CN115710553A

CN115710553A - Candida tropicalis and application thereof

Info

Publication number: CN115710553A
Application number: CN202211565532.0A
Authority: CN
Inventors: 陈盈如
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-02-24

Abstract

The invention discloses candida tropicalis and application thereof, and relates to the technical field of microorganisms. The Candida tropicalis (Candida tropicalis) disclosed by the invention is Candida tropicalis LABOFMIC-308, is preserved in China center for type culture Collection in 9 months and 9 days in 2022, and has a preservation number of CCTCC NO: M20221391. Experiments show that LABOFMIC-308 has effects of promoting cell proliferation, repairing skin barrier, keeping moisture, improving skin antibacterial ability, and resisting aging, and can be used for preparing food, medicine, cosmetic, etc.

Description

Candida tropicalis and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to candida tropicalis and application thereof.

Background

Various bacteria, fungi, viruses, and small arthropods such as mites live on human skin, and these microorganisms and small arthropods are grouped together to form a skin microorganism group or a skin flora. The skin microbial group, cells, sweat, sebum and other substances on the surface of the skin and the external environment form an ecological system together, and the ecological system plays an important role in maintaining the health of the skin of a host. The resident microorganisms can directly or indirectly secrete antimicrobial peptides by regulating keratinocytes of the skin so as to resist colonization of external pathogenic bacteria; can also decompose keratinocyte fragments or lipid, assist in emulsifying a skin lipid membrane, and maintain the acidic environment of the epidermis to resist the colonization of external pathogenic bacteria.

Most of the microorganisms living on the skin behave symbiotically or synergetically under steady-state conditions, and the skin microorganisms play an important role in the maturation and homeostasis of skin immunity. Recent studies have demonstrated that skin microbiota regulates the expression of various innate factors, and that skin resident bacteria are not merely passive resident bacteria; they actively participate in host immunity through an intact skin barrier. The skin's innate immune system in combination with the skin's microflora is the body's barrier against pathogenic and opportunistic pathogens.

The skin microorganisms synthesize active metabolites on the skin surface, such as short chain fatty acids, B vitamins, etc., and regulate the physiological functions of the skin. An increasing number of studies have shown that an imbalance in the skin's micro-ecology (also known as an imbalance in the flora) leads to a range of skin diseases, such as atopic dermatitis, acne, seborrheic dermatitis, psoriasis, opportunistic infections, etc. When beneficial bacteria are reduced or even eliminated, the skin has various problems of reduced resistance, easy inflammation, infection, allergy, acne growth, water and oil imbalance and the like. 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 this, the invention aims to provide a candida tropicalis strain and application thereof.

The invention provides Candida tropicalis (Candida tropicalis), which is Candida tropicalis LABOFMIC-308 and has been preserved in China center for type culture Collection (CCTCC for short, with the address of eight-channel 299 in Wuchang district, wuhan university, zip code 430072) in 9 months and 9 days in 2022, and the preservation number is CCTCC NO: M20221391.

Another object of the present invention is to provide the use of Candida tropicalis as described above for the preparation of a product for improving skin conditions.

Preferably, the improvement of skin condition includes at least one of promotion of cell proliferation, repair of skin barrier, moisturization, improvement of skin antibacterial ability, anti-aging.

In some embodiments, the promoting cell proliferation comprises promoting proliferation of skin keratinocytes and/or promoting proliferation of fibroblasts, wherein the rate of proliferation of skin keratinocytes is: 10.19 to 15.97 percent, and the proliferation rate of the fibroblast proliferation promoting agent is 8.97 to 14.81 percent.

In some embodiments, the repairing the skin barrier comprises up-regulating the expression of barrier repair-associated genes comprising FLG and OVOL1.

In some embodiments, said moisturizing comprises up-regulating the expression of moisturizing-related genes comprising AQP3 and/or GBA.

In some embodiments, the increasing the skin antibacterial ability is up-regulating the expression of an antibacterial peptide-related gene including at least one of S100A7, S100A8 and LCN-2.

In some embodiments, the anti-aging comprises up-regulating expression of a first set of related genes comprising extracellular matrix related genes, apoptosis-inhibiting related genes, autophagy related genes, antioxidant cellular related genes, immunomodulatory factor related genes, and cell growth factor related genes, and down-regulating expression of a second set of related genes comprising extracellular matrix-degrading related genes and apoptosis-related genes.

In some embodiments, the anti-aging further comprises at least one of inhibiting collagenase activity, scavenging DPPH radicals, and anti-oxidation.

In some embodiments, the product is a food product, a pharmaceutical product, or a cosmetic product.

The invention discloses Candida tropicalis LABOFMIC-308 with a preservation number of CCTCC NO: M20221391. Experiments show that the Candida tropicalis LABOFMIC-308 has the functions of promoting cell proliferation, repairing skin barrier, moisturizing, improving skin antibacterial ability and resisting aging, and can be used for preparing food, medicines, cosmetics and the like.

Biological preservation Instructions

Candida tropicalis LABOFMIC-308 is preserved in China center for type culture Collection (CCTCC, address: wuhan university, wuhan's Wuchang district eight-way No. 299) in 2022, 9 months and 9 days, and the preservation number is CCTCC NO: M20221391.

Detailed Description

The invention provides candida tropicalis and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. 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 ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides Candida tropicalis LABOFMIC-308, which is derived from the epidermis of Yangshan juicy peaches and is identified as Candida tropicalis (Candida tropicalis) through a fungal ITS gene sequence. The strain is in a single cell, spherical or egg-spherical shape under a microscope; growing on a wort agar plate to form white round colonies with soft and smooth surfaces or partial wrinkles, no luster and flat centers, uniform texture and irregular edges; the culture medium shows uniform turbid growth in the wort liquid culture medium, and the optimal growth temperature is 28 ℃.

Candida tropicalis (Candida tropicalis), depository: china center for type culture Collection, address: wuhan city wuchang district eight-way 299, wuhan university, preservation date: 9 months and 9 days in 2022, the preservation number is CCTCC NO: M20221391.

In the present invention, the promotion of cell proliferation includes promotion of proliferation of skin keratinocytes and/or fibroblasts. The present invention takes HaCaT keratinocytes as a subject, and studies the cell proliferation promoting effect of candida tropicalis. The results show that the candida tropicalis can promote and repair HaCaT keratinocyte damage caused by SDS, improve the cell proliferation rate, and promote the proliferation rate to be 10.19-15.97%. In the present invention, the promotion of cell proliferation includes promotion of proliferation of skin fibroblasts. The present invention studies the cell proliferation promoting effect of Candida tropicalis by using HFF cells as subjects. The result shows that the Candida tropicalis can promote the proliferation of HFF cells, and the proliferation promoting rate is 8.97-14.81%.

In the invention, repairing the skin barrier comprises repairing SDS-induced cell damage and/or up-regulating the expression of barrier repair related genes; the barrier repair-associated gene includes at least one of FLG and OVOL1. Furthermore, according to the invention, haCaT cells are taken as a subject, and the SDS damage repair capacity of the Candida tropicalis is researched, and the result shows that the Candida tropicalis can repair cell damage caused by SDS, improve the cell proliferation rate and up-regulate the expression of barrier repair related genes of non-damaged cells.

In the present invention, the moisturizing includes, but is not limited to, up-regulating the expression of moisturizing-related genes, including, but not limited to, AQP3 and/or GBA. According to the invention, haCaT cells are taken as a subject, the skin moisturizing effect of the candida tropicalis is researched, and the result shows that the candida tropicalis can up-regulate the expression of the moisturizing related gene AQP3 and/or GBA and promote the skin cell moisturizing.

According to the invention, haCaT keratinocytes are taken as a subject, and the influence of the Candida tropicalis on the expression of genes related to HaCaT keratinocyte antimicrobial peptides is researched. The result shows that the candida tropicalis can up-regulate the expression of antibacterial peptide related genes S100A7, S100A8 and LCN-2 and improve the antibacterial capacity of the skin.

In the present invention, the anti-aging includes at least one of the following: up-regulating expression of an extracellular matrix synthesis-associated gene comprising at least one of TIMP1, LN, COL13A1, SMAD3, and COL3 A1; down-regulating expression of a degraded extracellular matrix-related gene, the degraded extracellular matrix-related gene comprising at least one of MMP family genes; up-regulating the expression of cell antioxidant related genes NRF2 and SIRT-1; down-regulating expression of apoptosis-related genes, including any one or more of the Caspase families; up-regulating the expression of autophagy-related gene LC 3B; up-regulating the expression of cell growth factor related genes VEGF, FGF1, FGF2, FGF21, HGF; inhibiting collagenase activity; removing DPPH free radicals; antioxidant ability.

In order to investigate the anti-aging effect of candida tropicalis, indexes related to cell aging, including at least one of extracellular matrix synthesis, extracellular matrix degradation, cellular anti-oxidation, apoptosis, autophagy, a cell growth factor, collagenase activity inhibition, DPPH radical scavenging and anti-oxidation capacity, are measured.

According to the invention, haCaT keratinocytes are taken as a subject, and the influence of the Candida tropicalis on synthesis of genes related to extracellular matrix of the HaCaT keratinocytes is researched. The result shows that the candida tropicalis can up-regulate the expression of a gene TIMP1 related to extracellular matrix synthesis and promote the synthesis of extracellular matrix.

The invention takes HFF fibroblasts as a subject, and researches the influence of the candida tropicalis on the synthesis and degradation of related genes of HFF extracellular matrix. The results show that the candida tropicalis can up-regulate the expression of extracellular matrix synthesis related genes LN, COL13A1, SMAD3 and COL3A1, promote the synthesis of extracellular matrix, down-regulate and degrade the expression of extracellular matrix related genes MMP3, MMP8 and MMP10, and inhibit the degradation of extracellular matrix.

According to the invention, haCaT keratinocytes are taken as a subject, and the influence of the Candida tropicalis on the expression of the HaCaT keratinocyte antioxidant related genes is researched. The result shows that the candida tropicalis can up-regulate the expression of the anti-oxidation related gene NRF2 and enhance the antioxidant capacity of cells.

The invention takes HaCaT keratinocytes as a subject, and researches the influence of the Candida tropicalis on the HaCaT keratinocyte autophagy. The result shows that the candida tropicalis can up-regulate the expression of the autophagy-related gene LC3B and promote autophagy of cells to eliminate aged cells.

According to the invention, haCaT keratinocytes are taken as a subject, and the influence of the Candida tropicalis on HaCaT keratinocyte growth factor related gene expression is researched. The result shows that the candida tropicalis can up-regulate the expression of the vascular endothelial growth factor related gene VEGF and promote cell growth.

The invention takes HFF fibroblasts as subjects and researches the influence of the candida tropicalis on the apoptosis of the HFF cells. The result shows that the candida tropicalis can reduce the expression of apoptosis related genes Caspase3 and Caspase9 and inhibit apoptosis.

The invention takes HFF fibroblasts as a subject, and researches the influence of the candida tropicalis on the inhibition of the apoptosis of the HFF. The result shows that the candida tropicalis can up-regulate the expression of the apoptosis-related gene BCL-2 and inhibit apoptosis.

The invention takes HFF fibroblasts as a subject and researches the influence of the candida tropicalis on the expression of the anti-oxidation related genes of the HFF keratinocytes. The result shows that the candida tropicalis can up-regulate the expression of the oxidation-resistant related gene SIRT-1 and enhance the oxidation resistance of cells.

The invention takes HFF fibroblasts as a subject and researches the influence of the candida tropicalis on the expression of genes related to the HFF fibroblast immune regulatory factors. The result shows that the Candida tropicalis can up-regulate the expression of the gene MOR related to the immune regulation factor and enhance the immune regulation capability of cells.

The invention takes HFF fibroblasts as a subject and researches the influence of the candida tropicalis on the expression of genes related to the HFF fibroblast growth factors. The result shows that the candida tropicalis can up-regulate the expression of fibroblast growth factor related genes FGF1, FGF2, FGF21 and HGF and promote cell growth.

According to the invention, the collagenase is taken as a subject, and the detection of the Candida tropicalis on the activity of the collagenase is researched. The result shows that the candida tropicalis can inhibit the activity of the collagen enzyme and reduce the degradation of the collagen.

According to the invention, candida tropicalis is taken as a subject, and detection of DPPH free radical elimination and total antioxidant capacity by the Candida tropicalis is researched. The result shows that the candida tropicalis can eliminate DPPH free radicals, has antioxidant capacity and reduces oxidative damage of cells.

The invention also provides the use of the candida tropicalis LABOFMIC-308 in the above technical scheme for the preparation of a product for improving skin conditions, wherein the candida tropicalis LABOFMIC-308 is present in the application or product in a form of live or dead or subjected to intermittent sterilization, or in a form of a lysate and/or extract of thallus, or in a form of a yeast product or in a form of a supernatant or a derivative, and the derivative is preferably selected from the following forms: metabolites, metabolic biological products, exosomes, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: supernatant, bacterial lysate.

Further, the product comprises a flora containing the candida tropicalis;

furthermore, the product also comprises a microbial inoculum prepared from the candida tropicalis and a lysate, an extract and a metabolite thereof, or a microbial inoculum prepared from a flora containing the candida tropicalis, which is not limited in the invention.

Further, the product also includes the candida tropicalis or lysate, extract and metabolite thereof, which is not limited in the invention. The invention is not limited in this regard.

The formulation of the product includes, but is not limited to, at least one of creams, emulsions, oils, mists, gels, powders, and lyophilizates, which is not limited in this respect.

The invention also provides a method for improving the skin state, which comprises the steps of smearing, externally applying, fumigating or injecting by using the product in the technical scheme, and the method is not limited by the invention.

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-308

Sampling on the surface of Yangshan juicy peach. Properly processing the sample, uniformly mixing the sample in normal saline by shaking, taking the supernatant, streaking the supernatant on a malt extract agar plate, culturing the malt extract agar plate at the constant temperature of 28 ℃ for 12 to 16 hours, and selecting white colonies to repeatedly inoculate and screen until uniform single colonies are obtained, wherein the single colonies are named as LABOFMIC-308.

Gram staining microscopic examination: the strain LABOFMIC-308 is in the shape of single cell, sphere or egg sphere under a microscope; growing on a wort agar plate to form white round colonies with soft and smooth surfaces or partial wrinkles, no luster and flat centers, uniform texture and irregular edges; the culture medium shows uniform turbid growth in the wort liquid culture medium, and the optimal growth temperature is 28 ℃.

Example 2 nucleic acid identification of LABOFMIC-308

1. Fungal ITS Gene sequence analysis

Picking single colony to be placed in wort liquid culture medium, culturing overnight at 28 ℃, then centrifuging at 12000 ℃ for 1min to collect thallus, and operating according to the steps of the yeast genome DNA extraction kit. The primers adopt saccharomycete universal primers ITS1 and ITS4, a PCR amplification system is a 15 mu L system, and the pre-denaturation is carried out for 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; extension at 72 ℃ for 7min.

2. As a result, the

The homology comparison (BLASTN) between the sequencing result of the PCR product and the standard sequence published in GenBank results in that the LABOFMIC-308 strain is Candida tropicalis (Candida tropicalis).

Example 3LABOFMIC-308 promotion of SDS-induced HaCaT Damage repair assay in human immortalized keratinocytes

1. Preparation of first supernatant of LABOFMIC-308

Selecting Candida tropicalis LABOFMIC-308 single colony in malt wort liquid culture medium, shake culturing at 40 deg.c for 16-18 hr, detecting with enzyme labeling instrument, diluting with PBS to regulate OD ₆₀₀ And (3) centrifuging for 2min at 12000 rpm to separate and precipitate thalli, taking out the thalli, inactivating the thalli at 121 ℃ for 30min under high pressure, and filtering the thalli by using a 0.22-micron filter membrane to obtain a first supernatant, wherein the first supernatant is the inactivated supernatant.

2. Experiment for promoting HaCaT cell repair

Inoculation of HaCaT cells (5X 10) ⁴ cells/well) to 96-well plates and cultured overnight until cells adhere. SDS was prepared at 50. Mu.g/ml, and 100. Mu.l of 5% carbon dioxide was added to each well and incubated at 37 ℃ for 8 hours. 5% primary supernatant (control group replaced primary supernatant with equal volume of PBS) was added to each well for 24h of incubation. Mu.l of CCK-8 solution was added to each well, incubated for 4h, and absorbance A at 450nm was measured.

The formula for calculating the cell proliferation rate and the results are shown in the following table:

the above results show that the first supernatant of LABOFMIC-308 can promote repair of SDS-induced HaCaT keratinocyte damage, i.e., promote cell proliferation rate, which is 10.19% to 15.97%.

Example 4LABOFMIC-308 promotion of HaCaT Barrier repair-related Gene expression experiment

1. Preparation of LABOFMIC-308 first supernatant and thallus lysate

Preparation of the first supernatant referring to example 3;

the preparation method of the thallus lysate comprises the following steps:

and (3) cleaning the centrifugally precipitated thalli in the embodiment 3 twice by PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial sludge, resuspending the crushed bacterial sludge to the volume of centrifugation by PBS, crushing by ultrasonic waves for 20 minutes, and inactivating at 121 ℃ for 30min under high pressure to obtain a thalli lysate.

2. Experiment for promoting HaCaT barrier repair related gene expression

Inoculation of human immortalized keratinocytes HaCaT (2 ml/well, 5X 10 content) ⁵ Cells) to a 6-well plate, and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator until the cells adhere to the wall. Respectively adding 5% (V/V) of the first supernatant and 10% (V/V) of the thallus lysate (equal volume of PBS is used for replacing the first supernatant/thallus lysate in a control group respectively), culturing for 24 hours, adding the lysate, extracting total RNA of cells, performing reverse transcription to cDNA after detecting the concentration and the purity of the RNA, and detecting the expression of FLG and OVOL1 genes by adopting real-time qPCR (quantitative polymerase chain reaction) with GAPDH (GAPDH) as an internal reference gene. The PBS-treated group added with the same volume was used as a control (relative gene expression fold F = 1) and 2 was used ^-ΔΔCT F value was calculated for each sample.

The formula: f =2 ^-ΔΔCT Wherein:

△CT _{experiment of} ＝CT _{Experiment of} -CT _{Internal reference (experiment)} ；

△CT _Control ＝CT _Control -CT _{Internal reference (contrast)} ；

△△CT＝△CT _{Experiment of} -△CT _Control 。

The results are shown in the following table:

in vitro cell experiments show that the first supernatant and the thallus lysate of the candida tropicalis LABOFMIC-308 have the function of up-regulating the expression of the skin barrier repair related factor filaggrin gene FLG and the OVO-like transcription factor 1 gene OVOL1, and the gene expression level is up-regulated by 1.14 to 1.48 times. It is indicated that LABOFMIC-308 has the function of promoting skin barrier repair.

Example 5LABOFMIC-308 Up-regulating HaCaT moisturizing-related Gene expression experiment

1. Preparation of LABOFMIC-308 first supernatant and thallus lysate

The preparation method refers to example 3 and example 4.

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 6-well plate, 5% carbon dioxide incubator 37 ℃ overnight until cells adhere. Respectively adding 5% (V/V) of the first supernatant, adding 10% (V/V) of inactivated thallus (the control group respectively replaces the first supernatant/thallus lysate with PBS of the same volume), culturing for 24h, adding the 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 the 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 The F value of each sample was calculated and the results are shown in the following table:

in vitro cell experiments show that the first supernatant and the thallus lysate of the candida tropicalis LABOFMIC-308 have the function of up-regulating the expression of a water channel protein 3 gene AQP3 and a glucocerebrosidase gene GBA related to moisture preservation, and the gene expression level is up-regulated by 1.02 to 1.78 times. It is shown that LABOFMIC-308 has skin moisturizing effect.

Example 6LABOFMIC-308 Regulation of photoaging HaCaT extracellular matrix/autophagy/antioxidant/growth factor-related Gene expression experiments

1. Preparation of LABOFMIC-308 thallus lysate

The preparation method refers to example 4.

2. HaCaT cell preparation and ultraviolet ray damage

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. Total dose of 2J/cm was applied to cells in the wells ² Ultraviolet UVB radiation damage.

3. LABOFMIC-308 addition

Separately, 10% (V/V) of the cell lysate was added to the stimulated HaCaT cells (control group replaced cell lysate with an equal volume of PBS). Each group 3 was incubated overnight at 37 ℃.

4. qPCR method for detecting relative expression multiple of extracellular matrix/autophagy/antioxidation/growth factor 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 TIMP1, an autophagy related gene LC3B, an antioxidant related gene NRF2 and a cell growth factor VEGF 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 thalli lysate up-regulates extracellular matrix gene TIMP1, autophagy-related gene LC3B, antioxidant-related gene NRF2 and cell growth factor VEGF. The results are shown in the following table:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has the effects of up-regulating expression of a tissue metalloproteinase inhibitor 1 gene TIMP1 related to HaCaT extracellular matrix, a cell autophagy-related microtubule-related protein 1 light chain 3 beta gene LC3B, an oxidation-resistant related nuclear factor E2-related factor 2 gene NRF2 and a growth factor-related vascular endothelial growth factor gene VEGF, and the relative expression multiple of the genes is 1.16-1.98.

Example 7LABOFMIC-308 promotes the proliferation of human fibroblast HFF

1. Preparation of LABOFMIC-308 thallus lysate

The preparation method refers to example 4.

2. HFF cell preparation and LABOFMIC-308 addition

HFF cells cultured with DMEM were seeded at 100 ul/well (3X 10 cells per well) ⁴ Cells) were transferred to 96-well plates and cultured overnight until cells were adherent. Formulation 200. Mu. M H ₂ O ₂ Mu.l of the culture medium was added to each well, and the culture was carried out at 37 ℃ for 1 hour in a 5% carbon dioxide incubator.The original medium was discarded, washed twice with PBS, 100. Mu.l of fresh medium was added, and 10% (V/V) of cell lysate was added to each well (control group replaced cell lysate with PBS of equal volume). And culturing for 24h. After adding 10. Mu.l of CCK-8 solution to each well, incubating for 4h, and detecting the absorbance A at 450nm, the calculation formula and results are as follows:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has the effect of promoting human fibroblast HFF proliferation, and the proliferation rate is 8.97-14.81%.

Example 8LABOFMIC-308 Regulation of oxidative damage HFF extracellular matrix/immunomodulatory factor/antioxidant/apoptosis/cell growth factor-related Gene expression assay

1. Preparation of LABOFMIC-308 first supernatant and thallus lysate

The preparation method refers to example 3 and example 4.

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-308 addition

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

4. qPCR method for detecting relative expression multiple of extracellular matrix/immunoregulatory factor/antioxidation/apoptosis/cell growth factor 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 the cells by using GAPDH as an internal reference gene and adopting real-time qPCR (quantitative polymerase chain reaction)The expression of the matrix related genes LN, COL13A1, SMAD3 and COL3A1, the immune regulatory factor related gene MOR, the antioxidation related gene SIRT-1, the apoptosis-related gene BCL-2, the cell growth factor related genes FGF1, FGF2, FGF21 and HGF, the MMP family of degradation extracellular matrix related genes and the Caspase family of cell apoptosis-related genes. Relative expression multiple F =1 of control group gene, using 2 ^-ΔΔCT The F value of each sample was calculated.

The first supernatant fluid is used for up-regulating extracellular matrix related genes LN, SMAD3 and COL13A1, an immunoregulation factor related gene MOR, an antioxidation related gene SIRT-1, an apoptosis inhibiting gene BCL-2 and cell growth factor related genes FGF1, FGF2, FGF21 and HGF; down-regulating and degrading MMP family of extracellular matrix related genes and Caspase family of apoptosis related genes, and the results are shown in the following table:

the thalli lysate up-regulates extracellular matrix related genes COL3A1, immunoregulation factor related genes MOR, cell growth factor related genes FGF1, FGF2, FGF21 and HGF; down-regulating and degrading apoptosis-related gene Caspase family. The results are shown in the following table:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has the effects of up-regulating the expression of laminin LN related to HFF extracellular matrix, collagen alpha 1 chain gene COL13A1 of XIII, collagen SMAD3 gene SMAD3 and collagen alpha 1 chain gene COL3A1 of III, beta-endorphin receptor gene MOR related to immunoregulatory factor, sirtuin family protein gene SIRT-1 related to antioxidation, B-lymphoma-2 gene BCL-2 related to inhibition of apoptosis and fibroblast growth factor related genes FGF1, FGF2, FGF21 and hepatocyte growth factor gene HGF, and the relative expression multiple of the genes is 1.20-2.46 times; has the functions of down-regulating matrix metalloproteinase family gene MMP related to degradation of extracellular matrix and cell apoptosis related cysteine proteinase family gene Caspase with relative expression multiple of 0.09-0.77.

Example 9LABOFMIC-308 experiments on the Up-regulated HaCaT antimicrobial peptide-related Gene expression

1. Preparation of LABOFMIC-308 first supernatant and thallus lysate

The preparation method refers to example 3 and example 4.

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-308 addition and LPS stimulation

5% (V/V) of the first supernatant and 10% (V/V) of the cell lysate were added to HaCaT cells cultured overnight (in the control group, the first supernatant/cell lysate was replaced with PBS of equal volume). After 2h, 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 20h.

4. qPCR method for detecting relative expression multiple of antibacterial peptide related gene

Removing the culture medium of the cells, adding a lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, and detecting the expression of genes S100A7, S100A8 and LCN-2 by using a real-time qPCR (quantitative polymerase chain reaction) by using GAPDH as an internal reference gene. Control with an equal volume of PBS-treated group (gene relative expression fold F = 1) using 2 ^-ΔΔCT The F value of each sample was calculated.

The first supernatant liquid is used for up-regulating antibacterial peptide related genes S100A7 and S100A8, and the results are shown in the following table:

the bacterial lysate up-regulates antibacterial peptide related genes S100A7, S100A8 and LCN-2. The results are given in the following table:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has the effect of promoting the expression of antibacterial peptide related genes psoriatin S100A7, calgranulin A S A8 and lipocalin-2 LCN-2, and the relative expression multiple of the genes is 1.09-1.67. It is indicated that LABOFMIC-308 has the function of improving skin ability.

Example 10LABOFMIC-308 inhibition of collagenase Activity assay

1. Preparation of first supernatant of LABOFMIC-308

The preparation method refers to example 3.

2. Collagenase activity inhibition assay

mu.L of 0.2% gelatin (collagen) solution and 50. Mu.L of 0.1mol/L Tris-HCl (containing 50mmol/L CaCl 2) solution at pH7.5 were mixed, 25. Mu.L of the first supernatant (control group replaced the first supernatant with an equal volume of wort liquid medium), and 25. Mu.L of collagenase solution were added to react at 37 ℃ for 0.5h. After the reaction is stopped, the activity of each group of collagenase is determined by ninhydrin color development at 593nm, and the collagenase inhibition rate is calculated.

The calculation formula and the result of the activity inhibition rate of the collagen enzyme are shown in the following table:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has the effect of inhibiting the activity of collagenase, and the inhibition rate is 10.63% -11.77%.

Example 11 Effect of LABOFMIC-308 on scavenging free radicals

1. LABOFMIC-308 second supernatant preparation

Selecting Candida tropicalis LABOFMIC-308 single colony in malt juice liquid culture medium, culturing at 28 deg.C and 160r/min in shaking bed for 16-18 h, detecting with enzyme labeling instrument, diluting with PBS, and adjusting OD ₆₀₀ Centrifuging at 12000 rpm for 2min to separate and precipitate thallus, deactivating at 121 deg.C under high pressure for 30min, and filtering with 0.22 μm filter membraneIs a second supernatant, which is an inactivated supernatant.

2. Detection of DPPH free radical clarity in second supernatant of LABOFMIC-308

The reagent preparation and detection method are carried out according to the instruction of the detection kit for the scavenging capability of the Solebao DPPH free radical. The 536nm absorbance of each sample was measured, averaged and the clearance of each sample calculated.

The calculation formula and the result are shown in the following table:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has the effect of eliminating DPPH free radicals, and the free radical clearance rate is 91.56-92.15%.

Example 12LABOFMIC-308 Total antioxidant Capacity determination experiment

1. Preparation of second supernatant of LABOFMIC-308

The preparation process is as in example 11.

2. Determination of Total Oxidation resistance

The reagent preparation and detection method is carried out according to the specification of a Solebao total antioxidant capacity (T-AOC) detection kit, wherein the total volume of the reaction is 0.204ml, and the volume of a sample in the reaction is 0.006ml. Measuring 593nm absorption A of each sample according to the formula

And (4) calculating to obtain an X value by A test-A blank =6.1464X-0.0009, and further calculating to obtain the total antioxidant capacity.

The formula for calculating the total antioxidant capacity and the results are shown in the following table:

in vitro cell experiments show that the Candida tropicalis LABOFMIC-308 has an antioxidant effect, and the total antioxidant capacity is 3.638-3.808 mu mol/mL.

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

1. A strain of Candida tropicalis (Candida tropicalis) is Candida tropicalis LABOFMIC-308, which is preserved in China center for type culture Collection in 9 months and 9 days in 2022, and the preservation number is CCTCC NO: M20221391.

2. Application of Candida tropicalis with preservation number of CCTCC NO: M20221391 in preparing products for improving skin conditions.

3. The use of claim 2, wherein the improvement in skin condition comprises at least one of promoting cell proliferation, repairing skin barrier, moisturizing, enhancing skin antibacterial ability, anti-aging.

4. Use according to claim 3, wherein said promotion of cell proliferation comprises promotion of proliferation of skin keratinocytes and/or promotion of proliferation of fibroblasts, said promotion of proliferation of skin keratinocytes having a proliferation rate of: 10.19 to 15.97 percent, and the proliferation rate for promoting the proliferation of the fibroblasts is 8.97 to 14.81 percent.

5. The use of claim 3, wherein said repairing the skin barrier comprises up-regulating the expression of barrier repair-related genes, said barrier repair-related genes comprising FLG and OVOL1.

6. The use according to claim 3, wherein moisturizing comprises up-regulating the expression of moisturizing-related genes comprising AQP3 and/or GBA.

7. The use according to claim 3, wherein the improvement of skin antibacterial ability is the up-regulation of the expression of antibacterial peptide-related genes comprising at least one of S100A7, S100A8 and LCN-2.

8. The use of claim 3, wherein said anti-aging comprises up-regulating the expression of a first set of related genes comprising extracellular matrix related genes, apoptosis-inhibiting related genes, autophagy related genes, antioxidant cellular related genes, immunomodulatory factor related genes, and cell growth factor related genes, and down-regulating the expression of a second set of related genes comprising extracellular matrix-degrading related genes and apoptosis-related genes.

9. The use of claim 3, wherein the anti-aging further comprises at least one of inhibiting collagenase activity, scavenging DPPH free radicals, and anti-oxidation.

10. Use according to any one of claims 2 to 9, wherein the product is a food product, a pharmaceutical product or a cosmetic product.