A white tea fermenting liquid with anti-wrinkle and firming efficacy, and a preparation method and application thereof

Abstract

The application discloses a preparation method of Bai Tiyin fermentation liquor with anti-wrinkle and firming effects, which comprises the following steps: preparing a fermentation substrate by mixing Baihelinguozha, white truffle and Chinese Stachyurus praecox with water, inoculating lactobacillus into the fermentation substrate, and carrying out fermentation culture and sterilization; wherein Baihelinguozha, white truffle and Chinese Stachyurus praecox are fermented with water, and the mass percentage of Baihelinguozha, white truffle and Chinese Stachyurus praecox is 1:(1-10):(0.1-2). The Bai Tiyin fermentation liquor prepared by the method has ideal anti-wrinkle, firming, antioxidant and soothing and anti-allergy effects, and has the advantages of simple preparation process, no use of any organic reagent, energy saving and environmental protection, high safety, and meeting the requirements of modern skin external agents on functionality and safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation, and particularly relates to a white factor fermentation liquor with anti-wrinkle and firming effects, and a preparation method and application thereof. BACKGROUND

[0002] Skin aging is part of human aging, caused by multiple factors of endogenous and exogenous, mainly including the regulation of skin aging process by genes, free radical damage and photoaging effect, etc. Modern people pay more and more attention to skin care, and the demand for skin care products with anti-inflammatory, antioxidant and anti-aging functions is also increasing.

[0003] Rhinacanthus nasutus, also known as Xianhecao, has the shape of a white crane flying with its wings spread, and has the effects of activating the immune system, enhancing immunity, reducing liver fire, and detoxifying and relieving swelling. At present, the research on Rhinacanthus nasutus is basically in the fields of food and pharmacy, focusing on the extraction process and chemical components of Rhinacanthus nasutus, and there is little research on the fermentation preparation of Rhinacanthus nasutus.

[0004] White truffle is a large block fungus of ascomycetes, also known as truffle. Studies have shown that white truffle contains rich polysaccharides, proteins, amino acids and polyunsaturated fatty acids, as well as rich minerals and sterols. Due to its complex composition, if fermentation is not proper, it is likely to be decomposed, causing resource waste.

[0005] S.chinensis Franch. belongs to Stachyuraceae and Stachyurus Sieb.et Zucc., and is a small grass with the stem pith used for medicine, having the effects of diuresis, lactation promotion and damp-heat clearing. At present, the research on S.chinensis Franch. is basically in the field of pharmacy, mainly focusing on the extraction process, chemical components and pharmacological effects of S.chinensis Franch., and there is no report on the fermentation of S.chinensis Franch. and its application in the field of skin care products.

[0006] Therefore, there is an urgent need in the art to develop a preparation method capable of simultaneously fermenting Rhinacanthus nasutus, white truffle and S.chinensis Franch., and the product can be applied in the field of skin external agents, significantly improving its bioavailability and anti-wrinkle and firming cosmetic effects. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the defects in the prior art that white crane ganoderma grass is mainly applied in the field of medicine, the application field is limited, and there is a lack of fermentation process; truffle fermentation is difficult to control, and Chinese stachys affinis lacks research in the field of fermentation, and to provide a white factor fermentation liquor with anti-wrinkle and firming effects, and a preparation method and application thereof. The white factor fermentation liquor prepared by the present application has ideal antioxidant performance, anti-wrinkle and firming effects, the preparation process is simple, no organic reagent is used in the preparation process, has high safety, and meets the requirements of modern skin external agents on functionality and safety.

[0008] The present application solves the above technical problems by adopting the following technical solutions:

[0009] The present application provides a preparation method of a white factor fermentation liquor with anti-wrinkle and firming effects, which comprises the following steps: preparing a fermentation substrate by mixing white crane ganoderma grass, white truffle and Chinese stachys affinis with water, inoculating lactobacillus into the fermentation substrate, and carrying out fermentation culture and sterilization.

[0010] The white crane ganoderma grass, the white truffle and the Chinese stachys affinis are fermented with water, and the mass percentage of the white crane ganoderma grass, the white truffle and the Chinese stachys affinis is 1:(1-10):(0.1-2).

[0011] In some embodiments, the mass percentage of the white crane ganoderma grass, the white truffle and the Chinese stachys affinis is 1:(1-4):(0.5-2), and preferably, the mass percentage of the white crane ganoderma grass, the white truffle and the Chinese stachys affinis is 1:3:0.5.

[0012] The mixing is conventional in the art, and generally, uniform mixing is sufficient.

[0013] The white crane ganoderma grass is subjected to conventional crushing and sieving operations in the art to obtain white crane ganoderma grass powder, and the particle size of the sieving is 50-100 mesh, and preferably 80 mesh.

[0014] The white truffle is subjected to conventional crushing and sieving operations in the art to obtain white truffle powder, and the particle size of the sieving is 50-100 mesh, and preferably 80 mesh.

[0015] The Chinese stachys affinis is subjected to conventional crushing and sieving operations in the art to obtain Chinese stachys affinis powder, and the particle size of the sieving is 50-100 mesh, and preferably 80 mesh.

[0016] The water is deionized water.

[0017] The fermentation substrate can also be subjected to conventional operations including sterilization before use.

[0018] The sterilization conditions and methods can be conventional conditions and methods for such operations in the art, and can generally be high-temperature sterilization methods.

[0019] When the fermentation substrate is sterilized by the high-temperature sterilization method, the sterilization temperature can be conventional for such operations in the art, and is preferably 110-125°C, and more preferably 115-121°C. When the fermentation substrate is sterilized by the high-temperature sterilization method, the sterilization time can be conventional for such operations in the art, and is preferably 15-35 min, and more preferably 20-35 min, for example 30 min. When the fermentation substrate is sterilized by the high-temperature sterilization method, the sterilization pressure can be conventional for such operations in the art, and is preferably 0.1-0.14 MPa, and more preferably 0.1-0.13 MPa, for example 0.12 MPa.

[0020] According to conventional practice in the art, the sterilization operation can further include a cooling operation, which can generally be cooling to room temperature.

[0021] In some embodiments, the Lactobacillus is inoculated in the form of a Lactobacillus bacterial solution.

[0022] In some embodiments, the Lactobacillus can include Lactobacillus bulgaricus, and preferably the Lactobacillus bulgaricus deposited on October 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC) at 1st North Chengxi Road, 3rd Building, Chaoyang District, Beijing, China, with a postal code of 100101, and with a deposit number of CGMCC No. 28800.

[0023] When the Lactobacillus bulgaricus is used, the Lactobacillus bulgaricus can be added in the form of a Lactobacillus bulgaricus bacterial solution according to conventional practice in the art, and the concentration of the Lactobacillus bulgaricus in the Lactobacillus bulgaricus bacterial solution can be 10 8 - 10 12 CFU / mL, and preferably 10 9 - 10 11 CFU / mL.

[0024] In the fermentation method, the number of Lactobacillus bulgaricus inoculated per unit volume of the fermentation substrate, based on the amount of water added, can be conventional in the art, and is preferably 10 7 - 10 9 CFU / mL, and more preferably 10 8 CFU / mL.

[0025] The temperature of the fermentation culture is 37-43℃, preferably 38℃.

[0026] The time of the fermentation culture is 15-24h, preferably 20h.

[0027] The fermentation culture is aerobic fermentation, preferably static fermentation.

[0028] The sterilization condition and method can be conventional condition and method in the art, and can be high temperature sterilization method.

[0029] When the high temperature sterilization method is used for the sterilization, the temperature of the sterilization can be conventional temperature in the art, preferably 110-125℃, more preferably 115-121℃. When the high temperature sterilization method is used for the sterilization, the time of the sterilization can be conventional time in the art, preferably 15-35min, more preferably 20-35min, for example 30min. When the high temperature sterilization method is used for the sterilization, the pressure of the sterilization can be conventional pressure in the art, preferably 0.1-0.14Mpa, more preferably 0.1-0.13MPa, for example 0.12MPa.

[0030] According to the convention in the art, the sterilization operation can further include cooling operation, which can be cooling to room temperature.

[0031] In some embodiments, the mixing operation can further include secondary sterilization and / or adding preservative according to the convention in the art.

[0032] The secondary sterilization condition and method can be conventional condition and method in the art, and can be high temperature sterilization method.

[0033] When the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization can be conventional temperature in the art, preferably 80-95℃, more preferably 90-95℃. When the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization can be conventional time in the art, preferably 15-35min, more preferably 20-35min, for example 30min. The pressure of the secondary sterilization can be conventional time in the art, preferably 0.1-0.14Mpa, more preferably 0.1-0.13MPa, for example 0.12MPa.

[0034] In the process of adding the preservative, the preservative can include at least one of 1,3-propanediol, p-hydroxyacetophenone and 1,3-propanediol according to the convention in the art. When the preservative includes the 1,3-propanediol, the p-hydroxyacetophenone and the 1,2-hexanediol, the 1,3-propanediol can account for 1% to 2% by mass of the material prepared after mixing; the 1,2-hexanediol can account for 0.3% to 2% by mass of the material prepared after mixing; the p-hydroxyacetophenone can account for 0.3% to 2% by mass of the material prepared after mixing; preferably, the 1,3-propanediol accounts for 1.5% by mass of the material prepared after mixing, the p-hydroxyacetophenone accounts for 0.5% by mass of the material prepared after mixing, and the 1,2-hexanediol accounts for 0.5% by mass of the material prepared after mixing.

[0035] In the process of adding the preservative, the mixing can be performed under the convention in the art, and the temperature of the mixing can be the temperature conventionally used in the art for such operation, preferably 60 to 80°C, more preferably 70 to 80°C, for example 75°C.

[0036] The present application also provides a white tea ferment liquor with anti-wrinkle and firming effects, which is prepared by the method for preparing a white tea ferment liquor with anti-wrinkle and firming effects as described above.

[0037] The present application also provides use of the white tea ferment liquor with anti-wrinkle and firming effects as described above directly as a product, as an additive or as a base in the preparation of a skin external agent.

[0038] In some embodiments, the white tea ferment liquor with anti-wrinkle and firming effects can be at least one of an antioxidant active ingredient, a soothing and anti-sensitivity active ingredient and a moisturizing active ingredient in the skin external agent.

[0039] The anti-wrinkle and firming active ingredient can be a soothing and anti-sensitivity active ingredient with the activity of inhibiting hyaluronidase.

[0040] The present application also provides a skin external agent including the white tea ferment liquor with anti-wrinkle and firming effects as described above.

[0041] In some embodiments, the skin external agent can further include active ingredients conventionally used in the art, which can generally include any one or more of whitening active ingredients, anti-inflammatory active ingredients, anti-sensitivity active ingredients and antioxidant active ingredients.

[0042] In some embodiments, the skin external agent can include a mask, an essence or a toner according to the convention in the art.

[0043] In some embodiments, the white truffle fermentation broth having anti-wrinkle and firming efficacy can account for 5% to 99%, preferably 60% to 99%, of the mass percentage of the skin external preparation.

[0044] In some embodiments, the room temperature generally refers to 15 to 40°C.

[0045] On the basis of common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, thereby obtaining preferred examples of the present application.

[0046] The reagents and raw materials used in the present application are commercially available.

[0047] The positive progress effect of the present application is that the white truffle fermentation broth having anti-wrinkle and firming efficacy prepared by the present application has ideal anti-wrinkle and firming efficacy, antioxidant efficacy, and soothing and anti-allergic efficacy, the preparation process is simple, no organic reagent is used, it is energy-saving and environmentally friendly, has high safety, and meets the demand of modern skin external preparations for functionality and safety. BRIEF DESCRIPTION OF DRAWINGS

[0048] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The drawings, together with the following detailed description, form a part of the specification and are included to further illustrate preferred embodiments of the present application and to explain the principles and advantages of the present application. In the drawings:

[0049] Figure 1 The comparative diagram of the collagen content improvement of the products prepared in Examples 1 to 4 or Comparative Examples 1 to 4;

[0050] Figure 2 The comparative diagram of the elastase inhibition ability of the products prepared in Examples 1 to 4 or Comparative Examples 1 to 4;

[0051] Figure 3 The comparative diagram of the mitochondrial activity improvement ability of the products prepared in Examples 1 to 4 or Comparative Examples 1 to 4. DETAILED DESCRIPTION

[0052] The present application will be further described below by way of examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples, if no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the goods.

[0053] The experimental methods used in the following examples are conventional methods, unless otherwise specified.

[0054] The source of white truffle in the following examples and comparative examples is commercially available.

[0055] The source of white truffle in the following examples and comparative examples is commercially available.

[0056] The source of Chinese Stachys joltana in the following examples and comparative examples is commercially available.

[0057] The preservation date of Lactobacillus bulgaricus in the following examples and comparative examples is October 27, 2023, the preservation unit name is China General Microbiological Culture Collection Center (CGMCC), the address is No. 3, Beichen West Road, Chaoyang District, Beijing, the postcode is 100101, and the preservation number is CGMCC NO. 28800.

[0058] Example 1

[0059] (1) The commercially available dried white crane ganoderma was crushed and sieved, and the crushed mesh size was 80 mesh. The commercially available dried Chinese Stachys joltana was crushed and sieved, and the crushed mesh size was 80 mesh. 2 g of white crane ganoderma powder, 6 g of white truffle and 1 g of Chinese Stachys joltana powder were mixed with 200 g of deionized water, wherein the mass ratio of white crane ganoderma powder, white truffle and Chinese Stachys joltana powder was 1:3:0.5; After mixing evenly, sterilization was carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min. After sterilization, it was cooled to room temperature. After cooling, 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid was inoculated, the viable bacterial count in the bacterial liquid was 10 10 CFU / mL, after inoculation of the bacterial liquid, culture was carried out in a incubator at 38°C for 20 h. After culture, sterilization was carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min. After sterilization, it was cooled to room temperature. White crane ganoderma compound fermentation liquor was prepared.

[0060] (2) 80 g of the white crane ganoderma compound fermentation liquor prepared above and 1.5 g of 1,3-propanediol were mixed evenly, and then sterilized at 90°C for 30 min. Then, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added at 75°C, and mixed evenly to prepare white factor fermentation liquor with anti-wrinkle and firming effects.

[0061] Example 2

[0062] Compared with Example 1, the only difference is that in step (1), the mass ratio of white crane ganoderma, white truffle and Chinese Stachys joltana is 1:1:0.5. The specific operation is as follows: 2 g of white crane ganoderma powder, 2 g of white truffle and 1 g of Chinese Stachys joltana powder were mixed with 200 g of deionized water, and other condition parameters were the same as those in Example 1.

[0063] (1) The commercially available dried white crane Ganoderma lucidum is crushed and sieved, and the crushed size is 80 mesh. The commercially available dried Chinese Stachys floridus is crushed and sieved, and the crushed size is 80 mesh. 2 g of white crane Ganoderma lucidum powder, 2 g of white truffle and 1 g of Chinese Stachys floridus powder are mixed with 200 g of deionized water, wherein the mass percentage of the three of white crane Ganoderma lucidum powder, white truffle and Chinese Stachys floridus powder is 1:3:0.5; after uniform mixing, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization, it is cooled to room temperature. After cooling, 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid is inoculated, the number of viable bacteria in the bacterial liquid is 10 10 CFU / mL, after inoculation of the bacterial liquid, culture is carried out in a culture box at 38 ℃ for 20 h, after culture, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization, it is cooled to room temperature. White crane Ganoderma lucidum compound fermentation liquor is prepared.

[0064] (2) The above-prepared 80 g of white crane Ganoderma lucidum compound fermentation liquor and 1.5 g of 1,3-propanediol are uniformly mixed, and then sterilized at a temperature of 90 ℃ for 30 min, and then cooled to 75 ℃. 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone are added and uniformly mixed to prepare white factor fermentation liquor with anti-wrinkle and firming effects.

[0065] Example 3

[0066] Compared with Example 1, the only difference is that the fermentation temperature is 43 ℃, the fermentation time is 15 h, and the other conditions are the same as those in Example 1.

[0067] (1) The commercially available dried white crane Ganoderma lucidum is crushed and sieved, and the crushed size is 80 mesh. The commercially available dried Chinese Stachys floridus is crushed and sieved, and the crushed size is 80 mesh. 2 g of white crane Ganoderma lucidum powder, 2 g of white truffle and 1 g of Chinese Stachys floridus powder are mixed with 200 g of deionized water, wherein the mass percentage of the three of white crane Ganoderma lucidum powder, white truffle and Chinese Stachys floridus powder is 1:3:0.5; after uniform mixing, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization, it is cooled to room temperature. After cooling, 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid is inoculated, the number of viable bacteria in the bacterial liquid is 10 10 CFU / mL, after inoculation of the bacterial liquid, culture is carried out in a culture box at 38 ℃ for 20 h, after culture, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization, it is cooled to room temperature. White crane Ganoderma lucidum compound fermentation liquor is prepared.

[0068] (2) The 80 g white crane ganoderma grass composite fermentation liquor prepared above and 1.5 g of 1,3-propanediol are mixed uniformly, then sterilized at a temperature of 90°C for 30 min, then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone are added and mixed uniformly to prepare white factor fermentation liquor with anti-wrinkle and firming efficacy.

[0069] Example 4

[0070] Compared with Example 1, the only difference is that in step (1), the mass ratio of white crane ganoderma grass, white truffle and Chinese flos genkwa is 1:2:1.5, 2 g of white crane ganoderma grass powder, 4 g of white truffle and 3 g of Chinese flos genkwa powder are added, and other condition parameters are the same as those in Example 1.

[0071] (1) The commercially available dried white crane ganoderma grass is crushed and sieved, and the crushed mesh size is 80 mesh. The commercially available dried Chinese flos genkwa is crushed and sieved, and the crushed mesh size is 80 mesh. 2 g of white crane ganoderma grass powder, 6 g of white truffle and 1 g of Chinese flos genkwa powder are mixed with 200 g of deionized water, wherein the mass percentage of the three of white crane ganoderma grass powder, white truffle and Chinese flos genkwa powder is 1:3:0.5; after being mixed uniformly, sterilization is carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min, and after sterilization is completed, cooling is carried out to room temperature. After cooling is completed, 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid is inoculated, the viable bacterial count in the bacterial liquid is 10 10 CFU / mL, after inoculation of the bacterial liquid, culture is carried out in a culture box at 38°C for 20 h, after culture is completed, sterilization is carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min, and after sterilization is completed, cooling is carried out to room temperature to prepare white crane ganoderma grass composite fermentation liquor.

[0072] (2) The 80 g white crane ganoderma grass composite fermentation liquor prepared above and 1.5 g of 1,3-propanediol are mixed uniformly, then sterilized at a temperature of 90°C for 30 min, then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone are added and mixed uniformly to prepare white factor fermentation liquor with anti-wrinkle and firming efficacy.

[0073] Comparative Example 1

[0074] Compared with Example 1, the only difference is that the fermentation strain is replaced by Lactobacillus acidophilus HH-LA26, and other condition parameters are the same as those in Example 1.

[0075] (1) The commercially available dried white crane Ganoderma lucidum is crushed and sieved, and the crushed size is 80 meshes. The commercially available dried Chinese Stachys floridana Schm-idt is crushed and sieved, and the crushed size is 80 meshes. 2 g of white crane Ganoderma lucidum powder, 6 g of white truffle and 1 g of Chinese Stachys floridana Schmid powder are mixed with 200 g of deionized water, wherein the mass percentage of the three of white crane Ganoderma lucidum powder, white truffle and Chinese Stachys floridana Schmid powder is 1:3:0.5; after being mixed uniformly, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization is completed, it is cooled to room temperature, 2 mL of Lactobacillus acidophilus HH-LA26 bacterial liquid is inoculated, the viable bacterial count in the bacterial liquid is 10 10 CFU / mL, after inoculation of the bacterial liquid, culture is carried out in a culture box at 38 ℃ for 20 h, after culture is completed, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization is completed, it is cooled to room temperature, and the white crane Ganoderma lucidum compound fermentation liquor is prepared.

[0076] (2) The above-prepared 80 g of white crane Ganoderma lucidum compound fermentation liquor and 1.5 g of 1,3-propanediol are mixed uniformly, and then sterilization is carried out at a temperature of 90 ℃ for 30 min, and then the temperature is lowered to 75 ℃, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone are added and mixed uniformly, and the white truffle fermentation liquor with anti-wrinkle and firming effects is prepared.

[0077] Comparative Example 2

[0078] Compared with Example 1, the difference is only that 2 g of white crane Ganoderma lucidum powder, 6 g of white truffle and 1 g of Tongtuo are added, and other conditions are the same as those in Example 1.

[0079] (1) The commercially available dried white crane Ganoderma lucidum is crushed and sieved, and the crushed size is 80 meshes. The commercially available dried Tongtuo is crushed and sieved, and the crushed size is 80 meshes. 2 g of white crane Ganoderma lucidum powder, 6 g of white truffle and 1 g of Tongtuo powder are mixed with 200 g of deionized water, wherein the mass percentage of the three of white crane Ganoderma lucidum powder, white truffle and Tongtuo powder is 1:3:0.5; after being mixed uniformly, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization is completed, it is cooled to room temperature, 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid is inoculated, the viable bacterial count in the bacterial liquid is 10 10 CFU / mL, after inoculation of the bacterial liquid, culture is carried out in a culture box at 38 ℃ for 20 h, after culture is completed, sterilization is carried out in a high-temperature sterilization pot at 121 ℃ and 0.12 MPa for 30 min, and after sterilization is completed, it is cooled to room temperature, and the white crane Ganoderma lucidum compound fermentation liquor is prepared.

[0080] (2) The 80 g white fungus and 1.5 g 1,3-propanediol are mixed uniformly, sterilized at 90°C for 30 min, then cooled to 75°C, 0.5 g 1,2-hexanediol and 0.5 g p-hydroxyacetophenone are added and mixed uniformly to obtain white factor fermentation liquor with anti-wrinkle and firming effects.

[0081] Comparative Example 3

[0082] Compared with Example 1, the only difference is that 4 g white truffle and 5 g Chinese flag pollen powder are added, and other conditions are the same as those in Example 1.

[0083] (1) The commercially available dry Chinese flag pollen is crushed and sieved, and the crushed mesh size is 80 mesh. 4 g white truffle and 5 g Chinese flag pollen powder are mixed with 200 g deionized water, wherein the mass ratio of the three is 4:5; after mixing uniformly, sterilization is carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min, and after sterilization, the temperature is cooled to room temperature. 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid is inoculated, the viable bacterial count in the bacterial liquid is 10 10 CFU / mL, after inoculation of the bacterial liquid, culture is carried out in a 38°C incubator for 20 h, and after culture, sterilization is carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min, and after sterilization, the temperature is cooled to room temperature to obtain white fungus and Chinese flag pollen compound fermentation liquor.

[0084] (2) The 80 g white fungus and 1.5 g 1,3-propanediol are mixed uniformly, sterilized at 90°C for 30 min, then cooled to 75°C, 0.5 g 1,2-hexanediol and 0.5 g p-hydroxyacetophenone are added and mixed uniformly to obtain white factor fermentation liquor with anti-wrinkle and firming effects.

[0085] Comparative Example 4

[0086] Compared with Example 1, 4 g white truffle and 5 g white fungus powder are added, and other conditions are the same as those in Example 1.

[0087] (1) The commercially available dry white fungus is crushed and sieved, and the crushed mesh size is 80 mesh. 5 g white fungus powder, 4 g white truffle and 200 g deionized water are mixed, wherein the mass ratio of the three is 1:3:0.5; after mixing uniformly, sterilization is carried out in a high-temperature sterilization pot at 121°C and 0.12 MPa for 30 min, and after sterilization, the temperature is cooled to room temperature. 2 mL of Lactobacillus bulgaricus CGMCC No. 28800 bacterial liquid is inoculated, the viable bacterial count in the bacterial liquid is 10 10CFU / mL, the bacteria liquid was inoculated and cultured in a 38℃ incubator for 20h, after the culture was finished, the bacteria liquid was sterilized in a high-temperature sterilization pot at 121℃ and 0.12MPa for 30min, and then cooled to room temperature to obtain the white huaishengcha composite fermentation liquid.

[0088] (2) 80g of the white huaishengcha composite fermentation liquid prepared above and 1.5g of 1,3-propanediol were uniformly mixed, sterilized at 90℃ for 30min, then cooled to 75℃, 0.5g of 1,2-hexanediol and 0.5g of p-hydroxyacetophenone were added and uniformly mixed to obtain the white factor fermentation liquid with anti-wrinkle and firming effects.

[0089] Effect implementation example 1

[0090] Collagen content determination

[0091] Experimental method: the anti-wrinkle and firming effects of the products prepared in the above examples 1-4 and comparative examples 1-4 were verified by determining the COL-I content in human skin fibroblasts.

[0092] Logarithmic phase human skin fibroblasts were taken, and the cell suspension was inoculated on a 6-well cell culture plate at a density of 250,000 / mL, 2mL of the cell suspension was added to each well, and the culture was performed for 12h. The experimental group, the blank group and the model group were set. Among them, the experimental group and the model group were irradiated with 18mJ / cm 2 UVA for 40min. After irradiation, the supernatant in the experimental group, the blank group and the model group was discarded, 2mL of DMEM solution was added to the blank group and the model group, and 2mL of the test solution (the product prepared in the above examples or comparative examples was configured into a test solution with a volume percentage of 2%, and the test solution needed to be filtered through a 0.22μm sterile filter membrane before use) was added to the experimental group. After adding, the cells were treated for 24h, the supernatant was discarded, the cells were washed with PBS for 2-3 times, then the cells were treated with cell lysis solution and transferred to a centrifuge tube, centrifuged at 10000r / min and 4℃ for 10min, and the supernatant was taken to obtain the cell lysis solution. 20μL of the cell lysis solution was used to detect the total protein content in the sample by using a BCA kit; the collagen content was determined according to the experimental operation of the ELISA kit instruction, and the OD value of each was measured at 450nm, the standard curve equation was Y=(A-D) / [1+(X / C) B ]+D, A=1.78854, B=1.23259, C=9.31934, D=-0.01557, R 2 =0.99859, the expression amount x of collagen was calculated according to the standard curve, and A=x / b was corrected with the total protein content b, and the experimental results are shown in Table 1 and Figure 1 ( Figure 1 middle, #p<0.001 means extremely significant decrease compared with the blank group; p>0.05, ns means no significant increase compared with the model group; *p<0.05 means increase compared with the model group, **p<0.01 means significant increase compared with the model group, ***p<0.001 means extremely significant increase compared with the model group.

[0093] Table 1

[0094]

[0095]

[0096] The above results show that the human skin fibroblast cells of the products prepared in Examples 1-4 of the present application have significantly higher collagen content than the comparative examples 1-4, and it can be seen that the present application has excellent anti-wrinkle and firming effects.

[0097] Effect Example 2

[0098] Elastase activity

[0099] Experimental method: Elastin plays a key role in skin youthfulness, and the absence of elastin is a major cause of skin aging. Inhibition of elastase activity can effectively slow down the decomposition of elastin and maintain the elasticity of the skin. Elastase can catalyze the hydrolysis of the hydroxyl group contained in the peptide bond of various amino acids, so when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, it produces a colored substance, and its absorbance value can be measured at 406 nm in a spectrophotometer. According to the change in absorbance value, the inhibitory effect of the test substance on elastase activity can be calculated to evaluate whether the test substance has anti-wrinkle and firming effects. Elastase, substrate, test sample and positive control are all prepared with Tris-HCl buffer.

[0100] Tris-HCl buffer (pH 8.0)

[0101] 1.1 mmol / L of substrate (N-succinyl-alanine-alanine-alanine-p-nitroaniline):

[0102] 4.5 mg was dissolved in 10 mL of Tris-HCl buffer (pH 8.0).

[0103] 2.2 μmol / L of elastase solution: 0.5 mg of elastase powder was dissolved in

[0104] 1 mL of Tris-HCl buffer (pH 8.0), and was prepared immediately before use.

[0105] 3. Positive control (EGCG: Epigallocatechin Gallate): 2 mg / ml, prepared according to the required amount of the test.

[0106] 4. Sample solution: diluted with Tris-HCl buffer to the corresponding volume fraction according to the test concentration.

[0107]

[0108] Elastase activity inhibition rate:

[0109] Inhibition rate % = (1 - (T - T0) / (C - C0)) x 100%

[0110] In the formula: T - sample group absorbance, i.e. the absorbance of the solution after the sample affects the elastase and substrate reaction; T0 - sample background group absorbance; C - enzyme reaction group absorbance, i.e. the absorbance of the elastase and substrate reaction without adding the sample; D - solvent background group absorbance.

[0111] The products prepared in Examples 1-4 and Comparative Examples 1-4 were subjected to elastase inhibition rate tests, and the results are shown in Table 2 and Figure 2 .

[0112] Table 2

[0113] Elastase inhibition rate (%) Example 1 51.28±1.96 Example 2 41.03±2.96 Example 3 48.29±1.96 Example 4 44.87±5.34 Comparative Example 1 20.51±5.18 Comparative Example 2 26.5±0.74 Comparative Example 3 33.33±0.74 Comparative Example 4 31.2±6.66

[0114] The above results show that the elastase rates of the products prepared in Examples 1-4 are significantly higher than those of Comparative Examples 1-4, indicating that the products prepared in the present application have excellent effects of slowing down the decomposition of elastin and maintaining the elasticity of the skin.

[0115] Effect Example 3

[0116] Mitochondrial activity

[0117] Experimental method: WST-8 in CCK-8 dyeing agent is reduced to highly water-soluble yellow formazan product under the action of electron carriers in cell mitochondria. The amount of formazan product generated can indirectly reflect the activity of mitochondria in cells.

[0118] The products prepared in the above examples and comparative examples were respectively configured into a 2% volume percentage test solution in a serum-free DMEM medium. The test solution needs to be filtered through a 0.22 μm sterile filter membrane.

[0119] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum. The cells were grown in a 37°C, 5% CO2 incubator, and when the cell fusion reached more than 85%, the logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion reaction was terminated with serum-containing DMEM. The cells were inoculated in a 96-well plate at a density of 8000 cells / well and incubated in a 37°C, 5% CO2 incubator overnight. The HaCaT cells were divided into blank groups and sample groups, with at least 3 replicates in each group. The blank groups were added with serum-free medium, and the sample groups were added with the samples 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, and the results are shown in Table 3 and Figure 3 .

[0120] Table 3

[0121] Mitochondrial activity (%) Blank group 100±3.1 Example 1 128.43±3.82 Example 2 113.45±5.65 Example 3 120.37±8.01 Example 4 117.02±5.53 Comparative Example 1 105.74±7.28 Comparative Example 2 108.05±6.73 Comparative Example 3 108.49±4.76 Comparative Example 4 108.78±7.97

[0122] The results show that the mitochondrial activity of the products prepared in Examples 1-4 is much higher than that of the products prepared in Comparative Examples 1-4. It can be seen that the products prepared in the present application have ideal cell metabolism promoting effects, and the fermentation culture temperature and strain have a great influence on the cell metabolism promoting performance of the final product.

[0123] Finally, it should be noted that the terms "comprising", "including", or any other variant thereof in the present application are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment.

[0124] Although the present application has been disclosed by the description of specific embodiments of the present application above, it should be understood that those skilled in the art can design various modifications, improvements or equivalents of the present application within the spirit and scope of the appended claims. These modifications, improvements or equivalents should also be considered to be included in the scope of the present application.

Claims

1. A method for preparing a fermented extract of white ginseng with anti-wrinkle and firming effects, characterized in that, It includes the following steps: preparing a fermentation substrate by mixing white crane lingzhi grass, white truffle and Chinese jasmine with water, inoculating lactobacillus into the fermentation substrate, fermenting and culturing, and sterilizing. The mass percentages of the white crane lingzhi grass, the white truffle, and the Chinese hibiscus flower are 1:(1~4):(0.5~2). The lactobacillus is Lactobacillus bulgaricus, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28800. The fermentation culture temperature is 37~43℃; The fermentation culture time is 15~24h.

2. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 1, characterized in that, The mass percentages of the white crane lingzhi grass, the white truffle, and the Chinese gypsy flower are 1:3:0.

5.

3. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 1, characterized in that, The fermentation method satisfies at least one of the following conditions: The white crane lingzhi grass is white crane lingzhi grass powder that has been crushed and sieved; The Chinese jasmine flower is Chinese jasmine flower powder that has been crushed and sieved. The water is deionized water.

4. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 3, characterized in that, The fermentation method satisfies at least one of the following conditions: The particle size of the white crane Ganoderma powder sieved is 50-100 mesh; The particle size of the powder of *Gynostemma pentaphyllum* is 50-100 mesh after sieving.

5. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 4, characterized in that, The fermentation method satisfies at least one of the following conditions: The particle size of the white crane Ganoderma powder sieved is 80 mesh; The particle size of the powder of *Gynostemma pentaphyllum* was 80 mesh when sieved.

6. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 1, characterized in that, The lactobacillus was inoculated in the form of lactobacillus bacterial solution.

7. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 6, characterized in that, The concentration of *Lactobacillus bulgaricus* in the bacterial culture is 10. 8 ~10 12 CFU / mL.

8. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 7, characterized in that, The concentration of *Lactobacillus bulgaricus* in the bacterial culture is 10. 10 CFU / mL.

9. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 1, characterized in that, The fermentation substrate also includes a sterilization process before use.

10. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 9, characterized in that, The sterilization is a high-temperature sterilization method; when the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 110~125℃, the sterilization time is 15~35min, and the sterilization pressure is 0.1~0.14Mpa.

11. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 10, characterized in that, When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 115~121℃, the sterilization time is 20~35min, and the sterilization pressure is 0.1~0.13MPa.

12. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 11, characterized in that, When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization time is 30 minutes and the sterilization pressure is 0.12 MPa.

13. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 1, characterized in that, The fermentation method satisfies at least one of the following conditions: The sterilization process also includes a cooling operation; The fermentation culture temperature was 38℃; The fermentation culture time is 20 hours; The fermentation culture is an aerobic fermentation.

14. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 13, characterized in that, The fermentation method satisfies at least one of the following conditions: The cooling refers to cooling to room temperature; The aerobic fermentation is static fermentation.

15. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 1, characterized in that, The mixing process also includes secondary sterilization and / or the addition of preservatives; During the addition of the preservative, the preservative includes at least one selected from 1,3-propanediol, p-hydroxyacetophenone, and 1,2-hexanediol; when the preservative includes 1,3-propanediol, p-hydroxyacetophenone, and 1,2-hexanediol, the 1,3-propanediol accounts for 1% to 2% of the mass percentage of the mixture obtained; the 1,2-hexanediol accounts for 0.3% to 2% of the mass percentage of the mixture obtained; and the p-hydroxyacetophenone accounts for 0.3% to 2% of the mass percentage of the mixture obtained. During the addition of the preservative, the mixing temperature is 60~80℃.

16. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 15, characterized in that, The secondary sterilization method is high-temperature sterilization. When the high-temperature sterilization method is used for secondary sterilization, the temperature of the secondary sterilization is 110~125℃; the time of the secondary sterilization is 15~35min; and the pressure of the secondary sterilization is 0.1~0.14Mpa. During the addition of the preservative, the 1,3-propanediol accounts for 1.5% of the mass of the mixture, the p-hydroxyacetophenone accounts for 0.5% of the mass of the mixture, and the 1,2-hexanediol accounts for 0.5% of the mass of the mixture. During the addition of the preservative, the mixing temperature is 70~80℃.

17. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 16, characterized in that, When the high-temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 115~121℃; the time of the secondary sterilization is 20~35min; and the pressure of the secondary sterilization is 0.1~0.13MPa. During the addition of the preservative, the mixing temperature is 75°C.

18. The method for preparing the white turmeric fermentation broth with anti-wrinkle and firming effects as described in claim 17, characterized in that, When the high-temperature sterilization method is used for the secondary sterilization, the secondary sterilization time is 30 minutes and the secondary sterilization pressure is 0.12 MPa.

19. A white teosin fermentation liquid with anti-wrinkle and firming effects, which is prepared by the method of preparing white teosin fermentation liquid with anti-wrinkle and firming effects according to any one of claims 1 to 18.

20. The use of the white turmeric ferment broth with anti-wrinkle and firming effects as described in claim 19, directly as a product, as an additive, or as a base in the preparation of topical skin agents.

21. The white turmeric fermentation liquid with anti-wrinkle and firming effects as described in claim 20, characterized in that, The fermented extract of white turmeric, which has anti-wrinkle and firming effects, is used as the anti-wrinkle active ingredient and / or firming active ingredient in the topical skin agent.

22. The white turmeric fermentation liquid with anti-wrinkle and firming effects as described in claim 21, characterized in that, The anti-wrinkle active ingredient is one that increases collagen content and / or inhibits antioxidant elastase activity; the firming active ingredient is one that enhances mitochondrial activity.

23. A topical skin agent comprising the fermented extract of white thyme as described in claim 19, which has anti-wrinkle and firming effects.

24. The topical skin agent as described in claim 23, characterized in that, The topical skin agent also includes any one or more of the following active ingredients: whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients, and antioxidant active ingredients.

25. The topical skin agent as described in claim 23, characterized in that, The topical skin agents include face masks, serums, or toners.

26. The topical skin agent as described in claim 23, characterized in that, The fermented extract of white turmeric with anti-wrinkle and firming effects accounts for 5% to 99% of the mass of the topical skin agent.

27. The topical skin agent as described in claim 26, characterized in that, The fermented extract of white turmeric with anti-wrinkle and firming effects accounts for 60% to 99% of the mass of the topical skin agent.

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