Preparation method and application of three-headed fruit fermentation liquor capable of regulating cell rhythm

Abstract

The invention discloses a preparation method and application of a three-thorn fruit fermentation broth capable of regulating cell rhythm, and the preparation method comprises the following steps: (1) adding nitraria fruit, yellow thorn fruit and prinsepia utilis royle into a fermentation enzymatic hydrolysate, and carrying out enzymolysis to prepare a fermentation substrate; and (2) inoculating schizophyllum commune into the fermentation substrate for fermentation, and sterilizing to obtain the schizophyllum commune fermentation substrate, wherein a preparation method of the fermented enzymatic hydrolysate comprises the following steps: adding water into a substrate, sterilizing to obtain a culture medium, inoculating, performing aerobic fermentation, and centrifuging, thereby obtaining the fermented enzymatic hydrolysate. The fermentation enzymatic hydrolysate comprises agaricus blazei fermentation enzymatic hydrolysate or saccharomyces cerevisiae fermentation enzymatic hydrolysate. According to the method, saccharomyces cerevisiae or agaricus blazei fermentation enzymatic hydrolysate is firstly used for enzymolysis extraction, wall breaking is conducted on plants preliminarily, active ingredients in the plants are released, then schizophyllum commune is further fermented, further release and conversion are achieved, ideal effects of oxidation resistance, moisturizing, soothing, tightening, whitening and the like are achieved by adjusting cell rhythm, fermentation conditions are mild, and the method is suitable for industrial production. Good application prospects are realized.

Description

Technical Field

[0001] The present application belongs to the technical field of cosmetic preparation, and in particular relates to a method for preparing a fermented liquid of Tripterygium wilfordii having the function of regulating cell rhythm and an application thereof. Background Art

[0002] In recent years, with the rapid development of the economy and the continuous improvement of the economic level, people's living standards have been greatly improved, and the pace of life has also accelerated significantly. Modern fast-paced life and high-intensity work force young people to bear huge work pressure, often stay up late, have irregular diets, and do not get enough rest. Therefore, it is very easy to cause dry and dehydrated skin, dull complexion, etc. Therefore, bad lifestyles and eating habits, mental stress and work pressure are extremely harmful to the skin.

[0003] White thorn is mostly distributed in arid areas. Its fruit has high nutritional and medicinal value. It has the effects of strengthening the stomach and spleen, nourishing yin and moistening the lungs, promoting blood circulation and digestion. It is mainly used by the people to treat spleen and stomach weakness, indigestion, neurasthenia and other symptoms.

[0004] Yellow thorn, Berberis dasystachya Maxim., Berberaceae, is a traditional wild berry used as medicine and food by some ethnic minorities, just like white thorn and sea buckthorn (black thorn). They are collectively called "Three Thorns of Qinghai". The Handbook of Commonly Used Chinese Herbal Medicines in Qinghai has a detailed record of yellow thorn bark (i.e. root bark): yellow thorn bark is the stem bark of the dicotyledonous plant medicine Berberis dasystachya Maxim., and the medical function of its fruit is also briefly described, which has the function of treating abdominal pain, indigestion, abdominal distension, dysentery, etc.

[0005] Qingci is a perennial shrub of the genus Flatwood of the Rosaceae family, also known as Qiangziguo. It mainly grows on hillsides and wastelands at an altitude of 1,800 to 3,200 meters in Yunnan, Guizhou and Sichuan. According to the "Southern Yunnan Materia Medica", Qingci tip tastes bitter and cold in nature. It mainly attacks all painful carbuncles and poisonous sores. If there is a thick one, it will come out, and if there is no thick one, it will disappear immediately and disperse tuberculosis. Experiments have confirmed that Qingci fruit is rich in vitamins A, D, E, K and a variety of fatty acids that are very important to human health, and is rich in calcium, phosphorus, potassium, sodium, zinc, iron, magnesium, aluminum, sulfur, copper, barium, nickel and other trace elements. It has obvious effects of clearing heat and detoxifying, promoting blood circulation and reducing swelling. In addition, the fatty acid structure contained in Qingci fruit is very close to the lipids in human skin, so it is easily absorbed by human skin.

[0006] In addition, some studies have used organic solvents to extract the active ingredients contained in the thorn fruit. However, during the use of organic solvents, it is very easy to damage the active ingredients, resulting in a decrease in efficacy. In addition, organic solvents are easy to remain during use, resulting in side effects of the product and irritation to the skin. The enzymes used in traditional enzymatic extraction methods are also obtained through the process of fermentation and purification of proteases. This process is relatively cumbersome and complicated, and also faces the risk of reduced protease activity.

[0007] At present, research on food toxicology, food health functions and pharmacology has been conducted on white thorn, yellow thorn and green thorn, but there is a lack of relevant reports in the field of cosmetics. In addition, organic solvents can easily damage active ingredients and cause side effects on the skin.

[0008] Therefore, there is an urgent need for a safe, residue-free preparation method that effectively utilizes the active ingredients in white thorn, yellow thorn and green thorn, applies them to cosmetics, and exerts synergistic effects. Summary of the invention

[0009] The technical problem to be solved by the present application is to overcome the defects that the existing research lacks a method for effectively combining the active ingredients of white thorn, yellow thorn and green thorn to exert synergistic effects, and most of the research on white thorn, yellow thorn and green thorn is concentrated in the fields of food and pharmacy, lacks a method for using them in the field of cosmetics, and the organic solvent process of extracting the active ingredients of the thorn fruit is easy to damage the active ingredients, and it is easy to leave residues during use, resulting in side effects and irritation of the product, and to provide a method for preparing a fermented liquid of three-thorn fruit with the function of regulating cell rhythm and its application.

[0010] This application adopts the following technical solutions to solve the above technical problems:

[0011] The present application provides a method for preparing a fermented liquid of Tripterygium wilfordii having the function of regulating cell rhythm, which comprises the following steps:

[0012] (1) adding white thorn fruit, yellow thorn fruit and green thorn fruit to a fermentation enzymatic hydrolyzate, and performing enzymatic hydrolysis to prepare a fermentation substrate;

[0013] (2) inoculating Schizophyllum corytatum into the fermentation substrate for fermentation, followed by sterilization to obtain the product;

[0014] The preparation method of the fermentation hydrolysate comprises: adding water to the substrate and sterilizing it to obtain a culture medium, aerobically fermenting it after inoculation, and centrifuging it; the fermentation hydrolysate comprises Agaricus blazei fermentation hydrolysate or Saccharomyces cerevisiae fermentation hydrolysate.

[0015] In some embodiments, in step (1), the Nitraria tangutorum fruit further comprises an operation of crushing the Nitraria tangutorum fruit to obtain Nitraria tangutorum fruit powder before use.

[0016] In some embodiments, in step (1), the yellow thorn fruit further comprises an operation of crushing to obtain yellow thorn fruit powder before use.

[0017] In some embodiments, in step (1), the prickle fruit further comprises the step of crushing the prickle fruit to obtain prickle fruit powder before use.

[0018] In some embodiments, in step (1), the mass ratio of the white thorn fruit, the yellow thorn fruit and the green thorn fruit is 1: (0.5-2): (0.5-2).

[0019] In some embodiments, in step (1), the mass ratio of the sum of the mass of the Nitraria tangutorum, the Psoralea tangutorum and the Psoralea tangutorum to the mass of the fermentation hydrolyzate is 1:(200-500).

[0020] In some embodiments, in step (1), the Agaricus blazei mushroom fermentation hydrolyzate is obtained by fermenting Agaricus blazei mushroom;

[0021] In some embodiments, in step (1), the Saccharomyces cerevisiae fermentation hydrolyzate is obtained by fermenting Saccharomyces cerevisiae;

[0022] In some embodiments, in step (1), the fermentation enzymatic solution is fermented using rice or potato as a substrate. The preservation date of the Agaricus blazei is September 13, 2021, the preservation number is CGMCC No. 23250, the classification name is Agaricus blazei, the preservation unit name is China Microbiological Culture Collection Center (CGMCC), the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, zip code 100101.

[0023] The Brazilian mushroom is added in the form of Brazilian mushroom liquid. The preparation method comprises: taking out the frozen liquid, thawing it at room temperature, taking 300 μL of the frozen liquid and inoculating it into 300 mL of PDA liquid culture medium, then culturing it at 28°C and 180 rpm for 3 days, then coating it on a PDA solid plate, placing it in a 28°C constant temperature incubator and culturing it for 2 days, then picking a single colony and placing it in 300 ml of PDA liquid culture medium, culturing it at 28°C and 180 rpm for 3 days, and using it as a seed liquid for subsequent use.

[0024] Among them, the brewing yeast includes brewing yeast YW-1; the preservation date of the brewing yeast YW-1 is March 27, 2019, the preservation number is CGMCC No.17452, the classification name is brewing yeast (Saccharomyces cerevisiae), the name of the preservation unit is the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC for short), and the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101.

[0025] Among them, the brewer's yeast is added in the form of a brewer's yeast liquid. When the brewer's yeast includes the brewer's yeast YW-1, the preparation method includes: taking out the frozen liquid, thawing it at room temperature, taking 300 μL of the frozen liquid and inoculating it in 300 mL of PDA liquid culture medium, then culturing it at 28°C and 180 rpm for 2 days, then spreading it on a PDA solid plate, placing it in a constant temperature incubator at 28°C and culturing it for 2 days, then picking a single colony in 300 ml of PDA liquid culture medium, culturing it at 28°C and 180 rpm for 2 days, and using it as a seed liquid for subsequent use.

[0026] In some embodiments, in step (1),

[0027] In some embodiments, in step (1), the mass ratio of the substrate to the water is 1:(20-50), preferably 1:20.

[0028] In some embodiments, in step (1), the sterilization is conventional in the art, generally high temperature sterilization. The sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C. The high temperature sterilization time can be a conventional time for such operations in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0029] In some embodiments, in step (1), the number of viable bacteria inoculated is 1 to 2×10 7 CFU / mL.

[0030] In some embodiments, in step (1), the sterilization further comprises a cooling operation, wherein the cooling is cooling to room temperature.

[0031] In some embodiments, in step (1), the conditions and methods of aerobic fermentation are conventional in the art, and aerobic fermentation is generally performed in a constant temperature oscillating box. When the constant temperature oscillating box is used for aerobic fermentation, the rotation speed of the constant temperature oscillating box is 150-200 rpm, preferably 180 rpm.

[0032] In some embodiments, in step (1), the temperature of the aerobic fermentation is 25-32°C, preferably 28°C.

[0033] In some embodiments, in step (1), the aerobic fermentation time is 36 to 60 hours, preferably 48 hours.

[0034] In some embodiments, in step (1), the process further includes centrifuging and removing mycelium after aerobic fermentation.

[0035] In some embodiments, in step (1), the conditions and methods of the enzymatic hydrolysis are conventional in the art, and the enzymatic hydrolysis is generally performed in a constant temperature shaking box.

[0036] Wherein, when the constant temperature oscillating box is used for the enzymatic hydrolysis, the rotation speed of the constant temperature oscillating box is 150-200 rpm, preferably 180 rpm.

[0037] Wherein, when the constant temperature oscillating box is used for the enzymolysis, the enzymolysis time is 1 to 2 hours, preferably 2 hours.

[0038] Wherein, when the enzymolysis is performed using the constant temperature oscillating box, the temperature of the enzymolysis is 25-30°C, preferably 28°C.

[0039] Wherein, when the enzymolysis is performed using the constant temperature oscillating box, the rotation speed of the enzymolysis is 150-200 rpm, preferably 180 rpm.

[0040] The enzymatic hydrolysis time is 1 to 2 hours, preferably 2 hours.

[0041] In some embodiments, in step (1), the enzymatic hydrolysis further includes sterilization.

[0042] The sterilization is conventional in the art, generally high temperature sterilization. The temperature of the high temperature sterilization can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C.

[0043] The high temperature sterilization time may be the conventional time for such operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0044] In some embodiments, in step (2), the Schizophyllum includes Schizophyllum commune Wbsh-01, the preservation date is March 9, 2023, the preservation number is CGMCC No.40388, the classification name is Schizophyllum commune, the preservation unit name is China National Microbiological Culture Collection Center (CGMCC for short), the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0045] In some embodiments, in step (2), the Schizophyllum sp. is added in the form of a Schizophyllum sp. liquid.

[0046] Among them, the preparation method of the schizophyllum bacterial liquid includes: taking out the frozen bacterial liquid, thawing it at room temperature, taking 300 μL of the frozen liquid and inoculating it in 300 mL of PDA liquid culture medium, then culturing it at 28°C and 180 rpm for 2 days, then spreading it on a PDA solid plate, placing it in a 28°C constant temperature incubator and culturing it for 2 days, then picking a single colony in 300 ml of PDA liquid culture medium, culturing it at 28°C and 180 rpm for 3 days, and using it as a seed bacterial liquid for subsequent use.

[0047] In some embodiments, in step (2), the fermentation is aerobic fermentation, and the conditions and methods of the aerobic fermentation are conventional in the art, and the aerobic fermentation is generally carried out in a constant temperature shaking box.

[0048] Wherein, the rotation speed of the constant temperature oscillation box is 150-200 rpm, preferably 180 rpm.

[0049] Wherein, the temperature of the aerobic fermentation is 25-30°C, preferably 28°C.

[0050] Wherein, the aerobic fermentation time is 2 to 3 days, preferably 3 days.

[0051] In some embodiments, in step (2), the viable count of the Schizophyllum is 1 to 2×10 8 CFU / mL, preferably 1×10 8 CFU / mL.

[0052] In some embodiments, in step (2), the sterilization is conventional in the art, generally high temperature sterilization. The sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C. The high temperature sterilization time can be a conventional time for such operations in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0053] In some embodiments, in step (2), the sterilization further includes centrifugal filtration, supernatant collection or sterilization operations.

[0054] Wherein, the particle size of the filtering membrane is 10 to 15 μm.

[0055] The sterilization is conventional in the art, generally high temperature sterilization. The temperature of the high temperature sterilization can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C.

[0056] The high temperature sterilization time may be the conventional time for such operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0057] The present application also provides a trifoliate thorn fruit fermentation liquid having the function of regulating cell rhythm, which is prepared by the preparation method of the trifoliate thorn fruit fermentation liquid having the function of regulating cell rhythm as described above.

[0058] The present application also provides a use of the above-mentioned fermented liquid of Tripterygium wilfordii having the function of regulating cell rhythm directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0059] In some embodiments, the fermented liquid of the thorn fruit having the function of regulating cell rhythm is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, cell rhythm regulating active ingredient, and whitening active ingredient of the skin external preparation;

[0060] Wherein, the antioxidant component is an antioxidant active component having the ability to scavenge hydroxyl free radicals or scavenge DPPH free radicals.

[0061] Wherein, the active ingredient for regulating cell rhythm is an active ingredient for regulating cell rhythm that increases the expression amount of CLOCK gene.

[0062] Wherein, the moisturizing active ingredient is a moisturizing active ingredient that can increase the expression of the HAS gene.

[0063] Wherein, the soothing active ingredient is a soothing active ingredient that can reduce the expression amount of TPRV1 gene.

[0064] Wherein, the firming active ingredient is a firming active ingredient that can increase the expression of ELN gene.

[0065] Wherein, the whitening active ingredient is a whitening active ingredient that has the ability to inhibit tyrosinase activity.

[0066] The present application also provides a skin external preparation, which includes the above-mentioned fermented liquid of the fruit of Tripterygium wilfordii having the function of regulating cell rhythm.

[0067] In some embodiments, the skin topical preparation may further include active ingredients conventionally used in the art, generally including at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0068] In some embodiments, the skin topical agent may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.

[0069] In some embodiments, the mass percentage of the fermented liquid of Tripterygium wilfordii having the function of regulating cell rhythm in the external skin preparation may be 5% to 99%, preferably 60% to 99%.

[0070] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0071] The reagents and raw materials used in this application are commercially available.

[0072] The positive progressive effect of the present application is that the present application utilizes brewer's yeast fermentation hydrolysate or Agaricus blazei fermentation hydrolysate to perform enzymatic extraction on the fruits of white thorn, yellow thorn and green thorn in steps, followed by fermentation; thereby effectively decomposing the active substances, combining with Schizophyllum to further release the fermentation substrate, and achieving the desired antioxidant, moisturizing, soothing, firming, whitening and other effects by regulating cell rhythms, and the fermentation conditions are mild, with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0074] in:

[0075] Figure 1 It is a comparison chart of hydroxyl radical scavenging ability in the embodiment and the comparative example;

[0076] Figure 2 It is a comparison chart of DPPH free radical scavenging ability in the embodiment and the comparative example;

[0077] Figure 3 The figure is a comparison chart of the relative expression of CLOCK gene in the embodiment and the comparative example;

[0078] Figure 4 It is a comparison diagram of the relative expression of HAS gene in the embodiment and the comparative example;

[0079] Figure 5 It is a comparison chart of the relative expression of TRPV1 gene in the embodiment and the comparative example;

[0080] Figure 6 It is a comparison chart of the relative expression of ELN gene in the embodiment and the comparative example;

[0081] Figure 7 It is a comparison chart of the tyrosinase activity inhibition ability in the embodiment and the comparative example. DETAILED DESCRIPTION

[0082] 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.

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0084] The green thorns in the following examples and comparative examples are from Dehou Town, Wenshan City, Yunnan Province.

[0085] The Xanthoceras spicata in the following examples and comparative examples was from Zhang County, Gansu Province.

[0086] The Nitraria tangutorum in the following examples and comparative examples was from Minqin County, Gansu Province.

[0087] The preservation date of the Brazilian mushroom in the following embodiments and comparative examples is September 13, 2021, the preservation number is CGMCC No. 23250, the classification name is Brazilian mushroom (Agaricus blazei), the name of the preservation unit is the General Microbiology Center of the China Microorganism Culture Collection Administration (referred to as CGMCC), and the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0088] The deposit date of the brewer's yeast YW-1 in the following examples and comparative examples is March 27, 2019, the deposit number is CGMCC No.17452, the classification name is Saccharomyces cerevisiae, the name of the depository is the General Microbiological Center of the China Microbiological Culture Collection Administration (referred to as CGMCC), and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0089] The schizophyllum in the following embodiments and comparative examples include schizophyllum Wbsh-01, the deposit date is March 9, 2023, the deposit number is CGMCC No.40388, the classification name is Schizophyllum commune, the name of the depository is the General Microbiological Center of the China National Committee for the Preservation of Microorganisms (referred to as CGMCC), and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101. The curvature of Lactobacillus in the following comparative example was deposited on August 30, 2021, the deposit number is CGMCC No.23317, the classification name is Lactobacillus curvatus, the name of the depository is the General Microbiological Center of the China National Committee for the Preservation of Microorganisms (referred to as CGMCC), the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101.

[0090] The preservation date of the capsule-coated yeast in the following comparative example is June 7, 2021, the preservation number is CGMCC No. 22671, the classification name is Saccharomycopsis fibuligera, the name of the preservation unit is the General Microbiology Center of China Culture Collection Administration (CGMCC for short), and the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0091] Example 1

[0092] (1) Cut the potatoes into pieces, weigh 15 g, add water to make pulp, and finally add water to 300 g, sterilize at 121°C for 30 min, and cool to 25°C to prepare a culture medium; then inoculate 3 g of Saccharomyces cerevisiae (YW-1) seed culture liquid, wherein the number of live bacteria of Saccharomyces cerevisiae inoculated in the culture medium is 10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 48 h, followed by centrifugation to remove the precipitate to obtain the potato Saccharomyces cerevisiae fermentation hydrolysate;

[0093] (2) Then, 0.6 g of white thorn fruit powder, 0.6 g of yellow thorn fruit and 0.6 g of green thorn fruit were weighed and added to 300 g of potato saccharomyces cerevisiae fermentation hydrolyzate, and the fermentation was performed at 28°C and 180 rpm for 2 h, followed by sterilization at 121°C for 30 min to obtain a fermentation substrate, and 1.5 g of Schizophyllum bacterial solution was added, wherein the number of live bacteria of Schizophyllum in the fermentation substrate was 10 8 CFU / mL, and then aerobically fermented at 28°C and 180rpm for 3 days. After the fermentation, the mycelium was removed by centrifugation, and then filtered through a 15μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30min.

[0094] Example 2

[0095] Compared with Example 1, the only difference is that in step (1), potatoes in the culture medium are replaced by rice, and other condition parameters are consistent with Example 1.

[0096] The preparation method of rice brewer's yeast fermentation hydrolyzate comprises: weighing 15g of rice, adding it into 300g of water, sterilizing it at 121°C for 30min, cooling it to 25°C, and preparing a culture medium; then inoculating 3g of brewer's yeast (YW-1) seed culture liquid, wherein the number of live yeast cells in the culture medium is 10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 48 h, followed by centrifugation to remove the precipitate to obtain rice brewer's yeast fermentation hydrolysate.

[0097] Example 3

[0098] Compared with Example 1, the only difference is that in step (1), the saccharomyces cerevisiae (YW-1) in the culture medium is replaced by Agaricus blazei, and the other condition parameters are the same as those in Example 1.

[0099] The preparation method of Agaricus blazei mushroom liquid comprises: taking out the frozen mushroom liquid of Agaricus blazei mushroom, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL of PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 3 days, then coating it on a PDA solid plate, placing it in a constant temperature incubator at 28° C. and culturing it for 2 days, then picking out a single colony and placing it in 300 ml of PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 3 days, and using it as Agaricus blazei mushroom seed liquid for subsequent use.

[0100] (1) Cut the potatoes into pieces, weigh 15 g, add water to make pulp, and finally add water to 300 g, sterilize at 121°C for 30 min, and cool to 25°C to prepare a culture medium; then inoculate 3 g of Agaricus blazei seed liquid, wherein the number of viable Agaricus blazei in the culture medium is 10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 48 h, followed by centrifugation to remove the precipitate to obtain the Agaricus blazei fermentation hydrolysate.

[0101] Example 4

[0102] Compared with Example 1, the only difference is that the fermentation temperature of the enzymatic hydrolyzate fermented by potato saccharomyces cerevisiae (YW-1) in step (1) is 23° C., and other condition parameters are consistent with those in Example 1. The specific operation is as follows:

[0103] (1) Cut the potatoes into pieces, weigh 15 g, add water to make pulp, and finally add water to 300 g, sterilize at 121° C. for 30 min, and cool to 25° C. to prepare a culture medium; then inoculate 3 g of brewer's yeast seed liquid, wherein the number of live yeast cells inoculated in the culture medium is 10 7 CFU / mL, and then aerobically fermented at 23°C and 180 rpm for 48 h, followed by centrifugation to remove the precipitate to obtain the potato yeast fermentation hydrolysate.

[0104] Example 5

[0105] Compared with Example 1, the only difference is that the fermentation time of the potato yeast fermentation hydrolyzate in step (1) is 36 hours, and the other condition parameters are consistent with Example 1. The specific operation is as follows:

[0106] (1) Cut the potatoes into pieces, weigh 15 g, add water to make pulp, and finally add water to 300 g, sterilize at 121°C for 30 min, and cool to 25°C to prepare a culture medium; then inoculate 3 g of Saccharomyces cerevisiae seed liquid, wherein the number of live Saccharomyces cerevisiae inoculated in the culture medium is 10 7 CFU / mL, and aerobic fermentation was carried out at 23°C and 180 rpm for 36 h, followed by centrifugation to remove the precipitate and obtain the potato yeast fermentation hydrolysate.

[0107] Comparative Example 1

[0108] Compared with Example 1, the only difference is that in step (1), the cerevisiae (YW-1) bacteria inoculated in the culture medium is replaced with Saccharomyces cerevisiae, and the other condition parameters are consistent with those in Example 1. The specific operation is as follows:

[0109] The preparation method of the button capsule film-coated yeast liquid includes: taking out the button capsule film-coated yeast frozen liquid, thawing it at room temperature, taking 300 μL of the frozen liquid and inoculating it in 300 mL of PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 2 days, then spreading it on a PDA solid plate, placing it in a constant temperature incubator at 28° C. and culturing it for 2 days, then picking a single colony in 300 ml of PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 3 days, and using it as a seed liquid for subsequent use.

[0110] (1) Cut the potato into pieces, weigh 15 g, add water to make pulp, and finally add water to 300 g, sterilize at 121° C. for 30 min, and cool to 25° C. to prepare a culture medium; then inoculate 3 g of the seed culture liquid of the capsule-covered yeast, wherein the number of viable bacteria of the capsule-covered yeast inoculated in the culture medium is 10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 48 hours, followed by centrifugation to remove the precipitate to obtain the potato capsule-coated yeast fermentation hydrolysate.

[0111] Comparative Example 2

[0112] Compared with Example 1, the only difference is that in step (1), the saccharomyces cerevisiae (YW-1) in the culture medium is replaced by Lactobacillus curvatus, and other condition parameters are consistent with those in Example 1. The specific operations are as follows:

[0113] The preparation method of the Lactobacillus curvatus bacterial liquid comprises: taking out the frozen bacterial liquid of Lactobacillus curvatus, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it in 300 mL of PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 2 days, then coating it on a PDA solid plate, placing it in a constant temperature incubator at 28° C. and culturing it for 2 days, then picking out a single colony and placing it in 300 ml of PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 3 days, and using it as a seed bacterial liquid for subsequent use.

[0114] (1) Cut the potato into pieces, weigh 15 g, add water to make pulp, and finally add water to 300 g, sterilize at 121° C. for 30 min, and cool to 25° C. to prepare a culture medium; then inoculate 3 g of Lactobacillus curvatus seed liquid, wherein the number of viable Lactobacillus curvatus inoculated in the culture medium is 10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 48 h, followed by centrifugation to remove the precipitate to obtain the fermentation hydrolysate of Lactobacillus curvatus.

[0115] Effect Example 1 Hydroxyl Radical Scavenging Rate

[0116] The Fenton reaction is used to generate hydroxyl radicals (·OH), and salicylic acid is added to the reaction system. ·OH reacts with salicylic acid to generate a colored compound 2,3-dihydroxybenzoic acid with characteristic absorption at 510nm. The fixed reaction time method is used to measure the absorbance of the reaction solution containing the analyte at 510nm, and compared with the blank solution to determine the scavenging effect of the analyte on ·OH.

[0117] The liquid to be tested is the product obtained in the above-mentioned embodiment and comparative example;

[0118] Add reagents to the test tube according to Table 1, and add 9mmol / L FeSO4, 9mmol / L salicylic acid ethanol solution, test solution, appropriate amount of deionized water and 8.8mmol / L H2O2 solution in sequence. Shake well, heat in a 37℃ water bath for 15 minutes, then take out and measure the absorbance A0 of the blank control and the absorbance A0 of the sample group. x and the background absorbance of the solution without H2O2 x0 When determining A0, the reference solution was a system without hydrogen peroxide. Three parallel experiments were performed in each group, and the absorbance values ​​were tested and the average values ​​were taken. The results are shown in Table 1. The hydroxyl radical scavenging rate of each group was calculated according to the following formula. The results are shown in Tables 2 and Figure 1 .

[0119] Hydroxyl radical scavenging rate = (A0-(A x -A x0 )) / A0×100%.

[0120] Table 1

[0121]

[0122] Table 2

[0123] serial number Hydroxyl free radical scavenging rate (%) Example 1 78.52 Example 2 66.69 Example 3 67.07 Example 4 64.18 Example 5 69.67 Comparative Example 1 51.65 Comparative Example 2 43.15

[0124] From the results, it can be seen that the hydroxyl radical scavenging rates of the products prepared in Examples 1 to 5 are much higher than those of the products prepared in Comparative Examples 1 to 2. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation temperature, time and strains selected for the enzymatic hydrolysate have a great influence on the antioxidant properties of the final product.

[0125] Effect Example 2 DPPH free radical scavenging rate

[0126] DPPH is a stable nitrogen-centered free radical that appears purple in organic solvents and has a maximum absorption at 517nm. In the DPPH experiment, when antioxidants are added to the DPPH solution, they can react with the DPPH free radical and capture the unpaired electrons of the free radical, causing the DPPH free radical to lose its free radical properties and change from purple to colorless. By comparing the changes in absorbance before and after the reaction, the antioxidant capacity of the sample can be quantitatively evaluated. This method is simple and rapid, and is widely used in industries such as food, medicine, and cosmetics to evaluate the antioxidant properties of products. Therefore, the scavenging effect of the sample on the DPPH free radical can be evaluated by measuring the changes in absorbance.

[0127] The specific experimental steps of the DPPH free radical scavenging experiment are as follows (the test solution is a test solution diluted with water in advance to a volume concentration of 25%):

[0128] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution and mix well (tube A1);

[0129] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution and mix well (tube A2);

[0130] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0131] (4) After the reaction was protected from light for 30 minutes, the absorbance of tubes A1, A2, and A3 was measured at 517 nm. The clearance rate was calculated as follows: clearance rate = [(A2 + A3) - A1] / A2 × 100%.

[0132] This experiment tested the DPPH free radical scavenging experiment on the embodiments and comparative examples. The results are shown in Tables 3 and Figure 2 .

[0133] Table 3

[0134]

[0135]

[0136] From the results, it can be seen that the DPPH free radical scavenging rates of the products prepared in Examples 1 to 5 are much higher than those of the products prepared in Comparative Examples 1 to 2. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation temperature, time and strains selected for the enzymatic hydrolysate have a great influence on the antioxidant properties of the final product.

[0137] Effect Example 3 Relative expression of mRNA-CLOCK, HAS, TRPV1, ELN genes

[0138] 4% sample was used to treat cells, and then TriQuick total RNA extraction reagent was used to extract RNA from HaCaT cells according to the instructions. FastQuant cDNA kit was used for cDNA first-strand synthesis reaction. Fast Super qPCR Master Mix and qRT-PCR were used to detect the cDNA obtained by further reverse transcription. Finally, the relative expression was calculated. The calculation formula is as follows: Expression ratio = 2^(-△△Ct) Where: △△Ct = △Ct of sample group - △Ct of blank group; △Ct = Ct value of gene to be tested - Ct value of reference gene. The specific primer sequences are shown in Table 4 below, and the test results are shown in Tables 5 to 8 and Figures 3 to 6 .

[0139] Table 4 qRT-PCR primer sequence list

[0140]

[0141] Table 5

[0142]

[0143]

[0144] Biological rhythms are essential for maintaining normal metabolic activities such as metabolism. The CLOCK gene expresses the core factor of biological rhythms, Clock, which plays an important role in regulating biological rhythms. By testing the effect of samples on CLOCK gene expression, we can evaluate whether the samples regulate the metabolic activities of cells through biological rhythms.

[0145] From Table 5 and Figure 3 The results show that the relative mRNA expression of CLOCK in Examples 1 to 5 is significantly higher than that in Comparative Examples 1 to 2. It can be seen that the products prepared in the examples of the present application have an ideal effect of regulating cell rhythms, and the fermentation temperature, time and bacterial species selected for the enzymatic hydrolyzate have a great influence on the effect of the final product in regulating cell rhythms.

[0146] Table 6

[0147]

[0148]

[0149] HAS is a gene related to the activity of hyaluronan synthase. A high level of HAS expression can promote the expression of hyaluronan synthase, increase the hyaluronic acid content in cells, and thus play a moisturizing role. The effect of testing the sample on HAS expression can further evaluate the moisturizing effect of the sample on the skin.

[0150] From Table 6 and Figure 4 The results show that the relative mRNA expression of HAS in Examples 1 to 5 is significantly higher than that in Comparative Examples 1 to 2. It can be seen that the products prepared in the examples of the present application have ideal anti-aging effects, and the fermentation temperature, time and strain selected for the enzymatic hydrolyzate have a great influence on the anti-aging effects of the final product.

[0151] Table 7

[0152] serial number Relative expression level of TPRV1 gene (%) Blank group 1.00 Model Group 1.39 VC 0.83 Example 1 0.35 Example 2 0.49 Example 3 0.58 Example 4 0.55 Example 5 0.57 Comparative Example 1 0.69 Comparative Example 2 0.80

[0153] TRPV1 is related to the human skin's perception of external stimuli. High expression of TRPV1 makes the skin more susceptible to external stimuli, causing itching, redness and pain. This indicator is often used to evaluate whether the sample has a soothing effect.

[0154] From Table 7 and Figure 5The results show that after the damaged cells were treated with the products prepared in Examples 1 to 5, the intracellular TRPV1 content was significantly reduced, which shows that the products prepared in Examples 1 to 5 have a good soothing effect. Compared with Comparative Examples 1 to 2, the expression level of TRPV1 in Examples 1 to 5 is lower, which shows that the products prepared in the examples of the present application have an ideal soothing effect, and the fermentation culture temperature, time and the selected strain of the enzymatic hydrolyzate have a great influence on the soothing effect of the final product.

[0155] Table 8

[0156] serial number ELN gene relative expression level (%) Blank group 1.00 Model Group 0.66 VC 1.07 Example 1 1.51 Example 2 1.53 Example 3 1.18 Example 4 1.21 Example 5 1.40 Comparative Example 1 0.92 Comparative Example 2 0.90

[0157] Elastin is an important component of the skin. It only accounts for 5% of the skin, but it is the key to stimulating the production of collagen and elastic fibers in the skin. Collagen, which accounts for 70% of the skin, determines the softness and fullness of the skin, and elastic fibers determine the firmness and elasticity of the skin. 90% of elastic fibers are composed of elastin. Testing the gene expression level of ELN can evaluate whether the sample has a firming effect.

[0158] From Table 8 and Figure 6 The results show that the relative mRNA expression of ELN in Examples 1 to 5 is significantly higher than that in Comparative Examples 1 to 2. It can be seen that the products prepared in the examples of the present application have ideal anti-aging effects, and the fermentation temperature, time and strain selected for the enzymatic hydrolyzate have a great influence on the firming effect of the final product.

[0159] Effect Example 4 In vitro whitening experiment

[0160] In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on DOPA to form DOPAQUINONE, which spontaneously undergoes a series of reactions and finally forms melanin. In a phosphoric acid solution at pH 6.8, tyrosinase can catalyze the conversion of DOPA into DOPAQUINONE, and the absorbance can be measured at 475nm on a spectrophotometer.

[0161] The raw materials with tyrosinase activity inhibition can reduce the conversion of dopa into dopaquinone, thereby reducing the absorbance value. According to the change of absorbance value, the inhibitory effect of the raw materials on tyrosinase activity is evaluated.

[0162] Culture cells

[0163] After B16 cells in the logarithmic growth phase were subcultured and adhered to the culture medium, the cells were directly cultured with DMEM medium containing α-MSH (final concentration 0.5 μM); when the cells grew to 80-90% (culture dish), 8 ml of PBS was added to wash the cells, and the process was repeated once;

[0164] Collect cells

[0165] (One large dish collects one tube of cell sediment) Then add 2mL PBS, scrape the cells with a cell scraper, and then collect them into a 15ml centrifuge tube; at the same time, rinse the culture dish with PBS solution and transfer the liquid to a 15ml centrifuge tube, then centrifuge at 1000rpm for 5-10min, then carefully discard the supernatant (keep 1ml), gently blow the cells to suspend, and transfer them to a 1ml Eppendorf tube, and centrifuge at 1000rpm for 5-10min, discard the supernatant, and keep the cell sediment for later use. Note: Try not to leave any liquid in the Eppendorf tube. If not used for a long time, freeze it at -80℃.

[0166] Lyse cells

[0167] Add 500 μL of cell lysis buffer to each tube of cells, vortex to mix, and lyse at -20°C for 30 min.

[0168] 4. Collect cell lysate

[0169] After lysis, centrifuge at 10,000 g for 30 min in a 4°C centrifuge, transfer the supernatant to a 1.5 ml Eppendorf tube and proceed to subsequent operations.

[0170] 5. Protein Content Determination

[0171] Take 10 μL of cell lysate and dilute it 10 times with PBS to a volume of 100 μL for protein content determination (finally incubate at 37°C with lid on for 30 min). Prepare standard solution according to the instructions, make curve, and calculate the protein content in cell lysate. Finally, dilute the protein content of cell lysate with PBS (pH 6.8) to the required concentration of 3.5 mg / mL.

[0172] The tyrosinase activity inhibition experiment was performed as shown in the table below and added into a 1.5 ml Eppendorf tube in sequence.

[0173] PBS phosphate buffer (pH 6.8).

[0174]

[0175] Calculate the tyrosinase inhibition rate:

[0176]

[0177] (1) Where:

[0178] T—absorbance value of the sample tube, i.e., absorbance value of the solution after the sample reacts with tyrosinase;

[0179] T0—sample background absorbance;

[0180] C—average absorbance value of enzyme reaction tube, i.e. absorbance value of tyrosinase and DOPA reaction without adding sample;

[0181] C0—Solvent background absorbance.

[0182] The results are shown in Table 9 and Figure 7 .

[0183] Table 9

[0184]

[0185]

[0186] From Table 9 and Figure 7 The results show that the tyrosinase activity inhibition rate in Examples 1 to 5 is significantly higher than that in Comparative Examples 1 to 2. It can be seen that the products prepared in the examples of the present application have ideal whitening effects, and the fermentation temperature, time and strains selected for the enzymatic hydrolysate have a great influence on the whitening effect of the final product.

[0187] 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.

[0188] 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 liquid of Psoralea corylifolia having the function of regulating cell rhythm, characterized in that: It includes the following steps: (1) adding white thorn fruit, yellow thorn fruit and green thorn fruit to a fermentation enzymatic hydrolyzate, and performing enzymatic hydrolysis to prepare a fermentation substrate; (2) inoculating Schizophyllum corytatum into the fermentation substrate for fermentation, followed by sterilization to obtain the product; The preparation method of the fermentation hydrolysate comprises: adding water to the substrate and sterilizing it to obtain a culture medium, aerobically fermenting it after inoculation, and centrifuging it; the fermentation hydrolysate comprises Agaricus blazei fermentation hydrolysate or Saccharomyces cerevisiae fermentation hydrolysate.

2. The method for preparing the fermented liquid of the thorn fruit having the function of regulating cell rhythm as claimed in claim 1, characterized in that: The method for preparing the fermented liquid of the thorny thorn fruit capable of regulating cell rhythm satisfies at least one of the following conditions: In step (1), the Nitraria tangutica fruit is crushed to obtain Nitraria tangutica fruit powder before use; In step (1), the yellow thorn fruit also includes an operation of crushing to obtain yellow thorn fruit powder before use; In step (1), the process of crushing the prickle fruit to obtain prickle fruit powder before use is also included; In step (1), the mass ratio of the white thorn fruit, the yellow thorn fruit and the green thorn fruit is 1: (0.5-2): (0.5-2); In step (1), the mass ratio of the sum of the mass of the white thorn fruit, the yellow thorn fruit and the green thorn fruit to the mass of the fermentation enzymatic hydrolyzate is 1: (200-500); In step (1), the Agaricus blazei mushroom fermentation enzymatic hydrolysate is obtained by fermenting Agaricus blazei mushroom; In step (1), the saccharomyces cerevisiae fermentation hydrolyzate is obtained by fermenting saccharomyces cerevisiae; In step (1), the fermentation hydrolyzate is fermented using rice or potato as a substrate.

3. The method for preparing the fermented liquid of the thorn fruit having the function of regulating cell rhythm as claimed in claim 2, characterized in that: The method for preparing the fermented liquid of the thorny thorn fruit capable of regulating cell rhythm satisfies at least one of the following conditions: The deposit date of the Agaricus blazei is September 13, 2021, the deposit number is CGMCC No. 23250, the classification name is Agaricus blazei, the depository is China General Microbiological Culture Collection Center (CGMCC), the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101; The brewer's yeast includes brewer's yeast YW-1. Preferably, the brewer's yeast YW-1 has a preservation date of March 27, 2019, a preservation number of CGMCC No.17452, a classification name of brewer's yeast (Saccharomyces cerevisiae), and a preservation unit name of the General Microbiology Center of China National Microbiological Culture Collection (CGMCC for short), and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a zip code of 100101.

4. The method for preparing the fermented liquid of the thorn fruit having the function of regulating cell rhythm as claimed in claim 1 or 2, characterized in that: The method for preparing the fermented liquid of the thorny fruit capable of regulating cell rhythm satisfies at least one of the following conditions: In step (1), the mass ratio of the substrate to the water is 1:(20-50), preferably 1:20; In step (1), the sterilization is high temperature sterilization, the sterilization temperature is 110°C to 125°C, preferably 110°C to 121°C, for example 121°C; the high temperature sterilization time is 20 to 45 minutes, preferably 25 to 40 minutes, for example 30 minutes; In step (1), the number of viable bacteria inoculated is 1 to 2×10 7 CFU / mL; In step (1), the sterilization further comprises a cooling operation, and preferably the cooling is cooling to room temperature; In step (1), the aerobic fermentation is carried out in a constant temperature oscillating box. Preferably, when the aerobic fermentation is carried out in the constant temperature oscillating box, the rotation speed of the constant temperature oscillating box is 150-200 rpm, more preferably 180 rpm. In step (1), the temperature of the aerobic fermentation is 25-32°C, preferably 28°C; In step (1), the aerobic fermentation time is 36 to 60 hours, preferably 48 hours; In step (1), the aerobic fermentation further includes the operation of removing mycelium by centrifugation.

5. The method for preparing the fermented liquid of Psoralea corylifolia having the function of regulating cell rhythm as claimed in claim 1, characterized in that: The method for preparing the fermented liquid of the thorny thorn fruit capable of regulating cell rhythm satisfies at least one of the following conditions: In step (1), the enzymolysis is carried out in a constant temperature oscillating box. Preferably, when the enzymolysis is carried out in the constant temperature oscillating box, the rotation speed of the constant temperature oscillating box is 150 to 200 rpm, preferably 180 rpm; In step (1), the enzymatic hydrolysis time is 1 to 2 hours, preferably 2 hours; In step (1), the temperature of the enzymatic hydrolysis is 25-30°C, preferably 28°C; in step (1), sterilization is also included after the enzymatic hydrolysis; Preferably, the sterilization is high-temperature sterilization, the temperature of the high-temperature sterilization is 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C; the time of the high-temperature sterilization is 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

6. The method for preparing the fermented liquid of Psoralea corylifolia having the function of regulating cell rhythm as claimed in claim 1, characterized in that: In step (2), the fermentation is aerobic fermentation, and the aerobic fermentation is carried out in a constant temperature shaking box; Preferably, the rotation speed of the constant temperature oscillating box is 150-200 rpm, preferably 180 rpm; Preferably, the temperature of the aerobic fermentation is 25-30°C, more preferably 28°C; Preferably, the aerobic fermentation time is 2 to 3 days, more preferably 3 days; Preferably, the viable count of the Schizophyllum is 1 to 2×10 8 CFU / mL, preferably 1×10 8 CFU / mL.

7. The method for preparing the fermented liquid of Psoralea corylifolia having the function of regulating cell rhythm as claimed in claim 1, characterized in that: In step (2), the sterilization further includes centrifugal filtration, supernatant collection or sterilization operations; Preferably, the filter membrane particle size is 10 to 15 μm; Preferably, the sterilization is high temperature sterilization; More preferably, the temperature of the high temperature sterilization is 110°C to 125°C, and further preferably 110°C to 121°C, for example 121°C; More preferably, the high temperature sterilization time is 20 to 45 minutes, and further more preferably 25 to 40 minutes, for example 30 minutes.

8. A fermented liquid of thorny thorn fruit having the function of regulating cell rhythm, which is prepared by the preparation method of the fermented liquid of thorny thorn fruit having the function of regulating cell rhythm as described above.

9. Use of a fermented liquid of Tripterygium wilfordii having the function of regulating cell rhythm directly as a product, as an additive or as a base in the preparation of a skin topical preparation; Preferably, the fermented liquid of the thorn fruit having the function of regulating cell rhythm is used as at least one of the antioxidant active ingredient, the active ingredient regulating cell rhythm, the moisturizing active ingredient, the soothing active ingredient, the firming active ingredient, and the whitening active ingredient of the skin external preparation; More preferably, the antioxidant component is an antioxidant active component having the ability to scavenge hydroxyl free radicals or scavenge DPPH free radicals; More preferably, the active ingredient for regulating cell rhythm is an active ingredient for regulating cell rhythm that increases the expression of CLOCK gene; More preferably, the moisturizing active ingredient is a moisturizing active ingredient that increases the expression of the HAS gene; More preferably, the soothing active ingredient is a soothing active ingredient that has the ability to reduce the expression of the TPRV1 gene; More preferably, the firming active ingredient is a firming active ingredient that increases the expression of ELN gene; More preferably, the whitening active ingredient is a whitening active ingredient that has tyrosinase inhibitory activity.

10. A skin external preparation comprising the fermented liquid of the thorn fruit having the function of regulating cell rhythm as claimed in claim 8; Preferably, the skin topical preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; More preferably, the skin external preparation includes but is not limited to a facial mask, an essence or a toner; More preferably, the mass percentage of the fermented liquid of the thorny thorn fruit capable of regulating cell rhythm in the skin external preparation is 5% to 99%, for example, 60% to 99%.

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