CN115948260A - Geotrichum galactose yeast and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a geotrichum galactose yeast and application thereof. The invention provides a geotrichum galactose yeast (Galactomyces geotrichum) with a preservation number of CCTCC NO: M20221326 and application thereof. Experiments show that the Geotrichum galactomannan provided by the invention can promote cell proliferation and improve skin barrier, has excellent effects on the aspects of skin moisturizing, anti-aging, allergy relieving and pain relieving, skin antibacterial capacity enhancing, hair shedding reduction, whitening and the like, and is suitable for preparing foods, medicines, cosmetics and the like with related skin functions.

Description

Geotrichum galactose yeast and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a geotrichum galactose yeast and application thereof.

Background

With aging, skin aging causes a series of changes in the elasticity, thickness, moisture content, etc. of the skin, resulting in the appearance of clinical symptoms such as wrinkles, sagging, age spots, etc. Skin glycation reactions are also known as maillard reactions. When the basal metabolism of the skin is slow, the body sugar is metabolized and accumulated in the human body, and the non-enzymatic glycosylation final substances are caused by the collagen in the skin. These materials eventually deposit in the dermis layer of the skin, leaving the skin dull, yellow and lacking in extensibility. In the research of aging mechanism, it is found that the autophagy can delay the functional degradation of organs, clear damaged proteins and lipids in cells, repair DNA, help cells to restore metabolic homeostasis and provide protection for photoaging damaged skin.

Sensitive skin is a sub-healthy state of the skin and refers in particular to a syndrome of common irritations with unpleasant sensations (including stinging, burning, pain, itching and tingling), especially of the face. Endorphins are neurotransmitters conducted between the immune system and the nervous system, and because the reactions of a plurality of sensitive symptoms of the skin are related to the interference of the nervous system, the pain of the sensitive symptoms can be controlled by improving the secretion of the endorphins, the micro-inflammatory reaction is reduced, and the skin can be recovered to be in a healthy state again.

Various bacteria, fungi, viruses, mites and the like inhabit on the skin of a human body to form a skin microorganism group or a skin flora, and the skin microorganism group or the skin flora plays an important role in maintaining the health of the skin of a host. These resident microorganisms can secrete antimicrobial peptides directly or indirectly by regulating keratinocytes of the skin to resist colonization by external pathogenic bacteria. An increasing number of studies have shown that an imbalance in the skin's microecological balance leads to a range of skin diseases, such as atopic dermatitis, acne, seborrheic dermatitis, psoriasis, opportunistic infections, etc.

Therefore, the probiotic related product developed by utilizing the skin microbial technology is provided, and has great commercial application value.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide geotrichum galactococcus and its application in products for promoting cell proliferation, improving skin barrier, moisturizing, anti-aging, soothing the nerves, enhancing skin antibacterial ability, reducing hair loss and whitening skin.

The invention provides a geotrichum galactose yeast (Galactomyces geotrichum) with a preservation number of CCTCC NO: M20221326.

Experiments show that the geotrichum galactopyranose yeast can promote cell proliferation and repair skin barriers, and has excellent effects in moisturizing, resisting aging, relieving allergy and pain, enhancing skin antibacterial ability, reducing hair loss, whitening and the like.

Further, the invention also provides application of the galactose yeast of geotrichum in preparing skin care products.

The invention provides a skin care product which is prepared from a raw material containing the geotrichum galactococcus.

Preferably, the skin care product comprises one or two of the following components (1) or (2):

(1) A killed bacterium and/or a killed supernatant of said geotrichum galactococcus;

(2) A culture, exosome, lysate, extract, fermentation filtrate and/or fermentation lysate of said geotrichum galactococcus.

Furthermore, the formulation of the skin care product includes at least one of creams, emulsions, oils, water aqua, gels, powders and freeze-drying, which is not limited in the present invention.

The invention also provides application of the skin care product in at least one of improving skin quality, resisting aging, reducing hair loss and whitening.

Further, the skin improvement product comprises at least one of the following i to v:

promoting proliferation of cells, wherein the cells are fibroblasts;

ii, repairing the skin barrier, wherein the repairing of the skin barrier is the promotion of the expression of a barrier repair related gene FLG;

iii, moisturizing, wherein the moisturizing is to promote the expression of a moisturizing related gene GBA;

iv, relieving allergy and easing pain, wherein the relieving allergy and easing pain is the promotion of the generation of beta-endorphin;

and v, enhancing the antibacterial ability of the skin, wherein the skin antibacterial ability is enhanced by promoting the expression of at least one of antibacterial peptide related genes S100A7, S100A8, DEFB4, LL-37 or LCN-2.

In some embodiments, the present invention uses HFF cells as the subject, and the cell proliferation promoting effect of the galactopyranosylyeast is studied. Results show that the geotrichum galactococcus can promote the proliferation of HFF fibroblasts, and the proliferation promoting rate is 14.56-15.92%.

In some embodiments, the invention takes HaCaT keratinocytes as a subject, and researches the barrier repair effect of the geotrichum galactococcus on skin, and the results show that the geotrichum galactococcus can up-regulate the expression of the barrier repair related gene of the cells.

In some embodiments, the HaCaT cell is used as the subject, the skin moisturizing effect of the geotrichum galactococcus is studied, and the result shows that the geotrichum galactococcus can up-regulate the expression of the moisturizing related gene GBA and promote the skin cell moisturizing.

In some embodiments, the invention is directed to HFF fibroblasts as subjects, and the effect of said geotrichum galactopyranosum on the production of β -endorphin by HFF fibroblasts is studied. The result shows that the geotrichum galactococcus can promote the generation of the beta-endorphin of HFF fibroblasts, and plays the roles of relaxing and easing pain.

In some embodiments, the present invention uses HaCaT keratinocytes as a subject, and studies the effect of said digycoctomyces galactans on the expression of HaCaT keratinocyte antimicrobial peptide-related genes. The result shows that the geotrichum galactococcus can up-regulate the expression of antibacterial peptide related genes S100A7, S100A8, DEFB4, LL-37 and LCN-2 and enhance the antibacterial capacity of the skin.

Further, the anti-aging comprises at least one of the following a) to h):

a) Promoting expression of an extracellular matrix synthesis-associated gene comprising at least one of SMAD3, LN, MKX, COL1A1, COL3A1, or COL13 A1;

b) Promoting the expression of MMP3 for degrading extracellular matrix related genes;

c) Promoting the expression of cell antioxidant related gene NRF2 and/or SIRT-1;

d) Promoting the expression of cell immune regulatory factor related gene MOR;

e) Inhibiting expression of apoptosis-related genes; the apoptosis-related gene comprises any one or more of BAX and/or Caspase families;

f) And promoting the expression of the autophagy-related gene LC3B.

g) Promoting expression of a cell growth factor-related gene comprising at least one of FGF1, FGF2, FGF21, or HGF.

h) And reducing the content of protein carbonyl.

In order to investigate the anti-aging effect of the Geotrichum galactosaccharomyces, the invention measures the indexes related to cell aging, wherein the indexes comprise at least one of extracellular matrix synthesis, extracellular matrix degradation, cellular antioxidation, cellular immunoregulatory factors, apoptosis, autophagy, cell growth factors and protein carbonyl content.

In some embodiments, the present invention is directed to HFF fibroblasts as subjects, and the effect of the geotrichum galactococcus on synthesis-related genes and degradation-related genes of HFF extracellular matrix is studied. The result shows that the geotrichum galactococcus can up-regulate the expression of extracellular matrix synthesis related genes SMAD3, LN, MKX, COL3A1 and COL13A1, promote the synthesis of the extracellular matrix, down-regulate the expression of the extracellular matrix related gene MMP3 and inhibit the degradation of the extracellular matrix.

In some embodiments, the present invention uses HaCaT keratinocytes as a subject, and studies the effect of said digomomyces galactans on the expression of genes involved in the oxidation resistance of HaCaT keratinocytes. The result shows that the geotrichum galactococcus can up-regulate the expression of an anti-oxidation related gene NRF2 and enhance the antioxidant capacity of cells.

In some embodiments, the present invention is a subject with an HFF fibroblast, and the effect of the digomogalactagogue on the expression of an antioxidant-related gene in the HFF fibroblast is studied. The result shows that the geotrichum galactococcus can up-regulate the expression of the anti-oxidation related gene SIRT-1 and enhance the antioxidant capacity of cells.

In some embodiments, the present invention relates to HFF fibroblasts, and the effect of the said geotrichum galactopyranose on the expression of an immune regulator-related gene of HFF fibroblasts is studied. The result shows that the said galactose saccharomycete can regulate up the expression of the gene MOR related to the immune regulator and strengthen the immune regulating capacity of cell.

In some embodiments, the present invention uses HFF fibroblasts as subjects and studies the effect of said digomogalactagopsis on the apoptosis of HFF cells. The result shows that the geotrichum galactococcus can reduce the expression of apoptosis related genes BAX, caspase-3 and Caspase-9 and inhibit apoptosis

In some embodiments, the present invention uses HaCaT keratinocytes as a subject, and the effect of said geotrichum galactococcus on the autophagy of HaCaT keratinocytes is studied. The result shows that the geotrichum galactococcus can up-regulate the expression of the autophagy-related gene LC3B and promote autophagy to eliminate aged cells.

In some embodiments, the present invention is a subject having an HFF fibroblast, and the effect of the above-described digomogalacturonan on the expression of an HFF cell growth factor-related gene is studied. The result shows that the geotrichum galactococcus can up-regulate the expression of cell growth factor related genes FGF1, FGF2, FGF21 and HGF and enhance the growth capacity of cells.

In some embodiments, the present invention uses HFF fibroblasts as a subject, and studies the effect of said geotrichum galactopyranose on the carbonyl content of HFF fibroblast protein. The result shows that the galactose yeast of geotrichum can reduce the content of protein carbonyl and reduce the aging caused by protein saccharification.

Further, the whitening comprises at least one of the following 1) to 7):

1) Inhibiting the expression of at least one of melanin synthesis related genes TRP-1, PI3K, AKT, GSK3 beta or MITF;

2) Inhibiting the expression of a gene ASIP related to the generation of the pheomelanin;

3) Inhibiting the expression of melanin secretion related gene EDN;

4) Inhibiting the expression of at least one of melanin transport related genes RAB27A, va or HMGB1;

5) Inhibiting the expression of melanosome maturation associated gene OA1 and/or MATP;

6) Inhibiting the expression of a gene E-cadherin related to the migration of melanocytes;

7) Inhibiting tyrosinase activity.

In order to investigate the whitening effect of the said digomomycete galactosaccharomyces, the present invention measured melanin and pheomelanin related indices, which included at least one of melanin secretion, melanin synthesis, pheomelanin production, melanin transport, melanosome maturation, melanocyte migration, and tyrosinase activity inhibition.

In some embodiments, the subject of the present invention is a875 human melanoma cell, and the effect of said geomycin galactococcus on a gene involved in melanin synthesis in a875 cell is studied. The result shows that the geotrichum galactococcus can reduce the expression of melanin synthesis related genes TRP-1, PI3K, AKT, GSK3 beta and MITF and reduce the synthesis of melanin.

In some embodiments, the present invention is directed to a875 human melanoma cell as a subject and the effect of said geotrichum galactococcus on the expression of a melanogenesis associated gene in a875 cell is studied. The result shows that the geotrichum galactococcus can down regulate the expression of a melanogenesis related gene ASIP and reduce the melanogenesis.

In some embodiments, the subject of the invention is a875 human melanoma cell, and the effect of said geomycin galactococcus on melanin secretion from a875 cell is studied. The result shows that the geotrichum galactococcus can reduce the expression of melanin secretory gene EDN and reduce the secretion of cell melanin.

In some embodiments, the present invention is directed to a875 human melanoma cell as a subject and the effect of said geomycin galactosaccharomyces on melanin transport in a875 cell is studied. The result shows that the geotrichum galactococcus can reduce the expression of melanin transport genes RAB27A, va and HMGB1 and reduce the transport of cell melanin.

In some embodiments, the present invention is directed to a875 human melanoma cell as a subject and the effect of said geomycotic galactosaccharomyces on the expression of genes associated with melanosome maturation of a875 cell is studied. The result shows that the geotrichum galactococcus can reduce the expression of genes OA1 and MATP related to melanosome maturation and reduce the maturation of the melanosome cells.

In some embodiments, the subject of the present invention is a875 human melanoma cell, and the effect of said geotrichum galactococcus on the expression of a875 cell migration-associated gene is studied. The result shows that the geotrichum galactococcus can down regulate the expression of a melanin migration related gene E-cadherin and reduce the migration of melanocytes.

In some embodiments, the invention uses tyrosinase as a subject, and the effect of said geotrichum galactopyranosum on tyrosinase is studied. The result shows that the geotrichum galactococcus can inhibit the activity of tyrosinase and reduce the synthesis of melanin.

Further, the hair loss reduction includes at least one of the following (1) to (4):

(1) promoting the expression of the hair follicle cell growth factor related gene IGF-1 and/or FGF-2;

(2) promoting the expression of the gene HSP27 related to the inhibition of hair follicle apoptosis;

(3) promoting the expression of the gene c-Myc related to the hair follicle cell cycle regulatory factor;

(4) and inhibiting the expression of BMP4 in the telogen phase of the hair follicle.

In order to explore the hair loss reducing effect of the Geotrichum galactosaccharomyces, the invention measures the indexes related to hair papilla cell HDPCs, wherein the indexes comprise at least one of hair follicle cell growth factor, hair follicle cell apoptosis inhibition factor, hair follicle cell cycle regulation factor and hair follicle resting period.

In some embodiments, the present invention uses HDPCS as a subject, and studies the effect of said digycoctomyces on a gene associated with a cell growth factor of HDPCs. The result shows that the geotrichum galactococcus can up-regulate the expression of genes related to the hair follicle cell growth factor IGF-1 and FGF-2, promote the cell proliferation of HDPCs and induce the hair follicle regeneration.

In some embodiments, the present invention provides HDPCs as subjects for studying the effect of said geotrichum galactococcus on apoptosis of HDPCs. The result shows that the geotrichum galactococcus can up-regulate the expression of the gene HSP27 related to hair follicle cell apoptosis, inhibit HDPCs apoptosis and prevent hair loss.

In some embodiments, the present invention provides HDPCs as a subject, and the effect of said digomomyces galactans on the expression of genes involved in the hair follicle cell cycle regulators of HDPCs cells is studied. The result shows that the geotrichum galactococcus can up-regulate the expression of the gene c-Myc related to the hair follicle cell cycle regulatory factor, promote the G1/S conversion of HDPCs cell cycle and promote the hair growth.

In some embodiments, the HDPCS subject of the present invention studies the effect of the digitoctomyces on the genes associated with telogen phase. The result shows that the geotrichum galactococcus can reduce the expression of gene BMP4 related to the hair follicle resting period of HDPCs, promote the HDPCs to enter the hair follicle growth period from the hair follicle resting period and promote the hair growth.

The invention also provides a method for improving skin condition, which comprises using the product. The application method comprises smearing, external application, fumigation or injection, and the invention is not limited to the method.

The geotrichum galactococcus provided by the invention has a preservation number of CCTCC NO: M20221326, can promote cell proliferation and improve skin barrier, has excellent effects on skin moisture retention, aging resistance, allergy relief, pain relief, skin antibacterial capability enhancement, hair loss reduction, whitening and the like, and is suitable for preparing foods, medicines, cosmetics and the like with related skin functions.

Biological preservation Instructions

Geotrichum galactophore LABOFMIC-303 (Galactomyces geotrichum LABOFMIC-303) was deposited at the China center for type culture Collection at 8.25.2022 with the accession number of CCTCC NO: M20221326, china, wuhan university.

Detailed Description

The invention provides a galactose yeast of geotrichum and application thereof, and a person skilled in the art can realize the galactose yeast by appropriately improving process parameters by referring to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The geotrichum galactophore strain LABOFMIC-303 is derived from Maotai town Maotai-flavor distiller' S yeast, and is identified as geotrichum galactophore (Galactomyces geotrichum) through 26S rDNA. The strain is in a single cell, oval, sausage or spherical shape under a microscope; growing on malt extract agar plate to form white to milky irregular villous colony; the culture medium shows uniform turbid growth in the wort liquid culture medium, and the optimal growth temperature is 17 ℃.

Geotrichum galactococcus (Galactomyces geotrichum) LABOFMIC-303, deposited Unit: 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: the preservation number is M20221326 at 25/8 in 2022 and CCTCC NO.

Further, the digitoctomyces labofomic-303 provided by the invention is present in the use or product according to the invention in a form that is live or dead or sterilized at intervals, 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 LABOFMIC-303

Sampling in Maotaizhen Maotai-town Maotai-flavor distiller's yeast. Streaking the sample on a wort agar plate, culturing at the constant temperature of 17 ℃ for 12-16h, selecting white colonies, repeatedly inoculating and screening until uniform single colonies are obtained, and naming the colonies as LABOFMIC-303.

Gram staining microscopy: the strain LABOFMIC-303 is in the shape of a single cell, a sausage, an oval or a sphere under a microscope; growing on a wort flat plate to form white villous irregular colonies, wherein the white to milky white colonies are similar to mould colonies in shape; the culture medium shows uniform turbid growth in the wort liquid culture medium, and the optimal growth temperature is 17 ℃.

Example 2 nucleic acid identification of LABOFMIC-303

1. 26S rDNA gene sequence analysis:

picking single colony to be placed in wort liquid culture medium, culturing overnight at 17 ℃, then centrifuging at 12000 ℃ for 2min to collect thalli, and operating according to the steps of the yeast genome DNA extraction kit. The primers adopt yeast universal primers NL1 and NL4, a PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 5min at 95 ℃; 1min at 94 ℃, 1min at 52 ℃, 90s at 72 ℃ and 36 cycles; extension at 72 ℃ for 10min.

2. Results

The homology comparison (BLASTN) of the PCR product sequencing results with the published standard sequences in GenBank gave that the strain LABOFMIC-303 was a strain of Geotrichum galbana (Galactomyces geotrichum).

Example 3 LABOFMIC-303 promotes HaCaT barrier repair-related gene expression experiment

1. Preparation of labofomic-303 fermentation filtrate:

selecting single colony of galactose yeast LABOFMIC-303 of Geotrichum to malt wort liquid culture mediumShaking culture at 17 deg.c for 16-18 hr, detection with enzyme labeling instrument and regulation of OD via PBS dilution ₆₀₀ Centrifuging at 12000 rpm for 2min to separate and precipitate thallus, collecting supernatant, inactivating at 121 deg.C for 30min under high pressure, and filtering with 0.22 μm filter membrane to obtain fermentation filtrate.

2. Experiment for promoting HaCaT barrier repair related gene expression

Inoculation of human immortalized keratinocytes HaCaT (2 ml/well, 5X 10% in it) ⁵ Cells) to 6-well plate, 5% carbon dioxide incubator 37 ℃ overnight until cells adhere. Adding fermentation filtrate of 5% (V/V) (replacing the fermentation filtrate with PBS of the same volume in the control group), culturing for 24h, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, performing reverse transcription to obtain cDNA, detecting FLG gene expression by using GAPDH as an internal reference gene and adopting real-time qPCR. Control groups treated with PBS of equal volume (relative gene expression fold F = 1) were each added with 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 expression result of the up-regulation and modification of the related genes in the fermentation filtrate of LABOFMIC-303 is shown in the following table:

in vitro cell experiments show that the fermentation filtrate of the geotrichum galactophore LABOFMIC-303 has the function of up-regulating the expression of a skin barrier repair related factor filaggrin gene FLG, and the gene expression level is up-regulated by 1.42 to 1.51 times. It is shown that LABOFMIC-303 has the effect of improving the skin barrier.

Example 4 experiment for up-regulating HaCaT moisturizing related gene expression by LABOFMIC-303

1. Preparation of labofomic-303 fermentation filtrate:

the preparation method refers to example 3.

2. Experiment for up-regulating HaCaT moisturizing 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 5% (V/V) of fermentation filtrate (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, using GAPDH as an internal reference gene, and detecting the expression of GBA gene by adopting real-time qPCR. Control with an equal volume of PBS-treated group (gene relative expression fold F = 1) using 2 ^-ΔΔCT F value was calculated for each sample.

The expression results of the LABOFMIC-303 fermentation filtrate for up-regulating the moisture-retention related genes are shown in the following table:

in vitro cell experiments show that the geotrichum galactophore LABOFMIC-303 disclosed by the invention has the effect of up-regulating the expression of moisturizing-related glucocerebrosidase gene GBA, and the gene expression level is up-regulated by 1.23-1.28 times. It is shown that LABOFMIC-303 has skin moisturizing effect.

Example 5: LABOFMIC-303 regulation photoaging HaCaT extracellular matrix/antioxidation/autophagy related gene expression experiment

1. Preparing LABOFMIC-303 fermentation filtrate and fermentation lysate:

selecting single colony of galactose yeast LABOFMIC-303 of Geotrichum to shake-culture in malt wort liquid culture medium at 17 ℃ for 16-18 h, detecting by a microplate reader, diluting with PBS and adjusting OD ₆₀₀ And (4) centrifuging at 12000 rpm for 2min to separate and precipitate thallus, taking out supernatant, inactivating at 121 ℃ for 30min under high pressure, and filtering with a 0.22-micron filter membrane to obtain fermentation filtrate. The centrifugally precipitated thalli is washed twice by PBS and then is added with liquid nitrogen for grinding and crushing, crushed bacterial mud is collected and is resuspended to the volume during centrifugation by PBS, and then is crushed by ultrasonic waves for 20 minutes at 121 ℃ for 30min for high-pressure inactivation, thus obtaining the fermentation lysate.

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. LABOFMIC-303 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 extracellular matrix/autophagy/antioxidation related genes

Removing a culture medium from 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, and detecting the expression of an extracellular matrix related gene COL1A1, an antioxidant related gene NRF2 and a cell autophagy related gene LC3B by using GAPDH as an internal reference gene and 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 up-regulates extracellular matrix related gene COL1A1 and antioxidant related gene NRF2. The results are shown in the following table:

the fermentation lysate up-regulates the antioxidant-related gene NRF2, the autophagy gene LC3B. The results are shown in the following table:

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in vitro cell experiments show that the geotrichum galactococcus LABOFMIC-303 has the effects of up-regulating expression of collagen type I alpha chain gene COL1A1 related to HaCaT extracellular matrix, nuclear factor E2 related factor 2 gene NRF2 related to antioxidation, and microtubule related protein 1 light chain 3 beta gene LC3B related to autophagy, and the relative expression multiple of the genes is 1.15-1.75.

Example 6: LABOFMIC-303 promotes proliferation of HFF in human fibroblasts

1. LABOFMIC-303 fermentation lysate preparation:

the preparation method is referred to example 5.

2. HFF cell preparation and LABOFMIC-303 addition

HFF cells cultured in DMEM were seeded at 100 ul/well (3X 10/well) ⁴ Cells) were transferred to 96-well plates and cultured overnight until cells attached. Prepare 200 μ 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. Discard the original culture medium, PBS wash two times, add 100u l new culture medium, each hole with 10% (V/V) fermentation lysate, control with equal volume of PBS instead of fermentation lysate, culture 24h. Mu.l of CCK-8 solution was added to each well, incubated for 4h, and absorbance A at 450nm was measured.

The formula and results of LABOFMIC-303 calculation for promoting HFF cell proliferation are shown in the following table:

in vitro cell experiments show that the geotrichum galactose yeast LABOFMIC-303 has the effect of promoting the proliferation of human fibroblast HFF, and the proliferation rate is 14.56-15.92%.

Example 7: LABOFMIC-303 regulation of oxidative damage HFF extracellular matrix/apoptosis/antioxidation/immunoregulation factor/cell growth factor related gene expression experiment

1. Preparation of labofomic-303 fermentation filtrate and fermentation lysate:

the preparation method refers to example 5.

2. HFF cell preparation and H ₂ O ₂ Inducing oxidative damage

The HFF cells cultured with DMEM were digested at 0.5 ml/well (2X 10 contained therein) ⁵ Cells) were seeded 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. LABOFMIC-303 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/apoptosis/antioxidation/immunoregulation factor/cell growth factor related gene

Removing a culture medium from the cells, adding a lysate, 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 LN, MKX, SMAD3, COL3A1 and COL13A1, antioxidant related genes SIRT-1, immune regulatory factor related genes MOR and cell growth factor related genes FGF1, FGF2, FGF21 and HGF by adopting real-time qPCR (quantitative polymerase chain reaction) by taking GAPDH as an internal reference gene; and degrading the expression of MMP family of extracellular matrix related genes, caspase family of apoptosis related genes and BAX. Relative expression multiple F =1 of control group gene, using 2 ^-ΔΔCT The F value of each sample was calculated.

The fermentation filtrate up-regulates extracellular matrix related genes LN, MKX, SMAD3 and COL13A1, antioxidant related genes SIRT-1, immunoregulatory factor related genes MOR, cell growth factor related genes FGF1, FGF2, FGF21 and HGF; down-regulating the degradation of extracellular matrix related gene MMP3, apoptosis related genes Caspase family and BAX, the results are shown in the following table:

the fermentation lysate up-regulates and inhibits extracellular matrix-related gene COL3A1, cell growth factor-related genes FGF21 and HGF; down-regulating apoptosis-related genes Caspase-3 and Caspase-9. The results are shown in the following table:

in vitro cell experiments show that the geotrichum galactophore LABOFMIC-303 of the invention has the effects of up-regulating the expressions of a laminin LN and a mohoke protein gene MKX related to an HFF extracellular matrix, a signal transduction protein gene SMAD3, a collagen type III alpha 1 chain COL3A1 and a collagen type XIII alpha 1 chain gene COL13A1, an immunoregulatory factor related beta-endorphin receptor gene MOR, an antioxidation related Sirtuins protein family gene SIRT-1, fibroblast growth factor genes FGF1, FGF2, FGF21 and a hepatocyte growth factor gene HGF, and the relative expression multiple of the genes is 1.11-4.89 times; has the functions of down-regulating matrix metalloproteinase family gene MMP3 related to degrading extracellular matrix, BCL2-Associated X protein gene BAX related to apoptosis and cysteine proteinase family gene Caspase, and has relative expression multiple of 0.14-0.85.

Example 8 experiment of LABOFMIC-303 reduction of protein carbonyl content in HFF

1. Preparation of labofomic-303 fermentation filtrate and fermentation lysate:

the preparation method refers to example 5.

2. HFF cell preparation and UV damage

HFF cells were digested and dispensed at 2 ml/well (8X 10 contents) ⁵ Cells) were inoculated into 6-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. The total dose of the cells in the wells is 2J/cm ² Ultraviolet UVB radiation damage.

3. LABOFMIC-303 addition

The fermentation filtrate 5% (V/V), fermentation lysate 10% (V/V) were added to the stimulated HFF cells (control group replaced equal volume of PBS for fermentation filtrate/fermentation lysate, respectively). Each group 3 was incubated overnight at 37 ℃.

4. Experiment for reducing content of protein carbonyl in HFF

The medium in the 6-well plate was discarded, washed with PBS, RIPA was added, after sufficient lysis, the supernatant was removed by centrifugation at 12000 rpm for 5 min. And (3) detecting the supernatant of the sample according to the specification of a Nanjing constructed protein quantitative (TP) determination kit, and calculating the protein concentration. And (4) detecting according to the specification of the Nanjing built protein carbonyl content test box, and calculating the protein carbonyl content.

The calculation formula and the results of LABOFMIC-303 for reducing the carbonyl content of HFF cellular protein are shown in the following table:

in vitro cell experiments show that the geotrichum galactose yeast LABOFMIC-303 has the effects of reducing the content of protein carbonyl and reducing glycated protein, and the reduction rate is 7.26-23.66%.

Example 9: experiment for increasing generation amount of beta-endorphin of oxidative damage HFF cells by LABOFMIC-303

1. Preparing LABOFMIC-303 fermentation filtrate:

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 2 ml/well (5X 10 cells contained therein) ⁵ Cells) were inoculated into 6-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.

4. LABOFMIC-303 addition

The fermentation filtrates were added to 5% (V/V) of the stimulated HFF cells, respectively (control groups replaced the fermentation filtrates with an equal volume of PBS, respectively). Each group 3 was incubated overnight at 37 ℃.

4. Sample collection

Cell culture supernatant: the treated cell culture supernatant was collected and centrifuged (1000 Xg, 20 min) and either immediately detected or stored at-20 ℃/-80 ℃.

5. Human beta-endorphin (beta-EP) enzyme-linked immunosorbent assay kit for detecting generation amount and improvement rate of beta-EP

The reagent preparation and detection method are carried out according to the specification of a Jianglai biological human beta-endorphin (beta-EP) enzyme-linked immunosorbent assay kit. And adding 50 mu l of cell culture medium supernatant sample (the sample to be detected is diluted by 1 time by using a universal diluent so as to reduce the error influence of the matrix effect on the test result, and finally, the sample concentration is multiplied by the corresponding dilution factor) and a corresponding kit detection reagent into a 96-well enzyme label plate. After the reaction system is added with the stop solution, the absorbance A at 450nm is immediately detected. And calculating the content and the improvement rate of the beta-endorphin in the sample supernatant according to a formula.

The formula for calculating the amount of beta-endorphin produced by HFF cells by LABOFMIC-303 is shown as follows:

in vitro cell experiments show that the geotrichum galactose yeast LABOFMIC-303 has the effect of promoting the generation of beta-endorphin, and the improvement rate is 101.31-102.71%.

Example 10: LABOFMIC-303 up-regulation HaCaT antibacterial peptide related gene expression experiment

1. Preparation of labofomic-303 fermentation filtrate and fermentation lysate:

the preparation method refers to example 5.

2. HaCaT cell preparation

HaCaT cells were digested and then digested at 0.5 ml/well (2X 10 contained therein) ⁵ Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator.

3. LABOFMIC-303 addition and LPS stimulation

Fermentation filtrate 5% (V/V), fermentation lysate 10% (V/V) were added to HaCaT cells cultured overnight (control group replaced equal volume of PBS for fermentation filtrate/fermentation lysate). After 2 hours, 0.5ml of LPS solution with a concentration of 0.2. Mu.g/ml was added to induce cell inflammation, and the cells were cultured in a 5% carbon dioxide incubator at 37 ℃ for 20 hours.

4. qPCR method for detecting relative expression multiple of antibacterial peptide 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 S100A7, S100A8, DEFB4, and cDNA by using GAPDH as an internal reference gene and adopting real-time qPCR (quantitative polymerase chain reaction),LL-37, LCN-2 gene expression. Control with equal volume of PBS treated group (relative gene expression fold F = 1) using 2 ^-ΔΔCT F value was calculated for each sample.

The fermentation filtrate is used for up-regulating antibacterial peptide related genes S100A7, S100A8 and DEFB4, and the results are shown in the following table:

fermentation lysates up-regulated the antimicrobial peptide-related genes S100A8, DEFB4, LL-37, LCN-2, and the results are shown in the following table:

in vitro cell experiments show that the fermentation filtrate of the geotrichum galactophore LABOFMIC-303 disclosed by the invention has the effect of up-regulating the expression of an antibacterial peptide related psoriatin gene S100A7, a calgranulin gene S100A8, a beta defensin 4 gene DEFB4, cathelicidin family antibacterial peptide LL-37 and a lipocalin-2 gene LCN-2. The relative expression multiple of the gene is 1.21-2.71.

Example 11 inhibition of tyrosinase Activity by LABOFMIC-303

1. Preparation of labofomic-303 fermentation filtrate:

selecting single colony of geotrichum galactose yeast LABOFMIC-303 to shake-culture in malt wort liquid culture medium at 17 ℃ for 16-18 h, detecting by an enzyme-linked immunosorbent assay, and diluting with malt wort liquid culture medium to adjust OD ₆₀₀ And (3) centrifuging at 12000 rpm for 2min to separate and precipitate thallus, taking out supernatant, inactivating at 121 deg.C for 30min under high pressure, and filtering with 0.22 μm filter membrane to obtain fermentation filtrate.

2. Tyrosinase activity inhibition assay

250U/mL tyrosinase, 6.0mM levodopa and 80mM PBS were prepared. To each well of the 96-well plate, 50. Mu.l PBS + 20. Mu.l fermentation filtrate + 50. Mu.l tyrosinase + 50. Mu.l L levodopa were sequentially added (control group replaced fermentation filtrate with equal volume of wort liquid medium). And (3) uniformly mixing the reaction system, standing for 15min in a dark place at room temperature, and detecting the absorbance A at 475nm by using an enzyme-labeling instrument.

The formula for calculating the tyrosinase inhibitory activity of the LABOFMIC-303 fermentation filtrate and the results are shown in the following table:

in vitro cell experiments show that the galactose yeast LABOFMIC-307 of the geotrichum has the function of inhibiting the activity of tyrosinase. The activity inhibition rate is 29.41-30.77%.

Example 12 labofomic-303 regulation of a875 human melanoma melanin secretion/melanin synthesis/melanogenesis/melanin transport/melanosome maturation/melanocyte migration-related gene expression experiments

1. Preparation of labofomic-303 fermentation filtrate and fermentation lysate:

the preparation method is referred to example 5.

2. Preparation and ultraviolet irradiation of A875 human melanoma cells

A875 human melanoma cells were digested and dispensed at 0.5 mL/well (8.5X 10 contents) ⁴ Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. Total dose of 1J/cm was applied to cells in the wells ² The ultraviolet UVB irradiation induces melanogenesis.

3. LABOFMIC-303 addition

5% (V/V) of the fermentation filtrate and 10% (V/V) of the fermentation lysate were added to the stimulated A875 human melanoma cells, respectively (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 melanin secretion/melanin synthesis/melanogenesis/melanin transport/melanosome maturation/melanocyte migration related genes

Removing culture medium from the above cells, adding lysis solution, extracting total RNA of cells, detecting RNA concentration and purity, reverse-transcribing to cDNA, detecting melanin synthesis related genes TRP-1, PI3K, AKT, GSK3 beta, MITF and melanin generation related genes ASIP and black by using GAPDH as reference gene and adopting real-time qPCRExpression of gene EDN related to secretion of melanin, gene RAB27A related to melanin transport, va, HMGB1, gene OA1 related to melanosome maturation, MATP, gene E-cadherin related to melanocyte migration. 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 melanin synthesis related genes TRP-1, PI3K and GSK3 beta; melanin secretion-related gene EDN; melanin transport-related genes RAB27A, va; melanosome maturation associated genes OA1, MATP; the expression of the gene E-cadherin related to the migration of melanocytes, and the results are shown in the following table:

the fermentation lysate can reduce melanin synthesis related genes PI3K, AKT, GSK3 beta and MITF; melanogenesis associated gene ASIP; melanin secretion-related gene EDN; melanin transport related genes RAB27A, va and HMGB1; melanosome maturation-related gene MATP; the expression of the gene E-cadherin related to the migration of melanocytes, and the results are shown in the following table:

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in vitro cell experiments show that the geotrichum galactophore LABOFMIC-307 has the effects of reducing the expression of tyrosinase related protein 1 gene TRP-1 related to melanin synthesis, microphthalmia related transcription factor gene MITF, phosphatidylinositol 3-kinase gene PI3K, protein kinase B gene AKT, glycogen synthase kinase 3 beta gene GSK3 beta, cerotic signal protein gene ASIP related to melanogenesis, endothelin gene EDN related to melanin secretion, GTPase family protein gene RAB27A related to melanin transport, myosin gene Va, high mobility family protein B1 gene HMGB1, melanosome maturation related melanosome membrane protein OA1, membrane related transport protein gene MATP and melanin cell migration related cadherin gene E-cadherin gene, and the relative expression fold of the genes is 0.38-0.81. The LABOFMIC-307 has whitening effect of inhibiting melanin and melanoidin related gene expression.

Example 13: LABOFMIC-303 gene expression experiment for regulating oxidative damage HDPCs human hair follicle papilla cell growth factor/hair follicle cell apoptosis/hair follicle cell cycle regulating factor/hair follicle resting phase related gene

1. Labofomic-303 fermentation lysate preparation:

the preparation method is referred to example 5.

2. HDPCs human hair follicle papilla cell preparation and H ₂ O ₂ Inducing oxidative damage

HDPCs cultured in fibroblast culture medium were digested and dispensed at 2 mL/well (3X 10 inclusion) ⁵ Cells) were inoculated into 6-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 Stimulation was performed, and after standing at 37 ℃ for 1h, PBS was washed twice and the cell culture medium was changed.

3. LABOFMIC-303 addition

10% (V/V) of the fermentation lysate was added to the stimulated HDPCs (control group replaced the fermentation lysate with an equal volume of PBS), and the 5% carbon dioxide incubator was incubated overnight at 37 ℃.

4. qPCR method for detecting relative expression multiple of hair follicle cell growth factor/inhibiting hair follicle cell apoptosis/hair follicle cell cycle regulating factor/hair follicle resting phase related gene

Removing a culture medium from 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, detecting related genes IGF-1 and FGF-2 of hair follicle cell growth factors by using GAPDH as an internal reference gene and adopting real-time qPCR (quantitative polymerase chain reaction), HSP27 related genes of hair follicle cell apoptosis inhibition and c-Myc related genes of hair follicle cell cycle regulatory factors; and the expression of the gene BMP4 related to the resting stage of the hair follicle. Relative expression multiple F =1 of control group gene, using 2 ^-ΔΔCT The F value of each sample was calculated.

The fermentation lysate up-regulates the expression of genes related to hair follicle cell growth factors IGF-1 and FGF-2, the gene HSP27 related to hair follicle cell apoptosis and the gene c-Myc related to hair follicle cell cycle regulatory factor; the expression of the gene BMP4 related to the resting stage of the hair follicle is down regulated, and the results are shown in the following table:

in vitro cell experiments show that the fermentation lysate of the galactose yeast LABOFMIC-303 of the geotrichum has the functions of up-regulating insulin-like growth factor 1 gene IGF-1 and fibroblast growth factor 2 gene FGF-2 related to HDPCs hair follicle cell growth factors, inhibiting heat shock protein 27 gene HSP27 related to hair follicle cell apoptosis, and inhibiting Myc family protein gene c-Myc related to hair follicle cell cycle regulation factors, wherein the relative expression multiple of the genes is 1.22-1.50; the expression of BMP4 of the bone morphogenetic protein 4 gene related to the resting stage of hair follicle development is reduced, and the relative expression multiple of the gene is 0.67 to 0.86.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. A Galactomyces geotrichum (Galactomyces geotrichum) having a collection number of CCTCC NO: M20221326.

2. Use of the galactose yeast strain of geotrichum as set forth in claim 1 for the preparation of a skin care product.

3. A skin care product characterized by being produced from a raw material comprising the Geotrichum galactosaccharomyces of claim 1.

4. The skin care product according to claim 3, wherein the skin care product comprises one or both of the following (1) or (2):

(1) The inactivated bacteria and/or supernatant of the Geotrichum galactococcus of claim 1;

(2) A culture, exosome, lysate, extract, fermentation filtrate and/or fermentation lysate of the geotrichum galactococcus of claim 1.

5. The skin care product according to claim 3 or 4, wherein the formulation of the skin care product comprises at least one of creams, emulsions, oils, mists, gels, powders, and lyophilizates.

6. Use of the skin care product of any one of claims 3 to 5 for at least one of improving skin quality, anti-aging, reducing hair loss, and whitening.

7. The use of claim 6, wherein said skin improvement comprises at least one of the following i to v:

promoting proliferation of cells, wherein the cells are fibroblasts;

ii, repairing the skin barrier, wherein the repairing of the skin barrier is the promotion of the expression of a barrier repair related gene FLG;

iii, moisturizing, wherein the moisturizing is to promote the expression of a moisturizing related gene GBA;

iv, relieving allergy and easing pain, wherein the relieving allergy and easing pain is the promotion of the generation of beta-endorphin;

and v, enhancing the antibacterial capacity of the skin, wherein the skin antibacterial capacity is enhanced by promoting the expression of at least one of antibacterial peptide related genes S100A7, S100A8, DEFB4, LL-37 or LCN-2.

8. The use of claim 6, wherein the anti-aging comprises at least one of the following a) to h):

a) Promoting expression of an extracellular matrix synthesis-associated gene comprising at least one of SMAD3, LN, MKX, COL1A1, COL3A1, or COL13 A1;

b) Promoting the expression of MMP3 for degrading extracellular matrix related genes;

c) Promoting the expression of the cell antioxidant related gene NRF2 and/or SIRT-1;

d) Promoting the expression of cell immune regulatory factor related gene MOR;

e) Inhibiting expression of apoptosis-related genes; the apoptosis-related gene comprises any one or more of BAX and/or Caspase families;

f) Promoting the expression of the autophagy-related gene LC 3B;

g) Promoting the expression of a cell growth factor-related gene comprising at least one of FGF1, FGF2, FGF21, or HGF;

h) And reducing the content of protein carbonyl.

9. The use according to claim 6, wherein the whitening comprises at least one of the following 1) to 7):

1) Inhibiting the expression of at least one of melanin synthesis related genes TRP-1, PI3K, AKT, GSK3 beta or MITF;

2) Inhibiting the expression of a melanogenesis related gene ASIP;

3) Inhibiting the expression of melanin secretion related gene EDN;

4) Inhibiting the expression of at least one of melanin transport related genes RAB27A, va or HMGB1;

5) Inhibiting the expression of melanosome maturation associated gene OA1 and/or MATP;

6) Inhibiting the expression of a gene E-cadherin related to the migration of melanocytes;

7) Inhibiting tyrosinase activity.

10. The use of claim 6, wherein the reduction of hair loss comprises at least one of the following (1) to (4):

(1) promoting the expression of hair follicle cell growth factor related genes IGF-1 and/or FGF-2;

(2) promoting the expression of the gene HSP27 related to the inhibition of hair follicle apoptosis;

(3) promoting the expression of the gene c-Myc related to the hair follicle cell cycle regulatory factor;

(4) and inhibiting the expression of BMP4 in the telogen phase of the hair follicle.