CN115725428A - Candida parapsilosis strain and application thereof - Google Patents

Abstract

The invention discloses a candida parapsilosis strain and application thereof, and relates to the technical field of microorganisms. The invention discloses a Candida parapsilosis strain (Candida parapsilosis), which is a Candida parapsilosis strain LABOFMIC-310, is preserved in China center for type culture collection at 9 months and 9 days in 2022, and has a preservation number of CCTCC NO: M20221393. Experiments show that LABOFMIC-310 has the functions of promoting cell proliferation, repairing skin barrier, resisting aging and improving cell antibacterial ability, and can be used for preparing food, medicine, cosmetics, etc.

Description

Candida parapsilosis strain and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a candida parapsilosis strain 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 on the surface of the skin, sweat, sebum and other substances and the external environment form an ecosystem together, and the ecological system plays an important role in maintaining the health of the skin of a host. These resident microorganisms can directly or indirectly secrete antimicrobial peptides by regulating keratinocytes of the skin to resist colonization by 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.

In recent years, the skin is found to secrete proteins and polypeptides with antibacterial effect, and the polypeptides with antibacterial effect are called antimicrobial peptides (AMPs), are effector molecules of a natural immune system, can contact microbial membranes and dissolve cells, and have broad-spectrum antimicrobial effect. In addition, they are involved in interfering with cellular proliferation, immune responses, wound healing, cytokine release, leukocyte chemotaxis, protease anti-protease balance, and other reaction processes.

Modulation of AMPs expression and secretion: the expression of AMPs genes is finely regulated and host defense responses cause AMPs to increase in expression in epithelial tissues or inflammatory cells. Different microorganisms stimulate the body's immune system to produce a spectrum of different antimicrobial peptides that are regulated by Toll signaling and the like. The host's natural immune system is capable of developing a broad defense mobilization mechanism against pathogenic microorganisms, including the recognition of pathogen-associated molecules, adaptive immune stimulation, and secretion of host defense substances. In addition, AMPs also play a role in inflammatory injury, maintenance of cell function and vascular proliferation.

The skin microorganism group synthesizes active metabolites on the skin surface, such as short chain fatty acid, B vitamins, etc., and regulates the physiological function of 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 the above, the invention provides a candida parapsilosis strain and an application thereof.

The invention provides a Candida parapsilosis strain (Candida parapsilosis), which is a Candida parapsilosis strain LABOFMIC-310 and has been preserved in China center for type culture Collection (CCTCC for short, with the address of eight No. 299 in Wuhan city Wuchang district, wuhan university, zip code 430072) in 2022, 9 months and 9 days, and the preservation number of the Candida parapsilosis strain is CCTCC NO: M20221393.

The invention also aims to provide application of the candida parapsilosis strain in preparing products for improving skin conditions.

In some embodiments, the improving skin condition is at least one of promoting cell proliferation, repairing skin barrier, anti-aging, increasing cell antibacterial ability.

In some embodiments, the pro-cell proliferation comprises an effect of promoting fibroblast proliferation.

In some embodiments, the repairing the skin barrier comprises up-regulating the expression of a barrier repair-associated gene; the barrier repair related gene is at least one of FLG, IVL and OCLN;

the anti-aging is the up-regulation of the expression of extracellular matrix related genes; the extracellular matrix-associated gene comprises at least one of COL1A1, LN, COL13A1, SPTSSA, MKX, and COL3 A1;

the anti-aging is the expression of up-regulated apoptosis-inhibiting related gene BCL-2;

the anti-aging is the expression of up-regulated cell autophagy related gene LC 3B;

the anti-aging is the expression of up-regulated cell antioxidant related genes NRF2, SIRT1 and SIRT3

The anti-aging is the expression of up-regulated immune regulatory factor related gene MOR

The anti-aging is the expression of up-regulating cell growth factor related genes VEGF, FGF1, FGF2, FGF21 and HGF.

In some embodiments, the anti-aging is down-regulation of expression of extracellular matrix-associated genes; the extracellular matrix-associated gene includes at least one of the MMP families;

the anti-aging is the expression of down-regulation apoptosis related genes; the apoptosis-related gene includes expression of BAX.

In some embodiments, the increasing the antibacterial ability of the cell is up-regulating the expression of antibacterial peptide related genes S100A7, S100A8, LCN-2.

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

Another object of the present invention is to provide a product for improving skin conditions, which is prepared from the raw materials comprising the above-mentioned Candida parapsilosis strain.

In some embodiments, the strain of candida parapsilosis in the product comprises one or both of the following (1) or (2):

(1) The flora and/or the microbial inoculum of the candida parapsilosis strain;

(2) A culture, exosome, lysate and/or extract of the above candida parapsilosis strain.

The invention discloses a Candida parapsilosis LABOFMIC-310 with a preservation number of CCTCC NO: M20221393. Experiments show that LABOFMIC-310 has the functions of promoting cell proliferation, repairing skin barrier, resisting aging and improving cell antibacterial ability, and can be used for preparing food, medicine, cosmetics, etc.

Biological preservation Instructions

Candida parapsilosis LABOFMIC-310 was deposited in China center for type culture Collection (CCTCC, address: wuhan university, wuhan's Wuchang region eight No. 299, no. 9) at 2022, 9 months, and the preservation number is CCTCC NO: M20221393.

Detailed Description

The invention provides a candida parapsilosis strain 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 expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The Candida parapsilosis strain LABOFMIC-310 is derived from Shandonglai Yang Qiuyue pear pericarp and is identified as Candida parapsilosis (Candida parapsilosis) through a fungal ITS gene sequence. The strain is in a unicellular individual, spherical or elliptical and the like under a microscope, and does not have bud multiplication and false hypha; growing on a wort agar plate to form a thick white round colony with a convex center, uniform texture and neat edge; the culture medium shows uniform turbid growth in the wort liquid culture medium, and the optimal growth temperature is 28 ℃.

Candida parapsilosis (Candida parapsilosis) LABOFMIC-310, depository: 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: 9 months and 9 days in 2022, the preservation number is CCTCC NO: M20221393.

Further, the present invention provides candida parapsilosis labofomic-310 in the use or product according to the invention, in the form of live or dead or tyndallized, or in the form of a lysate and/or extract, or in the form of a yeast product or in the form of a fermentation product 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 product, lysate.

The test materials adopted by the invention are all common commercial products, and can be purchased commercially, and the invention is further described by combining the following embodiments:

example 1: isolation of LABOFMIC-310

Sampling on the autumn moon pear peel. Properly processing the sample, uniformly mixing the sample in normal saline by shaking, taking the supernatant, streaking the supernatant on a malt extract agar solid plate, culturing the malt extract agar solid 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-310.

Gram staining microscopic examination: the strain LABOFMIC-310 is in a single-cell individual under a microscope, is spherical or elliptical and the like, and has no bud reproduction or pseudohypha; growing on a wort agar plate to form a thick white round colony with a convex center, uniform texture and neat edge; 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-310

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 yeast 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 for 7min at 72 ℃.

2. Results

The homology comparison (BLASTN) of the PCR sequencing results with the published standard sequences in GenBank gave the strain LABOFMIC-310 as Candida parapsilosis.

Example 3: LABOFMIC-310 gene expression experiment related to promotion of HaCaT barrier repair

1. Preparation of labofomic-310 fermentation product:

selecting a single colony of Candida parapsilosis LABOFMIC-310 to be cultured in a malt wort liquid culture medium for 16 to 18 hours in a shaking way at the temperature of 28 ℃, detecting by an enzyme-linked immunosorbent assay and usingOD adjustment by PBS dilution ₆₀₀ And (3) 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 0.22 μm filter membrane to obtain fermentation product.

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 fermentation product (the control group replaces the fermentation product with PBS of 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 FLG, IVL and OCLN genes by real-time qPCR. 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 The F value of each sample was calculated.

The formula: f =2 ^-ΔΔCT Wherein:

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

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

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

The results are shown in the following table:

expression of LABOFMIC-310 fermentation product up-regulated repair gene

In vitro cell experiments show that the fermentation product of the Candida parapsilosis LABOFMIC-310 has the function of up-regulating the expression of a skin barrier repair related factor filaggrin gene FLG, an endothelial protein gene IVL and a tight junction protein gene OCLN, and the gene expression level is up-regulated by 1.13 to 1.85 times. It is shown that LABOFMIC-310 has the effect of promoting skin barrier repair.

Example 4: LABOFMIC-310 experiment for regulating photoaging HaCaT extracellular matrix/autophagy/antioxidation/growth factor related gene expression

1. Labofomic-310 fermentation product and lysate preparation:

selecting a single colony of Candida parapsilosis LABOFMIC-310 in a malt juice liquid culture medium, carrying out shake cultivation for 16-18 h at 28 ℃, detecting by an enzyme labeling instrument, diluting by PBS and adjusting OD 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 product. Washing the centrifugally precipitated thalli twice by PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial mud, resuspending to the volume of centrifugation by PBS, crushing by ultrasonic waves for 20 minutes at 121 ℃, and inactivating at high pressure for 30 minutes to obtain a lysate.

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. The total dose of the cells in the wells is 2J/cm ² Ultraviolet UVB radiation damage.

3. LABOFMIC-310 addition

Fermentation product 5% (V/V), lysate 10% (V/V) were added to the stimulated HaCaT cells respectively (control group equal volume of PBS instead of fermentation product/lysate respectively). 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 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 an extracellular matrix related gene COL1A1, an autophagy related gene LC3B, an antioxidant related gene NRF2 and a 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 fermentation product up-regulates an extracellular matrix related gene COL1A1, an autophagy related gene LC3B and an antioxidant related gene NRF2. The results are given in the following table:

the lysate up-regulates the autophagy-related gene LC3B, the antioxidant-related gene NRF2 and the cell growth factor VEGF. The results are given in the following table:

in vitro cell experiments show that the Candida parapsilosis LABOFMIC-310 has the effects of up-regulating expression of a HaCaT extracellular matrix-related collagen type I alpha 1 chain gene COL1A1, 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 cell growth factor-related vascular endothelial growth factor gene VEGF, and the relative expression multiple of the genes is 1.15-1.76.

Example 5: LABOFMIC-310 promotes proliferation of HFF in human fibroblasts

1. Labofomic-310 lysate preparation:

the preparation method refers to example 4.

2. HFF cell preparation and LABOFMIC-310 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. 200. Mu. M H2O2 was prepared, and 100. Mu.l of 5% carbon dioxide was added to each well and cultured at 37 ℃ for 1 hour. The original medium was discarded, washed twice with PBS, 100. Mu.l of fresh medium was added, and 10% (V/V) of lysate was added to each well (control group replaced lysate with an equal volume of PBS). And culturing for 24h. Mu.l of CCK-8 solution was added to each well, incubated for 4h, and absorbance A at 450nm was measured.

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

LABOFMIC-310 promotes HFF cell proliferation

In vitro cell experiments show that the Candida parapsilosis LABOFMIC-310 has the effect of promoting human fibroblast HFF proliferation, and the proliferation rate is 25.17-32.43%.

Example 6: LABOFMIC-310 experiment for regulating oxidation injury HFF extracellular matrix/immunoregulatory factor/antioxidation/apoptosis/cell growth factor related gene expression

1. LABOFMIC-310 fermentation product and lysate preparation:

the preparation method refers to 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-310 addition

The fermentation product 5% (V/V), lysate 10% (V/V) were added to the stimulated HFF cells (control groups replaced equal volumes of PBS for fermentation product/lysate, 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 a lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, detecting extracellular matrix related genes LN, SPTSSA, COL13A1, MKX and COL3A1, inhibiting apoptosis related genes BCL-2, antioxidant related genes SIRT-1 and SIRT-3, immune regulatory factor related genes MOR, cell growth factor related genes FGF1, FGF2, FGF21 and HGF by using GAPDH as an internal reference gene, and degrading the expression of extracellular matrix related genes MMP family and cell apoptosis related genes BAX. Relative expression multiple F =1 of control group gene, using 2 ^-ΔΔCT F value was calculated for each sample.

The fermentation product up-regulates extracellular matrix related genes LN, COL13A1 and SPTSSA, inhibits apoptosis gene BCL-2, antioxidant related gene SIRT-1, immunoregulatory factor related gene MOR, cell growth factor related gene FGF21; down-regulating and degrading MMP family of extracellular matrix related genes and BAX of apoptosis related genes, and the results are shown in the following table:

cell lysate up-regulated extracellular matrix related genes SPTSSA, MKX and COL3A1, an immune regulatory factor related gene MOR, an antioxidation related gene SIRT-3, an apoptosis inhibiting gene BCL-2, and cell growth factor related genes HGF, FGF1 and FGF2; down-regulating and degrading MMP family of extracellular matrix related genes and BAX of apoptosis related genes, and the results are shown in the following table:

in vitro cell experiments show that the Candida parapsilosis LABOFMIC-310 has the functions of up-regulating the expression of laminin LN related to HFF extracellular matrix, collagen alpha 1 chain gene COL13A1 of XIII, serine palmitoyltransferase gene SPTSSA, mohokkin protein gene MKX, collagen alpha 1 chain gene COL3A1 of III, beta-endorphin receptor gene MOR related to immune regulatory factor, B-lymphocytoma-2 gene BCL-2 related to inhibition of apoptosis, sirtuins protein family genes SIRT-1 and SIRT-3 related to antioxidation and fibroblast growth factor related genes FGF1, FGF2, FGF21 and hepatocyte growth factor gene HGF, and the relative expression multiple of the genes is 1.15-2.49 times; has the functions of down-regulating matrix metalloproteinase family gene MMP related to degrading extracellular matrix and cell apoptosis related BCL2-Associated X protein gene BAX, and has the relative expression multiple of 0.43-0.80.

Example 7: LABOFMIC-310 up-regulation HaCaT antibacterial peptide related gene expression experiment

1. Labofomic-310 fermentation product and lysate preparation:

the preparation method refers to 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-310 addition and LPS stimulation

Fermentation product 5% (V/V), lysate 10% (V/V) were added to HaCaT cells cultured overnight (controls replaced fermentation product/lysate with equal volume of PBS, respectively). 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 S100A7, S100A8 and LCN-2 genes by adopting real-time qPCR (quantitative polymerase chain reaction) by taking 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 fermentation product up-regulates antibacterial peptide related genes S100A7 and S100A8, and the results are shown in the following table:

the lysate up-regulates the antimicrobial peptide related genes S100A7, S100A8, LCN-2. The results are shown in the following table:

in vitro cell experiments show that the Candida parapsilosis LABOFMIC-310 has the function of promoting the expression of antibacterial peptide related genes such as psoriatin S100A7, calgranulin A S A8 and lipocalin-2 LCN-2, and the relative expression multiple of the genes is 1.18-1.53. It shows that LABOFMIC-310 has the function of improving the antibacterial ability of cells.

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 Candida parapsilosis strain (Candida parapsilosis) LABOFMIC-310 is preserved in China center for type culture collection at 9 months and 9 days in 2022, and the preservation number is CCTCC NO: M20221393.

2. Use of a strain of candida parapsilosis according to claim 1 for the preparation of a product for improving skin conditions.

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

4. Use according to claim 3, wherein said pro-cell proliferation comprises the effect of promoting fibroblast proliferation.

5. The use of claim 3, wherein the repair of the skin barrier comprises up-regulating the expression of a barrier repair-related gene; the barrier repair related gene is at least one of FLG, IVL and OCLN;

the anti-aging is the up-regulation of the expression of extracellular matrix related genes; the extracellular matrix-associated gene comprises at least one of COL1A1, LN, COL13A1, SPTSSA, MKX, and COL3 A1;

the anti-aging is the expression of up-regulated apoptosis-inhibiting related gene BCL-2;

the anti-aging is the expression of up-regulated cell autophagy-related gene LC 3B;

the anti-aging is the expression of up-regulated cell antioxidant related genes NRF2, SIRT1 and SIRT3

The anti-aging is the expression of up-regulated immune regulatory factor related gene MOR

The anti-aging is the up-regulation of the expression of cell growth factor related genes VEGF, FGF1, FGF2, FGF21 and HGF.

6. The use according to claim 3, wherein the anti-aging is down-regulation of expression of extracellular matrix-associated genes; the extracellular matrix-associated gene includes at least one of the MMP families;

the anti-aging is the expression of down-regulation apoptosis related genes; the apoptosis-related gene includes expression of BAX.

7. The use of claim 3, wherein the improvement of the antibacterial ability of the cell is the up-regulation of the expression of antibacterial peptide related genes S100A7, S100A8, LCN-2.

8. Use according to any one of claims 1 to 7, wherein the product is a food product, a pharmaceutical product or a cosmetic product.

9. A product for improving skin conditions, made from a material comprising the candida parapsilosis strain of claim 1.

10. The product according to claim 9, wherein the strain of candida parapsilosis in the product comprises one or both of the following (1) or (2):

(1) A population and/or a bacterial agent of the candida parapsilosis strain of claim 1;

(2) A culture, exosome, lysate and/or extract of the Candida parapsilosis strain of claim 1.