakin related genesDSG1、DSG3、DSC1Expression of the genes. The control (relative gene expression fold f=1) was equal volume PBS treated group, using 2 -ΔΔCT The F value of each sample was calculated by the method.
Supernatant regulation of aging-associated genesCDSN、DSG3Is expressed by (a). The results are shown in the following table:
cell lysate regulating and updating aging cutin related genesDSG1、DSC1Is expressed by (a). The results are shown in the following table:
the results show that SEUNEU-124 has the functions of up-regulating and promoting the updating of keratinocyte related cornea chain protein geneCDSNUp-regulating the gene expression level by 1.21-1.31; and down-regulating desmosomal protein related gene desmosomal core protein 1 geneDSG1Desmosome core protein 3 geneDSG3、Keratin mucin geneDSCIThe expression effect and the gene expression quantity are regulated down by 0.57-0.80. Thus SEUNEU-124 has the effect of renewing aged cutin.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. Debaryomyces hanseniiDebaryomyces hansenii) The strain is Debaryomyces hansenii SEUNEU-124, and has been preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221433 in 2022 and 9 months and 14 days.
2. The use of debaryomyces hansenii according to claim 1 for the preparation of a medicament or cosmetic for improving skin conditions, wherein the improvement of skin conditions is at least one of promotion of skin keratinocyte HaCaT proliferation, repair of skin barrier, moisturization, anti-aging, whitening, blacking, renewal of aged keratinous tissue.
3. The use according to claim 2, wherein,
the repairing the skin barrier comprises restoring cell viability and/or up-regulating expression of a barrier repair related gene; the barrier repair related gene isOVOL1AndOCLNat least one of (a) and (b);
the moisturizing is up-regulating moisturizing related genesGBAIs expressed by (a);
the whitening and blacking effects are inhibition effect of cytomelanin generation.
4. The use according to claim 2, wherein the anti-aging is at least one of the following a) to g):
a) Up-regulating the expression of genes involved in extracellular matrix synthesis, includingTIMP1、SPTSSA、LN、MKXAndCOL13A1at least one of (a) and (b);
b) Down-regulating the expression of genes related to degradation of extracellular matrix; the genes related to the degradation of the extracellular matrix compriseMMPAt least one of the family genes;
c) Down-regulating expression of apoptosis-related genes; the apoptosis-related genes includeBAXAnd/orCaspaseAny one or more of the families;
d) Up-regulating apoptosis-inhibiting related genesBCL-2Is expressed by (a);
e) Up-regulating cell antioxidant related geneNRF2、PTENAnd/orSIRT-1Is expressed by (a);
f) Up-regulating cell immunoregulatory factor related geneMORExpression of (2)
g) Upregulation of cell growth factorsRelated genesFGF1、FGF2、FGF7、FGF21、HGFIs expressed by (a).
5. The use according to claim 2, wherein the whitening and blacking comprises at least one of the following a) to f):
a) Down-regulating melanin synthesis-related genesMITF、AKT、GSK3βAnd/orP13KIs expressed by (a);
b) Down-regulating melanogenesis-related genesASIPIs expressed by (a);
c) Down-regulating melanin secretion-related genesEDNIs expressed by (a);
d) Down-regulating melanin transport-related genesRAB27A、SLAC2A、VaAnd/orHMGB1Is expressed by (a);
e) Down-regulating melanosome maturation-related geneOA1、MATPIs expressed by (a);
f) Down-regulating melanocyte migration and apoptosis-related genesE-cadherinIs expressed by (a).
6. The use according to claim 2, wherein the up-regulation of the aging horny cell-associated gene promotes the up-regulation of the horny cell-associated geneCDSNDown-regulating desmin-related genesDSG1、DSG3、DSC1Is expressed by (a).
7. A pharmaceutical or cosmetic product for improving skin conditions, characterized by being prepared from a raw material comprising debaryomyces hansenii according to claim 1.
8. The pharmaceutical or cosmetic product according to claim 7, wherein the debaryomyces hansenii in the pharmaceutical or cosmetic product comprises:
the flora and/or inoculant of debaryomyces hansenii of claim 1.
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| WO2022145600A1 (en) * | 2020-12-28 | 2022-07-07 | 숙명여자대학교산학협력단 | Debaryomyces hansenii smfm201812-3 and smfm201905-5 strain for tenderizing meat and improving flavor of meat, and method for tenderizing meat and improving flavor of meat using same |
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