Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, and preparation method and application thereof

Abstract

The present application discloses a method for preparing a Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, comprising the following steps: inoculating thermophilic Streptococcus and Lactobacillus into a fermentation substrate, fermenting and culturing, and sterilizing; the fermentation substrate comprises Tremella fuciformis fruiting bodies and water; wherein the ratio of the number of viable bacteria of the thermophilic Streptococcus to the Lactobacillus is 1:(0.8-2.2). The Tremella fuciformis fermented liquid prepared in the present application has ideal antioxidant, anti-allergic and damaged cell repair, firming and anti-wrinkle effects, simple preparation process, mild fermentation conditions, low energy consumption, resource saving, no use of any organic reagents, high product safety, meeting the needs of modern society for the functionality and safety of skin topical preparations, and can be widely used in the field of skin topical preparations.

Description

Technical Field

[0001] The present application belongs to the technical field of cosmetics, and in particular relates to Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, as well as a preparation method and application. Background Art

[0002] At present, the research on the product of golden ear fruiting body is focused on separation and purification. Excessive separation and purification operations will lead to the continuous loss of active ingredients in golden ear fruiting body. In addition, the existing technology mostly uses organic solvents to extract the active ingredients of golden ear fruiting body. For example, the golden ear fruiting body extractor is extracted by alcohol precipitation method and purified by adsorbent. However, organic solvents can only dissolve the active ingredients in golden ear fruiting body that are soluble in organic solvents, resulting in the loss of other insoluble components of golden ear fruiting body, thereby reducing the resource utilization rate of golden ear fruiting body. Moreover, the organic solvent needs to be removed after extraction, which makes the process complicated and has adverse effects on the environment. There will be a problem of residual solvent in the final product, which reduces the safety of product use.

[0003] Therefore, there is an urgent need in the art to develop a method for preparing a Tremella fuciformis fruiting body extract that can efficiently utilize the active ingredients in Tremella fuciformis fruiting bodies, is simple to operate, can be used in the cosmetics field, has good cosmetic effects, and is highly safe to use. Summary of the invention

[0004] The technical problem to be solved by the present application is to overcome the defects that the preparation method of the Tremella fuciformis fruiting body product in the prior art cannot realize the extraction of all ingredients, resulting in reduced resource utilization of Tremella fuciformis fruiting body ingredients, and the use of organic solvents in the extraction process, which pollutes the environment, is complicated to operate, and reduces the safety of use due to solvent residues, and to provide Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, as well as a preparation method and application. The Tremella fuciformis fermented liquid provided in the present application has ideal antioxidant properties, and the preparation process is simple, energy consumption is low, no organic solvent is required, and the final product is highly safe to use, which meets the requirements of modern society for the efficacy and safety of cosmetics.

[0005] This application adopts the following technical solutions to solve the above technical problems:

[0006] The present application provides a method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, which comprises the following steps: inoculating thermophilic Streptococcus and Lactobacillus into a fermentation substrate, fermenting and culturing, and sterilizing; the fermentation substrate comprises Tremella fuciformis fruiting bodies and water;

[0007] Wherein, the ratio of the number of viable bacteria of the thermophilic streptococcus to that of the lactobacillus is 1:(0.8-2.2).

[0008] In some embodiments, the lactobacillus may include Lactobacillus plantarum and / or Lactobacillus crispatus.

[0009] Wherein, the Lactobacillus plantarum may include the Lactobacillus plantarum deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.28799.

[0010] The Lactobacillus plantarum is deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit date is October 27, 2023, and the deposit number is CGMCC No. 28799.

[0011] Wherein, the Lactobacillus crispatus may include the Lactobacillus crispatus which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.23317.

[0012] The Lactobacillus curvatus preservation unit is the General Microbiology Center of China Culture Collection Administration (CGMCC for short), the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the preservation date is August 30, 2021, and the preservation number is CGMCC No. 23317.

[0013] The Streptococcus thermophilus may include the Streptococcus thermophilus deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 28798.

[0014] The depository of the Streptococcus thermophilus is the General Microbiology Center of the China Culture Collection Administration (CGMCC for short), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit date is October 27, 2023, and the deposit number is CGMCC No. 28798.

[0015] The Lactobacillus paracasei may include Streptococcus thermophilus which is deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC No.23145.

[0016] The Lactobacillus paracasei is deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The deposit date is August 16, 2021, and the deposit number is CGMCC No. 23145.

[0017] In some embodiments, the lactobacillus can be added in the form of a lactobacillus bacterial solution according to the conventional art, and the concentration of live lactobacillus in the lactobacillus bacterial solution can be 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL, e.g. 10 8 CFU / mL.

[0018] In some embodiments, the thermophilic streptococcus can be added in the form of a thermophilic streptococcus liquid according to the conventional art, and the concentration of live thermophilic streptococci in the thermophilic streptococcus liquid can be 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL, e.g. 10 8 CFU / mL.

[0019] In some embodiments, the ratio of the number of live bacteria of the thermophilic streptococcus to that of the lactobacillus is 1:(1-2), preferably 1:1.5.

[0020] In some embodiments, the method for preparing the fermentation substrate comprises: mixing the Tremella fuciformis fruiting body with the water, and performing heating extraction under stirring conditions.

[0021] The temperature of the heating extraction may be 80-100°C, preferably 90-100°C, and more preferably 95-100°C.

[0022] The heating extraction time may be 50 to 80 minutes, preferably 60 minutes.

[0023] The stirring speed may be 180-250 rpm, preferably 100-200 rpm.

[0024] In some embodiments, the Tremella fuciformis fruiting bodies may be further crushed and sieved before use to obtain Tremella fuciformis fruiting body powder.

[0025] Wherein, the mesh number of the sieve used during the screening can be 100 to 200 meshes.

[0026] In some embodiments, the water may include deionized water and / or distilled water.

[0027] In some embodiments, the mass ratio of the Tremella fuciformis fruiting body to the water in the fermentation substrate may be 1:(100-300), preferably 1:(100-200).

[0028] In some embodiments, the fermentation substrate may be sterilized according to conventional procedures in the art before use. The method for sterilizing the fermentation substrate may be a high temperature sterilization method conventionally used in the art.

[0029] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 90-100°C, more preferably 95-100°C.

[0030] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 20 to 40 minutes, such as 30 minutes.

[0031] According to the common practice in the art, the fermentation substrate may be further cooled after being sterilized, generally to room temperature.

[0032] In some embodiments, the fermentation culture temperature may be 37-43°C.

[0033] In some embodiments, the fermentation culture time may be 10 to 20 hours, preferably 15 to 20 hours.

[0034] In some embodiments, the fermentation culture method may be static fermentation.

[0035] In some embodiments, the sterilization method may be a method conventionally used for such operations in the art, generally a high-temperature sterilization method.

[0036] When the high temperature sterilization method is used for the sterilization, the sterilization temperature may be 90-100°C, preferably 95-100°C.

[0037] When the high temperature sterilization method is used for the sterilization, the sterilization time can be 20 to 40 minutes, preferably 30 minutes.

[0038] In some embodiments, the sterilization operation may further include cooling and / or centrifugation, and collecting the supernatant.

[0039] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.

[0040] The centrifugal rotation speed may be a conventional rotation speed for such operation in the art, preferably 3000-5000 rpm, more preferably 4000-5000 rpm, for example 4800 rpm.

[0041] The centrifugation time may be the conventional time for such operation in the art, preferably 20 to 40 min, more preferably 30 to 40 min, for example 30 min.

[0042] Wherein, the centrifugal operation may further include at least one of filtration, secondary sterilization and mixing with a preservative.

[0043] The purpose of the filtration is to collect the filtrate and remove the precipitate. The pore size of the filter membrane used in the filtration process can be 0.4-0.9 μm, preferably 0.45-0.8 μm.

[0044] The secondary sterilization method may be a high temperature sterilization method conventionally used in this field for such operations.

[0045] When the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization can be a conventional temperature for such operations in the art, preferably 90-100°C, more preferably 95-100°C.

[0046] When the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization can be the conventional time for such operation in the art, preferably 20 to 40 minutes, more preferably 30 to 40 minutes.

[0047] During the mixing process with the preservative, the mixing temperature can be a conventional temperature for such operations in the art, preferably 50-80°C.

[0048] During the mixing with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional method in the art.

[0049] When the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after the centrifugation may be 0.5% to 1%.

[0050] When the preservative includes 1,2-hexanediol, the mass percentage of 1,2-hexanediol in the supernatant obtained after the centrifugation may be 0.5% to 2%.

[0051] The present application also provides Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, which is prepared by the preparation method of Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as described above.

[0052] The present application also provides the use of the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as described above directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0053] In some embodiments, the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects can be used as at least one of the antioxidant active ingredient, soothing active ingredient, skin repair active ingredient and firming and anti-wrinkle active ingredient in the skin external preparation.

[0054] Wherein, the antioxidant active ingredient is an antioxidant active ingredient with DPPH free radical scavenging effect, an antioxidant active ingredient with hydroxyl free radical scavenging effect;

[0055] The soothing active ingredient is a soothing active ingredient that has an effect of inhibiting hemolysis of erythrocytes and inhibiting the production of TRPV1 receptor protein;

[0056] The skin repair active ingredient is a skin repair active ingredient that has the effect of promoting lysosomal activity and mitochondrial activity;

[0057] The firming and anti-wrinkle active ingredient is a firming and anti-wrinkle active ingredient that has the function of inhibiting the activity of elastase.

[0058] The present application also provides a skin external preparation, which includes the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as described above.

[0059] In some embodiments, the skin topical preparation may further contain active ingredients, which may include at least one of antioxidant active ingredients, soothing active ingredients, skin repair active ingredients, and firming and anti-wrinkle active ingredients.

[0060] In some embodiments, the skin topical preparation may include but is not limited to a facial mask, essence or toner according to conventional methods in the art.

[0061] In some embodiments, the room temperature generally refers to 25-28°C.

[0062] In some embodiments, the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects accounts for 5% to 99% of the skin external preparation.

[0063] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0064] The reagents and raw materials used in this application are commercially available.

[0065] The positive progressive effects of the present application are that the Auricularia auricula fermentation liquid prepared by the present application has ideal antioxidant activity, soothing, skin repair and firming and anti-wrinkle effects, the preparation process is simple, the fermentation conditions are mild, the energy consumption is low, resources are saved, no organic reagents are used, the product is relatively safe to use, and it meets the needs of modern society for the functionality and safety of skin topicals, and can be widely used in the field of skin topicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0067] in:

[0068] Figure 1 A comparison chart of the DPPH free radical scavenging ability of the products obtained in Examples 1 to 7 or Comparative Examples 1 to 5;

[0069] Figure 2 A comparison chart of the hydroxyl radical scavenging abilities of the products obtained in Examples 1 to 7 or Comparative Examples 1 to 5;

[0070] Figure 3 This is a comparison chart of the hemolysis inhibition ability of cells treated with the products obtained in Examples 1 to 7 or Comparative Examples 1 to 5;

[0071] Figure 4 This is a comparison chart of intracellular lysosomal activity after cells were treated with the products obtained in Examples 1 to 7 or Comparative Examples 1 to 5;

[0072] Figure 5 This is a comparison chart of mitochondrial activity in cells after the cells were treated with the products obtained in Examples 1 to 7 or Comparative Examples 1 to 5;

[0073] Figure 6 This is a comparison chart of the inhibition rate of in vitro protease activity after cells were treated with the products prepared in Examples 1 to 7 or Comparative Examples 1 to 5;

[0074] Figure 7 This is a comparison chart of the TRPV1 receptor protein content after cells were treated with the products obtained in Examples 1 to 7 or Comparative Examples 1 to 5. DETAILED DESCRIPTION

[0075] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0077] The golden ear fruiting bodies in the following examples and comparative examples were purchased from Gutian Gaoting Industry and Trade Co., Ltd.

[0078] The thermophilic Streptococcus in the following examples is deposited in China General Microbiological Culture Collection Center with the deposit number CGMCC No.28798.

[0079] The plant lactobacillus in the following examples is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.28799.

[0080] The Lactobacillus curvatum in the following examples is deposited in China General Microbiological Culture Collection Center with the deposit number CGMCC No.23317.

[0081] The Lactobacillus bulgaricus in the following examples was purchased from Zhengzhou Hehe Bioengineering Technology Co., Ltd. and was numbered HH-LB57.

[0082] The Lactobacillus paracasei in the following examples is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.23145.

[0083] Example 1

[0084] (1) Screen clean and intact golden ear fruiting bodies, wash the golden ear fruiting bodies with water twice, crush them, and pass them through a 200-mesh sieve to obtain golden ear fruiting body powder. Take 3g of golden ear fruiting body powder and 300g of deionized water, heat and extract them at a temperature of 95°C and a rotation speed of 150rpm for 30min, then mix the whole, sterilize them in an autoclave at a temperature of 100°C for 30min, and cool them to room temperature after sterilization to obtain a fermentation substrate.

[0085] (2) The thermophilic Streptococcus (CGMCC No. 28798) and the plant lactobacillus (CGMCC No. 28799) were prepared to have a viable cell count of 10 8CFU / mL of bacterial liquid, inoculating 1.0 mL of thermophilic Streptococcus (CGMCC No. 28798) bacterial liquid and 1.0 mL of plant lactobacillus (CGMCC No. 28799) bacterial liquid into the fermentation substrate prepared in step (1), with the ratio of the number of live bacteria of the two being 1:1, shaking well, and standing for fermentation culture in a constant temperature incubator at a temperature of 37° C. for 15 h. After the fermentation culture is completed, placing the fermentation liquid in a high pressure sterilizer at a temperature of 95° C. for sterilization for 30 min. After the sterilization is completed, cooling the fermentation liquid to room temperature, centrifuging it, collecting the supernatant, the centrifugal speed is 4800 rpm, and the centrifugal time is 30 min. Filtering the supernatant to collect the filtrate, the pore size of the filter membrane used for filtration is 0.8 μm. Then, secondary sterilization was carried out in a high-pressure sterilizer at a temperature of 100°C for 30 minutes, and p-hydroxyacetophenone and 1,2-hexanediol were added at 75°C, with p-hydroxyacetophenone accounting for 5% of the mass percentage of the supernatant and 1,2-hexanediol accounting for 5% of the mass percentage of the supernatant. The mixture was mixed evenly to obtain a Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects.

[0086] Example 2

[0087] Compared with Example 1, the only difference is that the fermentation bacteria in step (2) are different, Lactobacillus plantarum (CGMCC No. 28799) is replaced by Lactobacillus curvatum (CGMCC No. 23317), and other condition parameters are the same as in Example 1.

[0088] Example 3

[0089] Compared with Example 1, the only difference is that the inoculum ratio of thermophilic Streptococcus and plantarum Lactobacillus in step (2) is different, the viable cell count ratio of the two is 2:3, and other condition parameters are the same as in Example 1.

[0090] Example 4

[0091] Compared with Example 1, the only difference is that the type of fermentation bacteria is different, specifically Lactobacillus bulgaricus, and other condition parameters are the same as Example 1.

[0092] Example 5

[0093] Compared with Example 1, the only difference is that the fermentation temperature in step (2) is 43° C., and other condition parameters are the same as Example 1.

[0094] Example 6

[0095] Compared with Example 1, the only difference is that the fermentation culture time in step (2) is 20 hours, and other condition parameters are the same as Example 1.

[0096] Example 7

[0097] Compared with Example 1, the only difference is that the type of fermentation bacteria is different, specifically Lactobacillus paracasei, and other condition parameters are the same as Example 1.

[0098] Comparative Example 1

[0099] Compared with Example 1, the only difference is that a single strain of bacteria, specifically Lactobacillus plantarum, is used for fermentation, and other condition parameters are the same as those in Example 1.

[0100] Comparative Example 2

[0101] Compared with Example 1, the only difference is that the fermentation bacteria are different, specifically thermophilic Streptococcus, and other condition parameters are the same as Example 1.

[0102] Comparative Example 3

[0103] Compared with Example 1, the only difference is that the fermentation strains are different, namely, Saccharomyces cerevisiae and Lactobacillus plantarum, and other condition parameters are the same as in Example 1.

[0104] Comparative Example 4

[0105] Compared with Example 1, the only difference is that the inoculum ratio of thermophilic Streptococcus and plantarum Lactobacillus in step (2) is different, the viable cell count ratio of the two is 1:3, and the other condition parameters are the same as in Example 1.

[0106] Comparative Example 5

[0107] Compared with Example 1, the only difference is that the inoculum ratio of thermophilic Streptococcus and plantarum Lactobacillus in step (2) is different, the viable cell count ratio of the two is 3:2, and other condition parameters are the same as in Example 1.

[0108] Effect Example 1

[0109] DPPH free radical scavenging assay

[0110] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0111] (1) Take an equal volume (1 mL) of the test solution (the example and the prepared product) and mix with 2×10 4 mol / L DPPH solution and mix well (tube A1);

[0112] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 4mol / L DPPH solution and mix well (tube A2);

[0113] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0114] (4) After 30 minutes of reaction in the dark, measure the absorbance of tubes A1, A2, and A3 at 517 nm. The clearance rate is calculated as follows:

[0115] Clearance rate = [(A2 + A3) A1] / A2 x 100%.

[0116] In this experiment, the products obtained in the examples and comparative examples were subjected to DPPH free radical scavenging test. The results are shown in Tables 1 and Figure 1 .

[0117] Table 1

[0118] DPPH free radical scavenging rate (%) Example 1 59.22 Example 2 53.10 Example 3 65.24 Example 4 55.78 Example 5 59.36 Example 6 67.87 Example 7 54.89 Comparative Example 1 22.17 Comparative Example 2 26.66 Comparative Example 3 40.16 Comparative Example 4 43.20 Comparative Example 5 34.52

[0119] The results show that the DPPH radical scavenging rates of the products prepared in Examples 1 to 7 are much higher than those of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation temperature and strains have a great influence on the antioxidant properties of the final products.

[0120] Effect Example 2

[0121] Hydroxyl radical scavenging assay

[0122] Hydroxyl radical generation system model: Under certain conditions, H2O2 and divalent iron ions are mixed to produce OH. The Fenton reaction is:

[0123] H2O2+Fe 2+ =OH. +OH-+Fe 3+

[0124] Hydroxyl radical is the most chemically active free radical among active oxygen species. It can react with almost any biological macromolecule in living cells at a very fast speed and is the free radical that is most harmful to the body. However, adding salicylic acid to the reaction system can effectively capture OH and produce a colored product, 2,3-dihydroxybenzoic acid.

[0125]

[0126] The colored product has a strong absorption peak at 510nm. If a test substance with the function of scavenging OH is added to the system, and the test substance captures OH more than salicylic acid, OH can be removed in time, thereby reducing the amount of colored product generated and causing the absorbance to decrease. Therefore, the fixed reaction time method is used to measure the absorbance of the reaction solution containing the test substance at 510nm and compare it with the blank solution, so that the scavenging effect of the test substance on OH can be determined.

[0127] According to the following table, the required reagents were prepared in advance, and the sample group was the above-mentioned embodiment and comparative example, and then the test was carried out according to Table 2. After the reaction was completed, if there was precipitation, it was detected after centrifugation. Calculation formula: Hydroxyl free radical scavenging rate % = (OD sample group-OD sample bottom value) / OD blank group × 100, the calculation results are shown in Table 3 and Figure 2 .

[0128] Table 2

[0129]

[0130] Table 3

[0131]

[0132]

[0133] The results show that the hydroxyl radical scavenging rates of the products prepared in Examples 1 to 7 are much higher than those of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation temperature and strains have a great influence on the antioxidant properties of the final products.

[0134] Effect Example 3

[0135] Erythrocyte hemolysis inhibition test

[0136] Sodium dodecyl sulfate (SDS) is an anionic surfactant and a classic skin irritant. It can cause skin barrier dysfunction and irritate the skin. SDS acts on red blood cells, causing the red blood cell membrane to rupture, releasing hemoglobin and causing hemolysis.

[0137] By establishing an SDS erythrocyte hemolysis model, the samples were added into the model, and the inhibition rate of the samples on SDS-induced erythrocyte hemolysis was evaluated by comparing with the model hemolysis, thereby evaluating the soothing effect of the samples.

[0138] 1. Preparation of Red Blood Cell Suspension (RBC)

[0139] 1.1 Cleaning

[0140] Fresh sheep blood was diluted with PBS solution at a dilution ratio of 2:5 in a 50mL centrifuge tube. The centrifuge tube was gently turned upside down to fully mix the blood and PBS. Subsequently, the supernatant (the yellowish white blood cell layer) was discarded at room temperature at 1000×g. PBS buffer was then added and repeated 1-2 times. This process can remove a large number of white blood cells, plasma and yellow debris.

[0141] 1.2 Preparation of red blood cell suspension (RBC)

[0142] Use a disposable pipette to transfer the blood cell pellet in the centrifuge tube to a new EP tube, take 1 mL of RBC pellet into a 50 mL EP tube, add 49 mL PBS buffer to dilute, and then gently shake to mix.

[0143] 250μL RBC was placed in a 1.5mL EP tube, and 750mL distilled water was added to make up to 1mL. Centrifuge at 10000×g for 1min to stop the incubation. After centrifugation, the supernatant was taken and the OD value was measured at 540nm. The OD value was 0.764 at 540nm.

[0144] 1.3 Testing process

[0145] ①Blank group: add 250μL RBC suspension and 750μL PBS buffer to 1.5mL EP tubes; ②Model control group: add 250μL RBC suspension and 750μL distilled water to 1.5mL EP tubes. ③Model group: add 735μL PBS buffer, 250μL RBC suspension and 15μL 0.1% SDS solution to 1.5mL EP tubes. ④Sample group: add the sample to be tested (final concentration is 20%) and 15μL 0.1% SDS solution to 1.5mL EP tubes, add PBS buffer to 750mL, and then add 250μL RBC suspension to 1mL.

[0146] The four groups of samples were placed in an incubator and incubated at 180 rpm and 37°C for 60 min. They were then taken out and centrifuged at 10,000 × g for 1 min to terminate the reaction. Photos were taken and 200 μL of the supernatant was aspirated into a 96-well plate. The absorbance was measured at 540 nm and the sample red blood cell hemolysis inhibition rate was calculated. The results are shown in Table 4 and Figure 3 .

[0147] 1) Hemolysis rate of model group % = (OD model - OD blank) / (OD model comparison - OD blank) * 100

[0148] 2) Hemolysis rate of sample group % = (OD sample group - OD sample control) / (OD model comparison - OD blank) * 100

[0149] 3) Sample hemolysis inhibition rate % = (hemolysis rate of model group - hemolysis rate of sample treatment group) / hemolysis rate of model group * 100.

[0150] Table 4

[0151]

[0152]

[0153] The results show that the erythrocyte hemolysis inhibition rates of the products prepared in Examples 1 to 7 are much higher than those of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have ideal soothing effects, and the fermentation culture temperature and strains have a great influence on the soothing properties of the final product.

[0154] Effect Example 4

[0155] Lysosome activity assay

[0156] The products obtained in the above examples and comparative examples were respectively prepared into 2% test solution in the experimental group using serum-free DMEM culture medium. The test solution was required to be filtered through a 0.22 μm sterile filter membrane.

[0157] Lysosomes participate in the apoptosis and phagocytosis of their own cells. After normal cells die from aging, they will be digested and degraded by lysosomes. At the same time, some biological macromolecules will be absorbed by lysosomes and degraded into small molecules. The level of lysosome activity can indirectly reflect cell metabolism. The enhancement of lysosome activity helps the self-metabolism ability of cells and promotes cell self-renewal. The proton gradient in the lysosome makes the pH value in the lysosome lower than that in the cytoplasm, so that neutral red can be charged and accumulated in the lysosome. When cell proliferation accelerates, the number of lysosomes also increases, and the amount of neutral red that can be ingested will increase.

[0158] HaCaT cells were seeded in a 96-well plate at a density of 8000 cells / well, 0.1 mL per well, and divided into a blank group and a sample group, with 3 parallels in each group. Serum-free culture medium was added to the blank group, and the sample group was added to the sample to be tested. After 24 hours of cell treatment, 10 μL of neutral red reagent was added for staining. After the reaction, 100 μL of lysis buffer was added and shaken. OD540nm was measured. Lysosomal activity % = sample group / blank group*100. The results are shown in Table 5 and Figure 4 .

[0159] Table 5

[0160] Lysosomal activity (%) Blank group 100 Example 1 144.16 Example 2 148.70 Example 3 153.25 Example 4 144.16 Example 5 142.86 Example 6 161.69 Example 7 150.00 Comparative Example 1 113.64 Comparative Example 2 106.49 Comparative Example 3 126.62 Comparative Example 4 130.52 Comparative Example 5 122.08

[0161] The results show that the lysosomal activity of the products prepared in Examples 1 to 7 is much higher than that of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have an ideal effect of promoting cell metabolic activity, and the fermentation temperature and strain have a great influence on the performance of the final product in promoting cell metabolic capacity.

[0162] Effect Example 5

[0163] WST-8 in CCK-8 stain is reduced to a highly water-soluble yellow formazan product (Formazan) by dehydrogenase in the cell mitochondria under the action of electron carriers. The amount of formazan produced can indirectly reflect the activity of mitochondria in the cell.

[0164] The products obtained in the above examples and comparative examples were respectively prepared into 2% test solution in the experimental group using serum-free DMEM culture medium. The test solution was required to be filtered through a 0.22 μm sterile filter membrane.

[0165] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum. The cells were grown in an incubator at 37°C and 5% CO2. When the cell fusion reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin, the digestion reaction was terminated with serum-containing DMEM, and the cells were inoculated in a 96-well plate at a density of 8,000 cells / well and cultured overnight in a 37°C, 5% CO2 incubator. The HaCaT cells were divided into a blank group and a sample group, with at least 3 parallels in each group. The blank group was added with serum-free culture medium, and the sample group was added with the sample to be tested. After 24 hours of cell treatment, 10 μL CCK-8 was added for staining, and the OD 450 nm determination, mitochondrial activity % = sample group / blank group*100, the results are shown in Table 6 and Figure 5 .

[0166] Table 6

[0167] Mitochondrial activity% Blank group 100 Example 1 128.35 Example 2 128.05 Example 3 128.94 Example 4 118.18 Example 5 117.72 Example 6 133.62 Example 7 113.63 Comparative Example 1 108.37 Comparative Example 2 100.58 Comparative Example 3 109.82 Comparative Example 4 107.51 Comparative Example 5 106.51

[0168] The results show that the mitochondrial activity of the products prepared in Examples 1 to 7 is much higher than that of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have an ideal effect of promoting cell metabolism, and the fermentation temperature and strain have a great influence on the cell metabolism promoting performance of the final product.

[0169] Effect Example 6

[0170] In vitro elastase activity inhibition assay

[0171] Elastin plays a key role in the youthfulness of the skin, and the lack of elastin is the main cause of skin aging. Inhibiting the activity of elastase can effectively slow down the decomposition of elastin and maintain the elasticity of the skin. Elastase can catalyze the hydrolysis of hydroxyl groups contained in the polypeptide bonds of various amino acids. Therefore, when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, a colored substance will be produced, and its absorbance value can be measured at 406nm in a spectrophotometer. According to the change in absorbance, the inhibitory effect of the test substance on the activity of elastase is calculated, and whether the test substance has anti-wrinkle and firming effects can be evaluated.

[0172] The test was carried out by adding reagents in sequence according to Table 7 below and incubating the reaction. The substrate was N-succinyl-alanine-alanine-alanine-p-nitroaniline. The calculation results are shown in Tables 8 and Figure 6 .

[0173] Table 7

[0174]

[0175] After the reaction was completed, the samples were spotted in a 96-well plate and the absorbance at 406 nm was measured.

[0176] Table 8

[0177]

[0178]

[0179] The results show that the elastase activity inhibition rates of the products prepared in Examples 1 to 7 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal anti-wrinkle and firming effects, and the fermentation temperature and strains have a great influence on the anti-wrinkle and firming properties of the final products.

[0180] Effect Example 7

[0181] TRPV1 content detection

[0182] TRPV1 is a receptor protein that is distributed in skin-related cells. TRPV1 is a switch for skin sensitivity and is closely related to burning and pain in the skin. It belongs to the transient receptor potential (TRP) superfamily and is a key ion channel for the production of hyperalgesia. It is a multiphasic receptor widely distributed in the peripheral nervous system and is mainly expressed in the terminals of medium and small sensory neurons in the central and peripheral regions.

[0183] Testing the TRPV1 content of a sample can assess whether the sample has a soothing effect.

[0184] The samples prepared in the above examples and comparative examples were respectively prepared into 2% test solution in the experimental group using serum-free DMEM culture medium. The test solution was required to be filtered through a 0.22 μm sterile filter membrane.

[0185] HaCaT cells in the logarithmic phase were taken, and the cell suspension was inoculated on a 6-well cell culture plate at a density of 250,000 / mL. 2 mL of cell suspension was added to each well and cultured for 12 hours. The experimental group and the model group were treated with 5 μM capsaicin solution for 24 hours, and the supernatant was discarded. 2 mL of DMEM solution was added to the blank group and the model group, and 4 mL of DMEM solution containing the sample (the sample volume percentage was 2%) was added to the experimental group to treat the cells for 24 hours. After the treatment, the supernatant was aspirated for use. The determination of TRPV1 was carried out according to the instructions of the ELISA kit (purchased from Wuhan Cloud-Clone Technology Co., Ltd.), and the OD values ​​were measured at 450 nm. The calculation results are shown in Tables 9 and Figure 7 .

[0186] Table 9

[0187]

[0188]

[0189] The results show that the relative expression of TRPV1 of the products prepared in Examples 1 to 7 is lower than that of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have ideal soothing effects, and the fermentation temperature and strains have a great influence on the soothing performance of the final product.

[0190] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0191] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, characterized in that: The method comprises the following steps: inoculating thermophilic streptococci and lactobacillus into a fermentation substrate, culturing by fermentation, and sterilizing; the fermentation substrate comprises Tremella fuciformis fruiting bodies and water; Wherein, the ratio of the number of viable bacteria of the thermophilic streptococcus to that of the lactobacillus is 1:(0.8-2.2); The lactobacillus includes Lactobacillus plantarum and / or Lactobacillus crispatus; the method for preparing the fermentation substrate includes: mixing the Tremella fuciformis fruiting body with the water, and performing heating extraction under stirring conditions.

2. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 1, characterized in that: The preparation method satisfies at least one of the following conditions: The concentration of live bacteria of the lactobacillus in the lactobacillus liquid is 10 6 ~10 10 CFU / mL; The live bacteria concentration of the thermophilic streptococcus in the thermophilic streptococcus liquid is 10 6 ~10 10 CFU / mL; The ratio of the number of live bacteria of the thermophilic streptococcus to that of the lactobacillus is 1:(1-2).

3. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 2, characterized in that: The preparation method satisfies at least one of the following conditions: The concentration of live bacteria of the lactobacillus in the lactobacillus liquid is 10 7 ~10 9 CFU / mL; The live bacteria concentration of the thermophilic streptococcus in the thermophilic streptococcus liquid is 10 7 ~10 9 CFU / mL; The ratio of the number of live bacteria of the thermophilic streptococcus to that of the lactobacillus is 1:1.

5.

4. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 3, characterized in that: The preparation method satisfies at least one of the following conditions: The live bacteria concentration of the lactobacillus in the lactobacillus liquid is 108 CFU / mL; The live bacterial concentration of the thermophilic streptococci in the thermophilic streptococci bacterial liquid is 108 CFU / mL.

5. The method for preparing Tremella fuciformis fermented liquid having moisturizing, soothing and firming effects as claimed in any one of claims 2 to 4, characterized in that: The preparation method satisfies at least one of the following conditions: The plant lactobacillus (Lactobacillus plantarum) includes the plant lactobacillus deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.28799; The Lactobacillus crispatus includes the Lactobacillus crispatus which is deposited in China General Microbiological Culture Collection Center and has a deposit number of CGMCC No.23317; The thermophilic streptococcus includes the thermophilic streptococcus deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 28798.

6. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 5, characterized in that: The method for preparing the fermentation substrate satisfies at least one of the following conditions: The temperature of the heating extraction is 80-100°C; The heating extraction time is 50 to 80 minutes; The stirring speed is 180-250 rpm; The golden ear fruiting bodies are further crushed and sieved before use to obtain golden ear fruiting body powder; The water includes deionized water and / or distilled water; The mass ratio of the Tremella fuciformis fruiting body to the water in the fermentation substrate is 1:(100-300); The fermentation culture temperature is 37-43°C; The fermentation culture time is 10 to 20 hours; The fermentation culture method is static fermentation; The sterilization method is high temperature sterilization; when the high temperature sterilization method is used for sterilization, the sterilization temperature is 90-100°C; when the high temperature sterilization method is used for sterilization, the sterilization time is 20-40 minutes; After the sterilization operation, the method further comprises cooling and / or centrifuging, collecting the supernatant and mixing with a preservative.

7. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 6, characterized in that: The method for preparing the fermentation substrate satisfies at least one of the following conditions: The temperature of the heating extraction is 90-100°C; The heating extraction time is 60min; The stirring speed is 100-200 rpm; The mesh size of the sieve used during the sieving is 100 to 200 meshes; The mass ratio of the Tremella fuciformis fruiting body to the water in the fermentation substrate is 1:(100-200); The fermentation culture time is 15 to 20 hours; When the high temperature sterilization method is used for the sterilization, the sterilization temperature is 95-100°C; When the high temperature sterilization method is used for the sterilization, the sterilization time is 30 minutes; The cooling is cooling to room temperature.

8. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 7, characterized in that: At least one of the following conditions is met: the temperature of the heating extraction is 95-100°C; The centrifugal speed is 3000-5000 rpm; The centrifugation time is 20 to 40 minutes; The centrifugation operation further includes at least one of filtration, secondary sterilization and mixing with a preservative.

9. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 8, characterized in that: At least one of the following conditions is met: The centrifugal speed is 4000-5000 rpm; The centrifugation time is 30 to 40 minutes; The pore size of the filter membrane used in the filtration process is 0.4 to 0.9 μm; The secondary sterilization method is a high temperature sterilization method; when the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 90 to 100° C.; when the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization is 20 to 40 minutes; During the mixing process with the preservative, the mixing temperature is 50 to 80° C.; during the mixing process with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.5% to 1%; when the preservative includes 1,2-hexanediol, the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.5% to 2%.

10. The method for preparing Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 9, characterized in that: At least one of the following conditions is met: The centrifugal speed is 4800 rpm; The centrifugal time is 30 min; The pore size of the filter membrane used in the filtration process is 0.45 to 0.8 μm; When the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 95-100° C.; when the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization is 30-40 minutes.

11. Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects, which is prepared by the preparation method of Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects according to any one of claims 1 to 10.

12. Use of the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 11 directly as a product, as an additive or as a base in the preparation of a skin external preparation.

13. The use according to claim 12, characterized in that The Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects is used as at least one of the antioxidant active ingredient, soothing active ingredient, skin repair active ingredient and firming and anti-wrinkle active ingredient in the skin external preparation.

14. The use according to claim 13, characterized in that The Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects satisfies at least one of the following conditions: The antioxidant active ingredients are antioxidant active ingredients with DPPH free radical scavenging effect and antioxidant active ingredients with hydroxyl free radical scavenging effect; The soothing active ingredient is a soothing active ingredient that has an effect of inhibiting hemolysis of erythrocytes and inhibiting the production of TRPV1 receptor protein; The skin repair active ingredient is a skin repair active ingredient that has the effects of promoting lysosomal activity and promoting mitochondrial activity; The firming and anti-wrinkle active ingredient is a firming and anti-wrinkle active ingredient that has the function of inhibiting the activity of elastase.

15. A skin external preparation, characterized in that: It comprises the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects as claimed in claim 11.

16. The external skin preparation according to claim 15, wherein The skin topical preparation satisfies at least one of the following conditions: The skin topical preparation further comprises at least one of an antioxidant active ingredient, a soothing active ingredient, a skin repair active ingredient, and a firming and anti-wrinkle active ingredient; The skin external preparation includes a facial mask, essence or toner; The mass percentage of the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects in the skin external preparation is 5% to 99%.

17. The external skin preparation according to claim 15, wherein The mass percentage of the Tremella fuciformis fermented liquid with moisturizing, soothing and firming effects in the skin external preparation is 60% to 99%.

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