CN114686525A

CN114686525A - Preparation method and application of yeast fermentation product for repairing skin barrier

Info

Publication number: CN114686525A
Application number: CN202111552435.3A
Authority: CN
Inventors: 徐梦漪; 林朝栋; 叶海敏; 何笙丽; 郑跃萍
Original assignee: Guangzhou Youke Biotechnology Co ltd
Current assignee: Guangzhou Youke Biotechnology Co ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-07-01
Anticipated expiration: 2041-12-17
Also published as: CN114686525B

Abstract

The invention relates to the field of C12N1/20, in particular to a preparation method and application of a yeast fermentation product for repairing skin barriers, which comprises the following steps: (1) inoculation, (2) fermentation, and (3) sterilization. According to experimental data, the GFF prepared by the invention can induce the expression of cell tight junction protein, improve the transmembrane resistance of cells and increase the intercellular compactness, thereby having an obvious skin tightening and pulling effect.

Description

Preparation method and application of yeast fermentation product for repairing skin barrier

Technical Field

The invention relates to the field of C12N1/20, in particular to a preparation method and application of a yeast fermentation product for repairing skin barriers.

Background

The human skin is the first barrier directly contacting with the outside, and the physical barriers established by combining procedures of differentiation, induction expression and the like of epidermal cells in the skin can effectively resist the invasion of external harmful substances or pathogens and can also effectively prevent the water loss in the skin. Aging and damage to the skin barrier not only can lead to skin problems such as skin blemishes, wrinkles, etc., but also can pose potential risks to human health.

CN109528607A discloses a skin barrier repair composition and a skin barrier repair product, which achieve the effect of repairing the skin barrier through various plant components such as lavender and sanchi flower, however, the action mechanism of the skin barrier repair composition can only be relieved from the perspective of moisturizing and moisturizing the epidermis, and the inner part of the barrier cannot be directly repaired from the aspect of inducing differentiation of epidermal cells.

CN112043667A discloses a composition for moisturizing and maintaining skin barrier and a preparation method and application thereof, and the composition is added with yeast schizophyllum commune and lotus seed fermentation liquor, has a certain skin barrier repair effect, but has unobvious long-term moisturizing and wrinkle removing effects and relatively single function.

Therefore, the composition which has the effects of long-acting water locking and wrinkle resistance on the basis of repairing the skin barrier has a very application prospect.

Disclosure of Invention

In a first aspect of the present invention, there is provided a method for preparing yeast ferment for repairing skin barrier, comprising the steps of: (1) inoculation, (2) fermentation, and (3) sterilization.

As a preferred embodiment, the culture is carried out for 36-40h at 28-33 ℃ after the inoculation.

As a preferred embodiment, the culture solution in the fermentation comprises a saccharide culture solution.

In a preferred embodiment, the saccharide in the saccharide culture solution comprises at least one of glucose, maltose and sucrose.

In order to reduce the color of the fermentation product after sterilization, it is preferable that the saccharide in the saccharide culture solution is sucrose.

As a preferred embodiment, the amount of inoculation in the fermentation is between 1 and 3%.

In a preferred embodiment, the ratio of fermentation aeration to volume of fermentation broth in the fermentation is 0.8 to 1.2.

As a preferred embodiment, the temperature of the sterilization is 70-80 ℃.

As a preferred embodiment, the preparation method comprises the following steps:

(1) inoculation: inoculating the strain into sterilized YPD culture solution, and shake-culturing at 28-33 deg.C for 36-40 hr to obtain seed solution;

the YPD culture solution is prepared from the following raw materials in percentage by weight: 1% of yeast extract powder, 2% of peptone, 2% of glucose and the balance of pure water;

the strain is Chinese hyphomycete, separated from Beijing wine medicine, and has the strain number of 244 and the dry standard number of 58743;

(2) fermentation: absorbing seed liquid and moving the seed liquid into the sterilized culture solution, carrying out shake culture at 28-33 ℃, wherein the rotating speed is 120-140rpm, the air flow/culture solution volume ratio is 0.8-1.2V/V/min, detecting that the viable count reaches more than 2E8CFU/ml after culturing for 24h, and the pH value is about 4.5, and stopping fermentation;

the inoculation amount of the seed liquid is 1-3% of the volume of the culture liquid;

the preparation raw materials of the culture solution comprise, by weight: 3% of saccharides, 0.5% of peptone, 0.5% of yeast extract powder, 0.3% of dipotassium phosphate, 0.1% of magnesium sulfate heptahydrate and the balance of pure water;

(3) and (3) sterilization: centrifuging the culture solution, separating thallus and fermentation liquid, heating the separated fermentation liquid to 70-80 deg.C, maintaining the temperature for 15min for sterilization, cooling to 65 deg.C, adding antiseptic, stirring, and filtering with 300 mesh filter screen to obtain yeast fermented product.

Tight junctions are an important intercellular structure in the skin barrier, tightening the skin by anchoring proteins parallel to each other on adjacent cell membranes, preventing other molecules from passing between them.

NMF (Natural Moisturizing factor) is a natural Moisturizing agent produced by skin in stratum corneum cells, is a core component for Moisturizing skin, and is mainly produced by degrading filaggrin induced by Caspase-14 in cells, so that the defect of Caspase-14 can cause the reduction of skin moisture content.

The applicant finds that the product GFF obtained by fermenting the filamentous fungi can obviously induce the expression of cell tight junction protein and the expression of Caspase-14, and the product GFF serving as an active ingredient is added into a skin care product and has the effects of long-acting water locking and wrinkle resisting on the basis of repairing skin barriers. GFF and its efficacy have not been reported in the prior art.

The second aspect of the invention provides application of the yeast fermentation product obtained by the preparation method of the yeast fermentation product for repairing the skin barrier in a skin care product, wherein the skin care product comprises the yeast fermentation product and a solvent.

In a preferred embodiment, the weight ratio of the yeast fermentation product to the solvent is (14-18): 1.

as a preferred embodiment, the solvent includes at least one of polyhydric alcohol, ethylhexylglycerin, hyaluronic acid.

Compared with the prior art, the invention has the following beneficial effects:

1. according to experimental data, the GFF prepared by the invention can induce the expression of cell tight junction protein, improve the transmembrane resistance of cells and increase the intercellular compactness, thereby having an obvious skin tightening and pulling effect.

2. According to experimental data, the GFF prepared by the invention can induce the expression of Caspase-14, improve the enzyme activity, promote the generation of NMF, strengthen the effects of water replenishing and locking from inside to outside and ensure that the skin keeps moist for a long time.

3. According to experimental data, the GFF prepared by the invention can improve the expression of filaggrin, enhance the skin barrier function and strengthen the hydration effect of the skin, and the transdermal water loss rate proves that the problem of water shortage and dryness of the skin caused by the damage of the barrier can be obviously improved.

4. According to experimental data, the GFF prepared by the invention has mild performance and no skin irritation, can be used as an active ingredient to be added into various skin care products, and has wide application prospect.

Drawings

FIG. 1 is a graph showing the results of an experiment for GFF-induced cell claudin expression.

FIGS. 2 to 3 are graphs showing the results of experiments in which GFF induces Caspase-14 expression.

FIGS. 4 to 5 are graphs showing the results of experiments for expressing GFF-Loricrin (LOR) which is a barrier function-promoting loricrin.

Fig. 6-8 are graphs showing the results of GFF experiments to increase skin hydration.

FIGS. 9-10 are graphs of skin elasticity test results.

FIGS. 11-12 are graphs showing the results of tests for increasing skin firmness.

FIG. 13 is a graph showing the results of the closed patch test on human skin.

Fig. 14 is a graph showing the results of the skin irritation test.

FIGS. 15-16 are graphs showing the results of evaluation of the barrier repair effect of volunteers.

FIGS. 17-22 are graphs showing the results of skin irritation tests.

FIGS. 23-24 are graphs showing the results of toxicology experiments.

Detailed Description

Example 1

In a first aspect, this embodiment provides a method for preparing yeast ferment for repairing skin barrier, comprising the following steps:

(1) inoculation: inoculating the strain into sterilized YPD culture solution, and performing shake culture at 30 ℃ for 38 hours to obtain seed solution;

(2) fermentation: absorbing seed liquid, transferring the seed liquid into a sterilized culture solution, performing shake culture at 30 ℃, wherein the rotation speed is 130rpm, the air flow/volume ratio of the culture solution is 1.0V/V/min, detecting that the number of viable bacteria is more than 2E8CFU/ml after culturing for 24 hours, and stopping fermentation when the pH is 4.5;

the inoculation amount of the seed solution is 2% of the volume of the culture solution;

the preparation raw materials of the culture solution comprise, by weight: 3% of sucrose, 0.5% of peptone, 0.5% of yeast extract powder, 0.3% of dipotassium phosphate, 0.1% of magnesium sulfate heptahydrate and the balance of pure water;

(3) and (3) sterilization: centrifuging the culture solution, separating thallus and fermentation liquid, heating the separated fermentation liquid to 75 deg.C, keeping the temperature for 15min for sterilization, cooling to 65 deg.C, adding antiseptic, stirring, and filtering with 300 mesh filter screen to obtain yeast fermented product (GFF).

In a second aspect of this embodiment, the application of the yeast ferment obtained by the method for preparing the yeast ferment for repairing the skin barrier in the skin care product is provided, wherein the skin care product comprises 96% of yeast ferment (GFF) and 4% of solvent by weight.

The solvent is dipropylene glycol, 1, 2-hexanediol and ethylhexylglycerin which are compounded in a weight ratio of 5:1: 0.1.

Comparative example 1

This comparative example provides a method for preparing yeast ferment for repairing skin barrier in the first aspect, and the specific embodiment is the same as example 1, except that the sucrose is replaced by glucose in the raw material for preparing the culture solution.

In a second aspect of the present comparative example, the application of the yeast fermentation product obtained by the method for preparing the yeast fermentation product for repairing the skin barrier in the skin care product is provided, and the specific implementation manner is the same as that in example 1.

Comparative example 2

The first aspect of the present comparative example provides a method for preparing a yeast fermentation product for repairing skin barrier, which is the same as example 1 except that in the sterilization of (3), the separated fermentation broth is heated to 90 ℃ and kept for 20min for sterilization.

Performance testing

The yeast fermentates obtained in the examples and comparative examples were placed in an environment of 40 ℃ for 12 hours and then in an environment of-5 ℃ for 12 hours, and the presence or absence of turbidity and stratification was observed.

	Stability of
		Example 1	Without change
Comparative example 1	Has slight turbidity
		Comparative example 2	With obvious turbidity and demixing

The yeast fermentation (GFF) prepared in example 1 was subjected to the following tests.

GFF-induced expression assay of cellular Claudin:

GFF was prepared into aqueous solutions of 1%, 10%, and 20% by mass, respectively, and a control group was purified water, and the conditions of GFF-induced cell fibronectin were tested by protein blotting, and the results of the test are shown in FIG. 1.

Immunohistochemistry and protein immunoblotting are used for detecting the expression of claudin and occludin protein of mouse skin lesion, and protein immunoblotting (Westernblot) is used for detecting the expression of claudin-7 and occludin protein of model skin lesion. Grinding and extracting skin tissue protein, quantifying by using a BCA kit, adding 5 times of loadingbuffer for high-temperature denaturation, performing electrophoresis and quick electrotransfer, sealing 5% skimmed milk powder at room temperature for 1h, diluting a primary antibody by using a phosphate Polysorbate Buffer Solution (PBST), diluting claudin-7 (1: 100), occludin (1: 1000), shaking overnight at 4 ℃, diluting a secondary antibody by using PBST (1: 1 ten thousand), and after incubating for 1h in a shaking table at room temperature in a dark place, sweeping a membrane to obtain an image. The software ImageJ performs grey value analysis on the bands.

In the blank group, both claudin-1 and claudin-7 marked the epidermal cell envelope, with continuous expression and complete network. occludin is mainly used for marking epidermal cell membranes, cytoplasm has a small amount of expression, and the expression is continuous; compared with the blank group, the expression of 3 proteins in the model group is remarkably reduced (P is less than 0.01), and the expression integrity is seriously damaged, wherein the occludin marks the abnormal localization of the whole epidermis. The test group showed that expression of claudin-1, occludin, was increased with better effect (P < 0.05).

The results of the experiment (see fig. 1) indicate that GFF can induce the expression of tight junction protein (TJ) in epidermal keratinocytes, enhancing skin barrier function.

The test expression objects of the tight junction protein are Occludin and claudin.

GFF-induced Caspase-14 expression assay:

western blotting detection of Caspase-14 expression: animal model skin is respectively treated by a blank group and a test group (5% GFF and 1% GFF) for 24 hours, protein is extracted after skin tissue grinding, the protein concentration is determined by a BCA method, about 20 mu g of total protein is subjected to 4mA constant current electrophoresis in 6% SDS-PAGE gel, the protein is transferred to a nitrocellulose membrane at constant voltage of 100V, PBS (phosphate buffer solution) containing 5% skimmed milk powder is sealed at 4 ℃ overnight, anti-Caspase-14 and beta-actin primary antibody (the dilution concentration is 1: 1000) are added for incubation for 2 hours at room temperature, after PBS is washed, goat anti-rabbit secondary antibody marked by HRP (horse radish peroxidase) at the ratio of 1: 3000 is added for incubation for 1 hour at room temperature, after PBS is washed, chemiluminescent agent is added, a biological image analyzer is used for imaging, and semi-quantitative analysis is carried out by taking beta-actin as an internal reference.

The results of the experiment (see FIGS. 2-3) show that: GFF has obvious effect of promoting the expression of Caspase-14 expression protein in skin, and GFF can improve the expression activity of Caspase-14.

GFF expression test for elevation of barrier function Loricrin (LOR):

detecting the expression of the loicrin marker by an immunohistochemical method, carrying out dewaxing and antigen retrieval on a model by a paraffin section, carrying out temperature sealing on a peroxidase closed liquid for 10min, incubating serum working solution at 37 ℃ for 30min, and incubating a loicrin (1: 200) primary antibody overnight; the lorarin was incubated with the enhanced enzyme-labeled immunoglobulin G (IgG) antibody for 1h at 37 ℃ and then developed into a color patch.

The results of the experiment (see FIGS. 4-5) show that: the expression of the loricrin in the skin lesion tissues of each group, blank group, is mainly expressed in the horny layer of the epidermis and is in continuous linear distribution. After the control group model is damaged, the expression level of the loicrin is obviously reduced, and the staining gradually reaches the granular layer; the test group 10% GFF increased the expression of loricrin, extended to the stratum corneum, and promoted the formation of stratum corneum barrier.

10% GFF improves LOR protein expression amount to 40.2-71.1%.

Test of GFF for improving skin moisture content:

human body test: the subjects were 35 years old, 5mL of the skin care product prepared in example 1 was applied to the skin once a day in the morning and evening for 14 days, and the skin surface changes were recorded by photographing, and the results of the experiment are shown in fig. 6 to 8.

Moisture content variation test: the experimental method refers to T/ZHCA005-2019, namely, the method for testing the influence of cosmetics on skin elasticity, and the experimental results before and after use are respectively shown in figures 6-7.

Transdermal water loss rate test: the experimental method refers to T/ZHCA003-2018 test method for testing moisture loss of affected skin of cosmetics, and the experimental result is shown in FIG. 8.

As can be seen from the figure, after the skin care product is used for 14 days, the transdermal water loss of the skin is obviously reduced, the water content of the skin is improved by 30-36%, and the dryness of the skin is improved.

5. Skin elasticity test

The Cutometer measures skin elasticity parameters such as total skin stretch and

tonicity characteristics

1, 4 and 24 hours after a single application and 2 weeks after cessation of use. Total elasticity measures total skin stretch, including viscous deformation, which represents the perceived elasticity of hydrated skin. Net elasticity is independent of viscous deformation; it is used to characterize the mechanisms of skin movement, in particular to test the skin's tonicity properties.

Both 5% and 10% GFF act on skin with a significant increase in total and net elasticity. The results are shown in FIGS. 9-10.

6. Test for improving skin firmness:

the test subjects were 35 years old, and 5mL of the skin care product prepared in example 1 was applied to the facial skin once a day, morning and evening for 14 days, and the results before and after application are shown in FIGS. 11-12, respectively.

7. Human skin closed patch test:

the experimental standard refers to the technical Specification for cosmetic safety 2015, the number of volunteers is 30, the age is 35-50 years, and the experimental result is shown in FIG. 13.

8. Skin irritation test:

the test standard refers to SN/T2329-2009 'cosmetic eye irritation/corrosiveness chick embryo chorioallantoic membrane test', a stimulation scoring method is adopted, the test time is 300s, and the test result is shown in figure 14.

9. Evaluation of volunteer barrier repair effect

Test objects: 25 female testers (age 18-40 years old)

Test area: face part

Test samples: facial mask control and facial mask containing 10.0% GFF

The facial mask reference substance is a facial mask without GFF.

The test method comprises the following steps: the facial mask reference substance and 10.0% GFF facial mask are applied to consumer by random half-face blind test for 10min, and then removed, and scored according to the given index of test table, and the performance of two facial masks is evaluated and compared. The evaluation results are shown in FIGS. 15 to 16.

10. Skin irritation test: the test reports are shown in FIGS. 17-22.

11. Toxicology experiments: the test reports are shown in FIGS. 23-24.

Claims

1. A preparation method of yeast leavening for repairing skin barrier is characterized by comprising the following steps: (1) inoculation, (2) fermentation, and (3) sterilization.

2. The method for preparing yeast ferment for repairing skin barrier according to claim 1, wherein the yeast ferment is cultured for 36-40h at 28-33 ℃ after inoculation.

3. The method for preparing yeast ferment for repairing skin barrier according to claim 2, wherein the culture solution in fermentation comprises sugar culture solution.

4. The method for preparing yeast fermentation product for repairing skin barrier according to claim 3, wherein the sugar in the sugar culture solution comprises at least one of glucose, maltose and sucrose.

5. The method for preparing yeast ferment for repairing skin barrier according to any one of claims 1 to 4, wherein the inoculation amount in the fermentation is 1 to 3 percent.

6. The method for preparing yeast ferment for repairing skin barrier according to any one of claims 1 to 5, wherein the ratio of fermentation aeration volume to fermentation broth volume in the fermentation is 0.8 to 1.2.

7. The method for preparing yeast ferment for repairing skin barrier according to any one of claims 1 to 6, wherein the temperature for sterilization is 70 to 80 ℃.

8. Use of the yeast ferment obtained by the method for preparing the yeast ferment for repairing the skin barrier according to any one of claims 1 to 7 in a skin care product, wherein the skin care product comprises the yeast ferment and a solvent.

9. The use of the yeast ferment obtained by the preparation method of the yeast ferment for repairing the skin barrier in the skin care product according to claim 8 is characterized in that the weight ratio of the yeast ferment to the solvent is (14-18): 1.

10. the use of yeast ferment obtained from the method for preparing a yeast ferment for repairing skin barriers according to claim 9 in skin care products, wherein the solvent comprises at least one of polyol, ethylhexylglycerin and hyaluronic acid.