Wickerhamiella submembranae and application thereof

Abstract

The application discloses a strain of submembranaceus wickerhamia and application thereof, and relates to the technical field of microorganisms. Wickerhamomyces subpelliculosus The strain is named as LABOFMIC-312, and the submembranaceus wickerhamia with a preservation number of CCTCC No:M 20221395 has been preserved in the China Center for Type Culture Collection on September 9, 2022. Experiments show that the LABOFMIC-312 has the functions of promoting proliferation of skin keratinocytes, repairing skin barrier, moisturizing, resisting aging and improving cell antibacterial capacity, and can be used for preparing medicines, cosmetics and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a Wickerhamomyces subpelliculosus and application thereof. BACKGROUND

[0002] The surface area of human skin is about 2 square meters, and if the hair follicles and sweat glands are taken into account, the surface area may reach 25 square meters. On such a huge organ, there are about 1 million microorganisms per square centimeter, which together with the skin form a miniature ecosystem. Most of the microorganisms on the skin are mutualistic and influence each other. On the one hand, our sebum and dead keratinocytes provide sufficient nutrients for microorganisms; on the other hand, the microflora maintains the normal structure of the skin, resists the invasion of external pathogens, and promotes the immune response and wound repair of the skin.

[0003] These microorganisms mainly exist on the surface of the skin and in skin appendages such as hair follicles, sweat glands and sebaceous glands. The most representative microorganisms include Propionibacterium, Corynebacterium, Staphylococcus and Malassezia. These resident microorganisms can directly or indirectly secrete antibacterial peptides by regulating keratinocytes to resist colonization of external pathogenic bacteria. Antibacterial peptides refer to all oligopeptides or polypeptides that can kill or inhibit bacteria, can contact microbial membranes, dissolve cells, and have broad-spectrum antimicrobial effects.

[0004] Resident bacteria can produce lipids, sterols and other substances to provide nutrients for body cells and participate in skin cell metabolism. Metabolic lipids can form an emulsified lipid film on the skin, which can prevent excessive evaporation of water and keep the skin moist. The importance of skin microecology lies in the dynamic balance of the entire microecological system. When the skin, environment and flora of the human body are in an uncoordinated state, the skin will raise an alarm and present skin problems or even skin diseases. Therefore, providing a probiotic-related product developed using skin microbial technology has great commercial application value. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a Wickerhamomyces subpelliculosus and application thereof.

[0006] One purpose of the present application is to provide a Wickerhamomyces subpelliculosus, which is named LABOFMIC-312 and has been preserved in the China Center for Type Culture Collection on September 9, 2022, with a preservation number of CCTCC No: M 20221395.

[0007] Preferably, the sample is taken from the peel of Qiuyue pear, and after appropriate treatment, the sample is shaken and mixed in physiological saline, and the supernatant is streaked on a malt agar plate. After constant temperature incubation at 28°C for 12 to 16 hours, milky white colonies are picked and repeatedly inoculated and screened until a uniform single colony is obtained, which is named LABOFMIC-312;

[0008] Under the microscope, it appears as a single cell, oval or spherical; when growing on a malt wort plate, it can form round colonies with a smooth, opaque surface, milky white, and neat edges; it grows uniformly turbidly in malt wort liquid culture medium.

[0009] Another object of the present invention is to provide a use of the above-mentioned Wickham's yeast in preparing a product for improving skin condition, wherein the improvement of skin condition includes at least one of promoting the proliferation of skin keratinocytes, repairing the skin barrier, moisturizing, anti-aging, and improving the antibacterial ability of cells.

[0010] In some embodiments, repairing the skin barrier comprises repairing SDS-induced cell damage and / or upregulating the expression of barrier repair-related genes FLG.

[0011] It should be noted that this embodiment studied the SDS-damage repair ability of the aforementioned Wickham's yeast using HaCaT keratinocytes as test subjects. The results showed that the aforementioned Wickham's yeast could repair SDS-induced cell damage, increase cell proliferation, and upregulate the expression of barrier-repair genes in non-damaged cells.

[0012] In some embodiments, the moisturizing is up-regulating the expression of moisturizing genes AQP3 and / or GBA.

[0013] It should be noted that this embodiment used HaCaT keratinocytes as test subjects to study the moisturizing effect of the above-mentioned Wickham's yeast on the skin. The results showed that the above-mentioned Wickham's yeast can upregulate the expression of the moisturizing genes AQP3 and / or GBA, promoting skin cell moisturization.

[0014] In some embodiments, the anti-aging effect includes upregulating the expression of at least one of the extracellular matrix genes SPTSSA, SMAD3, LN, MKX, COL1A1, COL3A1, and COL13A1, upregulating the expression of the apoptosis inhibitory gene BCL-2, upregulating the expression of the autophagy gene LC3B, upregulating the expression of at least one of the cellular antioxidant genes NRF2 and SIRT-3, upregulating the expression of the cellular immune regulatory factor gene MOR, or upregulating the expression of at least one of the cell growth factor genes FGF2, FGF21, and HGF.

[0015] In some embodiments, the anti-aging is at least one of down-regulating expression of at least one of MMP1, MMP3 of the MMP family of extracellular matrix degrading genes, or down-regulating expression of at least one of BAX and / or Caspase-3 of the Caspase family of apoptosis genes.

[0016] In some embodiments, the anti-aging is at least one of inhibiting collagenase activity, scavenging DPPH free radicals, and antioxidant capacity.

[0017] It should be noted that in order to explore the anti-aging effect of the above-mentioned sub-membrane Wickerhamiella domercqiae, the present embodiment measures the indicators related to cell aging, including at least one of promoting skin keratinocyte proliferation, promoting extracellular matrix synthesis, extracellular matrix degradation, cell antioxidant, cell immunomodulatory factor, cell autophagy, inhibiting cell apoptosis, cell growth factor, inhibiting collagenase activity, scavenging DPPH free radicals, and antioxidant capacity.

[0018] In the present embodiment, HaCaT keratinocytes are used as the test object to study the effect of the above-mentioned sub-membrane Wickerhamiella domercqiae on promoting cell proliferation. The results show that the sub-membrane Wickerhamiella domercqiae can promote the repair of HaCaT keratinocyte damage caused by SDS, and increase the cell proliferation rate by 11.90% to 13.21%.

[0019] In the present embodiment, HaCaT keratinocytes are used as the test object to study the effect of the above-mentioned sub-membrane Wickerhamiella domercqiae on the synthesis and degradation of extracellular matrix related genes of HaCaT keratinocytes. The results show that the sub-membrane Wickerhamiella domercqiae can up-regulate the expression of the extracellular matrix synthesis related gene COL1A1, promote the synthesis of extracellular matrix, and down-regulate the expression of the extracellular matrix degradation related gene MMP1, inhibit the degradation of extracellular matrix.

[0020] In the present embodiment, HFF fibroblasts are used as the test object to study the effect of the above-mentioned sub-membrane Wickerhamiella domercqiae on the synthesis and degradation of extracellular matrix related genes of HFF cells. The results show that the sub-membrane Wickerhamiella domercqiae can up-regulate the expression of the extracellular matrix synthesis related genes SPTSSA, SMAD3, LN, MKX, COL3A1, and COL13A1, promote the synthesis of extracellular matrix, and down-regulate the expression of the extracellular matrix degradation related gene MMP3, inhibit the degradation of extracellular matrix.

[0021] In the present embodiment, HaCaT keratinocytes are used as the test object to study the effect of the above-mentioned sub-membrane Wickerhamiella domercqiae on the expression of antioxidant related genes of HaCaT keratinocytes. The results show that the sub-membrane Wickerhamiella domercqiae can up-regulate the expression of the antioxidant related gene NRF2, and enhance the antioxidant capacity of the cells.

[0022] This study used HFF fibroblasts as the test subject to investigate the effect of the aforementioned Wickham yeast on the expression of antioxidant-related genes in HFF fibroblasts. The results showed that the aforementioned Wickham yeast could upregulate the expression of the antioxidant-related gene SIRT-3, enhancing the antioxidant capacity of the cells.

[0023] This study used HFF fibroblasts as the experimental subjects to investigate the effect of the aforementioned Wickhamella submembrane yeast on the expression of genes related to immune regulatory factors in HFF fibroblasts. The results showed that the aforementioned Wickhamella submembrane yeast could upregulate the expression of the immune regulatory factor-related gene MOR, thereby enhancing the immune regulatory capacity of the cells.

[0024] This study used HaCaT keratinocytes as the experimental subjects to study the effect of the above-mentioned Wickham's yeast on HaCaT keratinocyte autophagy. The results showed that the above-mentioned Wickham's yeast can upregulate the expression of the autophagy-related gene LC3B, promoting autophagy to eliminate aging cells.

[0025] In this embodiment, HFF fibroblasts were used as test subjects to study the effect of the above-mentioned Wickham's yeast on HFF cell apoptosis. The results showed that the above-mentioned Wickham's yeast could downregulate the expression of apoptosis-related genes BAX and Caspase-3, thereby inhibiting cell apoptosis.

[0026] In this embodiment, HFF fibroblasts were used as test subjects to study the effect of the above-mentioned Wickham's yeast on inhibiting apoptosis in HFF fibroblasts. The results showed that the above-mentioned Wickham's yeast could upregulate the expression of the apoptosis-related gene BCL-2, thereby inhibiting apoptosis.

[0027] This study used HFF fibroblasts as the test subject to investigate the effect of the aforementioned Wickham yeast on the expression of growth factor-related genes in HFF cells. The results showed that the aforementioned Wickham yeast could upregulate the expression of growth factor-related genes FGF2, FGF21, and HGF, thereby enhancing cell growth.

[0028] In this embodiment, collagenase was used as the test subject to study the effect of Wickham's yeast on the activity of collagenase. The results showed that Wickham's yeast could inhibit the activity of collagenase and reduce collagen degradation.

[0029] This embodiment used Wickham's yeast as a test subject to study its DPPH free radical scavenging and total antioxidant capacity. The results showed that Wickham's yeast can scavenge DPPH free radicals and has antioxidant capacity, reducing oxidative damage to cells.

[0030] In some embodiments, the improving the antimicrobial ability of cells is to upregulate the expression of antimicrobial peptide genes, and the antimicrobial peptide genes are composed of at least one of S100A7, S100A8 and DEFB4.

[0031] This study used HaCaT keratinocytes as test subjects to investigate the effect of the aforementioned Wickham yeast on the expression of antimicrobial peptide-related genes in HaCaT keratinocytes. The results showed that the aforementioned Pichia luwii could upregulate the expression of antimicrobial peptide-related genes S100A7, S100A8, and DEFB4, thereby enhancing the cells' antimicrobial capacity.

[0032] Another object of the present invention is to provide a product for improving skin condition, which is made from the above-mentioned Wickham's yeast raw material; the product is a cosmetic, and the Wickham's yeast in the product includes exosomes, lysates and / or extracts of Wickham's yeast.

[0033] Furthermore, the product includes a bacterial agent made from the above-mentioned Wickham's yeast and its lysate, extract, and metabolites.

[0034] Furthermore, the dosage form of the product includes at least one of creams, lotions, oils, aqueous solutions, gels, powders, and freeze-dried solutions.

[0035] Another object of the present invention is to provide a method for improving skin condition, which comprises using the product of the present invention. The method of using the product comprises applying, externally applying, fumigating or injecting.

[0036] The present invention discloses Wickerhamomyces subpelliculosus LABOFMIC-312, which has a deposit number of CCTCC No. M 20221395. Experiments have shown that LABOFMIC-312 has anti-aging and skin antibacterial properties, and can be used to prepare medicines, cosmetics, and the like.

[0037] Biological Deposit Description

[0038] Wickerhamomyces subpelliculosus LABOFMIC-312 was deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on September 9, 2022, and its deposit number is CCTCC No: M20221395. DETAILED DESCRIPTION

[0039] The present invention provides Wickham's yeast and its applications. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0040] The present invention discloses a Wickerhamomyces subpelliculosus yeast strain, LABOFMIC-312, sampled from the peel of Qiuyue pear fruit in Laiyang City, Shandong Province, and identified as Wickerhamomyces subpelliculosus by 26S rDNA analysis. Under a microscope, the strain appears single-celled and oval or spherical. When grown on a malt wort plate, it forms smooth, opaque, round colonies with a milky white surface and neat edges. In liquid malt wort culture medium, it exhibits uniform turbidity and has an optimal growth temperature of 28°C.

[0041] Wickerhamomyces subpelliculosus LABOFMIC-312, deposited at China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: September 9, 2022, deposit number: CCTCC NO: M20221395.

[0042] The submembrane Wickham yeast LABOFMIC-312 provided by the present invention is present in the application or product of the present invention in the form of living or dead or intermittently sterilized, or in the form of a lysate and / or extract, or in the form of a yeast product or a fermentation product or derivative. The derivative form is preferably selected from: metabolites, metabolic biological products, exosomes, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products and lysates.

[0043] The test materials used in the present invention are all common commercial products and can be purchased commercially. The present invention is further described below with reference to the following examples:

[0044] Example 1 Isolation of LABOFMIC-312

[0045] Sampling was done from the peel of Qiuyue pear. After proper treatment, the sample was shaken and mixed in physiological saline. The supernatant was streaked on a malt agar plate. After constant temperature incubation at 28°C for 12-16 hours, milky white colonies were picked and repeatedly inoculated and screened until a uniform single colony was obtained, which was named LABOFMIC-312.

[0046] Microscopic examination: The strain LABOFMIC-312 appears as a single cell, oval or spherical under a microscope; when grown on malt wort plates, it forms round colonies with smooth, opaque surfaces, milky white in color, and neat edges; it grows uniformly turbidly in malt wort liquid culture medium.

[0047] Example 2 Nucleic Acid Identification of LABOFMIC-312

[0048] 1. 26S rDNA gene sequence analysis:

[0049] Single colonies were picked and placed in malt extract liquid medium. After incubation at 28°C overnight, the cells were collected by centrifugation at 12,000 rpm for 2 minutes and the procedure was followed according to the yeast genomic DNA extraction kit. Yeast universal primers NL1 and NL4 were used, and the PCR amplification system consisted of a 50 μL system with a 95°C pre-denaturation for 5 minutes, 36 cycles of 94°C for 1 minute, 52°C for 1 minute, and 72°C for 90 seconds, followed by an extension at 72°C for 10 minutes.

[0050] 2. Results

[0051] The PCR product sequencing results were compared with the standard sequences published in GenBank by BLASTN, and it was concluded that the LABOFMIC-312 strain was Wickerhamomyces subpelliculosus.

[0052] Example 3: Experiment on the promotion of SDS-induced damage repair of human immortalized keratinocytes HaCaT by LABOFMIC-312

[0053] 1. Preparation of LABOFMIC-312 lysate:

[0054] Pick a single colony of Wickham yeast LABOFMIC-312 in malt extract liquid medium, culture it in a shaking incubator at 28℃ for 16-18h, detect it with a microplate reader and adjust the OD value by diluting it with PBS. 600 =0.2, centrifuged at 12000r / min for 2min to separate the bacterial precipitate, the centrifuged bacterial precipitate was washed twice with PBS and then added with liquid nitrogen to grind and crush, the crushed bacterial sludge was collected and resuspended with PBS to the volume at the time of centrifugation, and then ultrasonically crushed for 20min, 121℃, 30min, 0.28Mpa to inactivate, and the lysate was obtained.

[0055] 2. Promote HaCaT cell repair experiment

[0056] HaCaT cells (5×10 4Cells were plated (100 μL / well) in a 96-well plate and cultured overnight until adherent. Prepare 50 μg / mL SDS and add 100 μL to each well. Incubate in a 5% CO2 incubator at 37°C for 8 hours. Add 10% lysate to each well (an equal volume of PBS was used instead of lysate for the control group) and incubate for 24 hours. Add 10 μL of CCK-8 solution to each well and incubate for 4 hours. Absorbance A at 450 nm was measured.

[0057] The cell proliferation rate calculation formula and results are as follows:

[0058]

[0059] The results in the above table show that LABOFMIC-312 lysates can promote the damage of HaCaT keratinocytes induced by SDS and increase the cell proliferation rate, with the promoted proliferation rate ranging from 11.90% to 13.21%.

[0060] Example 4 Experiment on LABOFMIC-312 Promoting HaCaT Barrier Repair-Related Gene Expression

[0061] 1. Preparation of LABOFMIC-312 fermentation products:

[0062] Pick a single colony of Wickham yeast LABOFMIC-312 in malt extract liquid medium, culture it in a shaking incubator at 28℃ for 16-18h, detect it with a microplate reader and adjust the OD value by diluting it with PBS. 600 =0.2, centrifuged at 12000 r / min for 2 min to separate the bacteria and precipitate, took out the supernatant and inactivated it at 121°C, 30 min, 0.28 MPa, and filtered through a 0.22 μm filter membrane to obtain the fermentation product.

[0063] 2. Experiment on promoting HaCaT barrier repair-related gene expression

[0064] Immortalized human keratinocytes HaCaT (2 mL / well, containing 5 × 10 5 Cells) were plated in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator until the cells adhered. 5% (V / V) fermentation product was added (the control group used an equal volume of PBS instead of fermentation product). After culturing for 24 hours, lysis buffer was added to extract total cellular RNA. The RNA concentration and purity were tested and then reverse transcribed into cDNA. GAPDH was used as the internal reference gene, and real-time qPCR was used to detect the expression of FLG gene. The group treated with equal volume of PBS was used as the control (relative gene expression F=1), and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0065] Formula: F = 2 -ΔΔCT ,in:

[0066] △CT 实验=CT 实验 -CT 内参(实验) ;

[0067] △CT 对照 =CT 对照 -CT 内参(对照) ;

[0068] △△CT=△CT 实验 -△CT 对照 .

[0069] The results are shown in the table below:

[0070]

[0071] In vitro cell experiments showed that the fermentation product of the Wickham yeast LABOFMIC-312 of the present invention has the effect of upregulating the expression of the filaggrin gene FLG, a factor associated with skin barrier repair, with the gene expression level increased by 1.40 to 2.11 times. This shows that LABOFMIC-312 has the effect of promoting skin barrier repair.

[0072] Example 5 Experiment on upregulating the expression of HaCaT moisturizing-related genes by LABOFMIC-312

[0073] 1. Preparation of LABOFMIC-312 fermentation products and lysates:

[0074] Pick a single colony of Wickham yeast LABOFMIC-312 in malt extract liquid medium, culture it in a shaking incubator at 28℃ for 16-18h, detect it with a microplate reader and adjust the OD value by diluting it with PBS. 600 = 0.2, centrifuged at 12,000 rpm for 2 minutes to separate the bacterial pellet. The supernatant was removed and inactivated at 121°C for 30 minutes at 0.28 MPa, and filtered through a 0.22 μm filter to obtain the fermentation product. The centrifuged bacterial pellet was washed twice with PBS and then ground with liquid nitrogen. The crushed bacterial sludge was collected and resuspended in PBS to the volume used for centrifugation. The pellet was then ultrasonically disrupted for 20 minutes and inactivated at 121°C for 30 minutes at 0.28 MPa to obtain the lysate.

[0075] 2. Experiment on upregulating the expression of HaCaT moisturizing-related genes

[0076] Immortalized human keratinocytes HaCaT (2 mL / well, containing 5 × 10 5HaCaT cells were seeded into 6-well plates at 0.5 mL / well (containing 2 x 10 5 cells) and incubated at 37°C in a 5% CO2 incubator overnight until the cells adhered. Fermentation products 5% (V / V) and lysates 10% (V / V) were added, respectively, to the stimulated HaCaT cells (the control group was replaced with the same volume of PBS instead of the fermentation products / lysates). Each group had 3 replicates, and the cells were incubated at 37°C overnight. 2 -ΔΔCT The relative expression fold of the extracellular matrix / cell autophagy / antioxidant-related genes was detected by qPCR. 5 -ΔΔCT The results are shown in the following table: 600 5 -ΔΔCT

[0001] In vitro cell experiments showed that the submembrane Wickerhamomyces LABOFMIC-312 of the present application had the effect of up-regulating the expression of the moisturizing-related aquaporin 3 gene AQP3 and the glucocerebrosidase gene GBA, and the gene expression amount was up-regulated by 1.12-1.67 times. It was shown that LABOFMIC-312 had the effect of promoting skin moisturizing. Example 6: LABOFMIC-312 regulates the expression of extracellular matrix / antioxidant / cell autophagy-related genes in photoaged HaCaT cells

[0002] 1. Preparation of LABOFMIC-312 fermentation products and lysates:

[0003] The preparation method is as described in Example 5.

[0004] 2. Preparation of HaCaT cells and ultraviolet damage

[0005] HaCaT cells were seeded into 24-well plates at 0.5 mL / well (containing 2 x 10 5 cells) and incubated at 37°C in a 5% CO2 incubator overnight. The cells in the wells were irradiated with a total dose of 2 J / cm 2 of ultraviolet UVB.

[0006] 3. LABOFMIC-312 addition

[0007] Fermentation products 5% (V / V) and lysates 10% (V / V) were added to the stimulated HaCaT cells, respectively (the control group was replaced with the same volume of PBS instead of the fermentation products / lysates). Each group had 3 replicates, and the cells were incubated at 37°C overnight.

[0008] 4. Detection of the relative expression fold of extracellular matrix / cell autophagy / antioxidant-related genes by qPCR

[0009] After the above cells are discarded from the culture medium, a lysis solution is added to extract total RNA of the cells, and after detecting the RNA concentration and purity, reverse transcription is performed to cDNA, GAPDH is used as an internal reference gene, and real-time qPCR is used to detect the expression of extracellular matrix related gene COL1A1, antioxidant related gene NRF2, cell autophagy related gene LC3B, and extracellular matrix degradation related gene MMP1. The relative expression fold of the genes in the control group is F = 1, and the F value of each sample is calculated by using 2 -ΔΔCT Method.

[0089] The fermentation product up-regulates the extracellular matrix related gene COL1A1 and the cell autophagy gene LC3B. The results are shown in the following table:

[0090]

[0091] The lysate up-regulates the antioxidant gene NRF2 and the cell autophagy gene LC3B, and down-regulates the extracellular matrix degradation related gene MMP1. The results are shown in the following table:

[0092]

[0093] The in vitro cell experiment shows that the submembrane Wickerhamomyces LABOFMIC-312 of the present application has the effects of up-regulating the expression of the extracellular matrix related type I collagen alpha 1 chain gene COL1A1, the antioxidant related gene NRF2, and the cell autophagy related gene LC3B, and the relative expression fold of the genes is 1.21-1.88; and has the effect of down-regulating the expression of the extracellular matrix degradation related matrix metalloproteinase family gene MMP1, and the relative expression fold of the genes is 0.58-0.78.

[0094] Example 7: LABOFMIC-312 regulates the expression of oxidative damage HFF cell extracellular matrix / cell apoptosis / antioxidant / immune regulation factor / cell growth factor related genes

[0095] 1. Preparation of LABOFMIC-312 fermentation product and lysate:

[0096] The preparation method is referred to Example 5.

[0097] 2. Preparation of HFF cells and H2O2 induced oxidative damage

[0098] After the HFF cells cultured in DMEM are digested, 0.5 mL / well (containing 2 x 10 5 cells) is inoculated into a 24-well plate, and the plate is cultured in a 5% carbon dioxide incubator at 37°C overnight. H2O2 with a final concentration of 200 μM is added to each well for stimulation, and the plate is placed at 37°C for 1 h.

[0099] 3. LABOFMIC-312 addition

[0100] 5% (V / V) fermentation product and 10% (V / V) lysate were added to stimulated HFF cells (the control group used equal volumes of PBS instead of fermentation product / lysate), three replicates per group, and cultured at 37°C overnight.

[0101] 4. qPCR detection of relative expression of genes related to extracellular matrix / apoptosis / antioxidation / immune regulatory factors / inflammatory factors

[0102] After discarding the culture medium from the above cells, lysis buffer was added and total RNA was extracted from the cells. The RNA concentration and purity were tested and then reverse transcribed into cDNA. GAPDH was used as the internal reference gene. Real-time qPCR was used to detect the expression of extracellular matrix-related genes LN, MKX, COL3A1 and COL13A1, apoptosis-inhibiting gene BCL-2, antioxidant gene SIRT-3, immune regulatory factor-related gene MOR, cell growth factors FGF2, FGF21 and HGF; as well as extracellular matrix degradation-related gene MMP family, apoptosis-related gene Caspase family and BAX. The relative expression of the control gene was F = 1, and the expression of 2 was used. -ΔΔCT The F value of each sample was calculated by this method.

[0103] The fermentation product up-regulated the extracellular matrix-related genes LN, SMAD3, SPTSSA, MKX and COL13A1, inhibited the apoptosis gene BCL-2, the immune regulatory factor-related gene MOR, and the cell growth factors FGF2, FGF21 and HGF; and down-regulated the degradation of the extracellular matrix-related genes MMP family and the apoptosis-related gene BAX. The results are shown in the table below:

[0104]

[0105] The lysate up-regulated the inhibition of extracellular matrix-related genes SMAD3, SPTSSA, MKX, and COL3A1, the antioxidant-related gene SIRT-3, the immune regulatory factor-related gene MOR, the apoptosis-inhibiting gene BCL-2, the cell growth factor FGF21, and HGF; and down-regulated the MMP family of genes related to degradation of the extracellular matrix and the apoptosis-related gene Caspase-3. The results are shown in the table below:

[0106]

[0107] In vitro cell experiments show that the submembrane williamsmeat LABOFMIC-312 of the application has the effect of up-regulating the expression of HFF cell extracellular matrix-related laminin LN, Mohawk protein gene MKX, signal transduction protein gene SMAD3, serine palmitoyl transferase gene SPTSSA, collagen type III alpha 1 chain COL3A1 and collagen type XIII alpha 1 chain gene COL13A1, immune-regulating factor-related beta-endorphin receptor gene MOR, antioxidant-related Sirtuins protein family gene SIRT-1, apoptosis-inhibiting B lymphoma-2 gene BCL-2, and cell growth factor-related fibroblast growth factor genes FGF2, FGF21, hepatocyte growth factor gene HGF, with a gene relative expression multiple of 1.13-5.12; and has the effect of down-regulating the expression of extracellular matrix-degrading matrix metalloproteinase family gene MMP, apoptosis-related BCL2-Associated X protein gene BAX and caspase family gene Caspase, and cell inflammatory factor-related interleukin 6 gene IL-6, with a gene relative expression multiple of 0.34-0.85.

[0108] Example 8: LABOFMIC-312 inhibits collagenase activity experiment

[0109] 1. LABOFMIC-312 fermentation product preparation:

[0110] The preparation method is referred to Example 5.

[0111] 2. Collagenase activity inhibition rate detection

[0112] Mix 50 μL of 0.2% gelatin (collagen) solution and 50 μL of 0.1 moL / L, pH 7.5 Tris-HCL (containing 50 moL / L CaCL2) solution, add 25 μL of fermentation product (the control group is replaced with an equal volume of malt juice liquid medium instead of fermentation product), add 25 μL of collagenase solution, and react at 37°C for 0.5 h. After termination of the reaction, determine the collagenase activity of each group at 593 nm by ninhydrin coloration, and then calculate the collagenase inhibition rate.

[0113] The collagenase activity inhibition rate calculation formula and results are shown in the following table:

[0114]

[0115] In vitro cell experiments show that the submembrane williamsmeat LABOFMIC-312 of the application has the effect of inhibiting collagenase activity, with an inhibition rate of 10.63%-11.77%.

[0116] Example 9: LABOFMIC-312 free radical scavenging effect

[0117] 1. Preparation of LABOFMIC-312 fermentation product:

[0118] A single colony of the Saccharomyces cariocanus LABOFMIC-312 was picked and cultured in a malt juice liquid medium at 28°C for 16-18 hours. The enzyme label instrument was used for detection and the OD was adjusted by dilution with PBS. 600 The supernatant was removed and inactivated at 121°C for 30 minutes and 0.28 MPa. The fermentation product was obtained by filtration through a 0.22 μm filter membrane.

[0119] 2. DPPH free radical scavenging capacity detection of the LABOFMIC-312 fermentation product

[0120] The reagent preparation and detection method were performed according to the instructions of the DPPH free radical scavenging capacity detection kit. The absorbance at 536 nm of each sample was measured, the average value was calculated, and the scavenging rate of each sample was calculated.

[0121] The calculation formula and results are shown in the following table:

[0122]

[0123] In vitro cell experiments showed that the Saccharomyces cariocanus LABOFMIC-312 of the present application has the effect of scavenging DPPH free radicals, and the free radical scavenging rate is 80.32%-83.47%.

[0124] Example 10: Total antioxidant capacity determination experiment of LABOFMIC-312

[0125] 1. Preparation of LABOFMIC-312 fermentation product:

[0126] The preparation method is as described in Example 9.

[0127] 2. Total antioxidant capacity determination

[0128] The reagent preparation and detection method were performed according to the instructions of the total antioxidant capacity (T-AOC) detection kit. The total reaction volume was 0.204 mL, and the sample volume in the reaction was 0.006 mL. The absorbance at 593 nm of each sample was measured, the X value was calculated according to the formula: Ameasured-Ablank=6.1464X-0.0009, and the total antioxidant capacity was calculated.

[0129] The total antioxidant capacity calculation formula and results are shown in the following table:

[0130]

[0131] In vitro cell experiments show that the submembrane Wickham yeast LABOFMIC-312 of the present invention has an antioxidant effect, and the total antioxidant capacity is 2.890 μmoL / mL to 3.128 μmoL / mL.

[0132] Example 11 Experiment on upregulating HaCaT antimicrobial peptide-related gene expression by LABOFMIC-312

[0133] 1. Preparation of LABOFMIC-312 fermentation products and lysates:

[0134] The preparation method is as in Example 5.

[0135] 2. Preparation of HaCaT cells

[0136] HaCaT cells were digested and then diluted with 0.5 mL / well (containing 2×10 5 Cells were seeded into 24-well plates and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0137] 3. LABOFMIC-312 addition and LPS stimulation

[0138] 5% (v / v) fermentation product and 10% (v / v) lysate were added to HaCaT cells cultured overnight (the control group received an equal volume of PBS instead of fermentation product / lysate). Two hours later, 0.5 mL of 0.2 μg / mL LPS solution was added to induce cell inflammation, and the cells were incubated at 37°C in a 5% CO2 incubator for 20 hours.

[0139] 4. Detection of relative expression of antimicrobial peptide-related genes by qPCR

[0140] After discarding the culture medium from the above cells, lysis buffer was added to extract total cell RNA, and the RNA concentration and purity were tested before reverse transcription into cDNA. The expression of S100A7, S100A8, and LCN-2 genes was detected by real-time qPCR using GAPDH as the internal reference gene. The group treated with equal volume of PBS was used as the control (relative gene expression F = 1). -ΔΔCT The F value of each sample was calculated by this method.

[0141] The fermentation products up-regulated antimicrobial peptide-related genes S100A7 and DEFB4. The results are shown in the table below:

[0142]

[0143] The lysate up-regulated antimicrobial peptide-related genes S100A8 and DEFB4. The results are shown in the table below:

[0144]

[0145] In vitro cell experiments showed that the Wickham yeast LABOFMIC-312 of the present invention promoted the expression of antimicrobial peptide-related genes psoriasin S100A7, calgranulin A S100A8, and beta-defensin 4DEFB4, with relative gene expression levels ranging from 1.19 to 1.88. This indicates that LABOFMIC-312 has the effect of enhancing the antimicrobial ability of cells.

[0146] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Wickham's yeast ( Wickerhamomyces subpelliculosus ), the strain was named LABOFMIC-312 and was deposited in the China Center for Type Culture Collection on September 9, 2022, and its preservation number is CCTCCNo:M 20221395.

2. Use of the Wickham yeast of claim 1 in preparing a product for improving skin conditions, wherein: The product is a cosmetic, and the improvement of skin condition includes at least one of promoting the proliferation of skin keratinocytes, repairing the skin barrier, moisturizing, anti-aging, and improving the antibacterial ability of cells.

3. A product for improving skin condition, characterized in that: The product is made from the raw material of the submembranous Wickham yeast according to claim 1; the product is a cosmetic, and the submembranous Wickham yeast in the product includes the fermentation filtrate and fermentation lysate of the submembranous Wickham yeast.

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