Baijin fermentation liquor with anti-wrinkle and firming effects as well as preparation method and application of Baijin fermentation liquor

Abstract

The preparation method comprises the following steps: mixing rhinacanthus nasutus, white truffle and stachyurus chinensis with water to prepare a fermentation substrate, inoculating lactobacillus into the fermentation substrate, and carrying out fermentation culture and sterilization, so as to obtain the candida albicans fermentation liquid, the candida albicans fermentation liquid, the candida albicans fermentation liquid, the candida albicans fermentation liquid and the candida albicans fermentation liquid, the candida albicans fermentation liquid and the candida albicans fermentation liquid, wherein the rhinacanthus nasutus is prepared by fermenting rhinacanthus nasutus, white truffle, stachyurus chinensis and water, and the mass ratio of the rhinacanthus nasutus to the white truffle to the stachyurus chinensis is 1: (1-10): (0.1-2). The Baiji with the anti-wrinkle and firming effects prepared by the invention has ideal anti-wrinkle and firming effects, anti-oxidation effects and relieving and anti-allergy effects due to the fermentation liquor, the preparation process is simple, no organic reagent is adopted, the Baiji is energy-saving and environment-friendly, and the Baiji has relatively high safety and meets the requirements of modern skin external preparations on functionality and safety.

Description

Technical Field

[0001] The present application belongs to the field of fermentation technology, and in particular relates to a white grape fermentation liquid with anti-wrinkle and firming effects, and a preparation method and application thereof. Background Art

[0002] Skin aging is part of human aging, caused by a variety of endogenous and exogenous factors, including gene regulation of skin aging, free radical damage and photo-stimulated aging, 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 crane grass, is shaped like a flying white crane and has the effects of activating the immune system, enhancing immunity, reducing liver fire, and reducing swelling and detoxification. At present, the research on Rhinacanthus nasutus is mainly in the fields of food and medicine, focusing on the extraction process and chemical composition of Rhinacanthus nasutus, while there is little research on its fermentation to prepare fermented products.

[0004] White truffle is a large block-shaped fungus of the Ascomycetes family, also known as truffle. Studies have shown that white truffles are rich in polysaccharides, proteins, amino acids and polyunsaturated fatty acids, as well as minerals and sterols. Due to its complex composition, if it is not fermented properly, it is very likely to decompose, resulting in a waste of resources.

[0005] Chinese Stachyurus chinensis (S.chinensis Franch.) belongs to the Stachyurus family (Stachyuraceae), Stachyurus Sieb.et Zucc., which is endemic to East Asia. It is called "small tongcao". Its stem and pith are used as medicine, which has the effects of diuresis, lactation, and clearing dampness and heat. At present, the research on S.chinensis Franch. is basically in the field of pharmacy, mainly focusing on the extraction process, chemical composition and pharmacological effects of S.chinensis Franch., while the research on fermenting S.chinensis Franch. and using it in the field of skin care products has not been reported.

[0006] Therefore, there is an urgent need in the art to develop a preparation method that can simultaneously ferment Scutellaria baicalensis, white truffle and Chinese flag lily, and the product can be applied to the field of skin topical preparations to significantly improve its bioavailability and anti-wrinkle and firming beauty effects. Summary of the invention

[0007] The technical problem to be solved by this application is to overcome the defects of the prior art that white crane ganoderma is mainly used in the medical field, the application field is limited and there is a lack of fermentation technology; truffle fermentation is difficult to master, and there is a lack of research in the fermentation field of Chinese flag flower, and to provide a white ginseng fermentation liquid with anti-wrinkle and firming effects, and a preparation method and application thereof. The white ginseng fermentation liquid prepared in this application has ideal antioxidant properties, anti-wrinkle and firming effects, a simple preparation process, no organic reagents are used in the preparation process, and has high safety, which meets the requirements of modern skin topical preparations for functionality and safety.

[0008] This application adopts the following technical solutions to solve the above technical problems:

[0009] The present application provides a method for preparing a fermented liquor of white ginseng with anti-wrinkle and firming effects, which comprises the following steps: preparing a fermentation substrate by mixing white crane ganoderma, white truffle and Chinese flag flower with water, inoculating lactobacillus into the fermentation substrate, fermenting and culturing, and sterilizing;

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

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

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

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

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

[0015] The Chinese flagella flower is subjected to conventional crushing and sieving operations in the art to obtain Chinese flagella flower powder, and the sieved particle size is 50 to 100 meshes, preferably 80 meshes.

[0016] The water is deionized water.

[0017] The fermentation substrate may also be sterilized according to conventional procedures in the art before use.

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

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

[0020] Among them, according to the common practice in the art, the sterilization operation may 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 lactic acid bacteria liquid.

[0022] In some embodiments, the lactobacillus may include Lactobacillus bulgaricus. Preferably, the Lactobacillus bulgaricus is deposited on October 27, 2023. The name of the depository is China General Microbiology Center of China Culture Collection Administration (CGMCC for short), and the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is 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 the conventional art, and the concentration of the Lactobacillus bulgaricus in the Lactobacillus bulgaricus bacterial solution can be 10 8 ~10 12 CFU / mL, preferably 10 9 ~10 11 CFU / mL.

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

[0025] In the preparation method of the Baihe Lingzhi grass composite fermentation liquid, the fermentation culture temperature is 37-43°C, preferably 38°C;

[0026] The fermentation culture time is 15 to 24 hours, preferably 20 hours.

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

[0028] The sterilization conditions and methods may be conventional conditions and methods for such operations in the art, and may generally be high temperature sterilization.

[0029] When the high temperature sterilization method is used for the sterilization, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110-125°C, more preferably 115-121°C. When the high temperature sterilization method is used for the sterilization, the sterilization time can be a conventional time for such operations in the art, preferably 15-35 minutes, more preferably 20-35 minutes, for example 30 minutes. When the high temperature sterilization method is used for the sterilization, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, for example 0.12 MPa.

[0030] According to the common practice in the art, the sterilization operation may be further followed by a cooling operation, which may generally be cooling to room temperature.

[0031] In some embodiments, the mixing operation may further include secondary sterilization and / or adding preservatives according to conventional procedures in the art.

[0032] The conditions and methods for the secondary sterilization may be conventional conditions and methods for such operations in the art, and may generally be high-temperature sterilization.

[0033] 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 80-95°C, more preferably 90-95°C. When the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization can be a conventional time for such operations in the art, preferably 15-35 minutes, more preferably 20-35 minutes, for example 30 minutes. The pressure of the secondary sterilization can be a conventional time for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.1 3 MPa, for example 0.12 MPa.

[0034] In the process of adding the preservative, the preservative may include at least one of 1,3-propylene glycol, p-hydroxyacetophenone and 1,3-propylene glycol according to the conventional art. When the preservative includes the 1,3-propylene glycol, the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the 1,3-propylene glycol in the mixed material may be 1% to 2%; the mass percentage of the 1,2-hexanediol in the mixed material may be 0.3% to 2%; the mass percentage of the p-hydroxyacetophenone in the mixed material may be 0.3% to 2%; preferably, the mass percentage of the 1,3-propylene glycol in the mixed material is 1.5%, the mass percentage of the p-hydroxyacetophenone in the mixed material is 0.5%, and the mass percentage of the 1,2-hexanediol in the mixed material is 0.5%.

[0035] The process of adding the preservative can be carried out under mixing conditions according to the conventional conditions in the art. The mixing temperature can be the conventional temperature for such operations in the art, preferably 60-80°C, more preferably 70-80°C, for example 75°C.

[0036] The present application also provides a white glutinous rice fermentation liquid with anti-wrinkle and firming effects, which is prepared by the preparation method of the white glutinous rice fermentation liquid with anti-wrinkle and firming effects as described above.

[0037] The present application also provides a use of the above-mentioned fermented white grape extract with anti-wrinkle and firming effects directly as a product, as an additive or as a base in the preparation of a skin topical preparation.

[0038] In some embodiments, the white grape fermentation liquid with anti-wrinkle and firming effects can be used as at least one of the antioxidant active ingredient, soothing and anti-allergic active ingredient, and moisturizing active ingredient in the skin topical preparation.

[0039] Wherein, the anti-wrinkle and firming active ingredients may be soothing and anti-allergic active ingredients that inhibit the activity of hyaluronidase.

[0040] The present application also provides a skin external preparation, which includes the white grape fermentation liquid with anti-wrinkle and firming effects as described above.

[0041] In some embodiments, the skin topical preparation may further include active ingredients conventionally used in the art, generally including any one or more of whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0042] In some embodiments, the skin external preparation may include a facial mask, essence or toner according to conventional practices in the art.

[0043] In some embodiments, the mass percentage of the fermented liquor of white grape with anti-wrinkle and firming effects in the skin external preparation may be 5% to 99%, preferably 60% to 99%.

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

[0045] 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.

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

[0047] The positive progressive effect of the present application is that the white grape fermentation liquid with anti-wrinkle and firming effects prepared by the present application has ideal anti-wrinkle, firming, antioxidant and soothing and anti-allergic effects, the preparation process is simple, no organic reagents are used, it is energy-saving and environmentally friendly, has high safety, and meets the functionality and safety requirements of modern skin topicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The present application may be better understood by referring to the following description in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in 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. Among them:

[0049] Figure 1 This is a comparison chart of the improvement in collagen content of the products obtained in Examples 1 to 4 or Comparative Examples 1 to 4;

[0050] Figure 2 This is a comparison chart of the inhibitory ability of the products obtained in Examples 1 to 4 or Comparative Examples 1 to 4 on elastase;

[0051] Figure 3 This is a comparison chart of the ability of the products prepared in Examples 1 to 4 or Comparative Examples 1 to 4 to enhance mitochondrial activity. DETAILED DESCRIPTION

[0052] 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.

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

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

[0055] The sources of the white truffles in the following examples and comparative examples are commercially available.

[0056] The sources of the Chinese flag lily in the following examples and comparative examples are commercially available.

[0057] The preservation date of Lactobacillus bulgaricus in the following embodiments and comparative examples is October 27, 2023. The name of the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the preservation number is CGMCCNO.28800.

[0058] Example 1

[0059] (1) The commercially available dried white crane ganoderma lucidum was crushed and sieved to a mesh size of 80 mesh. The commercially available dried Chinese flag flower was crushed and sieved to a mesh size of 80 mesh. 2g of white crane ganoderma lucidum powder, 6g of white truffle and 1g of Chinese flag flower powder were mixed with 200g of deionized water, wherein the mass ratio of white crane ganoderma lucidum powder, white truffle and Chinese flag flower powder was 1:3:0.5; after uniform mixing, sterilized in a high-temperature autoclave at 121°C and 0.12MPa for 30min, cooled to room temperature after sterilization, and inoculated with 2mL of bulgaricus CGMCC No.28800 bacterial solution after cooling, the number of viable bacteria in the bacterial solution was 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0060] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid 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 Scutellaria baicalensis, white truffle and Chinese flagella is 1:1:0.5. The specific operation is: take 2g of Scutellaria baicalensis powder, 2g of white truffle, 1g of Chinese flagella powder and mix them with 200g of deionized water. The other conditions and parameters are the same as those in Example 1.

[0063] (1) The commercially available dried white crane ganoderma lucidum was crushed and sieved to a mesh size of 80 mesh. The commercially available dried Chinese flag flower was crushed and sieved to a mesh size of 80 mesh. 2g of white crane ganoderma lucidum powder, 2g of white truffle and 1g of Chinese flag flower powder were mixed with 200g of deionized water, wherein the mass percentage of white crane ganoderma lucidum powder, white truffle and Chinese flag flower powder was 1:3:0.5; after uniform mixing, sterilized in a high-temperature autoclave at 121°C and 0.12MPa for 30min, cooled to room temperature after sterilization, and inoculated with 2mL of bulgaricus CGMCC No.28800 bacterial solution after cooling, the number of viable bacteria in the bacterial solution was 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0064] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0065] Example 3

[0066] Compared with Example 1, the only difference is that the fermentation temperature is 43° C. and the fermentation time is 15 h. Other condition parameters are the same as Example 1.

[0067] (1) The commercially available dried white crane ganoderma lucidum was crushed and sieved to a mesh size of 80 mesh. The commercially available dried Chinese flag flower was crushed and sieved to a mesh size of 80 mesh. 2g of white crane ganoderma lucidum powder, 6g of white truffle and 1g of Chinese flag flower powder were mixed with 200g of deionized water, wherein the mass percentage of white crane ganoderma lucidum powder, white truffle and Chinese flag flower powder was 1:3:0.5; after uniform mixing, sterilized in a high-temperature autoclave at 121°C and 0.12MPa for 30min, cooled to room temperature after sterilization, and inoculated with 2mL of bulgaricus CGMCC No.28800 bacterial solution after cooling, the number of viable bacteria in the bacterial solution was 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 43°C for 15 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes. After the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0068] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0069] Example 4

[0070] Compared with Example 1, the only difference is that in step (1), the mass ratio of Scutellaria baicalensis, white truffle and Chinese flagella is 1:2:1.5, 2g of Scutellaria baicalensis powder, 4g of white truffle and 3g of Chinese flagella powder are added, and the other conditions and parameters are the same as those in Example 1.

[0071] (1) The commercially available dried white crane ganoderma lucidum was crushed and sieved to a mesh size of 80 mesh. The commercially available dried Chinese flag flower was crushed and sieved to a mesh size of 80 mesh. 2g of white crane ganoderma lucidum powder, 6g of white truffle and 1g of Chinese flag flower powder were mixed with 200g of deionized water, wherein the mass percentage of white crane ganoderma lucidum powder, white truffle and Chinese flag flower powder was 1:3:0.5; after uniform mixing, sterilized in a high-temperature autoclave at 121°C and 0.12MPa for 30min, cooled to room temperature after sterilization, and inoculated with 2mL of bulgaricus CGMCC No.28800 bacterial solution after cooling, the number of viable bacteria in the bacterial solution was 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0072] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0073] Comparative Example 1

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

[0075] (1) The commercially available dried white crane ganoderma lucidum was crushed and sieved to a mesh size of 80 mesh. The commercially available dried Chinese flag flower was crushed and sieved to a mesh size of 80 mesh. 2g of white crane ganoderma lucidum powder, 6g of white truffle and 1g of Chinese flag flower powder were mixed with 200g of deionized water, wherein the mass percentage of white crane ganoderma powder, white truffle and Chinese flag flower powder was 1:3:0.5; after uniform mixing, sterilized in a high-temperature autoclave at 121°C and 0.12MPa for 30min, cooled to room temperature after sterilization, and inoculated with 2mL of Lactobacillus acidophilus HH-LA26 bacterial solution after cooling, and the number of viable bacteria in the bacterial solution was 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0076] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0077] Comparative Example 2

[0078] Compared with Example 1, the only difference is that 2g of Scutellaria baicalensis powder, 6g of white truffle and 1g of Rhizoma Cyperi are added, and other conditions and parameters are the same as those in Example 1.

[0079] (1) The commercially available dried white crane ganoderma grass was crushed and sieved to a mesh size of 80 mesh, and the commercially available dried tongta wood was crushed and sieved to a mesh size of 80 mesh. 2g of white crane ganoderma grass powder, 6g of white truffle and 1g of tongta wood powder were mixed with 200g of deionized water, wherein the mass percentage of white crane ganoderma grass powder, white truffle and tongta wood powder was 1:3:0.5; after uniform mixing, sterilized in a high-temperature autoclave at 121°C and 0.12MPa for 30min, cooled to room temperature after sterilization, and inoculated with 2mL of bulgaricus CGMCC No.28800 bacterial solution after cooling, and the number of viable bacteria in the bacterial solution was 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0080] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0081] Comparative Example 3

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

[0083] (1) Crush and sieve the commercially available dried Chinese truncatum to a mesh size of 80. Take 4g of white truffle and 5g of Chinese truncatum powder and mix them with 200g of deionized water, wherein the mass ratio of white truffle to Chinese truncatum powder is 4:5; after mixing evenly, sterilize in a high-temperature sterilizer at 121°C and 0.12MPa for 30min, cool to room temperature after sterilization, and inoculate 2mL of Lactobacillus bulgaricus CGMCC No.28800 bacterial solution after cooling, and the number of viable bacteria in the bacterial solution is 10 10 CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0084] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0085] Comparative Example 4

[0086] Compared with Example 1, 4 g of white truffle and 5 g of Scutellaria baicalensis powder were added, and other parameters were the same as those in Example 1.

[0087] (1) The commercially available dried white crane ganoderma grass was crushed and sieved to a mesh size of 80. 5 g of white crane ganoderma grass powder, 4 g of white truffle and 200 g of deionized water were mixed, wherein the mass ratio of white crane ganoderma grass powder, white truffle and Chinese flag flower powder was 1:3:0.5; after being evenly mixed, sterilized in a high temperature sterilizer at 121°C and 0.12MPa for 30 min, cooled to room temperature after sterilization, and inoculated with 2 mL of bulgaricus CGMCC No.28800 bacterial solution after cooling, and the number of viable bacteria in the bacterial solution was 10 10CFU / mL, after the bacterial solution was inoculated, it was cultured in an incubator at 38°C for 20 hours, and after the culture was completed, it was sterilized in a high-temperature sterilizer at 121°C and 0.12MPa for 30 minutes, and after the sterilization was completed, it was cooled to room temperature to obtain the Baihe Lingzhi grass composite fermentation liquid.

[0088] (2) 80 g of the above-prepared Baihe Lingzhi grass composite fermentation liquid and 1.5 g of 1,3-propylene glycol were mixed evenly, and then sterilized at 90°C for 30 min, and then cooled to 75°C, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added, and mixed evenly to obtain a Baihe Lingzhi grass fermentation liquid with anti-wrinkle and firming effects.

[0089] Effect Example 1

[0090] Collagen content determination

[0091] Experimental method: This experiment verifies the anti-wrinkle and firming effects of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4 by measuring the COL-I content in human skin fibroblasts.

[0092] Human skin fibroblasts in 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. Experimental group, blank group and model group were set up. Among them, the experimental group and model group were treated with 18mJ / cm 2 The cells were irradiated with UVA for 40 minutes. After irradiation, the supernatant in the experimental group, blank group and model group was discarded, 2 mL of DMEM solution was added to the blank group and model group, and 2 mL of the test solution was added to the experimental group (the product prepared in the above embodiment or comparative example was configured into a test solution with a volume percentage of 2% using DMEM, and the test solution needed to be filtered through a 0.22 μm sterile filter membrane before use). The cells were treated for 24 hours, the supernatant was discarded, and the cells were washed 2 to 3 times with PBS. Then, the cells were treated with cell lysis solution and transferred to a centrifuge tube. Centrifuged at 10000 r / min and 4°C for 10 minutes, and the supernatant was taken to obtain the cell lysate. 20 μL of cell lysate was taken to detect the total protein content in the sample using a BCA kit; the determination of collagen content was carried out according to the instructions of the ELISA kit, and each OD value was measured at 450 nm. The standard curve equation is Y=(AD) / [1+(X / C) B ]+D, A=1.78854, B=1.23259, C=9.31934, D=-0.01557, R 2 =0.99859, the collagen expression x was calculated according to the standard curve, and corrected with the total protein content b to obtain A=x / b. The experimental results are shown in Table 1 and Figure 1 ( Figure 1 middle, #p<0.001, indicating an extremely significant decrease compared with the blank group; p>0.05, ns, indicating no significant increase compared with the model group; *p<0.05, indicating an increase compared with the model group, **p<0.01, indicating a significant increase compared with the model group, ***p<0.001, indicating an extremely significant increase compared with the model group).

[0093] Table 1

[0094]

[0095]

[0096] The above results show that the human skin fibroblasts obtained in Examples 1 to 4 of the present application have significantly higher collagen content than those in Comparative Examples 1 to 4, which shows 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 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, it will produce colored substances, and its absorbance 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. Elastase, substrate, test sample and positive control are all prepared with Tris-HCl buffer.

[0100] Tris-HCl buffer (pH 8.0)

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

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

[0103] 2.2 μmol / L elastase solution: weigh 0.5 mg of elastase powder and dissolve it in

[0104] 1mL Tris-HCl buffer (pH 8.0) for later use. Prepare 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: Dilute to the corresponding volume fraction with Tris-HCl buffer according to the test concentration.

[0107]

[0108] Elastase activity inhibition rate:

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

[0110] In the formula: T-sample group absorbance, that is, the absorbance of the solution after the sample affects the reaction of elastase and substrate; T0-sample background group absorbance; C-enzyme reaction group absorbance, that is, the absorbance of the reaction of elastase and substrate when no sample is added; D-solvent background group absorbance.

[0111] In this experiment, the elastase inhibition rate test was conducted on the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4. The results are shown in Tables 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 rate of the products prepared in Examples 1 to 4 of the present application is significantly higher than that of Comparative Examples 1 to 4. It can be seen 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 dye 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 generated can indirectly reflect the activity of mitochondria in cells.

[0118] 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.

[0119] 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 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 to 4 is much higher than that 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 an ideal effect of promoting cell metabolism, and the fermentation temperature and strains have a great influence on the cell metabolism performance of the final product.

[0123] 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.

[0124] 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 a fermented white grape liquor having anti-wrinkle and firming effects, characterized in that: The method comprises the following steps: mixing white crane ganoderma, white truffle and Chinese flag flower with water to prepare a fermentation substrate, inoculating lactobacillus into the fermentation substrate, fermenting and culturing, and sterilizing; Among them, the white crane ganoderma, white truffle and Chinese flagella are fermented with water, and the mass percentage of the white crane ganoderma, the white truffle and the Chinese flagella is 1: (1-10): (0.1-2).

2. The method for preparing the fermented white grape extract having anti-wrinkle and firming effects according to claim 1, characterized in that: The mass percentage of the White Crane Ganoderma Lucidum, the white truffle, and the Chinese Flagstone Flower is 1:(1-4):(0.5-2). Preferably, the mass percentage of the White Crane Ganoderma Lucidum, the white truffle, and the Chinese Flagstone Flower is 1:3:0.

5.

3. The method for preparing the fermented white grape extract with anti-wrinkle and firming effects according to claim 1, characterized in that: The fermentation method satisfies at least one of the following conditions: The white crane ganoderma lucidum grass is a white crane ganoderma lucidum grass powder that has been crushed and sieved. Preferably, the particle size of the white crane ganoderma grass powder after sieving is 50 to 100 meshes, preferably 80 meshes; The Chinese flag flower is a crushed and sieved Chinese flag flower powder, and the sieved Chinese flag flower has a particle size of 50 to 100 meshes, preferably 80 meshes; The Chinese flag flower is crushed and sieved Chinese flag flower powder. Preferably, the particle size of the sieved Chinese flag flower powder is 50-100 meshes, preferably 80 meshes; The water is deionized water.

4. The method for preparing the fermented white grape extract having anti-wrinkle and firming effects according to claim 1, characterized in that: The lactobacillus includes Lactobacillus bulgaricus; Preferably, the lactobacillus is inoculated in the form of a lactobacillus bacterial liquid; Preferably, the Lactobacillus bulgaricus is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No.28800; Preferably, when the Lactobacillus bulgaricus is used, the concentration of the Lactobacillus bulgaricus in the Lactobacillus bulgaricus bacterial solution may be 10 8 ~10 12 CFU / mL, preferably 10 10 CFU / mL.

5. The method for preparing the fermented white grape extract with anti-wrinkle and firming effects according to claim 1, characterized in that: The fermentation substrate further includes a sterilization operation before use; Preferably, the sterilization is a high-temperature sterilization method; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110-125°C, and more preferably 115-121°C; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15-35 minutes, and more preferably 20-35 minutes, for example 30 minutes; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.14Mpa, and more preferably 0.1-0.13MPa, for example 0.12MPa.

6. The method for preparing the fermented white grape extract having anti-wrinkle and firming effects according to claim 1, characterized in that: The fermentation method satisfies at least one of the following conditions: The sterilization further includes a cooling operation, preferably, the cooling is cooling to room temperature; The fermentation temperature is 37-43°C, preferably 38°C; The fermentation culture time is 15 to 24 hours, preferably 20 hours; The fermentation culture is aerobic fermentation, and preferably, the aerobic fermentation is static fermentation.

7. The method for preparing the fermented white grape extract having anti-wrinkle and firming effects according to claim 1, characterized in that: The mixing operation further includes secondary sterilization and / or adding preservatives; Preferably, 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 110-125°C, more preferably 115-121°C; the time of the secondary sterilization is 15-35min, more preferably 20-35min, for example 30min; the pressure of the secondary sterilization is 0.1-0.14Mpa, more preferably 0.1-0.1 3MPa, for example 0.12MPa; In the process of adding the preservative, the preservative includes at least one of 1,3-propylene glycol, p-hydroxyacetophenone and 1,3-propylene glycol; when the preservative includes the 1,3-propylene glycol, the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the 1,3-propylene glycol in the mixed material may be 1% to 2%; the mass percentage of the 1,2-hexanediol in the mixed material may be 0.3% to 2%; the mass percentage of the p-hydroxyacetophenone in the mixed material may be 0.3% to 2%; preferably, the mass percentage of the 1,3-propylene glycol in the mixed material is 1.5%, the mass percentage of the p-hydroxyacetophenone in the mixed material is 0.5%, and the mass percentage of the 1,2-hexanediol in the mixed material is 0.5%; The process of adding the preservative can be carried out under mixing conditions according to the conventional art, and the mixing temperature is preferably 60-80°C, more preferably 70-80°C, for example 75°C.

8. A fermented white glutinous rice liquid with anti-wrinkle and firming effects, which is prepared by the method for preparing the fermented white glutinous rice liquid with anti-wrinkle and firming effects described in any one of 1 to 7.

9. A use of the fermented liquor of white grape with anti-wrinkle and firming effects as claimed in claim 8 directly as a product, as an additive or as a base in the preparation of a skin external preparation; Preferably, the fermented liquor of white grape with anti-wrinkle and firming effects is used as the anti-wrinkle active ingredient and / or firming active ingredient in the skin external preparation; More preferably, the anti-wrinkle active ingredient is an ingredient that increases collagen content and / or inhibits antioxidant elastase activity; More preferably, the firming active ingredient is an ingredient that increases mitochondrial activity.

10. The present application also provides a skin external preparation, which comprises the white grape fermentation liquid with anti-wrinkle and firming effects as claimed in claim 8; Preferably, the skin topical preparation further comprises active ingredients conventionally used in the art, which may generally include any one or more of whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients; Preferably, the skin external preparation may include a facial mask, essence or toner according to the conventional art; Preferably, the mass percentage of the fermented liquor of white grape with anti-wrinkle and firming effects in the skin external preparation may be 5% to 99%, preferably 60% to 99%.

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