Application of preparing cosmetics by adopting lysate of eurotium cristatum and cosmetics

Abstract

The invention belongs to the technical field of biology, and discloses application of a lysate of eurotium cristatum to preparation of cosmetics. A lysate product of the bacterium has one or more of a whitening effect, a repairing effect, an anti-oxidation effect and a soothing effect. Meanwhile, the invention further provides cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the use of the lysate of Eurotium cristatum for preparing cosmetics and cosmetics. Background Art

[0002] This application is based on the priority of the prior application with the application number 202410518179.3 and the theme of a strain of Eurotium cristatum and uses.

[0003] Eurotium cristatum, commonly known as "golden flower", is a fungus belonging to the genus Eurotium of the family Trichocomaceae in the order Eurotiales. It consists of an ascocarp and hyphae, has low nutritional requirements, can grow on potato dextrose agar medium and Rose Bengal medium, has strong adaptability, can utilize a variety of nitrogen sources and carbon sources, and is the dominant strain during the fermentation process of Fuzhuan tea. It obtains nutrients from tea leaves and through its own metabolism, produces various enzymes to catalyze the transformation of various substances in tea, forming the unique color, aroma and taste qualities of Fuzhuan tea. Therefore, it is an important index for evaluating the quality of Fuzhuan tea.

[0004] In the research on this bacterium, we studied its effects on health aspects such as reducing blood lipid and alleviating fatty liver.

[0005] This case mainly studies the application of this bacterium in cosmetics. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of the lysate of Eurotium cristatum for preparing cosmetics, and the lysate of this bacterium has one or more of the effects of whitening, repairing, antioxidant and soothing.

[0007] At the same time, the present invention also provides cosmetics.

[0008] To achieve the above purpose, the present invention provides the following technical solution: the use of the lysate of Eurotium cristatum for preparing cosmetics;

[0009] The Eurotium cristatum has the taxonomic name Eurotium cristatum, and the preservation number is: CGMCC NO: 40145; the preservation date is: March 10, 2022, and the preservation unit is the General Microbiology Center of the China Microbial Culture Collection Center.

[0010] In the above use, the lysate is one of a water-soluble product and an alcohol-soluble product.

[0011] In the above use, the lysate is an alcohol-soluble product, and the cosmetics are cosmetics with soothing effects and / or antioxidant effects.

[0012] The relevant properties of the alcohol-soluble product can be seen in the test reports numbered 2502WH100-01A and 2502WH100-01B issued by Guangzhou Huamiao Testing Services Co., Ltd. The test report of 2502WH100-01A is for the antioxidant efficacy test of cosmetics, and the test report of 2502WH100-01B is for the soothing efficacy test of cosmetics; the relevant test contents can be seen later.

[0013] In the above uses, the lysate is a water-soluble product, and the cosmetic is a cosmetic having at least one of the functions of whitening, repairing, antioxidant, and soothing.

[0014] The relevant properties of the water-soluble product can be seen in the test reports numbered 2502WH100-01C, 2502WH100-01D, 2502WH100-01E, 2502WH100-01F, and 2502WH100-01G issued by Guangzhou Huamiao Testing Services Co., Ltd. The test report of 2502WH100-01C is for the whitening efficacy test of cosmetics

[0015] , the test report of 2502WH100-01D is for the whitening efficacy test of cosmetics; the test report of 2502WH100-01E is for the soothing efficacy test of cosmetics; the test report of 2502WH100-01F is for the antioxidant efficacy test of cosmetics; the test report of 2502WH100-01G is for the repair efficacy test of cosmetics; the relevant test contents can be seen later.

[0016] At the same time, the present invention also discloses a cosmetic containing the lysate of Eurotium cristatum.

[0017] In the above cosmetic, the lysate is one of a water-soluble product and an alcohol-soluble product.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] In the relevant applications of the Eurotium cristatum of the present invention in cosmetics, whether it is an alcohol-soluble product or a water-soluble product, it has excellent application performance related to cosmetics, which greatly expands the relevant applications of Eurotium cristatum in the cosmetic field. Description of the Drawings

[0020] Figure 1 It is a bar chart of the test results of the antioxidant efficacy test of cosmetics;

[0021] Figure 2 It is a bar chart of the test results of the soothing efficacy test of cosmetics;

[0022] Figure 3 It is a bar chart of the test results of the whitening efficacy test of cosmetics;

[0023] Figure 4 Test results of cell viability assay for whitening efficacy testing of cosmetics;

[0024] Figure 5 Results of determination of relative melanin synthesis content in B16 cells for whitening efficacy testing of cosmetics;

[0025] Figure 6 Test results of cell viability assay for soothing efficacy testing of cosmetics;

[0026] Figure 7 Results of determination of histamine content in RBL-2H3 cells for soothing efficacy testing of cosmetics;

[0027] Figure 8 Test results of cell viability assay for antioxidant efficacy testing of cosmetics;

[0028] Figure 9 ROS fluorescence image for antioxidant efficacy testing of cosmetics;

[0029] Figure 10 Test results for antioxidant efficacy testing of cosmetics;

[0030] Figure 11 Test results of cell viability assay for repair efficacy testing of cosmetics;

[0031] Figure 12 Result image of fibroblast scratch assay for repair efficacy testing of cosmetics;

[0032] Figure 13 Test results for repair efficacy testing of cosmetics;

[0033] Figure 14 Sample solution diagram after being dissolved in alcohol. Specific implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] For the relevant results of the isolation and identification of Eurotium cristatum, the tea fermentation of different strains, and the tea fermentation of different tea species, reference can be made to the prior application of the present applicant, with the application number CN202411467852.1 (priority number CN202410518179.3), and the theme is a strain of Eurotium cristatum and its uses.

[0036] This part focuses on discussing its relevant effects;

[0037] The Eurotium cristatum used in the present invention are all the preserved strains of the present invention, and their appearance is golden yellow.

[0038] The alcohol-soluble cell lysate of the first part of Eurotium cristatum

[0039] The sample is a yellow powder, which is sent to Guangzhou Huamiao Testing Service Co., Ltd. for alcohol dissolution operation; and the test reports numbered 2502WH100-01A and 2502WH100-01B are issued.

[0040] The alcohol dissolution operation detected by 2502WH100-01A is as follows: Dilute with 95 vol% ethanol to a sample concentration of 3%, and take the supernatant for testing after standing;

[0041] The alcohol dissolution operation detected by 2502WH100-01B is as follows: Dilute with absolute ethanol to a sample concentration of 3%, and take the supernatant for testing after standing.

[0042] During the alcohol dissolution process, the sample showed an insoluble state ( Figure 14 ), therefore, the detection was carried out by dispersing it in alcohol, standing, and then filtering. Undoubtedly, in industrial production, other methods can be used for further dissolution operations, which can significantly improve the efficacy of the product.

[0043] Next, the following will discuss the two test items one by one;

[0044] 1.1 Cosmetic antioxidant efficacy test (2502WH100-01A)

[0045] Detection basis: HMC-WI-030 DPPH free radical scavenging rate operation instruction

[0046] Detection results and conclusions: According to the laboratory method (HMC-WI-030 DPPH free radical scavenging rate), the antioxidant efficacy of the sample was tested by the laboratory method; the test results showed that: the DPPH free radical scavenging rate of the positive control > 50%, the reaction system was effective, when the test sample was diluted into a 3% ethanol solution, the DPPH free radical scavenging rate was 96.932%, showing a significant difference compared with the negative control (P<0.05); indicating that the sample has a certain antioxidant efficacy;

[0047] Test purpose and principle

[0048] Excessive production of free radicals will lead to natural aging and photoaging of the skin, resulting in wrinkles. Therefore, the ability to scavenge free radicals is one of the important indicators for evaluating anti-aging and antioxidant cosmetics (raw materials).

[0049] This test refers to the laboratory method (HMC-WI-030 DPPH free radical scavenging rate). The DPPH free radical scavenging rate test results of the test sample are compared with those of the negative control. If the scavenging rate of the test sample is higher than that of the negative control and there is a significant difference (P<0.05), it can be considered that the test sample has a certain antioxidant effect.

[0050] This test method is an in vitro method and is applicable to evaluating cosmetics that claim to achieve antioxidant effects by scavenging free radicals.

[0051] Instrument and equipment

[0052] BSA224S analytical balance

[0053] L6s ultraviolet spectrophotometer

[0054] Reagents

[0055] DPPH (1,1-diphenyl-2-picrylhydrazine), 98%

[0056] Test method

[0057] (1) Treatment of control and test samples

[0058] Sample group: Dilute with 95% ethanol to a sample concentration of 3%, let it stand, and take the supernatant for testing;

[0059] Positive control (vitamin E, purity ≥96%): Dilute with 95% ethanol to a positive control concentration of 0.1%;

[0060] Negative control: 95% ethanol.

[0061] Test operation steps

[0062] Set up sample tubes, sample background tubes, DPPH tubes and solvent background tubes. Three parallel tubes need to be set up for each group. Add different reagent solutions to the four groups respectively, shake gently, and let it stand at room temperature for 5 minutes. Transfer the reaction solutions of each group into 1 cm cuvettes and measure the absorbance at 517 nm.

[0063] Calculation formula

[0064]

[0065] In the formula: T—the absorbance of the sample tube, that is, the absorbance of the solution after the sample reacts with DPPH;

[0066] T0—the absorbance of the sample background;

[0067] C—the average value of the absorbance of the DPPH tube three times, that is, the absorbance of the DPPH solution without adding the sample;

[0068] C0—Absorbance value of the solvent background.

[0069] Test results

[0070] Sample group: 96.932 ± 0.917%;

[0071] Negative control: -2.152 ± 1.717%;

[0072] Positive control: 96.427 ± 0.096%;

[0073] The bar chart of the above results can be seen Figure 1 .

[0074] Conclusion: According to the laboratory method (HMC-WI-030 DPPH free radical scavenging rate), the antioxidant efficacy of the sample was tested by the laboratory method; the test results showed that: the DPPH free radical scavenging rate of the positive control > 50%, the reaction system was effective. When the test sample was diluted into a 3% ethanol solution, the DPPH free radical scavenging rate was 96.932%, showing a significant difference compared with the negative control (P < 0.05); indicating that the product has a certain antioxidant efficacy.

[0075] 1.2 Test on the soothing efficacy of cosmetics

[0076] Detection basis: HMC-WI-029 "Hyaluronidase Inhibition Rate" (2502WH100-01B)

[0077] Detection results and conclusion: According to the laboratory method (HMC-WI-029 hyaluronidase inhibition rate), the soothing efficacy of the sample was tested by the laboratory method; the test results showed that: the hyaluronidase inhibition rate of the positive control > 50%, the reaction system was effective. When the sample concentration was 3%, the hyaluronidase inhibition rate was 34.533%, showing a significant difference compared with the negative control (P < 0.05), indicating that the sample has a certain soothing efficacy.

[0078] Test purpose and principle

[0079] Hyaluronidase is a specific lyase of hyaluronic acid and is a participant in allergic reactions, with a strong correlation with the release of histamine by mast cells. Whether the test sample has a soothing efficacy can be judged by the hyaluronidase inhibition rate. The higher the hyaluronidase inhibition rate, the stronger the soothing efficacy of the substance, and vice versa.

[0080] This test referred to the laboratory method (HMC-WI-029 hyaluronidase inhibition rate), and compared the hyaluronidase inhibition rate test results of the test sample with those of the negative control. If the inhibition rate of the test sample was higher than that of the negative control and had a significant difference (P < 0.05), it could be considered that the test sample had a certain soothing efficacy.

[0081] This test method is an in vitro method and is applicable to evaluating cosmetics that claim to achieve a soothing effect by inhibiting hyaluronidase.

[0082] Test Materials and Methods

[0083] Instruments and Equipment

[0084] BSA224S Analytical Balance

[0085] L6s UV Spectrophotometer

[0086] Reagents

[0087] Hyaluronidase, BR

[0088] Sodium Hyaluronate, BR

[0089] Test Method

[0090] (1) Treatment of Control and Test Samples

[0091] Sample Group: Dilute with absolute ethanol to a sample concentration of 3%, let stand, and take the supernatant for testing;

[0092] Positive Control (Dipotassium Glycyrrhizinate, purity ≥ 98%): Dilute with pure water to a positive control concentration of 3%;

[0093] Negative Control: Absolute ethanol.

[0094] (2) Test Operation Steps

[0095] Set up a sample group, a sample background group, a solvent group, and a solvent background group. Each group needs to have 3 parallels. Add different reagent solutions to the four groups respectively, shake well, let stand at room temperature for 30 min for color development, and measure the absorbance value at a wavelength of 528 nm with a UV spectrophotometer.

[0096] (3) Calculation Formula

[0097]

[0098] In the formula: A — is the absorbance of the reaction solution without the sample; B — is the absorbance of the reaction solution without the sample and the enzyme;

[0099] C — is the absorbance of the reaction solution containing the sample and the enzyme; D — is the absorbance of the reaction solution containing the sample and without the enzyme.

[0100] Test Results

[0101] Sample Group: 34.533 ± 0.833%;

[0102] Negative Control: -2.050 ± 1.280;

[0103] Positive control: 68.338 ± 1.371;

[0104] The bar chart of the above results can be seen Figure 2 ;

[0105] Test conclusion

[0106] According to the laboratory method (HMC-WI-029 hyaluronidase inhibition rate), the soothing effect of the sample was tested by laboratory method; the test results showed that the hyaluronidase inhibition rate of the positive control > 50%, the reaction system was effective. When the sample concentration was 3%, the hyaluronidase inhibition rate was 34.533%, which had a significant difference compared with the negative control (P < 0.05), indicating that the sample had a certain soothing effect.

[0107] The second part is the water-soluble lysate of Eurotium cristatum (2502WH100-01C)

[0108] The sample was a yellow powder, which was sent to Guangzhou Huamiao Testing Service Co., Ltd. for water-soluble operation; and the test reports numbered 2502WH100-01C, 2502WH100-01D, 2502WH100-01E, 2502WH100-01F, and 2502WH100-01G were issued.

[0109] During the water-soluble process, the sample showed an insoluble state. Therefore, it was detected by the method of dispersing it in water, standing still, and then filtering. Undoubtedly, in industrial production, other methods can be used for further dissolution operations to significantly improve the efficacy of the product.

[0110] Next, the following will discuss the five detection items one by one;

[0111] 2.1 Cosmetic whitening effect test

[0112] Detection basis: HMC-WI-032 "Tyrosinase inhibition rate"

[0113] Detection results and conclusions: According to the laboratory method (HMC-WI-032 tyrosinase inhibition rate), the whitening effect of the sample was tested by laboratory method; the test results showed that the tyrosinase inhibition rate of the positive control > 50%, the reaction system was effective. When the test sample was diluted into a 3% aqueous solution, the tyrosinase inhibition rate was 9.648%, which had a significant difference compared with the negative control (P < 0.05); indicating that the sample had a whitening effect.

[0114] Test purpose and principle

[0115] In skin melanin biosynthesis, tyrosinase is the key enzyme that acts on dopa to form dopaquinone, which then spontaneously undergoes a series of reactions to finally form melanin. In a phosphate solution with a pH of 6.8, tyrosinase can catalyze the conversion of dopa into dopaquinone, and the absorbance value can be measured at 475 nm using a spectrophotometer. Cosmetics with inhibitory effects on tyrosinase activity can reduce the conversion of dopa into dopaquinone, thereby decreasing the absorbance value. Based on the change in the absorbance value, the inhibitory effect of the cosmetics on tyrosinase activity can be evaluated.

[0116] In this experiment, referring to the laboratory method (tyrosinase inhibition rate of HMC-WI-032), the tyrosinase inhibition rate test results of the test sample were compared with those of the negative control. If the inhibition rate of the test sample is higher than that of the negative control and there is a significant difference (P < 0.05), it can be considered that the test sample has a whitening effect.

[0117] This test method is an in vitro method and is applicable to evaluating cosmetics that claim to achieve a whitening effect by inhibiting tyrosinase activity.

[0118] Test Materials and Methods

[0119] Instrumentation

[0120] BSA224S Analytical Balance

[0121] RT-6100 Microplate Reader

[0122] Reagents

[0123] Polyphenol Oxidase (Mushroom), BR

[0124] L-DOPA, BR

[0125] Test Method

[0126] (1) Treatment of Control and Test Samples

[0127] Sample Group: Dilute the sample with pure water to a concentration of 3%, perform ultrasonic extraction and then centrifugation, and take the supernatant for testing;

[0128] Positive Control (Kojic Acid, purity ≥ 96%): Dilute with pure water to a concentration of 0.1% for the positive control;

[0129] Negative Control: Pure water.

[0130] Test Operating Procedures

[0131] Set up sample tubes, sample background tubes, enzyme reaction tubes, and solvent background tubes. Three parallel tubes should be set up for each group. Add different reagent solutions to the four groups respectively, shake gently, and let stand at room temperature for 5 minutes. Transfer the reaction solutions of each group into 1 cm cuvettes and measure the absorbance value at 475 nm.

[0132] Calculation formula

[0133]

[0134] Where: T—the absorbance value of the sample tube, i.e., the absorbance value of the solution after the sample reacts with tyrosinase;

[0135] T0—the background absorbance value of the sample;

[0136] C—the average value of the absorbance values of the enzyme reaction tubes three times, i.e., the absorbance value of the reaction of tyrosinase and dopa without adding the sample;

[0137] C0—the background absorbance value of the solvent.

[0138] Test results

[0139] Sample group: 9.648 ± 1.726%;

[0140] Negative control: -4.661 ± 1.025%;

[0141] Positive control: 98.564 ± 0.785%;

[0142] The bar chart of the above test results can be seen Figure 3 ;

[0143] Test conclusions

[0144] According to the laboratory method (HMC-WI-032 tyrosinase inhibition rate), the whitening effect of the sample was tested by the laboratory method; the test results showed that: the tyrosinase inhibition rate of the positive control > 50%, the reaction system was effective, when the test sample was diluted into a 3% aqueous solution, the tyrosinase inhibition rate was 9.648%, and there was a significant difference compared with the negative control (P < 0.05); it indicated that the sample had a whitening effect.

[0145] 2.2 Test of the whitening effect of cosmetics (2502WH100-01D)

[0146] Detection basis: HMC-WI-068 "Cell Melanin Synthesis Inhibition Experiment"

[0147] Detection results and conclusions: According to the laboratory method (HMC-WI-068 cell melanin synthesis inhibition experiment), when the concentration of the test sample was 0.25% and 0.5%, the cell activity was relatively high, and the relative content of cell melanin synthesis was 70.76% and 67.25% respectively. Among them, the test samples at 0.25% and 0.5% had a significant effect on inhibiting cell melanin synthesis (P < 0.05), that is, the test samples at 0.25% and 0.5% concentrations had an obvious ability to inhibit cell melanin synthesis, indicating that the test sample had a whitening effect.

[0148] Test purpose and principle

[0149] B16 mouse melanoma cells are commonly used cell models for studying melanogenesis. By measuring the change in the amount of melanin synthesis after the action of whitening raw materials on melanocytes, the whitening efficacy of cosmetics is evaluated.

[0150] This experiment refers to the laboratory method (HMC-WI-068 cell melanin synthesis inhibition experiment), and compares the results of cell melanin synthesis measurement between the test sample and the negative control. If the test sample can detect the effect of inhibiting melanin synthesis in B16 cells and has a significant difference compared with the negative control (P<0.05), it can be considered that the test sample has whitening efficacy.

[0151] This test method is an in vitro method, providing a method for detecting the relative content of melanin synthesis in B16 cells, which can be used as one of the evidence supports for the claim of whitening efficacy of cosmetic raw materials and finished products.

[0152] Test Materials and Methods

[0153] Instrumentation

[0154] RT-6100 Microplate Reader

[0155] Cells Used in the Experiment

[0156] B16 mouse melanoma cells

[0157] Test Methods

[0158] (1) Treatment of Test Samples

[0159] Sample Group: Dilute the sample with pure water to a 15% solution, then filter it through a 0.22 μm filter, and collect the filtrate as the sample mother liquor.

[0160] Negative Control: Basic medium.

[0161] (2) Test Operating Procedures

[0162] Cell Viability Test: Take B16 cells and seed them in a 96-well plate. After 24 h, aspirate the culture medium, add the basic medium containing different concentrations of the test sample. After 24 h, detect the OD490nm by the MTT method, and analyze the effect of the test sample on the viability of B16 cells through t-test.

[0163] Determination of the Relative Content of Melanin Synthesis in B16 Cells: Take B16 cells and seed them in a 6-well plate. After culturing for 24 h, replace the medium with the basic medium containing different concentrations of the test sample. After two more medium replacements, wash the cells twice with PBS. Add 200 μL of 0.25% trypsin to each well to digest the cells, collect the cells into a centrifuge tube, and centrifuge for 5 minutes. Add 200 μL of melanin extraction solution to each tube, shake well, transfer to a 96-well plate, and detect the absorbance at 405 nm for each well with a microplate reader.

[0164] Calculation formula

[0165] Relative content of melanin synthesis (%) = T * 100 / C;

[0166] T - Absorbance of the test sample well;

[0167] C - Average of three absorbances of the negative control group;

[0168] Test results

[0169] Cell viability test

[0170] Before testing the relative content of melanin synthesis in sample cells, cell viability screening is first carried out to obtain the optimal test concentration, as Figure 4 shown.

[0171] Test for relative content of melanin synthesis in cells

[0172] After screening the concentration through cell viability, the optimal test concentration is selected to measure the relative content of melanin synthesis in B16 cells, and the effect of the test sample on melanin synthesis in B16 cells is obtained, as Figure 5 shown.

[0173] Test conclusion

[0174] From Figure 4 , when the concentrations of the test sample are 0.25% and 0.5%, the cell viability > 90%;

[0175] According to the laboratory method (HMC-WI-068 Inhibition experiment of melanin synthesis in cells), when the concentrations of the test sample are 0.25% and 0.5%, the cell viability is relatively high, and the relative contents of melanin synthesis in cells are 70.76% and 67.25% respectively. Among them, the test samples with concentrations of 0.25% and 0.5% have a significant effect on inhibiting melanin synthesis in cells (P < 0.05), that is, the test samples at concentrations of 0.25% and 0.5% have an obvious ability to inhibit melanin synthesis in cells, indicating that the test sample has a whitening effect.

[0176] 2.3 Test for soothing effect of cosmetics (2502WH100 - 01E)

[0177] Detection basis: HMC-WI-121 Determination of histamine content in IgE-induced RBL-2H3 cells

[0178] Test Results and Conclusions: According to the laboratory method (HMC-WI-121 IgE-induced RBL-2H3 cells - Histamine Content Determination), when the concentrations of the test samples were 0.0625% and 0.125%, the cell viability was relatively high, and the cell histamine inhibition rates were 11.96% and 39.70% respectively, showing significant differences compared with the negative control (P<0.01). That is, at the concentrations of 0.0625% and 0.125%, the test samples had the ability to significantly inhibit the increase in cell histamine content, indicating that the test samples had a soothing effect.

[0179] Test Objectives and Principles

[0180] RBL-2H3 cells can be used as a cell model to study the inhibition of histamine content in cosmetics. By inducing RBL-2H3 cells with IgE, the histamine contents of the negative control and the test sample groups were measured to evaluate whether the test samples had an effect on inhibiting histamine content. The determination of histamine content was performed by enzyme-linked immunosorbent assay (ELISA).

[0181] This test referred to the laboratory method (HMC-WI-121 IgE-induced RBL-2H3 cells - Histamine Content Determination), and compared the results of cell histamine content determination between the test samples and the negative control. If the cell histamine content of the test sample was lower than that of the negative control and had a significant difference (P<0.05), it could be considered that the test sample had a soothing effect.

[0182] This test method was an in vitro method, providing a method for detecting the histamine content in RBL-2H3 cells, which could be used as one of the evidence supports for the claim of the soothing effect of cosmetic raw materials and finished products.

[0183] Test Materials and Methods

[0184] Instrumentation

[0185] RT-6100 Microplate Reader

[0186] Cells Used in the Experiment

[0187] Rat basophilic granulocytes: RBL-2H3 cells

[0188] Test Methods

[0189] (1) Treatment of Test Samples

[0190] Sample Group: The sample was diluted with pure water to a 15% solution, and then filtered through a 0.22 μm filter. The filtrate was collected as the sample stock solution.

[0191] Negative Control Group: Basal culture medium (for cell viability test); Basal culture medium containing inducer (for histamine content determination).

[0192] (2) Test Operating Procedures

[0193] Cell viability assay: Seed RBL-2H3 cells in a 96-well plate. After 24 h, aspirate the culture medium, and add basal medium containing test samples at different concentrations. After 24 h, detect the OD490nm by the MTT method, and analyze the effect of the test samples on the viability of RBL-2H3 cells by t-test;

[0194] Determination of histamine content: Seed RBL-2H3 cells in a 96-well plate. After culturing for 24 h, replace the medium with basal medium containing test samples at different concentrations. After 24 h, collect the cell supernatant and centrifuge. Aspirate the supernatant, and determine the histamine content in the cell culture supernatant using an ELISA kit to obtain the effect of the test samples on the histamine content of RBL-2H3 cells.

[0195] Calculation formula

[0196] Histamine inhibition rate (%) = (histamine content in negative control group - histamine content in sample group) * 100 / histamine content in negative control group

[0197] Test results

[0198] Cell viability assay

[0199] Before testing the histamine content of the sample cells, perform cell viability screening to obtain the optimal test concentration, as Figure 6 shown;

[0200] Test for histamine content in cells

[0201] After screening out the concentration through cell viability, select the optimal test concentration and perform the determination of histamine content in RBL-2H3 cells to obtain the effect of the test sample on the histamine content of RBL-2H3 cells, as Figure 7 shown.

[0202] Test conclusion

[0203] From Figure 6 , when the concentrations of the test samples are 0.0625% and 0.125%, the cell viability > 90%;

[0204] According to the laboratory method (HMC-WI-121 IgE-induced RBL-2H3 cells - determination of histamine content), when the concentrations of the test samples are 0.0625% and 0.125%, the cell viability is relatively high, and the histamine inhibition rates of the cells are 11.96% and 39.70% respectively, showing significant differences compared with the negative control (P < 0.01). That is, when the concentrations of the test samples are 0.0625% and 0.125%, the test samples have the ability to significantly inhibit the increase in histamine content in cells, indicating that the test samples have a soothing effect.

[0205] 2.4 Antioxidant efficacy test of cosmetics (2502WH100-01F)

[0206] Detection basis: HMC-WI-122 In vitro Keratinocyte Reactive Oxygen Species (ROS) Inhibition Test

[0207] Detection results and conclusions: According to the laboratory method (HMC-WI-122 Cosmetics Antioxidant Efficacy Test - In vitro Keratinocyte Reactive Oxygen Species (ROS) Inhibition Test), when the concentrations of the test samples were 0.25% and 0.5%, the cell viability was relatively high, and the ROS inhibition rates of the cells were 62.83% and 63.17% respectively, with significant differences compared with the negative control (P<0.0001). That is, at the concentrations of 0.25% and 0.5%, the test samples had an obvious effect of inhibiting cellular ROS, indicating that the test samples had antioxidant efficacy.

[0208] Test purposes and principles

[0209] Reactive oxygen species (ROS) in organisms can damage genetic materials and proteins, generate lipofuscin, and destroy collagen, ultimately causing skin aging and relaxation.

[0210] Intracellular reactive oxygen species can oxidize non-fluorescent 2',7'-dichlorofluorescein diacetate (DCFH) to generate fluorescent substances, and the intensity of green fluorescence is proportional to the intracellular ROS level.

[0211] This test referred to the laboratory method (HMC-WI-122 Cosmetics Antioxidant Efficacy Test - In vitro Keratinocyte Reactive Oxygen Species (ROS) Inhibition Test). By inducing an increase in the intracellular ROS content of human keratinocytes with hydrogen peroxide (H2O2), and by comparing the changes in the fluorescence intensity of the intracellular fluorescent dye after administration of the negative control and the test samples, the ROS inhibition rate was calculated. If the intracellular ROS content of the test samples was lower than that of the negative control and had a significant difference (P<0.05), it could be considered that the test samples had antioxidant efficacy.

[0212] Test materials and methods

[0213] Instrumentation

[0214] MF52-N Inverted Fluorescence Microscope

[0215] Cells used in the experiment

[0216] Keratinocytes: HaCaT cells

[0217] Test methods

[0218] (1) Treatment of test samples

[0219] Sample group: Dilute the sample with pure water to a 15% solution, then filter it with a 0.22 μm filter, and collect the filtrate as the sample stock solution.

[0220] (2) Experimental grouping

[0221] Negative control group (NC): Cells (+) H2O2 (+) Fluorescent probe (+) Sample (-);

[0222] Positive control group (PC): Cells (+) H2O2 (+) Fluorescent probe (+) Vitamin E (+);

[0223] Sample group (S): Cells (+) H2O2 (+) Fluorescent probe (+) Sample (+)

[0224] Naked cell group: Cells (-) H2O2 (-) Fluorescent probe (+) Sample (-)

[0225] (3) Experimental operation steps

[0226] Cell viability assay: Take cells and seed them in 96-well plates. After 24 h, aspirate the culture medium, add basal medium containing different concentrations of the test sample. After 24 h, detect the OD490nm by MTT method, and analyze the effect of the test sample on cell viability through t-test.

[0227] Determination of ROS inhibition rate: After the experimental grouping and drug administration, place the plates in a CO2 incubator for 24 h. After incubation, except for the blank group (BC) and the naked cell wells, the remaining wells need to be induced with ROS using a stimulator and then loaded with a fluorescent probe. After incubation in the CO2 incubator, under an inverted fluorescence microscope, using the FITC channel, with an incident light wavelength of 525 nm, an excitation light wavelength of 488 nm, and a 20x objective lens, set the same exposure time and take pictures.

[0228] Calculation formula

[0229] ROS inhibition rate (%) = 100% * (1 - (T - C0) / (C - C0))

[0230] Wherein:

[0231] T - The average value of the fluorescence intensity of the sample group in 3 times;

[0232] C - The average value of the fluorescence intensity of the negative control group in 3 times;

[0233] C0 - The average value of the fluorescence intensity of the naked cell group in 3 times.

[0234] Experimental results

[0235] Cell viability assay

[0236] Before determining the ROS inhibition rate of the test sample on cells, first perform cell viability screening to obtain the optimal concentration for testing, as Figure 8 shown.

[0237] ROS fluorescence image

[0238] ROS fluorescence image reference Figure 9 ;

[0239] Negative control group (NC): 0.00 ± 1.75%;

[0240] Positive control group (PC): 75.16 ± 1.46%;

[0241] Sample group (0.25%): 62.83 ± 1.11%;

[0242] Sample group (0.5%): 63.17 ± 2.07%;

[0243] The above results reference Figure 10 ;

[0244] Test conclusion

[0245] From Figure 8 , when the concentration of the test sample is 0.0625%, the cell viability > 90%;

[0246] According to the laboratory method (HMC-WI-122 Cosmetics Antioxidant Efficacy Test - In Vitro Keratinocyte Reactive Oxygen Species (ROS) Inhibition Test), when the concentration of the test sample is 0.25% and 0.5%, the cell viability is relatively high, and the cell ROS inhibition rates are 62.83% and 63.17% respectively, and there is a significant difference compared with the negative control (P < 0.0001). That is, the test sample has an obvious effect of inhibiting cell ROS at the concentrations of 0.25% and 0.5%, indicating that the test sample has antioxidant efficacy.

[0247] 2.5 Cosmetics Repair Efficacy Test (2502WH100 - 01G)

[0248] Detection basis: HMC-WI-043 Fibroblast Scratch Assay

[0249] Detection results and conclusions: According to the laboratory method (HMC-WI-043 Fibroblast Scratch Assay), after 24 hours, the scratch healing degree of fibroblasts in the positive control is relatively high, and the healing rate is 89.70%; the scratch healing degree of fibroblasts in the negative control is relatively low, and the healing rate is 17.20%; when the concentration of the test sample is 0.25% and 0.5%, the cell viability is relatively high, and the scratch healing rates of fibroblasts after 24 hours are 40.42% and 32.58% respectively. The samples with concentrations of 0.25% and 0.5% have a promoting effect on the scratch healing of fibroblasts, indicating that the samples have repair efficacy.

[0250] Test purpose and principle

[0251] When a wound occurs, fibroblasts are regulated by cytokines and extracellular matrix, etc., and rapidly proliferate and migrate, form granulation tissue to fill the wound, secrete collagen and ultimately participate in scar formation; fibroblasts can be used as a cell model to study the repair ability of cosmetics. By using the degree of scratch healing of fibroblasts after the administration of the test substance and the negative control, the efficacy of the test substance in terms of repair can be evaluated.

[0252] Test Materials and Methods

[0253] Instrumentation

[0254] MF52-N Inverted Fluorescence Microscope

[0255] Cells Used in the Experiment

[0256] Mouse fibroblasts: 3T3 cells

[0257] Test Methods

[0258] Treatment of Test Samples

[0259] Sample Group: Dilute the sample with pure water to a 15% solution, and then filter it through a 0.22 μm filter. Collect the filtrate as the sample mother liquor.

[0260] Negative Control: Basal medium (cell viability assay); incomplete medium (scratch assay).

[0261] Positive Control: Basal medium.

[0262] Test Operating Procedures

[0263] Cell Viability Assay: Seed 3T3 cells in a 96-well plate. After 24 h, aspirate the medium and add basal medium containing test samples at different concentrations. After 24 h, detect the OD490nm by the MTT method, and analyze the effect of the test samples on the viability of 3T3 cells by t-test.

[0264] Fibroblast Scratch Assay: Seed fibroblasts in a 6-well plate and culture for 24 h. Then, use a sterile pipette tip to draw a line in the middle of the 6-well plate to create a scratch, and aspirate the culture medium. Add basal medium (positive control), incomplete medium (negative control), and the corresponding sample group medium respectively. Observe the cell healing situation at two time points, 0 h and 24 h, and take pictures.

[0265] Calculation Formula

[0266]

[0267] Test Results

[0268] Cell Viability Assay

[0269] Before conducting the scratch test on the sample cells, cell viability screening was first performed to obtain the optimal test concentration, as Figure 11 shown.

[0270] Scratch test diagram of cells

[0271] The scratch test of fibroblasts in each group is as Figure 12 shown.

[0272] Test results

[0273] Scratch healing rate at 24h

[0274] Sample (0.25%): 40.42 ± 1.15%;

[0275] Sample (0.5%): 32.58 ± 1.75%;

[0276] Negative control: 17.20 ± 0.51%;

[0277] Positive control: 89.70 ± 0.32%.

[0278] The above results refer to Figure 13 ;

[0279] Test conclusion

[0280] From Figure 11 , when the test sample concentrations are 0.25% and 0.5%, the cell viability > 90%;

[0281] According to the laboratory method (HMC-WI-043 fibroblast scratch test), the fibroblast scratch healing degree of the positive control is higher after 24h, and the healing rate is 89.70%; the fibroblast scratch healing degree of the negative control is lower after 24h, and the healing rate is 17.20%; when the test sample concentrations are 0.25% and 0.5%, the cell viability is higher, and the fibroblast scratch healing rates after 24h are 40.42% and 32.58% respectively. The samples with concentrations of 0.25% and 0.5% have a promoting effect on fibroblast scratch healing, indicating that the samples have a repair effect.

[0282] In summary, the lysate of the present invention, whether it is an alcohol-soluble product or a water-soluble product, has excellent application properties related to cosmetics, which greatly expands the related applications of Eurotium cristatum in the field of cosmetics.

[0283] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims concerned.

Claims

1. Use of the lysate of Eurotium cristatum for preparing cosmetics; The Eurotium cristatum has a taxonomic name of Eurotium cristatum, and the preservation number is: CGMCC NO: 40145; the preservation date is March 10, 2022, and the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

2. The use according to claim 1, characterized in that, The lysate is one of a water-soluble product and an alcohol-soluble product.

3. The use according to claim 2, characterized in that, The lysate is an alcohol-soluble product, and the cosmetics are cosmetics with a soothing effect and / or an antioxidant effect.

4. The use according to claim 2, characterized in that, The lysate is a water-soluble product, and the cosmetics are cosmetics with at least one of the effects of whitening, repair, antioxidant, and soothing.

5. A cosmetic, characterized in that, Contains the lysate of Eurotium cristatum.

6. The cosmetic according to claim 5, characterized in that, The lysate is one of a water-soluble product and an alcohol-soluble product.

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