Fermentation method of potentilla anserina, fermentation liquor and application thereof in cosmetics

Abstract

The present application provides a fermentation method of Potentilla anserina, a fermentation liquor and application thereof in cosmetics, and belongs to the technical field of biological fermentation. The fermentation method of Potentilla anserina provided by the present application comprises the following steps: grinding and sieving tuber of Potentilla anserina, adding the sieved powder into a culture medium at an addition amount of 1%-3% w / v, sterilizing the culture medium, inoculating a strain into the sterilized culture medium at an inoculation amount of 8%-12% v / v, and fermenting the culture medium to obtain the fermentation liquor of Potentilla anserina after sterilization and filtration. The content of potentilline acid in the fermentation liquor of Potentilla anserina prepared by the fermentation method provided by the present application is significantly improved. Moreover, the fermentation liquor has good anti-inflammatory, antioxidant, moisturizing, anti-wrinkle, anti-aging and antibacterial effects, and is a kind of cosmetic raw material with good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological fermentation, and particularly relates to a fermentation method of Potentilla anserina, a fermentation liquor and application of the fermentation liquor in cosmetics. BACKGROUND

[0002] Potentilla anserina, also known as P. anserina, is a perennial herbaceous plant of Rosaceae. The tuberous roots of P. anserina contain polysaccharides, succinic acid, triterpenoids, polyphenols, flavanols, flavonoids, triterpenoids and other active substances, which make P. anserina have the effects of clearing heat and detoxifying, cooling blood and stopping dysentery. In addition, P. anserina contains vitamins, magnesium, zinc, potassium and calcium elements, has high medical and nutritional value, and has the functions of invigorating the stomach and spleen, generating saliva and quenching thirst, and tonifying qi and blood. The whole grass of P. anserina in bloom can also be used for astringent hemostasis, relieving cough and expectorating sputum, and also for tonifying.

[0003] As a unique plant with good medicinal value in China, P. anserina has attracted more and more attention from researchers. Chen et al. [1] The pharmacological effects of P. anserina were studied, and it was found that P. anserina played a protective role in protecting liver damage, antioxidant, anti-hypoxia, enhancing immune function, anti-inflammatory and anti-tumor. A few studies found that P. anserina could improve cardiovascular diseases and protect damaged myocardial tissue. Zhang et al. [2] It was found that pretreatment with P. anserina could effectively reduce acute myocardial ischemia-reperfusion injury in mice, maintain normal cardiac function, and thus play a protective role on the heart. Another study proved that the ethanol extract of P. anserina could significantly inhibit myocardial cell apoptosis caused by acute myocardial ischemia-reperfusion, and inhibit the expression of caspase3, caspase9 mRNA level and protein level. Choi H et al. [3] It was found that P. anserina could solve the skin problems caused by wearing a mask, including enhancing moisture, strengthening skin barrier and relieving itching.

[0004] As a medicinal and edible plant, P. anserina has high nutritional value and medical and health care functions, is rich in resources, has great development and utilization potential, but there are few related products, and antioxidant fermented products are scarce. At present, there are few fermentation processes of P. anserina using strains, and most of them are carried out saccharification reaction to promote the extraction of effective components of P. anserina, but the preparation process is complicated, and the saccharification reaction is not easy to control. Therefore, it is urgent to provide a simple and convenient Pteridophyte fermentation product with high antioxidant activity.

[0005] Morikawa T, Ninomiya K, Imura K, et al. Hepatoprotective triterpenes from traditional Tibetan medicine Potentilla anserina [J]. Phytochemistry, 2014, 102: 169-181.

[0006] Li L, Zhang L, Gong HY, et al. Anti-hypoxic and antioxidant mechanisms of ethanol extract from Rumex japonicus Houtt [J]. Chinese Journal of Food Hygiene, 2005, 17(4): 306.

[0007] Choi H, Ha JH, Kang HC, et.al. The Protective Effects of Moisturizer Containing Potentilla anserina Extract in the Topical Treatment of Skin Damage Caused by Masks [J]. Int J Mol Sci. 2023 Sep 19;24(18): 14294. SUMMARY

[0008] Based on the problems and deficiencies in the prior art, the present application aims to provide a fermentation method of Potentilla anserina, a fermentation liquid and its application in cosmetics. The fermentation method of Potentilla anserina provided by the present application grinds the tubers of Potentilla anserina into powder and sieves them, then adds the sieved powder to the culture medium at an addition amount of 1%-3% w / v, sterilizes it, inoculates the strain into the sterilized liquid at an inoculation amount of 8%-12% v / v for fermentation, and filters after sterilization to obtain the Potentilla anserina fermentation liquid. The Potentilla anserina fermentation liquid prepared by the fermentation method provided by the present application has a significantly improved Potentilla acid content. And has good anti-inflammatory, antioxidant and antibacterial effects, is a cosmetic raw material with good application prospect.

[0009] The technical scheme of the present application is as follows:

[0010] On the one hand, the present application provides a fermentation method of Potentilla anserina, which comprises the following steps:

[0011] S1, grinding and sieving the tubers of Potentilla anserina, and taking the sieved powder;

[0012] S2, adding the sieved powder to the culture medium, and obtaining a sterilized liquid after sterilization;

[0013] S3, inoculating the strain into the sterilized liquid of step S2, and obtaining the Potentilla anserina fermentation liquid after fermentation and sterilization and filtration.

[0014] Specifically, the sieving in step S1 is sieving through a 60-80 mesh sieve.

[0015] Preferably, the sieving in step S1 is sieving through an 80 mesh sieve.

[0016] Specifically, the amount of the undersize powder added in step S2 is 1%-3% w / v.

[0017] Preferably, the amount of the undersize powder added in step S2 is 2% w / v.

[0018] Specifically, the medium in step S2 includes one or more of anhydrous glucose, soluble starch, peptone, and yeast powder.

[0019] Further specifically, the medium in step S2 includes a red ganoderma basic medium, a purple ganoderma basic medium, a cordyceps militaris basic medium, a cicada worm cordyceps basic medium, a saccharomyces cerevisiae basic medium, and a bifidobacterium basic medium.

[0020] Preferably, the preparation method of the red ganoderma basic medium is weighing 3.5 g of anhydrous glucose, 0.5 g of peptone, 0.25 g of yeast powder, and 100 mL of pure water in a 250 mL conical flask, and sterilizing at 121°C for 20 min to obtain the red ganoderma basic medium.

[0021] Preferably, the preparation method of the purple ganoderma basic medium is weighing 3.5 g of soluble starch, 0.5 g of peptone, 0.75 g of yeast powder, and 100 mL of pure water in a 250 mL conical flask, and sterilizing at 121°C for 20 min to obtain the purple ganoderma basic medium.

[0022] Preferably, the preparation method of the cordyceps militaris basic medium is weighing 2.0 g of anhydrous glucose, 3.0 g of peptone, 0.75 g of yeast powder, 0.05 g of magnesium sulfate, 0.005 g of potassium dihydrogen phosphate, and 100 mL of pure water in a 250 mL conical flask, and sterilizing at 121°C for 20 min to obtain the cordyceps militaris basic medium.

[0023] Preferably, the preparation method of the cicada worm cordyceps basic medium is weighing 3.5 g of anhydrous glucose, 0.5 g of peptone, 0.25 g of yeast powder, and 100 mL of pure water in a 250 mL conical flask, and sterilizing at 121°C for 20 min to obtain the cicada worm basic medium.

[0024] Preferably, the preparation method of the saccharomyces cerevisiae basic medium is weighing 1.0 g of anhydrous glucose, 1.0 g of peptone, 0.5 g of yeast powder, and 100 mL of pure water in a 250 mL conical flask, and sterilizing at 121°C for 20 min to obtain the saccharomyces cerevisiae basic medium.

[0025] Preferably, the preparation method of the Bifidobacterium base medium is as follows: 2.0 g of anhydrous glucose, 1.0 g of proteose peptone, 0.75 g of yeast powder, 0.01 g of soluble starch, 0.5 g of sodium chloride, 100 mL of pure water are weighed in a 250 mL conical flask, and then high-temperature sterilization is performed at 121°C for 20 min to obtain the Bifidobacterium base medium.

[0026] Specifically, the inoculation amount of the strain in step S3 is 8%-12% v / v.

[0027] Preferably, the inoculation amount of the strain in step S3 is 10% v / v.

[0028] Specifically, the strain in step S3 includes but is not limited to Ganoderma lucidum strain, Ganoderma sinense strain, Cordyceps militaris strain, Cordyceps cicadae strain, Saccharomyces cerevisiae strain or Bifidobacterium strain.

[0029] Preferably, the strain number of the Ganoderma lucidum strain is CICC-14042.

[0030] Preferably, the strain number of the Ganoderma sinense strain is CICC-51332.

[0031] Preferably, the strain number of the Cordyceps militaris strain is CICC-14013.

[0032] Preferably, the strain number of the Cordyceps cicadae strain is CICC-81169.

[0033] Preferably, the strain number of the Saccharomyces cerevisiae strain is CGMCC 2.3880.

[0034] Preferably, the strain number of the Bifidobacterium strain is CICC-6068.

[0035] Further preferably, the strain in step S3 is the Saccharomyces cerevisiae strain, and the number is CGMCC 2.3880.

[0036] Specifically, the OD600 of the Ganoderma lucidum strain, the Ganoderma sinense strain, the Cordyceps militaris strain and the Cordyceps cicadae strain is greater than or equal to 1.

[0037] Specifically, the concentration of the Saccharomyces cerevisiae strain and the Bifidobacterium strain is 10 8 -10 9 CFU / mL.

[0038] Preferably, the concentration of the Saccharomyces cerevisiae strain and the Bifidobacterium strain is 10 8 CFU / mL.

[0039] Specifically, the fermentation in step S3 is at a temperature of 25-30 DEG C, and the fermentation time is 96-120h.

[0040] Preferably, the fermentation in step S3 is at a temperature of 25 DEG C, and the fermentation time is 96h.

[0041] Preferably, the sterilization in step S3 is 121 DEG C whole sterilization for 20 min.

[0042] Preferably, the filtration in step S3 is filtration through a 0.45 mu filter plate.

[0043] The beneficial effects of the present application are:

[0044] 1. The present application uses fermentation method to ferment Potentilla anserina, and the content of potentilline in the fermentation product is significantly improved compared with single extract.

[0045] 2. The fermentation liquid Potentilla anserina prepared by the method provided by the present application has significant antioxidant, anti-inflammatory and antibacterial effects. The DPPH free radical clearance rate reaches 91.43+3.39%, the COX-2 cyclooxygenase inhibition rate reaches 87.36+0.16%, the inhibition rate on Propionibacterium acnes reaches 94.11+1.67%, and the inhibition rate on Staphylococcus aureus reaches 58.93+0.56, which has significant antibacterial effect. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The figure is a standard detection spectrum of potentilline. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0048] Preparation of basic medium in basic experiment example 1

[0049] 1. Red Ganoderma basic medium: weigh 3.5g anhydrous glucose, 0.5g peptone, 0.25g yeast powder, 100mL pure water in a 250mL conical flask, and sterilize at 121 DEG C for 20 min to obtain the red Ganoderma basic medium.

[0050] 2. Purple Ganoderma basic medium: weigh 3.5g soluble starch, 0.5g peptone, 0.75g yeast powder, 100mL pure water in a 250mL conical flask, and sterilize at 121 DEG C for 20 min to obtain the purple Ganoderma basic medium.

[0051] 3. Cordyceps militaris basic medium: 2.0 g of anhydrous glucose, 3.0 g of proteose peptone, 0.75 g of yeast powder, 0.05 g of magnesium sulfate, 0.005 g of potassium dihydrogen phosphate, 100 mL of pure water were weighed in a 250 mL conical flask, and the cordyceps militaris basic medium was obtained by sterilizing at 121°C for 20 min.

[0052] 4. Cordyceps cicadae basic medium: 3.5 g of anhydrous glucose, 0.5 g of proteose peptone, 0.25 g of yeast powder, 100 mL of pure water were weighed in a 250 mL conical flask, and the cordyceps cicadae basic medium was obtained by sterilizing at 121°C for 20 min.

[0053] 5. Saccharomyces cerevisiae basic medium: 1.0 g of anhydrous glucose, 1.0 g of proteose peptone, 0.5 g of yeast powder, 100 mL of pure water were weighed in a 250 mL conical flask, and the saccharomyces cerevisiae basic medium was obtained by sterilizing at 121°C for 20 min.

[0054] 6. Bifidobacterium basic medium: 2.0 g of anhydrous glucose, 1.0 g of proteose peptone, 0.75 g of yeast powder, 0.01 g of soluble starch, 0.5 g of sodium chloride, 100 mL of pure water were weighed in a 250 mL conical flask, and the bifidobacterium basic medium was obtained by sterilizing at 121°C for 20 min.

[0055] Culture of strains in basic experiment example 2

[0056] 1. Ganoderma lucidum strain: purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC-14042.

[0057] 2. Ganoderma sinense strain: purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC-51332.

[0058] 3. Cordyceps militaris strain: purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC-14013.

[0059] 4. Cordyceps cicadae strain: purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC-81169.

[0060] 5. Saccharomyces cerevisiae strain: purchased from the China General Microbiological Culture Collection Center, with strain preservation number CGMCC 2.3880.

[0061] 6. Bifidobacterium strain: purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC-6068.

[0062] Example 1

[0063] The tuber of Potentilla anserina L. (also known as "goumian fruit") with full particles, no mildew, and no obvious deterioration is screened, crushed by a pulverizer, and passed through an 80-mesh sieve. The undersize portion is taken, added to the red ganoderma basic medium at an addition amount of 2%, sterilized at 121°C for 20 min, inoculated with 10% red ganoderma spores, cultured at a fermentation temperature of 25°C for 96 h under basic culture conditions, sterilized at 121°C for 20 min after fermentation, filtered through a 0.45-μm filter plate, and a Potentilla anserina L. fermentation liquor is obtained.

[0064] Example 2

[0065] The tuber of Potentilla anserina L. (also known as "goumian fruit") with full particles, no mildew, and no obvious deterioration is screened, crushed by a pulverizer, and passed through an 80-mesh sieve. The undersize portion is taken, added to the red ganoderma basic medium at an addition amount of 2%, sterilized at 121°C for 20 min, inoculated with 10% red ganoderma spores, cultured at a fermentation temperature of 25°C for 96 h under basic culture conditions, sterilized at 121°C for 20 min after fermentation, filtered through a 0.45-μm filter plate, and a Potentilla anserina L. fermentation liquor is obtained.

[0066] Example 3

[0067] The tuber of Potentilla anserina L. (also known as "goumian fruit") with full particles, no mildew, and no obvious deterioration is screened, crushed by a pulverizer, and passed through an 80-mesh sieve. The undersize portion is taken, added to the red ganoderma basic medium at an addition amount of 2%, sterilized at 121°C for 20 min, inoculated with 10% red ganoderma spores, cultured at a fermentation temperature of 25°C for 96 h under basic culture conditions, sterilized at 121°C for 20 min after fermentation, filtered through a 0.45-μm filter plate, and a Potentilla anserina L. fermentation liquor is obtained.

[0068] Example 4

[0069] The tuber of Potentilla anserina L. (also known as "goumian fruit") with full particles, no mildew, and no obvious deterioration is screened, crushed by a pulverizer, and passed through an 80-mesh sieve. The undersize portion is taken, added to the red ganoderma basic medium at an addition amount of 2%, sterilized at 121°C for 20 min, inoculated with 10% red ganoderma spores, cultured at a fermentation temperature of 25°C for 96 h under basic culture conditions, sterilized at 121°C for 20 min after fermentation, filtered through a 0.45-μm filter plate, and a Potentilla anserina L. fermentation liquor is obtained.

[0070] Example 5

[0071] The tuber of Potentilla anserina L. (also known as "goumian fruit") with full particles, no mildew, and no obvious deterioration is screened, crushed by a pulverizer, and passed through an 80-mesh sieve. The undersize portion is taken, added to the red ganoderma basic medium at an addition amount of 2%, sterilized at 121°C for 20 min, inoculated with 10% red ganoderma spores, cultured at a fermentation temperature of 25°C for 96 h under basic culture conditions, sterilized at 121°C for 20 min after fermentation, filtered through a 0.45-μm filter plate, and a Potentilla anserina L. fermentation liquor is obtained. 8Saccharomyces cerevisiae species of CFU / mL, fermentation temperature 25℃, basic culture conditions for 96h, after fermentation, 121℃ whole sterilization 20min, 0.45μm filter plate filtration, get the Potentilla feruginea fermentation broth.

[0072] Example 6

[0073] Screening of full particles, no mildew, no obvious deterioration of Potentilla feruginea tuber, by pulverizer crushing, 80 mesh screen, take the screen part, 2% addition, add it into the basic culture medium of Bifidobacterium, 121℃ high temperature sterilization 20min after putting to normal temperature, inoculation 10% concentration 10 8 CFU / mL of Bifidobacterium species, fermentation temperature 25℃, basic culture conditions for 96h, after fermentation, 121℃ whole sterilization 20min, 0.45μm filter plate filtration, get the Potentilla feruginea fermentation broth.

[0074] Comparative example 1

[0075] Screening of full particles, no mildew, no obvious deterioration of Potentilla feruginea tuber, by pulverizer crushing, 80 mesh screen, take the screen part, 2% addition, heating reflux method, extraction 210min, by medium speed qualitative filter paper filtration, get the Potentilla feruginea extract.

[0076] Comparative example 2

[0077] Screening of full particles, no mildew, no obvious deterioration of Potentilla feruginea tuber, by pulverizer crushing, 80 mesh screen, take the screen part, 10% addition, add it into the basic culture medium of Saccharomyces cerevisiae, 121℃ high temperature sterilization 20min after putting to normal temperature, inoculation 1.5% concentration 10 8 CFU / mL of Saccharomyces cerevisiae species, fermentation temperature 25℃, basic culture conditions for 96h, after fermentation, 121℃ whole sterilization 20min, 0.45μm filter plate filtration, get the Potentilla feruginea fermentation broth.

[0078] Comparative example 3

[0079] Screening of full particles, no mildew, no obvious deterioration of Potentilla feruginea tuber, by pulverizer crushing, 80 mesh screen, take the screen part, 10% addition, add it into the basic culture medium of Saccharomyces cerevisiae, 121℃ high temperature sterilization 20min after putting to normal temperature, inoculation 10% concentration 10 8 CFU / mL of Saccharomyces cerevisiae species, fermentation temperature 25℃, basic culture conditions for 96h, after fermentation, 121℃ whole sterilization 20min, 0.45μm filter plate filtration, get the Potentilla feruginea fermentation broth.

[0080] Comparative example 4

[0081] The tubers of Potentilla anserina with full granules, no mildew, no obvious deterioration were selected, crushed by a pulverizer, passed through an 80 mesh sieve, and the undersize portion was taken. The Potentilla anserina was added into a basic culture medium of Saccharomyces cerevisiae at an addition amount of 2%, sterilized at 121°C for 20 min, and then placed at room temperature. A strain of Saccharomyces cerevisiae with a concentration of 10 8 CFU / mL was inoculated at 15%. The fermentation was carried out at 25°C under basic culture conditions for 96 h. After the fermentation was completed, the whole was sterilized at 121°C for 20 min, filtered through a 0.45 μm filter plate, and a fermentation liquor of Potentilla anserina was obtained.

[0082] Comparative Example 5

[0083] The tubers of Potentilla anserina with full granules, no mildew, no obvious deterioration were selected, crushed by a pulverizer, passed through an 80 mesh sieve, and the undersize portion was taken. The Potentilla anserina was added into a basic culture medium of Saccharomyces cerevisiae at an addition amount of 2%, sterilized at 121°C for 20 min, and then placed at room temperature. A strain of Saccharomyces cerevisiae with a concentration of 10 8 CFU / mL was inoculated at 15%. The fermentation was carried out at 25°C under basic culture conditions for 96 h. After the fermentation was completed, the whole was sterilized at 121°C for 20 min, filtered through a 0.45 μm filter plate, and a fermentation liquor of Potentilla anserina was obtained.

[0084] Determination of Potentilla Acid Content in Potentilla anserina Fermentation Liquor by High Performance Liquid Chromatography

[0085] The Potentilla acid content in the Potentilla anserina fermentation liquor prepared in Examples 1-6 and Comparative Examples 2-5 and the Potentilla anserina extract prepared in Comparative Example 1 was detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows.

[0086] An Agilent 1260 chromatograph was used, a ZORBAX SB-C18 (4.6 mm x 250 mm, 5 μm) was used as the chromatographic column, the column flow rate was 0.8 mL / min, the column temperature was 35°C, the wavelength was 210 nm, and the sample injection amount was 10 μL. The mobile phase A was 0.2% acetic acid water, the mobile phase B was acetonitrile, and the gradient of the mobile phase A and the mobile phase B is shown in Table 1. The peak time of Potentilla acid was 24.121 min, and is shown in Figure 1 .

[0087] Table 1 Gradient of Mobile Phase A and Mobile Phase B

[0088] Time min A％ B％ 0 95 5 5 95 5 30 70 30 50 60 40 60 50 50 70 50 50 80 40 60 90 30 70 100 30 70

[0089] Table 2 Potentilla Acid Content (mg / mL)

[0090] Sample name Trifolin content (mg / mL) Example 1 0.2146 Example 2 0.2225 Example 3 0.1722 Example 4 0.1764 Example 5 0.3814 Example 6 0.2075 Comparative Example 1 0.0826 Comparative Example 2 0.1238 Comparative Example 3 0.1494 Comparative Example 4 0.1357 Comparative Example 5 0.1559

[0091] The determination results are shown in Table 2. Compared with the comparative example 1, the contents of the potentilla acid in the fermenting liquids of the potentilla chinensis prepared in the examples 1-6 are all significantly improved, which indicates that the fermentation process provided in the present application can significantly improve the content of the potentilla acid in the potentilla chinensis raw material, and the effect of the example 5 is the best, and the content of the potentilla acid is increased to 4.6 times, up to 0.3814 mg / mL.

[0092] Experimental example 2 DPPH free radical scavenging test

[0093] 1. Preparation of DPPH ethanol solution

[0094] 20 mg of DPPH was weighed, dissolved in anhydrous ethanol and diluted in a 250 mL volumetric flask, and the concentration of DPPH was prepared to be 2×10 -4 mol / L; store at 0-4℃ in the dark, and use it immediately, which is effective within 4 hours. (The positive control is vitamin C).

[0095] 2. Preparation of the test solution

[0096] The potentilla chinensis fermenting liquids prepared in the examples 1-6 and the potentilla chinensis extract prepared in the comparative example 1 were taken, and the test solutions with the concentrations of 0.25 mg / ml, 0.5 mg / mL, 0.1 mg / mL and 2 mg / mL were prepared by using pure water.

[0097] 3. Experimental steps

[0098] The reagents were added according to Table 3:

[0099] 1) 1 mL of the test solution was mixed with 1 mL of 2×10 -4 mol / L DPPH solution (A tube);

[0100] 2) 1 mL of anhydrous ethanol was mixed with 1 mL of 2×10 -4 mol / L DPPH solution (B tube);

[0101] 3) 1 mL of anhydrous ethanol was mixed with 1 mL of the test solution (C tube);

[0102] Table 3: Reagent ratio table

[0103] No. DPPH solution Anhydrous ethanol Test solution Total volume A 1 mL —— 1 mL 2 mL B 1 mL 1 mL —— 2 mL C —— 1 mL 1 mL 2 mL

[0104] 4) After 30 min of light-free reaction, the absorbance values of the A, B and C tubes were measured at 517 nm;

[0105] 5) DPPH free radical inhibition rate calculation formula: DPPH inhibition rate (%) = (B+C-A) / B

[0106] 4. DPPH free radical inhibition rate detection results

[0107] The test results are shown in Table 4. Compared with Comparative Example 1, the DPPH free radical scavenging rates of the Potentilla feru DPPH fermentation liquids prepared in Examples 1-6 are all significantly improved, indicating that the fermentation process provided by the application can significantly improve the antioxidant effect of Potentilla feru raw materials. The DPPH inhibition effect of Example 5 at 10% concentration is the most significant, and the DPPH free radical scavenging rate reaches 91.43±3.39%, which has a significant antioxidant effect.

[0108] Table 4 DPPH free radical scavenging

[0109] Sample 1％ 3％ 5％ 10％ Example 1 34.06±4.10％ 53.06±0.85％ 61.97±0.73％ 79.55±4.99％ Example 2 45.63±4.18％ 55.97±3.47％ 64.04±0.17％ 87.49±0.92％ Example 3 25.97±2.75％ 42.08±0.89％ 70.44±0.09％ 81.86±2.54％ Example 4 38.30±5.18％ 43.68±2.84％ 52.17±2.41％ 70.52±1.36％ Example 5 52.28±3.89％ 63.56±3.40％ 77.10±3.96％ 91.43±3.39％ Example 6 49.35±3.18％ 56.37±3.97％ 62.22±3.31％ 88.27±0.95％ Comparative Example 1 15.07±2.80％ 16.79±4.11％ 19.17±1.12％ 30.06±6.55％

[0110] Experimental Example 3 Verification of COX-2 Cyclooxygenase Activity Inhibition

[0111] The inhibition rate of different samples on COX-2 was tested by using a COX-2 kit, and the specific operation was according to the use instruction provided by the manufacturer:

[0112] 1. Prepare the sample culture medium: dilute the sample with DMSO to the concentration to be tested (100 μg / mL).

[0113] 2. Reagent preparation:

[0114] (1) Melt all reagents except rhCOX-2 to room temperature, centrifuge the solution to the bottom of the tube, and mix well for standby. COX-2 Probe, COX-2 Cofactor (50X) and COX-2 Substrate (50X) are prepared in DMSO, and can be promoted to melt at 37°C water bath for 0.5-2 min. After use, it should be immediately stored at -20°C in the dark.

[0115] (2) Preparation of COX-2 Cofactor working solution: prepare an appropriate amount of COX-2 Cofactor working solution according to the proportion of 5 microliters of COX-2 Cofactor working solution required for each sample. Take an appropriate amount of COX-2 Cofactor (50X), and dilute it with COX-2 Assay Buffer according to the proportion of 1:49. For example, 4 microliters of COX-2 Cofactor (50X) is added to 196 microliters of COX-2 Assay Buffer to prepare 200 microliters of COX-2 Cofactor working solution. The prepared COX-2 Cofactor working solution can be stored at 4°C and can only be used on the same day.

[0116] (3) Preparation of COX-2 working solution: Prepare the COX-2 working solution according to the proportion of 5 μl per sample. Take the appropriate amount of rhCOX-2 (25X) and dilute it with COX-2 Assay Buffer according to the proportion of 1:24. For example, 8 μl of rhCOX-2 (25X) is added to 192 μl of COX-2 Assay Buffer to prepare 200 μl of COX-2 working solution. The prepared COX-2 working solution can be temporarily stored on ice bath, and the enzyme activity is basically stable within 1 hour. Note: All operations involving COX-2 should be performed on ice.

[0117] (4) Preparation of COX-2 Substrate working solution: Prepare the COX-2 Substrate working solution according to the proportion of 5 μl per sample. Take the appropriate amount of COX-2 Substrate (50X) and add an equal volume of Substrate Buffer, mix well by vortexing. Then dilute the mixture with Milli-Q grade pure water or heavy distilled water according to the proportion of 1:24, and mix well by vortexing. For example, 20 μl of COX-2 Substrate (50X) is added to 20 μl of Substrate Buffer, vortexed, and then 960 μl of Milli-Q grade pure water or heavy distilled water is added, and mixed well by vortexing. Finally, 1 ml of COX-2 Substrate working solution is obtained. The prepared COX-2 Substrate working solution can be temporarily stored on ice bath, and is relatively stable within 1 hour. Note: The COX-2 Substrate working solution can also be prepared during the 10-minute incubation at 37°C during sample detection.

[0118] (5) Preparation of positive inhibitor Celecoxib solution: The positive control inhibitor Celecoxib provided in this kit has a concentration of 100 μM and is prepared in DMSO. It can be diluted to the required concentration or concentration gradient using the same solvent as the inhibitor to be tested as needed. Generally, the IC50 of Celecoxib is about 10 nM-100 nM.

[0119] 3. Sample detection:

[0120] (1) According to Table 5, set up control wells and sample wells using a 96-well black plate, and add samples and solutions in turn according to the table below. After adding the sample to be tested, mix well and incubate at 37°C for 10 minutes.

[0121] Table 5 Reagent Proportioning Table

[0122]

[0123] Note: *Sample solvent refers to the solvent used to prepare and dilute the inhibitor to be tested.

[0124] (2) Add COX-2 Probe 5 μl to each well.

[0125] (3) Add COX-2 Substrate working solution 5 μl to each well quickly, mix well. Note: The reaction will start after adding COX-2 Substrate working solution. If the number of wells is large, the operation can be done at low temperature or using a dispenser to reduce the error caused by the time difference of adding COX-2 Substrate working solution between wells. Mixing can also be done on a plate shaker.

[0126] (4) Incubate at 37℃ for 5 minutes in the dark, then measure the fluorescence. The excitation wavelength is 560 nm and the emission wavelength is 590 nm. If the fluorescence reading is low, the incubation time can be appropriately extended to 10-20 minutes.

[0127] 4. Calculation:

[0128] (1) Calculate the average fluorescence value of each sample well and the blank control well, which can be recorded as RFU blank control, RFU 100% enzyme activity control, RFU positive inhibitor control and RFU sample, respectively. RFU, Relative Fluorescence Unit.

[0129] (2) Calculate the inhibition percentage of each sample. The calculation formula is as follows:

[0130] Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) x 100%.

[0131] 5. COX-2 inhibition rate detection results

[0132] The results show (Tables 6-7) that compared with Comparative Example 1, the COX-2 cyclooxygenase inhibition rates of the Potentilla anserina fermentation liquids prepared in Examples 1-6 are significantly improved, indicating that the fermentation process can significantly enhance the antioxidant effect of Potentilla anserina raw materials, and Example 5 has the most significant COX-2 inhibition effect at a concentration of 10%, with a COX-2 cyclooxygenase inhibition rate of 87.36 ± 0.16%, having a significant anti-inflammatory efficacy.

[0133] Table 6 Inhibition rate of Examples 1-6 and Comparative Example 1 on COX-2 at a concentration of 0.5%

[0134] COX-2 inhibition rate (%) SD (%) Example 1 35.82 0.03 Example 2 39.33 0.01 Example 3 38.96 0.2 Example 4 32.53 0.12 Example 5 53.66 0.06 Example 6 49.97 0.07 Comparative Example 1 17.79 0.05

[0135] Table 7 Inhibition rate of Examples 1-6 and Comparative Example 1 on COX-2 at a concentration of 1.0%

[0136]

[0137]

[0138] Inhibitory effect of Experiment 4 on Propionibacterium acnes

[0139] 1. Culture of Propionibacterium acnes

[0140] Shake the culture medium with Propionibacterium acnes until no white bacterial clumps precipitate at the bottom. Use a sterile syringe pipette to transfer 200μL-800μL of the bacterial suspension into a new culture medium and incubate at 37°C in a biochemical incubator. After 4-5 days of incubation, subculture.

[0141] 2. Microbroth method for testing the antibacterial activity of samples

[0142] (1) Preparation of fresh culture medium

[0143] Transfer the sterile vesicle culture medium from the anaerobic bottle to a sterile 50 mL centrifuge tube. Centrifuge the sterile vesicle culture medium at 3500 rpm for 10 min at 25°C. Transfer the supernatant to another sterile 50 mL centrifuge tube for later use.

[0144] For the samples, each sample was diluted with fresh culture medium to 100 μL / mL (10%), 70 μL / mL (7%), 50 μL / mL (5%), 30 μL / mL (3%), and 10 μL / mL (1%).

[0145] (2) Extraction of Propionibacterium acnes

[0146] Gently mix the culture medium with Propionibacterium acnes and transfer to a sterile 50 mL centrifuge tube. Incubate at 25°C.

[0147] Centrifuge at 1000 rpm for 30 seconds, transfer the supernatant to a sterile 50 mL centrifuge tube, and incubate at 25°C.

[0148] Centrifuge at 3500 rpm for 10 min.

[0149] (3) Determination of bacterial concentration

[0150] Turn on the microplate reader and set the wavelength to 600 nm. Transfer 400 μL of the bacterial culture to a 1.5 mL sterile centrifuge tube. Transfer 200 μL of the bacterial culture from the 1.5 mL centrifuge tube containing 400 μL of bacterial culture to a standard 96-well plate. Measure the OD600 value of the bacterial culture at 600 nm. If the OD600 value is >0.5, dilute with sterile PBS.

[0151] (Exploration can be carried out at 2x, 4x, 6x, and 8x.) When the OD value is equal to 0.5, the next step of the experiment can be carried out.

[0152] (4) Dilution of bacterial culture

[0153] Calculate the required volume of bacterial suspension. Dilute the bacterial suspension (OD600 = 0.5) 160 times with fresh culture medium.

[0154] (5)Planning

[0155] In a sterile 96-well plate, 100 μL of diluted bacterial suspension was added to each well, followed by 100 μL of samples at different concentrations. 100 μL of samples at different concentrations plus 100 μL of P. acnes medium served as a control group. 200 μL of P. acnes medium without the addition of P. acnes served as a blank control. 100 μL of diluted bacterial suspension...

[0156] Use 100 μL of vesicular culture medium as a negative control and 100 μL of diluted bacterial suspension + 100 μL of 200 μg / mL penicillin sodium as a positive control, with 3 replicates for each.

[0157] Tear open the anaerobic gas-generating bag and place it in a 2.5L anaerobic container, then place a sterile 96-well culture plate in the 2.5L anaerobic container. After culturing for 24 hours, measure the absorbance at 600nm.

[0158] (6) After the plate is laid, it is placed in an anaerobic tank and incubated in an incubator for 24 hours. The OD value at 600 nm is then measured.

[0159] (7) Inhibition rate of Propionibacterium acnes:

[0160]

[0161] The inhibition rates (%) of Examples 1-6 and Comparative Example 1 against Propionibacterium acnes are shown in Table 8.

[0162] Table 8 Inhibition rate against Propionibacterium acnes

[0163] Sample concentration 1％ 3％ 5％ 10％ Example 1 42.03±1.17％ 60.69±1.62％ 64.38±2.34％ 75.02±3.87％ Example 2 30.48±0.06％ 62.63±0.82％ 73.34±2.15％ 84.07±0.82％ Example 3 42.71±2.20％ 59.18±4.27％ 74.68±3.86％ 82.21±1.67％ Example 4 39.07±6.56％ 65.39±2.91％ 68.99±4.27％ 76.77±4.34％ Example 5 45.60±5.11％ 71.24±3.50％ 81.68±3.44％ 94.11±1.67％ Example 6 43.35±3.67％ 62.36±2.12％ 77.49±2.20％ 81.86±1.58％ Comparative Example 1 12.03±4.08％ 20.69±2.27％ 34.38±1.55％ 55.02±4.33％

[0164] The results showed that, compared with Comparative Example 1, the fermentation broth of *Potentilla chinensis* prepared in Examples 1-6 all showed a significant increase in the inhibition rate of *Propionibacterium acnes*, indicating that the fermentation process can significantly enhance the inhibitory effect of harmful bacteria on *Potentilla chinensis* raw materials. In particular, Example 5 showed the most significant inhibitory effect of harmful bacteria at a concentration of 10%, with an inhibition rate of 94.11 ± 1.67% against *Propionibacterium acnes*.

[0165] Experimental Example 5: Inhibitory effect against Staphylococcus aureus

[0166] 1. Culture of Staphylococcus aureus

[0167] Mix the medium with S. aureus until there is no white precipitate at the bottom. Using a sterile pipette, transfer 200-800 μL of the bacterial solution into a new medium and incubate at 37°C in a biochemical incubator. After 4-5 days of incubation, perform subculturing.

[0168] 2. Microdilution broth method to test the antibacterial activity of the sample

[0169] (1) Preparation of fresh medium

[0170] Transfer the sterile medium into a sterile 50 mL centrifuge tube. Centrifuge the sterile medium at 3500 rpm for 10 min at 25°C. Transfer the supernatant into another sterile 50 mL centrifuge tube for later use.

[0171] For the sample, dilute each sample of fresh medium to 100 μL / mL (10%), 50 μL / mL (5%), and 10 μL / mL (1%).

[0172] (2) Extraction of S. aureus

[0173] Gently mix the medium with S. aureus and transfer it into a sterile 50 mL centrifuge tube. Centrifuge at 1000 rpm for 30 s at 25°C. Transfer the supernatant into a sterile 50 mL centrifuge tube and centrifuge at 3500 rpm for 10 min at 25°C.

[0174]

[0175] (3) Determination of the concentration of the bacterial solution

[0176] Turn on the microplate reader and set the wavelength to 600 nm. Take 400 μL of the bacterial solution into a 1.5 mL sterile centrifuge tube. Take 200 μL of the bacterial solution from the 1.5 mL centrifuge tube containing 400 μL of the bacterial solution and place it into a regular 96-well plate. Measure the OD600 value of the bacterial solution at 600 nm. If the OD600 value is >0.5, dilute it with sterile PBS.

[0177] (Exploration can be performed at 2-fold, 4-fold, 6-fold, and 8-fold) When the OD value is equal to 0.5, proceed to the next step.

[0178] (4) Dilution of the bacterial solution

[0179] Calculate the volume of the bacterial suspension required. Dilute the bacterial suspension (OD600=0.5) 3000-fold with fresh medium.

[0180] (5) Plating

[0181] ​In a sterile 96-well culture plate, 100 μL of diluted bacterial suspension was added to each well of the test wells, and 100 μL of samples of different concentrations, 100 μL of samples of different concentrations + 100 μL of LB medium were added as a control group, 200 μL of LB broth medium without P. acnes was added as a blank control, 100 μL of diluted bacterial suspension + 100 μL of LB broth medium was added as a negative control, and 100 μL of diluted bacterial suspension + 100 μL of 200 μg / mL penicillin sodium was added as a positive control, and each was prepared in triplicate.

[0182] The sterile 96-well culture plate was placed in a 37°C incubator. After 12 hours of incubation, the absorbance was measured at 600 nm.

[0183] (6) After plating, the OD value at 600 nm was measured after 12 hours of incubation in an incubator.

[0184] (7) Staphylococcus aureus inhibition rate:

[0185]

[0186] The results of the inhibition rates of Staphylococcus aureus of Examples 1-6 and Comparative Example 1 are shown in Table 9.

[0187] Table 9 Inhibition rate of Staphylococcus aureus

[0188] Sample concentration 1％ 3％ 5％ 10％ Example 1 5.89±0.38％ 10.07±2.44％ 24.10±0.79％ 30.16±3.75％ Example 2 3.88±1.44％ 11.36±1.62％ 19.57±2.73％ 26.72±0.80％ Example 3 13.08±3.93％ 21.20±2.04％ 25.64±3.45％ 26.54±3.87％ Example 4 12.85±2.98％ 19.57±1.74％ 24.79±9.13％ 32.59±1.26％ Example 5 21.50±2.05％ 29.26±1.20％ 36.85±1.52％ 58.93±0.56％ Example 6 21.71±0.93％ 27.16±1.96％ 35.44±2.05％ 48.58±0.92％ Comparative Example 1 0.24±2.91％ 10.98±1.29％ 18.72±2.61％ 24.08±6.78％

[0189] The results show that the inhibition rates of Staphylococcus aureus of the Potentilla anserina fermentation broth prepared in Examples 1-6 are significantly improved compared to Comparative Example 1, indicating that the fermentation process can significantly enhance the harmful bacteria activity inhibition effect of Potentilla anserina raw materials, and the harmful bacteria activity inhibition effect of Example 5 at 10% concentration is the most significant, with a Staphylococcus aureus inhibition rate of 58.93 ± 0.56%.

[0190] Experimental Example 6 Moisturizing effect

[0191] 6.1 Initial data collection

[0192] In the test environment, wait quietly for 20-30 min, and let the MPA-10 test the water content of the stratum corneum of the skin. The test area is the inner side of the forearm, and each test area is tested 3 times to take the average value, which is recorded as T0.

[0193] 6.2 Apply the test substance

[0194] (1) During the test period, the subjects applied the basic gel formula containing 5% test sample to the inner side of the forearm and the face according to the divided areas, and the subjects did not use any other cosmetics in the test area during the test period. The specific content of the basic gel formula is shown in Table 10 below:

[0195] Table 10 Base gel formulation

[0196]

[0197] (2) The test period is 2h, and during the test period, the subjects are prohibited from eating, drinking, playing, and other activities that cause strong changes in skin moisture.

[0198] 6.3 Data collection at different time periods

[0199] Before product use, 1h, 2h after product use, the skin stratum corneum moisture content of the test area on the inner forearm is tested, and the test results are recorded as T0, T2, T4. Each test area is tested 3 times and the average value is taken. The results of improving the skin stratum corneum moisture content of Examples 1-6 and Comparative Example 1 are shown in Table 11.

[0200] Table 11 Results of improving the skin stratum corneum moisture content

[0201]

[0202]

[0203] The results show that, compared with Comparative Example 1, the ferments of Potentilla freyniana prepared in Examples 1-6 can significantly improve the skin stratum corneum moisture content, indicating that the fermentation process can significantly improve the moisturizing and hydrating effect of Potentilla freyniana raw materials, and the moisturizing and hydrating effect of Example 5 at 10% concentration is the most significant, and after 4 hours, the skin stratum corneum moisture content is improved by 52.66%

[0204] Experimental Example 7 Elastase inhibition effect (anti-wrinkle firming)

[0205] 1. Preparation of Tris-HCl solution: Prepare 2 mmol / L Tris-HCl solution, and adjust the pH to 8.0 with 1 mol / L and 0.1 mol / L NaOH solution. Store at 4°C after use.

[0206] 2. Preparation of elastase solution: Prepare 1 U / mL elastase solution with 2 mmol / L Tris-HCl solution. If there is any remaining after use, store at -20°C.

[0207] 3. Preparation of substrate solution: Prepare 0.5 mg / mL substrate solution with 2 mmol / L Tris-HCl solution. If there is any remaining after use, store at -4°C.

[0208] 4. Preparation of test solution:

[0209] (1) Take the fermenting liquid of Potentilla atrosanguinea prepared in Example 1-6 and the extract liquid sample of Potentilla atrosanguinea prepared in Comparative Example 1, and use 2 mmol / L Tris-HCl solution to dissolve and prepare 10% test solution.

[0210] (2) Take the oil-soluble sample, dissolve in DMSO, and add to 2 mmol / L Tris-HCl solution to prepare 2% DMSO solution.

[0211] 5. Experimental procedure:

[0212] In the 96-well plate, add each reagent according to Table 12.

[0213] (1) A: 20 μL elastase solution + 10 μL test solution + 40 μL substrate solution + 80 μL Tris-HCl solution

[0214] (2) B: 20 μL elastase solution + 10 μL test solution + 120 μL Tris-HCl solution

[0215] (3) C: 20 μL elastase solution + 90 μL Tris-HCl solution + 40 μL substrate solution

[0216] Table 12 Reagent ratio table

[0217] No. Elastase solution Test solution 2 mmol / L Tris-HCl Substrate solution Total volume A 20 μL 10 μL 80 μL 40 μL 150 μL B 20 μL 10 μL 120 μL — 150 μL C 20 μL — 90 μL 40 μL 150 μL

[0218] First, add the elastase solution and the test solution (or Tris-HCl solution) and incubate for 15 min, then add the substrate solution and the buffer and incubate for 50 min, and measure the absorbance value at 410 nm.

[0219] Elastase inhibition rate calculation formula: Inhibition rate (%) = 100 x [1- (A-B) / C]

[0220] The results of the elastase inhibition rate of Example 1-6 and Comparative Example 1 are shown in Table 13.

[0221] Table 13 Elastase inhibition rate (%)

[0222] Sample name Concentration Inhibition rate (%) Example 1 10％ 52.35±2.6 Example 2 10％ 71.24±3.08 Example 3 10％ 34.63±0.66 Example 4 10％ 30.11±0.13 Example 5 10％ 75.39±2.95 Example 6 10％ 40.44±0.30 Comparative Example 1 10％ 19.87±0.32 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparative Example 1 Rutin 2 mmol / L 65.26±4.21

[0223] The results show that compared with Comparative Example 1, the elastase inhibition rate of the Potentilla atrosanguinea fermenting liquid prepared in Example 1-6 is significantly improved, indicating that the fermentation process can significantly improve the anti-wrinkle tightening effect of Potentilla atrosanguinea raw materials, and the elastase inhibition effect of Example 5 at 10% concentration is the most significant, with an elastase inhibition rate of 75.39 ± 2.95%.

[0224] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application shall be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A method for fermenting Potentilla anserina, characterized in that, The fermentation method comprises the following steps: S1, grinding and sieving the Potentilla anserina rhizome to obtain the sieved powder; S2, adding the sieved powder to the culture medium to obtain the sterilized liquid after sterilization; S3, inoculating the strain into the sterilized liquid of step S2, and obtaining the Potentilla anserina fermentation liquid after fermentation and sterilization and filtration; The addition amount of the sieved powder in step S2 is 1%-3% w / v; The inoculation amount of the strain in step S3 is 8%-12% v / v; The strain in step S3 is the Ganoderma lucidum strain, the Ganoderma cochileum strain, the Cordyceps militaris strain, the Cordyceps cicadae strain, the Saccharomyces cerevisiae strain or the Bifidobacterium strain. The fermentation temperature in step S3 is 25-30℃, and the fermentation time is 96-120h.

2. The fermentation process according to claim 1, characterized in that, The addition amount of the sieved powder in step S2 is 2% w / v.

3. The fermentation process of claim 1, wherein, The inoculation amount of the strain in step S3 is 10% v / v.

4. The fermentation process of claim 1, wherein, The culture medium in step S2 comprises one or more of anhydrous glucose, soluble starch, peptone and yeast powder.

5. The Potentilla anserina fermentation liquid prepared by the fermentation method of any one of claims 1-4.

6. The use of the Potentilla anserina fermentation liquid of claim 5 in the preparation of cosmetics.

7. Use according to claim 6, characterized in that, The cosmetics have anti-inflammatory, antioxidant or antibacterial efficacy.

8. A cosmetic product, characterized by, The cosmetics comprise the Potentilla anserina fermentation liquid of claim 5.

Images