Preparation method and application of nitraria tangutorum fermentation liquor with whitening and tightening effects

Abstract

The invention discloses a preparation method and application of nitraria tangutorum fermentation liquor with whitening and tightening effects, and the preparation method comprises the following steps: (1) crushing nitraria tangutorum fruits, adding water to obtain a fermentation substrate A, inoculating schizophyllum commune into the fermentation substrate A, and carrying out primary fermentation to obtain nitraria tangutorum fruit fermentation liquor; and (2) adding nitraria tangutorum leaves into the nitraria tangutorum fruit fermentation liquid to obtain a fermentation substrate B, and inoculating the fermentation substrate B with the schizophyllum commune again for secondary fermentation to obtain the nitraria tangutorum fruit fermentation liquid. The nitraria fruits and the nitraria leaves are subjected to stage fermentation, in the primary fermentation process, active substances in the nitraria fruits are released, in the secondary fermentation process, the nitraria leaves are added into nitraria fruit fermentation liquor, the active substances in the nitraria leaves are further extracted, and decomposition of the active substances in the nitraria leaves is avoided. By means of multiple fermentation, release of active substances in the nitraria tangutorum fermentation liquor is improved, ideal whitening, tightening and other effects are achieved, and the nitraria tangutorum fermentation liquor has good application prospects.

Description

Technical Field

[0001] The present application belongs to the technical field of cosmetic preparation, and in particular relates to a preparation method and application of a Nitraria tangutorum fermentation liquid having whitening and firming effects. Background Art

[0002] In recent years, with the rapid development of my country's economy and the continuous improvement of 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. Modern scientific research has proved that white thorn contains rich anthocyanins, polyphenols, flavonoids and other ingredients, which have excellent skin care effects. However, the current research focuses on the use of organic solvents to extract the active ingredients contained in white thorn. During the use of organic solvents, it is very easy to damage the active ingredients, resulting in a decrease in efficacy; moreover, organic solvents are easy to remain during use, resulting in side effects of the product and irritation to the skin.

[0004] Therefore, there is an urgent need for an extraction method that can effectively utilize the active ingredients contained in Nitraria tangutorum and apply them to cosmetics, with excellent efficacy and no chemical residue. Summary of the invention

[0005] The technical problem to be solved by the present application is to overcome the defects that the current stage of research in the prior art focuses on extracting the active ingredients contained in Nitraria tangutorum by using organic solvents, lacks effective utilization methods, and the organic solvent extraction process causes irritation to the product, and to provide a preparation method and application of Nitraria tangutorum fermentation liquid with whitening and firming effects.

[0006] This application adopts the following technical solutions to solve the above technical problems:

[0007] The present application provides a method for preparing a Nitraria tangutorum fermented liquid having whitening and firming effects, which comprises the following steps:

[0008] (1) crushing the Nitraria tangutorum fruit and adding water to obtain a fermentation substrate A, inoculating Schizophyllum schizonepeta into the fermentation substrate A for preliminary fermentation, and obtaining a Nitraria tangutorum fruit fermentation liquid;

[0009] (2) adding Nitraria tangutorum leaves to the Nitraria tangutorum fermentation liquid to obtain a fermentation substrate B, and inoculating the fermentation substrate B again with the Schizophyllum sp. to perform secondary fermentation to obtain the product.

[0010] In some embodiments, in step (1), the mass ratio of the Nitraria tangutorum fruit to the water is 0.5 to 1:100, preferably 1:100.

[0011] In some embodiments, in step (1), the method for preparing the fermentation substrate A comprises: uniformly mixing the Nitraria tangutorum powder and the water.

[0012] In some embodiments, in step (1), the fermentation substrate A is further sterilized before use.

[0013] Wherein, the method for sterilizing the fermentation substrate A can generally be a high temperature sterilization method.

[0014] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C.

[0015] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0016] In some embodiments, in step (1), the Schizophyllum commune is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, 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 Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC for short), and the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0017] In some embodiments, in step (1), the Schizophyllum sp. is added in the form of a Schizophyllum sp. bacterial liquid.

[0018] The preparation method of the schizophyllum liquid includes: inoculating the schizophyllum into a PDA liquid culture medium, then culturing at 28°C and 180rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28°C constant temperature incubator and culturing it for 7 days, then picking a single colony into 300ml PDA liquid culture medium, culturing it at 28°C and 180rpm for 7 days, and using it as the schizophyllum liquid for subsequent use.

[0019] In some embodiments, in step (1), based on the water in the fermentation substrate A, the number of viable bacteria of the Schizophyllum inoculated per unit mass of the water is 1 to 3×107 CFU / mL, preferably 3×10 7 CFU / mL.

[0020] In some embodiments, in step (1), the preliminary 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.

[0021] Wherein, when the constant temperature oscillation box is used for the aerobic fermentation, the rotation speed of the constant temperature oscillation box is 150-200 rpm, preferably 180 rpm.

[0022] In some embodiments, in step (1), the temperature of the initial fermentation is 25-32°C, preferably 28°C.

[0023] In some embodiments, in step (1), the initial fermentation time is 2 to 4 days, preferably 3 days.

[0024] In some embodiments, in step (1), after the initial fermentation, the process further includes centrifuging to remove mycelium.

[0025] The centrifugal rotation speed may be a conventional rotation speed for such operation in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm.

[0026] The centrifugation time may be the conventional time for such operation in the art, preferably 20 to 45 min, more preferably 25 to 40 min, for example 30 min.

[0027] In some embodiments, in step (2), the Nitraria tangutorum leaf is Nitraria tangutorum leaf powder.

[0028] In some embodiments, in step (2), the mass ratio of the Nitraria tangutorum leaf to the Nitraria tangutorum fruit fermentation liquid is 0.5-1:100, preferably 0.5:100.

[0029] In some embodiments, in step (2), the method for preparing the fermentation substrate B comprises: uniformly mixing the Nitraria tangutorum powder and the Nitraria tangutorum fermentation liquid.

[0030] In some embodiments, in step (2), the fermentation substrate B is further sterilized before use.

[0031] Wherein, the method for sterilizing the fermentation substrate A can generally be a high temperature sterilization method.

[0032] 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 100° C. to 110° C., more preferably 110° C.

[0033] When the high temperature sterilization method is used for the sterilization, the sterilization time can 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.

[0034] In some embodiments, in step (2), the secondary 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.

[0035] Wherein, when the constant temperature oscillation box is used for the aerobic fermentation, the rotation speed of the constant temperature oscillation box is 150-200 rpm, preferably 180 rpm.

[0036] In some embodiments, in step (2), the temperature of the secondary fermentation is 25-32°C, preferably 28°C.

[0037] In some embodiments, in step (2), the secondary fermentation time is 2 to 4 days, preferably 3 days.

[0038] In some embodiments, in step (2), based on the water in the fermentation substrate B, the number of viable bacteria of the Schizophyllum inoculated per unit mass of the water may be 0.5 to 1.5×10 7 CFU / mL, preferably 1.5×10 7 CFU / mL.

[0039] In some embodiments, in step (2), the secondary fermentation further comprises at least one of centrifugation, filtration to remove supernatant and sterilization.

[0040] The centrifugal rotation speed may be a conventional rotation speed for such operation in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm.

[0041] The centrifugation time may be the conventional time for such operation in the art, preferably 20 to 45 min, more preferably 25 to 40 min, for example 30 min.

[0042] Wherein, the filter membrane is 10-15 μm, preferably 15 μm.

[0043] The sterilization method may generally be high temperature sterilization.

[0044] When the high temperature sterilization method is used for the sterilization, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 100° C. to 110° C., more preferably 110° C.

[0045] When the high temperature sterilization method is used for the sterilization, the sterilization time can 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.

[0046] The present application also provides a Nitraria tangutorum fermented liquid with whitening and firming effects, which is prepared by the preparation method of the Nitraria tangutorum fermented liquid with whitening and firming effects as described above.

[0047] The present application also provides a use of the above-mentioned Nitraria tangutorum fermented liquid with whitening and firming effects directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0048] In some embodiments, the Nitraria tangutorum fermented liquid with whitening and firming effects is used as at least one of the antioxidant active ingredient, anti-sugar active ingredient, anti-aging active ingredient, and whitening active ingredient of the skin external preparation.

[0049] Wherein, the antioxidant component is an antioxidant component having the function of scavenging free radical DPPH.

[0050] Wherein, the anti-sugar active ingredient is an anti-sugar active ingredient having the function of inhibiting the content of AGEs.

[0051] Wherein, the anti-aging active ingredient is an anti-aging active ingredient that can improve the activity of SOD.

[0052] Wherein, the whitening active ingredient is a whitening active ingredient that has the ability to inhibit tyrosinase activity.

[0053] The present application also provides a skin external preparation, which comprises the Nitraria tangutorum fermented liquid having whitening and firming effects as described above.

[0054] 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.

[0055] In some embodiments, the topical skin preparation may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.

[0056] In some embodiments, the mass percentage of the Nitraria tangutorum fermented liquid with whitening and firming effects in the skin external preparation may be 5% to 99%, preferably 60% to 99%.

[0057] 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.

[0058] The reagents and raw materials used in this application are commercially available.

[0059] The positive and progressive effects of the present application are as follows: the present application uses a step-by-step fermentation method to perform multi-stage fermentation on the fruits and leaves of Nitraria tangutica, and during the primary fermentation process, the active substances in the Nitraria tangutica fruit are effectively released, and during the secondary fermentation process, Nitraria tangutica leaves are added to the Nitraria tangutica fruit fermentation liquid to further extract the active substances in the Nitraria tangutica leaves, and to avoid the decomposition of the active substances in the Nitraria tangutica leaves. The present application uses a multiple fermentation method to further improve the release of active substances in the Nitraria tangutica fermentation liquid, achieve ideal anti-aging, anti-oxidation, whitening, firming and other effects, and the fermentation conditions are mild, with good application prospects. At the same time, the prepared product can be used directly as a cosmetic or added to a basic cosmetic formula, and has good antioxidant, anti-aging and whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] 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.

[0061] in:

[0062] Figure 1 It is the effect diagram of DPPH free radical scavenging in the embodiment;

[0063] Figure 2 This is a diagram showing the effect of AGEs inhibition in the embodiment;

[0064] Figure 3 This is a diagram showing the effect of in vitro elastase inhibition in the examples;

[0065] Figure 4 This is a diagram showing the relative expression of ELN mRNA in the examples;

[0066] Figure 5 This is the effect diagram of the relative expression of NRF2 mRNA in the embodiment;

[0067] Figure 6 This is a diagram showing the activity of SOD enzyme (superoxide dismutase) in the embodiment;

[0068] Figure 7 It is a diagram showing the effect of inhibiting tyrosinase activity in the examples. DETAILED DESCRIPTION

[0069] 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.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0071] The raw materials Nitraria tangutorum fruit and Nitraria tangutorum leaf in the following examples are from Minqin County, Gansu Province.

[0072] Example 1

[0073] (1) After the white thorn fruit was crushed, 3 g was weighed and added to 300 g of water, followed by sterilization at 121°C for 30 min and cooling to 25°C to obtain fermentation substrate A. Then, 3 g of Schizophyllum seed liquid was inoculated, wherein the number of live Schizophyllum inoculated was 3×10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 3 days, followed by centrifugation to remove the mycelium to obtain the white thorn fruit fermentation liquid.

[0074] (2) 1.5 g of Nitraria tangutorum leaf powder was added to 300 g of Nitraria tangutorum fruit fermentation liquid, followed by sterilization at 110°C for 30 min and cooling to 25°C to obtain fermentation substrate B. Subsequently, 1.5 g of Schizophyllum seed liquid was inoculated and subjected to secondary aerobic fermentation at 28°C and 180 rpm for 2 days. The number of live bacteria of the inoculated Schizophyllum was 1.5×10 7 CFU / mL, after the fermentation, the mycelium was removed by centrifugation, and then finely filtered through a 15 μm filter membrane, the supernatant was taken, and finally sterilized at 110 °C for 30 min.

[0075] Example 2

[0076] Compared with Example 1, the only difference is that the time of the initial fermentation is different, the time of the aerobic fermentation is 2 days, and the other conditions are the same as those in Example 1. The specific operations are as follows:

[0077] (1) After the white thorn fruit was crushed, 3 g was weighed and added to 300 g of water, followed by sterilization at 121°C for 30 min and cooling to 25°C to obtain fermentation substrate A. Then, 3 g of Schizophyllum seed liquid was inoculated, wherein the number of live Schizophyllum inoculated was 3×10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 2 days, followed by centrifugation to remove the mycelium to obtain the white thorn fruit fermentation liquid.

[0078] (2) 1.5 g of Nitraria tangutorum leaf powder was added to 300 g of Nitraria tangutorum fruit fermentation liquid, followed by sterilization at 110°C for 30 min and cooling to 25°C to obtain fermentation substrate B. Subsequently, 1.5 g of Schizophyllum schizophyllum seed liquid was inoculated and subjected to secondary aerobic fermentation at 28°C and 180 rpm for 2 days. The number of live Schizophyllum schizophyllum inoculated was 1.5 × 10 7 CFU / mL, after the fermentation, the mycelium was removed by centrifugation, and then finely filtered through a 15 μm filter membrane, the supernatant was taken, and finally sterilized at 110 °C for 30 min.

[0079] Example 3

[0080] Compared with Example 1, the only difference is that the time of secondary fermentation is different, the time of aerobic fermentation is 1 day, and other conditions are the same as those in Example 1. The specific operation is as follows:

[0081] (1) After the white thorn fruit was crushed, 3 g was weighed and added to 300 g of water, followed by sterilization at 121°C for 30 min and cooling to 25°C to obtain fermentation substrate A. Then, 3 g of Schizophyllum seed liquid was inoculated, wherein the number of viable bacteria of the inoculated Schizophyllum was 3×10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 2 days, followed by centrifugation to remove the mycelium to obtain the white thorn fruit fermentation liquid.

[0082] (2) 1.5 g of Nitraria tangutorum leaf powder was added to 300 g of Nitraria tangutorum fruit fermentation liquid, followed by sterilization at 110°C for 30 min and cooling to 25°C to obtain fermentation substrate B. Subsequently, 1.5 g of Schizophyllum schizophyllum seed liquid was inoculated and subjected to secondary aerobic fermentation at 28°C and 180 rpm for 1 day. The number of live bacteria of the inoculated Schizophyllum schizophyllum was 1.5 × 10 7 CFU / mL, after the fermentation, the mycelium was removed by centrifugation, and then finely filtered through a 15 μm filter membrane, the supernatant was taken, and finally sterilized at 110 °C for 30 min.

[0083] Example 4

[0084] Compared with Example 1, the only difference is that the amount of Nitraria tangutorum leaf powder added in the secondary fermentation substrate is 0.6 g, and the other conditions are the same as those in Example 1. The specific operation is as follows:

[0085] (1) After the white thorn fruit was crushed, 3 g was weighed and added to 300 g of water, followed by sterilization at 121°C for 30 min and cooling to 25°C to obtain fermentation substrate A. Then, 3 g of Schizophyllum seed liquid was inoculated, wherein the number of viable bacteria of the inoculated Schizophyllum was 3×10 7 CFU / mL, and then aerobically fermented at 28°C and 180 rpm for 2 days, followed by centrifugation to remove the mycelium to obtain the white thorn fruit fermentation liquid.

[0086] (2) 0.6 g of Nitraria tangutorum leaf powder was added to 300 g of Nitraria tangutorum fruit fermentation liquid, followed by sterilization at 110°C for 30 min and cooling to 25°C to obtain fermentation substrate B. Subsequently, 1.5 g of Schizophyllum schizophyllum seed liquid was inoculated and subjected to secondary aerobic fermentation at 28°C and 180 rpm for 2 days. The number of viable bacteria of the inoculated Schizophyllum schizophyllum was 1.5×10 7 CFU / mL, after the fermentation, the mycelium was removed by centrifugation, and then finely filtered through a 15 μm filter membrane, the supernatant was taken, and finally sterilized at 110 °C for 30 min.

[0087] Effect Example 1 DPPH free radical scavenging rate

[0088] 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.

[0089] 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%):

[0090] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution and mix well (tube A1);

[0091] (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);

[0092] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0093] (4) After 30 minutes of reaction in the dark, measure the absorbance of tubes A1, A2, and A3 at 517 nm. The clearance rate is calculated as follows: clearance rate = [(A2 + A3) - A1] / A2 × 100%.

[0094] This experiment tested the DPPH free radical scavenging experiment on the embodiments and comparative examples. The results are shown in Tables 1 and Figure 1 .

[0095] Table 1

[0096] serial number DPPH free radical scavenging rate (%) Example 1 89.03 Example 2 88.16 Example 3 85.99 Example 4 86.17

[0097] The results show that the DPPH free radical scavenging rates in Examples 1 to 4 are all above 85%, showing a relatively ideal antioxidant effect.

[0098] Effect Example 2 Anti-glycation

[0099] The impact of advanced glycation end products (AGEs) on the skin is mainly reflected in the fact that they can cause skin aging and dull complexion. AGEs are the end products of non-enzymatic glycosylation reactions (Maillard reactions), which refer to stable covalent adducts generated by the spontaneous reaction of macromolecules such as proteins, lipids or nucleic acids with glucose or other reducing monosaccharides without the participation of enzymes. This test evaluates the ability of the sample to clear advanced glycation end products by detecting the AGES inhibition rate through the advanced glycation end product (AGEs) clearance test.

[0100] Simulated saccharification reaction inhibition system

[0101] A mixed solution containing 80 mg / mL bovine serum albumin and 240 mg / mL glucose was prepared using PBS and filtered through a 0.22 μm filter membrane as a 2x saccharification reaction solution. Each group of reaction systems was prepared according to Table 3. The final concentration of bovine serum albumin in each group of reaction systems was 40 mg / mL, the final concentration of glucose was 120 mg / mL, and the final concentration of the sample or positive control was as shown in Table 2.

[0102] Table 2 AGEs removal test reaction system

[0103]

[0104] After mixing evenly, incubate at 55°C for 4 days. Use PBS instead of the sample as a negative control, 10% aminoguanidine hydrochloride solution as a positive control, and PBS instead of the glycosylation reaction solution as a control system. After the reaction is completed, the incubated solution is cooled to room temperature, centrifuged at 2000r / min for 5min, the supernatant is filtered through a 0.22μm filter membrane, and 200μL of the reaction solution is added to a 96-well plate in sequence. Use a fluorescent microplate reader to detect at an excitation wavelength of 320nm and an emission wavelength of 460nm, and the AGEs inhibition rate is calculated according to the following formula.

[0105]

[0106] Where:

[0107] A-fluorescence intensity of the saccharification system with added test substance;

[0108] B-fluorescence intensity of PBS solution with added test substance;

[0109] The results are shown in Table 3 and Figure 2 .

[0110] Table 3

[0111] serial number AGEs inhibition rate (%) Example 1 95.38 Example 2 94.37 Example 3 92.34 Example 4 93.72

[0112] The results showed that the AGEs inhibition rates in Examples 1 to 4 were all above 90%, showing a relatively ideal anti-glycation effect.

[0113] Effect Example 3 In vitro elastase inhibition

[0114] Elastin plays a key role in the youthfulness of the skin, and the lack of elastin is the main cause of skin aging. Inhibiting the activity of elastase can effectively slow down the decomposition of elastin and maintain the elasticity of the skin. Elastase can catalyze the hydrolysis of hydroxyl groups contained in the polypeptide bonds of various amino acids. Therefore, when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, a colored substance will be produced, and its absorbance value can be measured at 406nm in a spectrophotometer. According to the change in absorbance, the inhibitory effect of the test substance on the activity of elastase is calculated, and whether the test substance has anti-wrinkle and firming effects can be evaluated.

[0115] During the assay, the reaction was carried out in a test tube, 50 μL of 1 μmol / L elastase solution was added to each well, and 400 μL of the sample to be tested was added. In a constant temperature water bath at 25°C for 15 minutes; then, if there was precipitation after the reaction was completed, centrifuge. The absorbance at 406 nm was measured in a 96-well plate, see Table 4.

[0116] Table 4

[0117]

[0118] Elastase activity inhibition rate:

[0119]

[0120] Where:

[0121] T-absorbance value of sample group, i.e., absorbance value of solution after sample affects the reaction between elastase and substrate; T0-absorbance value of sample background group;

[0122] C- absorbance value of enzyme reaction group, i.e. absorbance value of elastase and substrate reaction without adding sample; C0- absorbance value of solvent background group.

[0123] The results are shown in Table 5 and Figure 3 .

[0124] Table 5

[0125]

[0126]

[0127] The results showed that the in vitro elastase inhibition rates in Examples 1 to 4 were all above 90%.

[0128] Effect Example 4 ELN mRNA relative expression

[0129] 2% 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. The specific primer sequences are shown in Table 6 below, and the test results are shown in Tables 7 and Figure 4 .

[0130] Table 6 qRT-PCR primer sequence list

[0131]

[0132] Table 7

[0133]

[0134] The results showed that compared with the model group, the relative expression level of ELN gene in Examples 1 to 4 was significantly increased, which shows that the examples can significantly improve the anti-aging effect. Figure 4 **p<0.01, indicating that there is a significant statistical difference compared with the model group, which is significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the control group.

[0135] Effect Example 5 Relative expression of NRF2 mRNA

[0136] 2% 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. The specific primer sequences are shown in Table 8 below, and the test results are shown in Table 9 and Figure 5 .

[0137] Table 8 qRT-PCR primer sequence list

[0138]

[0139] Table 9

[0140]

[0141] The results showed that compared with the model group, the relative expression level of NRF-2 gene in Examples 1 to 4 was significantly increased, which shows that the examples can enhance the anti-aging effect. Figure 5 **p<0.01, indicating that there is a significant statistical difference compared with the model group, which is significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the control group.

[0142] Effect Example 6 SOD enzyme (superoxide dismutase) activity determination

[0143] This experiment uses Hacat cells from the Chinese Science Cell Bank to verify the cellular SOD enzyme activity of the products obtained in the above examples and comparative examples.

[0144] 1. The density of HaCaT cells is 500,000 / well (6-well plate). When the cell density reaches 80%, the culture medium is discarded;

[0145] 2. Wash twice with PBS, then add 1 mL of PBS, cover the blank well with tin foil and place in a UVB light box for 30 seconds;

[0146] 3. After irradiation, discard PBS, then wash with PBS, add 2mL of 2% sample and serum-free, and the positive control is 50μg / mL VC;

[0147] 4. Place in a 37°C incubator and incubate for 24 hours;

[0148] 5. Aspirate and discard the supernatant;

[0149] 6. Wash twice with pre-cooled PBS, add 100 μl of SOD sample preparation solution, pipette to fully lyse the cells, centrifuge at 12,000 g for 5 minutes at 4°C, and take the supernatant as the sample to be tested.

[0150] 7. Reagent preparation

[0151] 1) Preparation of WST-8 / enzyme working solution: Evenly mix 6040μl SOD detection buffer, 320μl WST-8 and 40μl enzyme solution to prepare WST-8 / enzyme working solution.

[0152] 2) Preparation of reaction start working solution: dilute 1 μl reaction start solution (40X) by adding 39 μl SOD detection buffer and mix well to obtain the reaction start working solution.

[0153] 8. Sample determination

[0154] Use a 96-well plate to set up sample wells and various blank control wells according to the table below. Add the sample to be tested and other solutions in sequence according to the table below. Add the reaction starter working solution and mix thoroughly. Incubate at 37℃ for 30 minutes. Measure the absorbance at 450nm.

[0155] Calculation formula: Inhibition percentage = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%

[0156] SOD enzyme activity units in the sample to be tested = inhibition percentage / (1-inhibition percentage) units

[0157] The calculation results are shown in Table 10 and Figure 6 .

[0158] Table 10

[0159] serial number SOD activity (U / mL) Control group 1.19 Model Group 0.81 VC (positive control) 1.15 Example 1 1.12 Example 2 0.98 Example 3 1.18 Example 4 0.94

[0160] The results showed that compared with the model group, the SOD activity in Examples 1 to 4 was significantly increased, which shows that the Examples can increase the content of anti-aging active ingredients. Figure 6 In the table, **p<0.01 indicates that there is a significant statistical difference compared with the model group, which is significantly reduced; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group, which is extremely significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the control group).

[0161] Effect Example 7 In vitro whitening test

[0162] 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.

[0163] 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.

[0164] Culture cells

[0165] 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;

[0166] Collect cells

[0167] (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℃.

[0168] Lyse cells

[0169] Add 500 μL of cell lysis buffer to each tube of cells, vortex to mix, and lyse at -20°C for 30 min.

[0170] 4. Collect cell lysate

[0171] 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.

[0172] 5. Protein Content Determination

[0173] 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.

[0174] The tyrosinase activity inhibition experiment was performed as shown in the table below and added into a 1.5 ml Eppendorf tube in sequence.

[0175] PBS phosphate buffer (pH 6.8).

[0176]

[0177] Calculate the tyrosinase inhibition rate:

[0178]

[0179] (1) Where:

[0180] T—absorbance value of the sample tube, i.e., absorbance value of the solution after the sample reacts with tyrosinase;

[0181] T0—sample background absorbance;

[0182] C—average absorbance value of enzyme reaction tube, i.e. absorbance value of tyrosinase and DOPA reaction without adding sample;

[0183] C0—Solvent background absorbance.

[0184] The results are shown in Table 11 and Figure 7 .

[0185] Table 11

[0186] serial number Tyrosinase activity inhibition rate (%) Example 1 74.49 Example 2 67.08 Example 3 71.60 Example 4 63.37

[0187] The results show that the tyrosinase activity inhibition rate in Examples 1 to 4 is stable at more than 60%, showing a relatively ideal whitening effect.

[0188] 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.

[0189] 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 Nitraria tangutorum fermented liquid having whitening and firming effects, characterized in that: The steps include: (1) crushing the Nitraria tangutorum fruit and adding water to obtain a fermentation substrate A, inoculating Schizophyllum schizonepeta into the fermentation substrate A for preliminary fermentation, and obtaining a Nitraria tangutorum fruit fermentation liquid; (2) adding Nitraria tangutorum leaves to the Nitraria tangutorum fermentation liquid to obtain a fermentation substrate B, and inoculating the fermentation substrate B again with the Schizophyllum sp. to perform secondary fermentation to obtain the product.

2. The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects according to claim 1, characterized in that: The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects satisfies at least one of the following conditions: In step (1), the mass ratio of the Nitraria tangutorum fruit to the water is 0.5 to 1:100, preferably 1:100; In step (1), the method for preparing the fermentation substrate A comprises: uniformly mixing the Nitraria tangutorum powder and the water; In step (1), the fermentation substrate A is further sterilized before use; Preferably, when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C; Preferably, when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

3. The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects according to claim 1, characterized in that: The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects satisfies at least one of the following conditions: In step (1), the schizophyllum is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, 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 Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC for short), the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101; In step (1), the Schizophyllum fungi are added in the form of Schizophyllum fungi liquid; In step (1), based on the water in the fermentation substrate A, the number of viable bacteria of the Schizophyllum inoculated per unit mass of the water is 1 to 3×10 7 CFU / mL, preferably 3×10 7 CFU / mL.

4. The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects according to claim 1, characterized in that: The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects satisfies at least one of the following conditions: In step (1), the preliminary fermentation is aerobic fermentation, and the aerobic fermentation is carried out in a constant temperature oscillation box; preferably, when the aerobic fermentation is carried out in the constant temperature oscillation box, the rotation speed of the constant temperature oscillation box is 150-200 rpm, preferably 180 rpm; In step (1), the temperature of the initial fermentation is 25-32°C, preferably 28°C; In step (1), the initial fermentation time is 2 to 3 days, preferably 3 days; In step (1), after the initial fermentation, the process further includes centrifuging to remove the residue and mycelium; Preferably, the centrifugal speed is 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm; Preferably, the centrifugation time is 20 to 45 min, more preferably 25 to 40 min, for example 30 min.

5. The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects according to claim 1, characterized in that: The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects satisfies at least one of the following conditions: In step (2), the Nitraria tangutica leaf is Nitraria tangutica leaf powder; In step (2), the mass ratio of the Nitraria tangutorum leaf to the Nitraria tangutorum fruit fermentation liquid is 0.5-1:100, preferably 0.5:100; In step (2), the method for preparing the fermentation substrate B comprises: uniformly mixing the Nitraria tangutorum powder and the Nitraria tangutorum fermentation liquid; In step (2), the fermentation substrate B is further sterilized before use. Preferably, the method for sterilizing the fermentation substrate A is generally a high-temperature sterilization method; when the high-temperature sterilization method is used for the sterilization, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 100° C. to 110° C., more preferably 110° C.; when the high-temperature sterilization method is used for the sterilization, the sterilization time may 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; In step (2), the secondary fermentation is aerobic fermentation, and the aerobic fermentation is carried out aerobic fermentation in a constant temperature shaking box; Preferably, when the constant temperature oscillating box is used for the aerobic fermentation, the rotation speed of the constant temperature oscillating box is 150-200 rpm, preferably 180 rpm; In step (2), the temperature of the secondary fermentation is 25-32°C, preferably 28°C; In step (2), the secondary fermentation time is 2 to 4 days, preferably 3 days; In step (2), based on the water in the fermentation substrate B, the number of viable bacteria of the Schizophyllum inoculated per unit mass of the water is 0.5 to 1.5×10 7 CFU / mL, preferably 1.5×10 7 CFU / mL.

6. The method for preparing the Nitraria tangutorum fermented liquid having whitening and firming effects according to claim 5, characterized in that: The secondary fermentation further includes at least one of centrifugation, filtering to remove supernatant and sterilization.

7. The method for preparing the Nitraria tangutorum fermented liquid having whitening and firming effects according to claim 6, characterized in that: The method for preparing the Nitraria tangutorum fermented liquid with whitening and firming effects satisfies at least one of the following conditions: The centrifugal speed is a conventional speed for such operations in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm; The centrifugation time is the conventional time for such operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes; The filter membrane of the filtration is 10-15 μm, preferably 15 μm; The sterilization method is high temperature sterilization; Preferably, when the high temperature sterilization method is used for the sterilization, the sterilization temperature is 100°C to 110°C, more preferably 110°C; Preferably, when the high temperature sterilization method is used for the sterilization, the sterilization time is 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

8. A Nitraria tangutorum fermented liquid with whitening and firming effects, which is prepared by the preparation method of the Nitraria tangutorum fermented liquid with whitening and firming effects as described above.

9. Use of a fermented liquid of Nitraria tangutorum with whitening and firming effects directly as a product, as an additive or as a base in the preparation of a skin topical preparation; Preferably, the Nitraria tangutorum fermented liquid with whitening and firming effects is used as at least one of the antioxidant active ingredient, anti-sugar active ingredient, anti-aging active ingredient, and whitening active ingredient of the skin external preparation; More preferably, the antioxidant component is an antioxidant component that has the ability to scavenge free radical DPPH; More preferably, the anti-sugar active ingredient is an anti-sugar active ingredient that has the ability to inhibit the content of AGEs; More preferably, the anti-aging active ingredient is an anti-aging active ingredient that has the ability to enhance SOD activity; More preferably, the whitening active ingredient is a whitening active ingredient that has tyrosinase inhibitory activity.

10. A skin external preparation comprising the Nitraria tangutorum fermented liquid with whitening and firming effects 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 Nitraria tangutorum fermented liquid with whitening and firming effects in the skin external preparation is 5% to 99%, such as 60% to 99%.

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